MXPA00004672A - Dna regulatory element for the expression of transgenes in neurons of the mouse forebrain - Google Patents

Abstract

The present invention provides for a recombinant nucleic acid molecule comprising a region of a calcium-calmodulin dependent kinase IIa promoter operatively linked to a gene of interest. The region of a calcium-calmodulin dependent kinase IIa promoter may comprise an 8.5 kilobase nucleic acid sequence which corresponds to the nucleic acid sequence of ATCC Accession No. 98582, designated pMM281. The present invention also provides a human cell line which has been stably transformed by a recombinant nucleic acid molecule comprising a gene of interest operatively linked to a nucleic acid encoding a calcium-calmodulin dependent kinase IIa promoter region which has a nucleotide sequence corresponding to the sequence of ATCC Accession No. 98581, designated pMM281. The present invention also provides for a transgenic nonhuman mammal whose germ or somatic cells contain a nucleic acid molecule which encodes a gene of interest under the control of CaMKIIa promoter (ATCC Accession No. 98583), introduced into the mammal, or an ancestor thereof, at an embryonic stage. Another embodiment of the present invention is a method of evaluating whether a compound is effective in treating symptoms of a neurological disorder in a subject, using the transgenic mammal as an in vivo test model.

Description

DNA REGULATOR ELEMENT FOR THE EXPRESSION OF TRANSGENES IN THE NEURONS OF THE MOUSE PROSENCELPHAL This application claims the priority of the application of United States Patent Serial No. 08 / 696,137, filed on November 12, 1997, the contents of which are incorporated herein by reference. The invention described herein is made with Government support under Grant No. 50733-03 from the National Institutes of Mental Health. Therefore, the American Government has certain rights in this invention.

BACKGROUND OF THE INVENTION \ Throughout this application, reference is made to several publications by author and date.

? The full citations of these publications can be found in an alphabetically arranged list at the end of the specification that immediately precedes the claims. The descriptions of these publications in their entireties are thus incorporated by reference in this application in order to more fully describe the state of the art as known to those skilled in the art as to the date of the invention described and claimed in the present .

The knowledge that memory has time-dependent phases dates from 1890 when William James first proposed a distinction between a primary or short-term memory, a memory that has to be maintained continuously in consciousness, and a secondary or long-term memory that can be separated from consciousness and can be recalled at will at a later time (James, 1890). According to James' vision, short-term memory P maintains information for a few seconds, while long-term memory holds information for extended periods of time. Subsequent experimental work suggested that these two phases of memory are usually in series and that the transition from short-term memory to long-term memory is facilitated by an increase in prominence or ^ • ^ number of training trials (Ebbinghaus, 1885; Weiskrantz, 1970; Craik and Lockhart, 1972; Wickelgren, 1983; Mandel et al., 1989). The distinction between these two main phases was placed on a firmer biochemical basis when it was found that long-term memory requires the synthesis of new proteins, whereas short-term memory does not require it (Davis and Squire, 1984). These biochemical studies also revealed that short-term memory often lasts many minutes, and therefore was more endured than the primary memory delineated by James. Therefore, these studies suggested that memory a The short term may have subdivisions in turn, and in addition to primary or working memory, there is an intermediate, subsequent stage, independent of protein synthesis, of short-term memory. Additional support for memory subcomponents also emerged from genetic studies in Drosophila and pharmacological studies 9 in rodents and chicks (McGaugh, 1968, Cherkin, 1969, Gibbs and Ng, 1977, Frieder and Allewis, 1982, Rosenz aig. et al., 1993; Tully et al., 1994; Zhao et al., 1995 a and b, Bennet et al., 1996). In addition to being able to distinguish temporal phases in memory storage, studies in humans and monkeys also delineated two distinct neural systems for long-term memory. ^^ term based on the types of information stored. The bilateral lesions of the temporal lobe, central revealed a deterioration in long-term memory, declarative memory for people, places and objects, but these injuries had no impact on the non-declarative memory for perceptual and motor skills. Particularly interesting was the finding that lesions of the temporal lobe system, central, that interfere with declarative memory, only interfere with the long-term form of this memory and not with the components of short-term memory, particularly not with working memory (Scoville and Milner, 1957; Mishkin, 1978; Zola-Morgan and Squire, 1985; Squire, 1987, Overman et al., 1990); Alvarez et al., 1994). These results indicate that structures in the temporal lobe, central, particularly the hippocampus, specifically favor long-term memory, but not some components of short-term memory.

SUMMARY OF THE INVENTION The present invention provides a recombinant nucleic acid molecule comprising an Il-dependent calcium-calmodulin kinase promoter region, operably linked to a gene of interest. The calcium-calmodulin-dependent kinase Il-a promoter region can comprise an 8.5 kilobase nucleic acid sequence corresponding to the nucleic acid sequence with Accession Number ATCC 98582, designated pMM403. The present invention also provides a human cell line that has been stably transformed from a recombinant nucleic acid molecule comprising a gene of interest operably linked to a nucleic acid encoding a calcium-dependent Il-a kinase promoter region. -calmodulin, which has a nucleotide sequence corresponding to the sequence with ATTC Accession Number 98581, designated pMM281. The present invention also relates to a non-human, transgenic mammal whose germ or somatic cells contain a nucleic acid molecule encoding a gene of interest under the control of a CaMKIIa promoter (Accession Number ATCC 98583), introduced into the mammal, or an ancestor thereof, in an embryric stage. Another embodiment of the present invention is a method for evaluating whether a compound is effective in the treatment of the symptoms of a neurological disorder in a subject, which comprises: (a) administering the compound to the non-human, transgenic mammal whose cells germ or somatic cells contain a nucleic acid molecule that codes for a gene of interest under the control of a Ca KIIa promoter, and (b) compare the neurological function of the mammal in the ^^ step (a) with the neurological function of the transgenic mammal in the absence of the compound, to thereby determine whether the compound is effective in the treatment of the symptoms of the neurological disorder in a subject.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1B. Expression of lacZ mRNA in the forebrain of the mouse (Figure 1A) schematic representation of the DNA constructs used for the generation of transgenic mice. Lac-CMK: the CaMKIIa promoter region of 8.5 kbp, as well as 84 nucleotides of the 5 'non-coding exon were fused to the lacZ gene of E. coli. The complete 3 '-UTR of the CaMKIIa mRNA was placed in the 3' direction of the lacZ coding region. lac-A: identical to lac-CMK except that the polyadenylation signal of bovine growth hormone was replaced by the 3'-UTR of CaMKII. nls-lac-CMK, the tet-O promoter (Craig et al., 1993) was linked to a modified lacZ ^ P gene with an in-phase function to the green fluorescent protein (GFP) and a nuclear localization sequence. (FIG. IB) Northern blot analysis of the poly (A) + RNA isolated from the forebrain of the lac-CMK and lac-A mice. Figures 2A-2B: Histochemistry of beta-galactosidase. Figures 3A-3D: Localization of mRNA in situ ^^ of lacZ in the hippocampus. (Figure 3A) in situ hybridization using the lac-Z specific oligonucleotide probes. SM (molecular extract), dendritic layer of dentate gyrus granule cells; SR (stratum radiatum), dendritic layer of the CAI pyramidal cells. (Figure 3B) X-gal staining of the hippocampus of horizontal sections of 20 μm as described in Figure 2. (Bar = 300 μm). Figures 4A-4D. Differential expression of beta-gal within dendrites. (Figure 4A) in situ hybridization against nls-lac-CMK mouse using a lacZ-specific probe. (Figure 4B) detection I histochemistry of ß-gal in the neuron nls-lac-CMK in culture. The MAP2 antibody specifically labels microtubules along the dendritic tree. Marking with MAP2 is indicated in red. The labeling of β-gal is shown in green. The arrows denote β-gal in presumptive dendritic spines. The arrowheads indicate areas of ß-gal staining of ^ P score along the dendrite (Figure 4D) expression of β-gal in a distant portion of the dendritic tree. The heads of the arrows denote scintillation areas of ß-gal. The open arrows show a dendrite that emerges from a neuron, which does not express the nls-lac-CMK transgene (Bar = 10 μM). Figures 5A-5B. Regulation of the transgene ^^ CaMKII-Asp286 with the tTA system. (Figure 5A) Strategy used to obtain transgenic expression regulated by forebrain-specific doxycycline. Two independent lines of transgenic mice are obtained, and the two transgenes are introduced into an individual mouse through pairing. (Figure 5B) Quantification by Southern blotting by RT-PCR of the expression CaMKII-Asp286 of the tet-O promoter, RT-PCR was performed on the total RNA of the forebrain and probed for the expression of the mutant mRNA of CaMKII -Asp286 as it is described (7, 21). Tgl, mouse that has only the CaMKII -transgen tTA promoter (line B). Tg2, mouse that has only the transgene tet-O-CaMKII -Asp286 (line 21). Tgq / Tg2, double transgenic mouse that has both the CaMKI I -transgen tTA promoter (line B) and the tet -O-CaMKII -Asp286 transgenes (line 21). Tgl / Tg2 + Dox, double transgenic mouse treated with doxycycline (2 mg / ml) plus 5% sucrose in drinking water for four weeks. ^ P Figures 6A-6B. Specific activation of the forebrain of a tet-0-lacZ transgene. (Figure 6A) Coronal section of the lac of the transgenic line B, doubled with X-Gal as described (Mayford et al., 1996). Ctx, cerebral cortex, Str, striatum; Hip, hippocampus, Amy, amygdala (Figure 6B) Coronal section obtained with X-Gal from the hippocampus from the translational Lacl and B lac2 lines ^ • ^ double. FALL, body layer of CAI cells; CA3, body layer of CA3 cells, OG, dentate gyrus. Figures 7A-7D. Regional distribution of CaMKII-Asp286 mRNA determined by in situ hybridization (Mayford et al., 1995). The sagittal, central sections of the transgenic, double B13, B21 and B22 lines showing the expression of the CaMKII -Asp286 transgene. B21 / amygdala shows a focus view of a coronal section of the mouse B21 double transgenic line.

Figure 8. Inversion of LTP deficit 10-Hz in CAI of hippocampal cuts. The field EPSP slopes before and after the 1-Hz tetanic stimulation were recorded and expressed as the percentage of the pre-tetanus baseline. The cross sections (400 μm bone) of the mouse hippocampus were prepared and placed in a cross-sectional chamber impregnated with artificial spinal brain fluids as described (Mayford et al., 1995). The post-synaptic potentials, field exciters (EPSP) were produced in response once per minute with bipolar stimulation electrodes, fine tungsten (pulse duration 0.05 ms). Stainless steel recording electrodes were placed in the stratum radiatum. The stimulation force was adjusted to produce 50% of the maximum EPSP obtainable in each cut. The synaptic response of the line ^? basal was collected for 20 minutes. Before tetanus. Tetanus was distributed at 10-Hz for 1.5 minutes at the same intensity as used in recording the baseline. For the treatment with doxycycline, the animals were administered doxycycline (1 mg / ml) at 5% sucrose in drinking water for 2 to 3 weeks, and then the sections were exposed to doxycycline (1 ng / ml) in the impregnated material. All animals were 2.5 to 6 months old at the time of recording or registration. Stimulation at 10 Hz for 1.5 minutes induced transient depression followed by potentiation in wild type mice (123 + 9 9% to 60 minutes after tetanus; n = 12 cuts, 6 mice (_). Tetanus (10 Hz) induced a mild depression in B13 double transgenic mice (89 ± 6% at 60 minutes after tetanus, n = 9 cuts, 3 mice) (") Treatment with doxycycline reversed the defect in B13 mice (132 ± 10%, n = 8 cuts, 4 mice) (•) Treatment with ^ doxycycline had no effect on synaptic potentiation in wild-type mice (122 + 6%, n = 16 cuts, 6 mice) ( V) Figures 9A-9D Reversible deficits in explicit memory learning in mice expressing the CaMKIIa transgene (Figure 9A) The Barnes circular labyrinth (Figure 9B) Percentage of B22 and wild type transgenic mice ^? they met the learning criteria in the circular labyrinth of Barnes. In the circular labyrinth of Barnes (Bach et al., 1995) the mice (2.5 to 6 months of age) were tested once a day until they met the criteria (five out of six sessions with three or fewer errors or until 40 days had elapsed). The order of the holes searched was recorded by an observer who did not know the genotype and condition of doxycycline, and these data was determined by the number of errors. The errors were defined as searches for any hole that does not have the tunnel behind it. The searches included pushing with the nose and deviations of the head over the hole. At the end of each session, the search strategy used was recorded by the observer. The spatial search strategy was operationally defined as the search for the escape tunnel with both error and distance scores = 3. The distance was calculated by counting the number of holes between the first hole searched within a session and the escape tunnel. An analysis of variance of a factor (ANOVA) (sex) did not reveal significant effect in sex for either wild-type transgenic mice, so that the data collapsed through this variable. For the error data, a three-factor ANOVA (genotype), doxycycline and session block) was used with a repeated measurement. For the data of the ^ spatial search strategy, the two groups of B22 transgenic mice were compared with a bidirectional ANOVA (doxycycline and session block) with a repeated measurement. An analysis of chi-squares revealed that the percentage of the B22 transgenics that acquire Barnes labyrinth (0%) was significantly different from the B22 transgenics in doxycycline of both wild type groups (X2 = 53.05, P <0.0001). Four groups of mice were tested: B22 transgenic (n = 6), B22 transgenic in doxycycline (1 mg / ml) for 4 weeks (n = 6), wild types (n = 8), and wild types in fdoxycycline (1 mg / ml) for 4 weeks (n = 7) (Figure 9C). Average of errors through the session blocks composed of five sessions. The values represent the mean of the ± SEM groups. A three-way ANOVA revealed a major effect of the genotype (F [1.23] = 4.28, P = 0.04). (Figure 9D). The percentage of sessions in which the spatial search strategy was used through the ^ session blocks by B22 transgenic mice. The values represent the group means ± SEM. A bidirectional ANOVA revealed a major, significant effect of doxycycline (F [l, 10] = 7.313, P 0.02). Figures 10A-10E. Reversible deficits in implicit memory learning in mice expressing the CaMKIIa transgene. The percentage of The time elapsed from freezing to context (Figure 10A) and suggestion (Figure 10B) 24 hours after training on lines B22 and B21. The values represent the group means ± SEM. A three-way ANOVA revealed a significant three-way interaction for the context (genotype per line by doxycycline) (F [1, 55] 9.177, P = 0.0037) and a significant bidirectional interaction for suggestion (line by genotype) (F [1, 55] = 5.087mp = 0.0281) Six groups of mice were tested: B22 transgenic (n = 6), B22 transgenic in doxycycline for four weeks (n = 11), transgenic B21 in doxycycline for four "weeks (n = 19), wild types (of both B22 and B21 lines) ( n = 11), and wild types (of both B22 and B21 lines) in doxycycline for 4 weeks (n = 8). (Figure 10C) Timeline illustrating the administration of doxycycline and behavioral training and testing. 10D) Context retention and suggested conditioning.Percentage of time elapsed from freezing to context and suggestion six weeks after training.The values represent the group means ± SEM.Scheffe test post-hoc analysis revealed that the mice transgenic B21 that s and switched to water were frozen significantly less in context than B21 transgenic mice in doxycycline (P = 0.01) and wildest types (P = 0.008) and significantly less at suggestion than B21 transgenic mice in doxycycline (P = 0.02) and wild types ( 0 = 0.0088). Three groups of mice were tested, transgenic B21 in doxycycline for four weeks before training and 6 weeks after training (n = 8), transgenic B21 in doxycycline for four weeks before training that were switched to water during the 6 weeks after the training (n = 8) and wild type mice (of the lines both B22 and B21, n = 19). (Figure 10E) The percentage of time elapsed from freezing to an intruder during the first 120 seconds after the mouse was exposed to a rat. The values represent the group means + SEM. Figures 11A-D. The calcineurin transgene is expressed in the forebrain hippocampus of CN98 mutant mice. Figure HA. Schematic representation of the construction of the calcineurin transgene used to generate CN98 mice. Figure 11B. Northern blot analysis of total RNA from CN98 mice. Figure 11C. Enzyme activity determined in extracts of the hippocampus of mice CN98 Dephosphorylation of the a32P substrate peptide was measured in the absence or presence of CaTA + EGTA. The values are the mean ± SEM. Wild type: 4.63 ± 0.44 nmol Pi / min / mg, n = 6; mutant CN98; 8.15 ± 0.57 nmol Pi / min / mg, n = 4, p < 0.001; wild type CN98 + EGTA: 0.427 ± 0.16 nmol Pi / min / mg, n = 6; mutant CN98 + EGTA: 0.32 ± 0.14 nmol Pi / min / mg, n = 4, p > 0.05. Figure 11D. Regional distribution of the calcineurin transgene in CN98 mice determined by in situ hybridization. Figures 12A-12F. Basal synaptic transmission and short-term forms of synaptic plasticity are not dramatically altered by overexpression of calcineurin. "Figure 12A: Entry-exit curve of the fEPSP slope (mV / ms) against the intensity of the stimulus (V) at the synapses of the CAl-collateral Schaffer pyramidal cells in CN98 and wild type mutant mice. as the mean ± SEM Figure 12B Graph of the amplitude of the discharge of pre-synaptic fiber (PSFV, mV) against slope fEPSP in the synapse of the CA-1 collateral cells of Schaffer from a random sample of slices of wildtype CN98 mutant mice Figure 12C.FePSP slope curve (mV / ms) against intensity (V) at the synapse of the CA-1 collateral pyramidal cells of CN98 mutant mice (13 cuts, 4 mice) ) and wild type (16 slices, 4 mice) in the presence of the DNQX agonist (10 μM) of the non-NMDA glutamate receptor and reduced MgSO (50 μM) .The data are presented as the mean ± SEM. synaptic responses mediated by the NMDA receptor r epresentative for one second, 100 Hz tetanus in wild-type cuts in mutants. The measurement bar is 50 ms and 5 mV. Figure 12D. Comparison of PTP in wild-type CN98 mutant mice. The PTP was caused by a one second series of 100 Hz, individual administered in the presence of 50 μm DL-AP5. The data are presented as the mean ± SEM of the normalized fEPSP slope. Figure 12E. Comparison of PPF in wild type CN98 mutant mice with intervals between stimuli of 20, 50, 100 and 250 ms. The data are presented as the mean ± SEM of the second response facilitation with respect to the first "16-cut response of 7 wild-type mice and 15 cuts of 6 mutant mice." Figure 12F. 1 Hz stimulation in CN98 wild type mice and mutants aged 3-4 weeks The data are represented as the mean ± SEM of the normalized fEPSP slope Figures 13A-13D.

^ Calcineurin inhibited L-LTP induced by four series of 100 Hz but not E-LTP induced by a 100 Hz series. Effect of overexpression of calcineurin on LTP in wild type animals CN98 (•) and mutants (o). LTP produced in response by (A) a single series of 100 Hz duration of one second, or (B) four series of 100 Hz separated by five minute intervals. Each point in the time paths represents the slope fEPSP of the mean + SEM normalized to the average of the slope fEPSP pre tetanos. The inserts show representative fEPSP traces just before tetanus and Figure 13A) '1 hour or Figure 13B) 3 hours later. Figure 13C) and Figure 13D): Drug was added at the indicated time in both panels at a concentration of 100 μM. Each point in the time paths represents the mean of the slope fEPSP ± SEM normalized to the mean of the slope pre-drug fEPSP. Inserts W show traces or indications of representative fEPSP just before the addition of the drug and 3 hours later. In (C) the decrease in the slopes of fEPSP caused towards the end of the application of Sp-cAMPS has been previously shown to reflect a decrease mediated by the adenosine-Al receptor transient in the release of glutamate (Frey et al., 1993). "J Figures 14A-F, Effects of protein synthesis and inhibitors of PKA in LTP four series and two series Figure 14A.LTP induced by four series of 100 Hz, with a five-minute inter-tetanus interval in the presence of anisomycin (_, 30 μM) or KT5720 (0, 1 μM) in hippocampal sections of wild type mice, drugs are added beginning 15 minutes before the first tetanus, and washed 15 minutes after the last tetanus. Each point over time represents the mean of the slope of fEPSP + SEM normalized to the mean of the pre-tetanus fEPSP slope. Figure 14B. Effects of prolonged pre-treatment of anisomycin in the LTP induced by four series of 100 Hz. Anisomycin (or, 30 μM) was added 60 minutes before the first tetanus, and washed 15 minutes after the last tetanus. Each point in the course of time represents the slope of fEPSP ± SEM normalized to the mean of the "slope pre-tetanus fEPSP." Without drug: 10 cuts, 8 mice; Anisomycin 4 cuts, 4 mice. Figure 14C. LPT introduced by two 100 Hz series, with an interval between stimuli of 20 seconds, in the presence or absence of anisomycin (or, 30 μM) in wild type hippocampal sections.

Each point in the course of time represents the mean of the slope ± SEM normalized to the mean of the pre-tetanus fEPSP slope. Without drug: 8 cuts, 5 mice, Anisomycin: 7 cuts, 4 mice. Figure 4 D. Effect of the PKA inhibitor, KT5720 (or, 1 μM) in LTP induced by two 100 Hz series in wild type hippocampal slices. Each point in the course of time represents the mean slope of the fEPSP ± SEM normalized to the mean of the pre-tetanus fEPSP slope. Figure 14E. LTP induced by two 100 Hz series in hippocampal slices of CN98 (o) and wild type (•) mutant mice. Each point in the course of time represents the mean of the slope fEPSP in ± SEM normalized to the mean of the ^ Ppendiente fEPSP pre-tetanus. Figure 14F. Effect of PKA inhibitor KT5720 (or, 1 μM) on LTP induced by two hippocampal slices from two 100 Hz series of CN98 mutant mice. Each point in the course of time represents the mean of the slope fEPSP ± SEM normalized to the mean of the slope fEPSP before tetanus. Figures 15A-C. The LTP-induced protocols of two and four series (Figure 15B and 15C), but not one series (Figure 15A), were reduced to wild type mice (•) and mice overexpressing the calcineurin transgene with the tTA system ( o).

Figure 14A. Wild type (•): 14 cuts, 9 mice; Tet-CN279 mutants (_): 6 cuts, 3 mice; '^ P Tet-CN273 (o) mutants: 4 cuts, 3 mice; Figure 15B. Wild type (•): 7 cuts, 4 mice; Tet-CN273 mutants (o): 6 cuts, 3 mice. Figure 15C. Wild type (•): 10 cuts, 8 mice; mutants Tet-CN279 (_) L 7 cuts, 4 mice. Figures 16A-D. The basal synaptic transmission and the short-term forms of synaptic plasticity are not altered by overexpression of calcineurin with the tTA system. Figure 16A. Entry-exit curve of the fEPSP slope (mV / ms) versus stimulus intensity (V) at the synapse of the CA-Collateral Schaffer pyramidal cells in mutant Tet-CN279 (9 cuts, V4 mice) and Tet-CN273 ( 20 cuts, 7 mice) and wild type mice (21 cuts, 9 mice). The data are presented as mean ± SEM. Figure 16B. Input-output curve of fEPSP (mV / ms) against intensity (V) at the synapse in the CAl-collateral cells of Schaffer in mutant Tet-CN279 (8 cuts, 4 mice) and Tet-CN273 ^ P (8 cuts) , 4 mice) and wild type mice (21 cuts, 8 mice) in the presence of the antagonist DNQX (10 μM) of the non-NMDA glutamate receptor and Reduced MgSO 4 (50 μM). The data are presented as mean ± SEM. Figure 16C. Comparison of PTP in mutant Tet-CN278 (6 cuts, 3 mice) and Tet-CN273 (8 cuts, 4 mice) and wild type mice (15 ^ ft cuts, 8 mice). The PTP was produced by a one second series of 100 Hz, individual, administered in the presence of 50 μM DL-AP5. The data are presented as mean ± SEM in the slope fEPSP normalized. Figure 16D. Comparison of PPF in mutant Tet-CN273 (9 cuts, 4 mice), and Tet-CN279 (13 cuts, 4 mice) and wild type mice (27 cuts, 10 mice) with intervals between stimuli of 20, 50, 100 and 250 ms The data are presented as the mean ± SEM of the facilitation of the second response in relation to the first response.

Figures 17A-B. Figure 17A. Caliculina A (750 nM) rescues the deficit in LTP induced by two ^ P 100 Hz series in Tet-CN279 mutant mice. Each point in the course of time represents the average of the slope fEPSP ± SEM normalized to the average of the pre-tetanus slope. Wild type (•), 7 cuts, 4 mice), Mutant with pre-treatment of caliculin A (o), 6 cuts, 3 mice), wild type with pre-treatment of caliculin A (U), 6 ^ P cuts, 3 mice ). Figure 17B. The deficit of LTP seen in the cuts of the Tet-CN279 mutants can be reversed by suppressing the expression of the transgene with doxycycline. Each point in the course of time represents the average of the slope fEPSP ± SEM normalized to the average of the pre-tetanus slope. Figure 18A-B. An independent phase of the B synthesis of protein, dependent on PKA of LTP, I-LTP exists in mouse hippocampus. Figure 18A-B. Schematic representation of the time course of the induced potentiation by protocol of a series (lower panel, Figure 18B) and four series (upper panel, Figure 18A). Figures 19A-C. CN98 mutant mice have damaged spatial memory in the labyrinth of Barnes when they are tested with a trial one day, but they have normal memory in a labyrinth suggestion version.

Figure 19A. Percentage of CN98 mice that acquired the spatial and suggestive versions of ^ P Barnes labyrinth with a 1-day trial. Figure 19B. The mean number of errors made by CN98 mice in the spatial version of Barnes' labyrinth with a 1-day trial. Figure 19C. The mean of errors made by CN98 mice in the version with suggestion of Barnes maze with a one-day trial. ^ P Figures 20A-C. CN98 mutant mice have a normal memory in the labyrinth of Barnes with four one-day trials. Figure 20A. Percentage of CN98 mice that acquired the spatial version of the Barnes labyrinth with four one-day trials. Figure 20B. The mean number of trials and days for acquisition for CN98 mice in the spatial j | B version of the Barnes maze with either one to four trials a day. Figure 20C. The average number of errors made by CN98 mice in the spatial version of Barnes' labyrinth with four attempts a day. Figure 21. CN98 mutant mice have short-term memory, normal in the task of recognizing a new object. A preference index (Pl) greater than 100 indicates preference for the new object during the test. A Pl equal to 100 indicates no preference, while a Pl less than 100 indicates a preference for the familiar object. Figures 22A-C. Regulated expression of ^ P transgene of calcineurin with the tTA system. Figure 22A. The strategy to obtain the expression regulated by doxycycline of the calcineurin transgene in mice. Mice of line B have the transgene of the CaMKIIa-tTa promoter and mice of the CN279 and CN273 lines, the transgene of the teto-CaM-AI promoter. Both transgenes are introduced into the ^ P same mouse through the coupling to generate the Tet-CN279 and Tet-CN273 mice. In mice Tet-CN279 and Tet-CN273, the expression of the calcineurin transgene is activated by tTA and can be repressed by doxycycline. Figure 22B. Analysis by transfer Northern of the total forebrain RNA of the wild-type and mutant Tet-CN279 and Tet-CN273 mice on or off doxycycline and RT-PCR of the total RNA of the forebrain of Tet-CN279 and Tet-CN273 mice, wild-type, mutant mice Tet-CN279 and Tet-CN273 in or out of doxycycline. Figure 22C. Enzyme activity determined in extracts of the hippocampus of Tet-CN279 and Tet-CN273 mice in or without doxycycline. Dephosphorylation of a labeled peptide substrate was measured in the absence or presence of the EGTA chelator of Ca2 + in wild type mice and mutants Tet-CN279 and Tet-CN273 in or outside doxycycline. The values are the average ± SEM. Wild type (Tet-CN279 and Tet-CN273): 3.58 ± 0.26 nmol Pi / min / mg, n = 6; mutant Tet-CN279: 7078 ± 0.70 nmol Pi / min / mg, n = 4, p > 0.0001; mutant Tet-CN273 8.39 ± 0.39 nmol Pi / min / mg, n = 3, p > 0.001; mutant Tet-CN279 in dox: 3.95 ± 0.48 nmol Pi / min / mg, n = 4, p > 0.05; mutant Tet-CN273 in dox: 4. 23 ± 0.36 nmol Pi / min / mg, n = 3, p > 0.05; wild type (Tet-CN279 + Tet-CN273) + EGTA: 0.432 ± 0. 11 nmol Pi / min / ng, n = 7; mutant (Tet-CN279 + Tet-CN273 + EGTA: 0.287 + 0.17 nmol Pi / min / mg, n = 7, p> 0.05) Figures 23A-D The expression of the calcineurin transgene is restricted mainly to the subfield I fell in the hippocampus of mutant mice Tet-CN279 and Tet-CN273 and was repressed by doxycycline. The regional distribution of the calcineurin transgene determined by in situ hybridization in wild-type mouse brain sagittal sections Tet-CN279, mutant Tet-CN279 and Tet-CN273 in or out of doxycycline. Figures 24A-G. Mutant mice CN98 and Tet-CN279 do not use the spatial search strategy. Figure 24A. Representative examples of the search strategies used in the spatial region of the Barnes circular maze task. Figures 24B-G. The use of the random search strategy for mice CN98 (B) and Tet-CN279 (C), of the serial search strategy for mice CN98 (D) and Tet-CN279 (E) and of the spatial search strategy for mice CN98 (F) and Tet-CN279 (G).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant nucleic acid molecule comprising a calcium-calmodulin dependent kinase IIOI promoter region, operably linked to a gene of interest. The calcium-calmodulin-dependent kinase promoter region Ila can comprise a nucleic acid sequence of 8.5 kilobases corresponding to the nucleic acid sequence of accession number ATCC 98582, which was deposited on November 11, 1997 under the clauses of the Treaty from Budapest with the American Culture Collection Species (see details later). The gene of interest may comprise a gene of acalcinurin, a gene comprised in brain function, a growth factor gene, an ion channel gene, a kinase gene, a neurotransmitter gene, a neurotrophic factor gene, a gene of phosphatase, a recombinant gene, an indicator gene, a receptor gene, a transcription factor transcription factor gene, a transcription factor gene. The neurotrophic factor may comprise the ciliary neurotrophic factor; nerve growth factor; neurotrophic factor 4/5; neurotrophic factor derived from the brain; or neurotrophic factor derived from glial cells. The neurotransmitter gene may comprise a serotonin gene, a dopamine gene, an epinephrine gene. One embodiment of the present invention is a line of human cells that has been stably transformed by a recombinant nucleic acid molecule comprising a gene of interest operatively linked to a nucleic acid encoding a kinase promoter region dependent on Calcium-calmodulin having a nucleotide sequence corresponding to a sequence of the accession number of ATCC 98581 or 98582, deposited on November 11, 1997. The gene of interest may be a gene of acalcinurin, a gene included in brain function , a gene for the growth factor, ^ a ion channel gene, a kinase gene, a neurotransmitter gene, a neurotrophic factor gene, a phosphatase gene, a recombinase gene, a reporter gene, a receptor gene, a transactivator transcription factor gene, a Gene of the transcription factor. The cell line can be a line of human neuronal cells. The present invention also relates to a transgenic non-human mammal whose germ or somatic cells contain a nucleic acid molecule encoding a gene of interest under the control of a CaMKIIa promoter (Accession Number ATCC 98583), introduced into the mammal, or in an ancestor of it, in an embryonic stage. The gene of interest may be a gene of acalcinurin, a gene involved in brain function, a gene of the growth factor, an ion channel gene, a kinase gene, a neurotransmitter gene, a neurotrophic factor gene, a gene of phosphatase, a recombinase gene, a reporter gene, a receptor gene, a ^ P transcription factor transcription factor gene, a transcription factor gene. The gene of interest can be any gene. The nucleic acid molecule that is the transgene of the transgenic non-human mammal may contain an appropriate piece of the DNA of the mammalian genomic clone designed by homologous recombination. Another embodiment of the present invention is a method for treating a neurological disorder in a subject, which comprises administering to the subject an effective amount of the recombinant nucleic acid comprising a calcium-calmodulin dependent kinase promoter region linked operably to a gene. of interest to express the gene of interest in the subject and thus treat the neurological disorder. The neurological disorder can be amnesia, Alzheimer's disease, amyotrophic lateral sclerosis, a brain injury, cerebral senility, chronic peripheral neuropathy, a cognitive disability, a degenerative disorder associated with learning, Down syndrome, dyslexia, amnesia or amnesia induced by electric shock, Guillain-Barre syndrome, head trauma, Huntington's disease, learning disability, memory deficiency, memory loss, mental illness, mental retardation, cognitive or memory dysfunction, dementia for several infarcts and senile dementia, myasthenia gravis, a neuromuscular ^ P disorder, Parkinson's disease, Pick's disease, a reduction in spatial memory retention, senility, or Turret's syndrome. Another embodiment of the present invention is a method for evaluating whether a compound is effective in the treatment of the symptoms of a neurological disorder in a subject comprising: (a) administering the compound to the non-human mammal, Transgenic ^ p whose germ or somatic cells contain a nucleic acid molecule that codes for a gene of interest under the control of a CaMKIIa promoter, and (b) compare the neurological function of the mammal in step (a) with the neurological function of the transgenic mammal in the absence of the compound, thereby determining whether the compound is effective in treating the symptoms of the neurological disorder in a subject. The neurological disorder may be amnesia, Alzheimer's disease, amyotrophic lateral sclerosis, brain injury, cerebral senility, chronic peripheral neuropathy, a cognitive disability, a degenerative disorder associated with learning, Down syndrome, dyslexia, amnesia or amnesia induced by electric shock, and Guillain-Barre syndrome, head trauma, Huntington's disease, a learning disability, a memory deficiency, memory loss, mental illness, mental retardation, cognitive or memory dysfunction, dementia for several infarcts and senile dementia, myasthenia gravis, a neuromuscular disorder, Parkinson's disease, Pick's disease, a reduction in the retention of spatial memory, senility, or Turret's syndrome. The compound can be an organic compound, a nucleic acid, a small molecule, an inorganic compound, a lipid, or a synthetic mk compound. The mammal can be a mouse, a sheep, a bovine, a canine, a pig or a primate. The subject can be a human. The administration may comprise intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; Liposome-mediated distribution; Gene bombardment; topical, nasal, oral, anal, ocular or otic distribution. The present invention provides a method for evaluating whether a compound is effective in treating symptoms of a neurological disorder of a subject comprising: (a) contacting a human neuronal cell of the human neuronal 9P cell line that has been transformed Stably by a recombinant nucleic acid molecule, comprising a gene of interest operably linked to a nucleic acid encoding a calcium-calmodulin-dependent kinase promoter region with the compound, and (b) comparing cell function, The neuronal cells of the neuronal cells in step (a) with cellular, neuronal function in the absence of the compound, thereby determining whether the compound is effective in the treatment of the symptoms of the neurological disorder. The present invention also provides a method for mitigating symptoms in a subject suffering from a neurological disorder, which comprises (??? administering to the subject an effective amount of the compound evaluated by the above methods in an amount effective to treat the symptoms in the subject suffering from a neurological disorder.) The cell population, neuronal may be a cell population, neuronal, aged, a population cellular, neuronal, electrically stimulated, or a cell population associated with a learning disability or a neurological disorder.The cellular, neuronal population can be from the CAI or CA3 region or the hippocampus.

As used herein, the term "Neural degradation" includes functional impairment of neuronal cells, characteristics of degeneration associated with age or characteristics of an association with a neurological disorder. "Neural degradation" also includes cognitive impairments that may be associated with aging, Alzheimer's, lateral sclerosis, amyotrophic, ^ P chronic peripheral neuropathy, use of drugs or alcohol, treatment by electro-shock or trauma, Guillain-Barre syndrome, Huntington's disease, a learning disability, a memory deficiency, a mental condition, myasthenia gravis, Parkinson's disease and reduction in the retention of spatial memory. As used herein, the term ^ B "learning disability" includes a deficit of memory or inability of the hippocampus concurrent with an electrophysiological deficit. As used in this, the term "stimulate a population of neuronal cells" includes electrical stimulation to an electrophysiological response elicited from the population of neuronal cells, treat the population of neuronal cells with a compound or a drug to produce a response, apply tetanus to the population of neuronal cells to produce an electrophysiological response, treating a subject with a compound, the compound that is capable of stimulating the neuronal cell of the subject or perfusing a solution containing a composition or compound on the population of neuronal cells. The answer may be a long-term potentiation of late phase, a long-term potentiation of early phase. The population of neuronal cells may be in a cutoff of ^ P hippocampus in. vi tro, in a subject in vi vo, or in another neuronal tissue. As used herein, the term "normal neuronal cell population" includes a population of neuronal cells derived from a subject that does not appear to have neuronal degradation due to aging, a neurological disorder, an inability to learn, exposure to trauma or ^ fc electric shock. As used herein, the term "cognitive disorder" includes a learning ability or a neurological disorder that may be Alzheimer's disease, a degenerative disorder associated with learning, a learning disability, memory or cognitive dysfunction, cerebral senility , dementia due to several infarcts and senile dementia, amnesia or amnesia induced by electric shock. Another embodiment of the present invention is a method for treating a subject with a cognitive memory disorder or learning disability, which comprises administering to the subject a therapeutically effective amount of a transgene capable of mitigating the symptoms of the cognitive disorder of the patient. memory or learning disability in the subject, thereby treating the cognitive memory disorder or the learning disability in the subject, wherein the transgene is made from a W construction derived from the CaMKIIa promoter (ATCC Accession Number 98581 or 98582, filed on November 11, 1997). The transgene may be associated with a suitable pharmaceutically acceptable carrier and administered intravenously or through the CSF for transient effects. The subject can be a mammalian subject or a human subject. Administration can be intralesional, intraperitoneal, intramuscular or intravenous fl) injection; infusion; distribution mediated by liposomes; Gene bombardment; topical, nasal, oral, anal, ocular or otic distribution. In the practice of any of the methods of the invention or preparation of any of the pharmaceutical compositions a "therapeutically effective amount" is an amount that is capable of mitigating the symptoms of the cognitive disorder of the subject's memory or learning. Therefore, the effective amount will vary with the subject being treated, as well as the condition in question. For the purposes of this invention, the methods of administration may include, but are not limited to, cutaneous, subcutaneous, intravenous, parenteral, oral, topical or aerosol administration. As used herein, the term "suitable pharmaceutically acceptable carrier" encompasses any of the pharmaceutically acceptable, normal carriers, such as phosphate buffered saline, water, emulsions, such as oil / water emulsion or a triglyceride emulsion, Various types of wetting agents, tablets, coated tablets and capsules. An example of a triglyceride emulsion, acceptable useful in the intravenous and intraperitoneal administration of the compounds is the triglyceride emulsion commercially known as Intralipid®. Typically, these carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talcum, vegetable fats or oils, gums, glycols, or other known excipients. These carriers may also include flavor and color additives or other ingredients. This invention also provides pharmaceutical compositions which include therapeutically effective amounts of protein compositions and compounds capable of mitigating the symptoms of cognitive memory disorder or learning in the subject of the invention together with diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. suitable for the treatment of neuronal degradation due to aging, a learning disability, or a neurological disorder. These compositions are liquids or formulations lyophilized or otherwise dried from P and include diluents of various buffer contents (eg, Tris-HCl., Acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts) solubilizing agents (e.g., glycerol, polyethylene glycerol), antioxidants (e.g., f ascorbic acid, sodium metabisulfite), preservatives ( for example, Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (eg, lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the compounds, formation of complexes with metal ions, or incorporation of the compound in or on preparations in the form of particles of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or in liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte spectra, or spheroplasts. These compositions will influence the physical state, solubility, stability, speed, and in vivo release and speed of depuration of the compound or compositions. The choice of compositions will depend on the physical and chemical properties of the compound capable of mitigating the symptoms of cognitive memory disorder or the inability to learn w in the subject. Controlled or sustained release compositions include formulation in lipophilic deposits (e.g., fatty acids, waxes, oils). Also comprised by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines) and the compound coupled to antibodies.^ fc induced against the tissue-specific receptor, ligands or antigens or coupled to ligands of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate protective coatings of particulate forms, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. Portions of the compound of the invention can be "labeled" by association with a detectable labeling substance (eg, radiolabeled with 125 I or biotinylated), to provide β / useful reagents in the detection and quantification of the compound or its cells that carry the receptor or its derivatives in solid tissue and fluid samples such as blood, brain spinal fluid or urine. When administered, compounds are often rapidly cleared from the circulation and therefore can produce a relatively short pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive compounds may be required to sustain therapeutic efficacy. Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol and polypropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone or polyproline are known to exhibit substantially prolonged half-lives in the blood after intravenous injection. that the corresponding unmodified compounds do (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). These modifications can also increase the solubility of the compound in the aqueous solution, eliminate aggregation, improve the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As the result, the desired biological activity may be achieved by the administration of these polymer-compound adducts, less frequently or at lower doses than with the unmodified compound. The binding of polyethylene glycol (PEG) to the compounds is particularly useful because PEG has very low toxicity in mammals (Carpenter et al., 1971). For example, a PEG adduct of adenosine deaminase was approved in the United States of America for use in humans for the treatment of severe combined immunodeficiency syndrome. A second advantage obtained by the conjugation of PEG is that of effectively reducing the immunogenicity and antigenicity of heterologous compounds. For example, a PEG adduct of a human protein may be useful for the treatment of the disease in other mammalian species without the (At the risk of activating a severe immune response.) The compound of the present invention capable of mitigating the symptoms of a cognitive memory or learning disorder may be distributed in a microencapsulation device to reduce or prevent the immune response of the host, against the compound or against cells that can produce the compound The compound of the present invention can also be distributed in a microencapsulated manner in a membrane, such as a liposome.

Polymers such as PEG can conveniently be attached to one or more residues of 'amino acids, reactive in a protein such as the alpha-amino group of the amino-terminal amino acid, the epsilon-amino groups of the side chains of lysine, the sulfhydryl groups of the side chains of cysteine, the carboxyl groups of the side chains of aspartyl and glutamyl, the alpha-carboxyl group of the carboxy-terminal amino acid, the lateral chains of tyrosine, or activated derivatives of the glycosyl chains attached to certain residues of asparagine, serine or threonine. Many activated forms of PEG suitable for direct reaction with proteins have been described. PEG reagents useful for reaction with the amino protein groups include active esters of carboxylic acid or derivatives of Carbonate, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole, or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing the maleimido or haloacetyl groups are useful reagents for the modification of the free sulfhydryl groups of the protein. Likewise, PEG reagents containing amino-hydrazine or hydrazine groups are useful for the reaction with the aldehydes generated by periodate oxidation of the carbohydrate groups in proteins.

In one embodiment, the compound of the invention is associated with a pharmaceutical carrier that includes a pharmaceutical composition. The pharmaceutical carrier can be a liquid and the pharmaceutical composition will be in the form of a solution. In another embodiment, the pharmaceutically acceptable carrier is a solid and the composition is in the form of a powder or tablet. In a further embodiment, the pharmaceutical carrier is a gel and the composition is in the form of a suppository or cream. In a further embodiment, the active ingredient can be formulated as a part of a transdermal, pharmaceutically acceptable patch.

Transgenic mice The methods used to generate transgenic mice are well known to those skilled in the art. For example, one can use the manual entitled "Manipulating the Mouse Embryo" by Brigid Hogan et al. (Ed. Cold Spring Harbor Laboratory) 1986. This invention further provides a transgenic non-human mammal whose germ or somatic cells contain a nucleic acid molecule comprising an 8.5 kb promoter region of the mouse CaMKIIa promoter, designated pMM403 (Accession Number ATCC 98582, filed November 11, 1997 ) which is operably linked to a gene of interest, introduced into the mammal, or an ancestor thereof, in one step. In one embodiment, the CaMKIIa promoter region of approximately 8.5 kb was granted Accession number ATCC 98582 which was deposited on November 11, 1997 with the American Species Crop Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 , USA, under the clause of the Budapest Treaty for the International flf Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Another embodiment of this invention is a non-produced 3 'region of the mouse CaMKII gene, designated pMM281, accession number of ATCC 98581, deposited on November 11, 1997, which was also deposited with the American Species Cultivation Collection under the Budapest Treaty clause for the ^ ft International Deposit Recognition Microorganisms for the Purposes of Patent Procedure. Another embodiment of this invention is a 3 'mouse intron designated pNN265, ATCC Accession Number 98582 which was deposited on November 11, 1997 with the American Species Crop Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland. 20852, USA, under the clause of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The gene of interest will be expressed under the control of the CaMKIIa promoter region, therefore the expression of the gene of interest will be located specifically to the hippocampal region of the mammalian brain. This invention provides a non-human, transgenic mammal whose cells can be transfected with a suitable vector with an appropriate sequence designed to reduce the expression levels of the deleterious genes in the hippocampus of the mammal. The non-human, transgenic mammal can be transfected with a suitable vector containing an appropriate piece of the genomic clone designed by homologous recombination. Alternatively, the non-human, transgenic mammal can be transfected with a suitable vector encoding an appropriate ribozyme or anti-sense molecule. See, for example, Leder and Stewart, U.S. Patent No. Aft 4,736,866 for methods for the production of a transgenic mouse. Transgenic mice have been generated using a construct which is an embodiment of the present invention (an 8.5 kb region of the CaMKIIa promoter which drives a gene of interest which may additionally comprise a 3 'untranslated region). For example, the gene of interest may be a lacZ gene, a CRE-recombinase gene, a transactivator transcription factor gene of tet-O-tetracycline, an akalcinurin gene, a phosphatase gene, or any gene included in brain function The gene of interest can be any gene capable of being expressed as a heterologous gene driven by the CaMKIIa promoter. The gene of interest may be a neurotrophic factor such as ciliary neurotrophic factor (see U.S. Patent No. 4,997,929); nerve growth factor (see, Patent of the United States No. 5,169,762); W A / 5 neurotrophic factor (see, PCT International Publication No. WO 92/05254); neurotrophic factor derived from the brain (see, U.S. Patent No. 5,180,820); neurotrophic factor derived from glial cells (see, PCT International Publication No. WO 93/06116) or any other neurotrophic factor (see Application European EP 0 386 752 Al). The descriptions of these publications in their entireties are thus incorporated by reference in this application in order to more fully describe the prior art as known to those skilled therein as to the date of the invention described and claimed in the present. The gene of interest may be a neurotrophic factor or a cytokine or a growth factor. These factors may include transforming growth factor-beta (TGF-β), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), NT-4, NT-5, NT-4/5, factor of nerve growth (NGF), activins, agrin, cell differentiation factor fl (CDF), glial cell growth factor (GGF), and the differentiation factor neu (NDF), ARIA, and heregulinas. The gene of interest may be a gene encoding a pharmaceutically important protein, a transcription factor, or an agonist, an antagonist, a kinase, a phosphatase, a nitric oxide synthase, CREB, a receptor, or a fl recombinase. The gene of interest can be any gene that codes for a protein that is functionally significant in brain functions, such as memory, cognitive functions and learning. The gene of interest may be a neurotransmitter gene. The neurotransmitter can be a serotonin gene, a dopamine gene or a ("Epinephrine." The gene of interest may be a gene encoding a neurotransmitter receptor protein.) The construction of the CaMKIIa promoter (which includes a CaMKII promoter region and a gene of interest) can be used to treat any disease where they are changed or alter the expression levels of genes in the hippocampus (forebrain), from those that will be presented under normal conditions. For example, ion channel levels or activities are not abnormal in some neurological disorders. Neurological disorders affecting the central nervous system, memory or cognitive functions can also be treated via the construction of the CaMKII promoter. These disorders can be the result of the normal aging process or result of nervous system damage from trauma, surgery, ischemia, infection or metabolic disease. The neurological disorder may be a neuromuscular ^ P disorder. Examples of neurological disorders include Alzheimer's disease, myasthenia gravis, Huntington's disease, Pick's disease, Parkinson's disease and Turret's syndrome. The gene of interest can be any gene that is identified or known that is included in the development of a neurological state. For example, genes that may be afflicted in Alzheimer's disease may be a gene of interest, see, for example, PCT Application No. PCT / EP93 / 03581, International Publication No. WO 94/13798. This invention provides a method for altering the expression of the neuroreceptor. In this method, the nucleic acid molecule comprising a promoter that is CaMKIIa that activates or activates the expression of a gene of interest is administered to a subject which can result in a change in the expression of the neuroreceptors. 6 § This invention provides the improvement of memory of a subject. fl) Another embodiment of this invention is wherein the gene of interest is a ribozyme that is capable of cleaving the mRNA that is produced by a neuronal cell. See, Ceche, et al., Patent of the States United No. 4,987,071; Altman et al., Patent of the United States No. 5,168,053; Haseloff et al., U.S. Patent No. 5,254,678, ^ Published European Application No. Hampel et al., EP 360,257. This invention also provides a replicable vector containing the CaMKIIa promoter sequence and a host cell containing this vector. This expression vector can be a prokaryotic expression vector, a eukaryotic expression vector, a mammalian expression vector, a yeast expression vector, or a baculovirus expression vector or an insect expression vector. Examples of these vectors include PKK233-2, pEUK-Cl, pREP4, pBlueBachHisA, pYES2, PSE280 or pEBVHis. Methods for the use of these replicable vectors can be found in Sambrook, et al., 1989 or in Kriegler 1990. The host cell can be a eukaryotic cell, a somatic cell, a germ, a neuronal cell, a myocyte, a cell of mammalian carcinoma, a lung cell, a prokaryotic cell, a virus packaging cell, or a barley cell. An "indicator molecule" as defined herein is a molecule or atom that, by its chemical nature, provides an identifiable signal that allows detection of the circular oligonucleotide. The reporter molecule can be encoded by a reporter gene. The detection can be either qualitative or quantitative. The present invention contemplates the use of any of the P indicator molecules commonly used and including radionucleotides, enzymes, biotins, psoralens fluorophores, chelated heavy metals and luciferin. The most commonly used indicator molecules are either enzymes, fluorophores, or radionucleotides linked to the nucleotides that are used in the synthesis of circular oligonucleotides.

Commonly used enzymes include peroxidase ^ k radish, alkaline phosphatase, glucose oxidase and a-galactosidase, among others. The substrates to be used with the specific enzymes are chosen in general because a colored product is formed in a detectable form by the enzyme acting on the substrate. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugate; for horseradish peroxidase, 1,2-phenylenediamine, 5-aminosalicylic acid or toluidine are commonly used. Methods for using these hybridization probes are well known and some examples of this methodology are provided by Sambrook et al., 1989.

Gene Therapy Several methods have been developed during the last decade for the transduction of genes in mammalian cells for potential use in gene therapy. In addition, the direct use of plasmid DNA has been used to transfer genes, fl) retroviruses, adenoviruses, parvo-viruses and herpes-viruses (Anderson et al., 1995, Mulligan, 1993, the contents of which are incorporated in their total in the present application). For the transfer of genes into ex vivo cells and the subsequent reintroduction into a host, retroviruses have been the vector of choice. The advantages are that the retrovirus infection is highly efficient and that the pro-virus ^ p generated after the infection is stably integrated into the host's DNA. However, a disadvantage is that stable integration requires cell division, and many of the hematopoietic progenitor cells, predecessors that will be the preferred targets of gene therapy, are not divided under the conditions used for infections and therefore do not incorporate the virus, or if they can not retain their potential to completely reconstitute themselves in a host. With all this and this problem, it is possible that the cells that initiate long-term culture, which can be transduced by the retroviruses, may be sufficient to re-populate compartment with cells that are going to have a relatively long and stable life. Many current gene therapy protocols use murine retroviral vectors to describe therapeutic genes in target cells; this process, which is called transduction, mimics the early events of the retroviral infection. The fl) crucial difference is that, different from the competent replication retroviruses, the genome of the vector packed inside the viral coat contains no genes for viral proteins and thus is incapable of replication. For example, a vector would be designed to have long, 3 'and 5' terminal repeat sequences necessary only for the integration of the viral DNA intermediate in the chromosome of the target host cell and a packaging signal that allows packaging in the structural viral proteins, supplied by the in trans packaging line (Miller, 1992; Wilson et al., 1990; the content of which is incorporated in its entirety in the present application.) Retroviral constructions are elaborated in which the DNA of the interest (that is, the gene that one wishes to be expressed under the control of the CaMKII 5 promoter, expression specifically localized to the forebrain, hippocampal regions) and inserted in the downstream direction of the CaMKIIa promoter to generate a vector. genomic is the terminal step for retroviral vectors, defective.They can not elaborate viral proteins in the cells transduced with the vector packed and therefore they can not produce the progeny virus. The retroviral constructs of the CaMKIIa promoter are transfected into virus packaging cell lines to generate infectious but not f ™ replicating virus particles. These virus packaging cell lines are known to those skilled in the art. Methods of cloning and retroviral infection of cell lines are well known to one skilled in the art and detailed protocols can be found in Kriegler, 1990. Production lines with high virus titers are chosen for their ability to transduce to the lines of neuronal, human cells resulting in the expression of the gene of interest in this cell line. There are several protocols for human gene therapy that have been approved for use by the Recombinant DNA Advisory Committee (RAC) that conforms to a general protocol of target cell infection and administration of transfected cells (see, for example, Blaese, RM, et al., 1990; Anderson, WF, 1992; Culver, KW et al., 1991). In addition, U.S. Patent No. 5,399,346 (Anderson, W. F. et al., March 21, 1995, U.S. Patent Application Serial No. 220,175) which describes the procedures for the transfer of retroviral genes. The contents of these support references are incorporated in their entirety in the present application. It may be necessary to select a particular sub-population in cells originally harvested for use in the infection protocol. Then, a retroviral vector containing the gene (s) of interest will be mixed in the medium of fl) culture. The vector binds to the surface of the subject's cells, enters the cells and inserts the gene of interest randomly into a chromosome. The gene of interest is now stably integrated and will remain in place and will pass to all fixed cells as the cells grow in number. The cells can be extended in the culture for a total of 9-10 days before the infusion of flfc (Culver et al., 1991). As the duration of time increases, the target cells are left in culture, the possibility of contamination also increases, therefore a shorter protocol will be more beneficial. In addition, the currently reported transduction efficiency of 10-15% is well below the linear transduction efficiency of 90-100% that will allow the elimination of the selection and expansion parts of the currently used protocols and reduce the opportunity of the contamination of target cells.

This invention provides the construction of retroviral vectors containing the cDNA for transactivation factor that is 3 'to the CaMKIIa gene. The efficiency of transduction of these vectors can be tested in cell culture systems. In one embodiment of the above method, the nucleic acid molecule is incorporated into a liposome to allow administration to the subject. fl) Methods of incorporation of nucleic acid molecules into liposomes are well known to those skilled in the art. In another embodiment of this method, the molecule can be distributed via transfection, injection or viral infection. Other methods of nucleic acid distribution and nucleic acid compositions as discussed herein include gene-mediated transfer.

Viral viruses, small particle bombardment, receptor-mediated endocytosis, and intralesional, intraperitoneal, or intramuscular injection. There are several protocols for human gene therapy that have been approved for use by the Recombinant DNA Advisory Committee (RAC) that are adapted to a general protocol of infection of target cells for administration of transfected cells, (see for example, Blaese, RM, et al., 1990; Anderson, WF, 1992; Culver, KW et al., 1991). In addition, U.S. Patent No. 5,399,346 (Anderson, W.F., et al., March 21, 1995, U.S. Patent Application Serial No. 220,175) describes those for the transfer of retroviral genes. The contents of these support references are incorporated in their entirety in the present application. Retrovirus-mediated gene transfer requires target cells that are undergoing cell division in order to achieve stable integration in this way, cells of a subject are often collected by removing the blood or bone marrow. Several methods have been developed during the last decade for the translation of genes into mammalian cells for potential use in gene therapy. In addition, the direct use of Plasmid DNA to transfer genes, retroviruses, adenoviruses, parvo-viruses and herpes-viruses (Anderson et al. ^ k al. , nineteen ninety five; Mulligan, 1993; The contents of which are incorporated in their entirety in the present application). Another embodiment of this invention is a method for inducing neuronal regeneration, which comprises administering to a subject an effective amount of the CaMKIIa promoter construct that drives a gene of interest and a pharmaceutically acceptable carrier to induce the formation of a synaptic junction between the neuron and a target cell. The target cell can be a neuronal cell, an endocrine cell, a muscle cell or any cell capable of forming a neuro-B) muscle junction. The expression of the gene of interest can facilitate the incorporation of grafts in nervous tissue or to promote the regeneration of the nerves after damage by trauma, infarction, infection or post-operatively. Alternatively, the transgenic non-human mammal can be transfected with an affected vector f) encoding an appropriate ribozyme or anti-sense molecule. See for example, Leder and Stewart, U.S. Patent No. 4,736,866 for methods for the production of a transgenic mouse. This anti-sense vector can be used as a gene therapy in humans to inhibit the expression of a gene in the forebrain. This invention is illustrated in the experimental details section that follows. These sections set forth to aid in the understanding of the invention, but are not proposed, nor should they be construed as, in any way limiting the invention as set forth in the claims that follow thereafter.

EXPERIMENTAL DETAILS Example 1: The 3'-untranslated region of CaMKIIa is ^ P a cis-action signal for the location and. Translation of mRNA in dendrites. Neural signaling requires that synaptic proteins are located properly within the cell and regulated there. In the neurons of mammals, polyribosomes are found not only in the cell body, but also fl) in the dendrites where they are concentrated inside or below the dendritic spine. The α-subunit of Ca2 + -dependent protein kinase II -calmodulin (CaMKIIa) is one of five mRNAs known to be present within dendrites, as well as in the body of neurons. This subcellular localization targeted by the mRNA for CaMKIIa provides a possible cellular biological mechanism to both control the distribution of the protein of similar or cognate CaMKIIa origin and to independently regulate the level of protein expression in the individual dendritic spines. To characterize the cis-action elements comprised in the localization of the dendritic mRNA, two lines of transgenic mice have been produced in which the CaMKIla promoter is used to drive the expression of a lacZ transcript, which is either continuous or lacks the 3 '-or translated region of the CaMKIIa gene. Although both lines of mice show expression in prosencephalon neurons that are parallel to the expression of the endogenous ÍCaMKIIa gene, only the lacZ transcripts that have the 3'-or translated region are located in the dendrites. The β-galactosidase protein shows a variable level of expression along the dendritic tree and within the dendritic corners, suggesting that the neurons can control the local biochemistry of the dendrite either through the differential location of the MRNA or variations in transductional efficiency at different sites along the dendrite. Polyribosomes are located within the re-neurons to the cell body, the proximal part of the axon, and throughout the full extent of dendritic clearance (Steward et al., (1982).

Within the dendrites, the polyribosomes are not randomly distributed, but rather appear to be concentrated within or below the dendritic spines (Steward et al., Prog. Brain Res., 1983; Cold Spring Harbor Symp. Quant. Biol. ., 1983). The dendritic spines are elaborations of the dendrite in which the excitatory synapses are formed. This concentration of the transductional machinery at the synaptic entry site suggests the possibility that the local concentration of ribosomes may function for the selective expression of certain gene products, which can be regulated in a specific manner of the synapse (Steward, 1992). The expression of the specific gene of the synapse can be presented by the selective selection of the specific mRNAs towards the specific dendritic spines along with the associated ribosomes. Alternatively, the mRNA for a given gene can be uniformly distributed to all the dendritic spines in a neuron, but the translation of that mRNA can be differentially regulated in the individual spines. Although the vast majority of neuronal mRNAs are restricted to the cell body, several mRNAs have been found in the dendrite as well as in the body. These include the mRNAs for protein 2 associated with microtubules (MAP2), the subunit of the protein kinase Ila dependent on Ca2 + - calmodulin, the IP3 receptor type II, and the genes of ? k unknown function designated L7 and ARC (Burgin et al., 1990; Furuichi et al., 1993; Garner et al., 1988; Lyford et al., 1995; Link et al., 1995, Bian et al., 1996; ). The molecular mechanisms responsible for the location of these mRNAs to dendrites are not known. However, dendritic mRNAs seem to be associated with some component of the cytoskeleton (Davis et al., 1987, Bassell et al, 1994). The fact is that only certain mRNAs are transported in the dendrites suggests that a cis-action signal, present only in those transcripts, mediates the selection to the target. This signal could be contained within the sequence or structure of the mRNA itself, or it could be carried within the nascent polypeptide chain. In the latter case, the complete complex consisting of the polyribosome, mRNA, and nascent peptide will be transported to the dendrite. The sub-unit a of the gene of the Ca2 + -dependent subunit protein-kinase II -calmodulin fl) (CaMKIIa) is expressed specifically to the neurons of the forebrain, where its mRNA is inside the dendrites as well as the body of the neuron. Cis-action elements have been characterized in transgenic mice, elements that measured the specific expression of the forebrain as well as the dendritic localization of the mRNA. A dendritically located lacZ gene shows an expression that is not ^ uniform along the length of the dendrite.

This suggests that the expression of the dendritically located mRNA is regulated either at the level of mRNA distribution or at the level of local translation.

Materials and Methods Transgen Construction. The promoter CaMKIIa was isolated from a cosmid library prepared from the spleen of the C57BL6J mouse using 0.4 kb Aval fragment comprising the transcription initiation region of the rat CaMKIIa gene (Sunyer et al., 1990). The full-length CaMKIIa cDNA was isolated from a mouse brain cDNA library (C57BL / 6J) using a rat CaMKIIa cDNA probe. The constructions were fined using normal techniques. The lacZ gene was obtained from a 3.5 kb HindIII / Dral fragment of lac pNSE (Forss-Petter, et al., 1990). The bovine polyadenylation signal was from pRC / CMV (Invitrogen®). The GFP gene was from pGFP-Cl (CLONTECH®). The nuclear localization signal was from the large t antigen of simian virus 40. It was inserted using synthetic oligonucleotides and consisted of the sequence SSDDEATADSQHSTPPKKKRKVEDP. Transgenic mice were produced by injection of DNA into embryos B6CBA F2 or B6 / SJL3F using normal techniques. Northern blot analysis was generated using 4μg of the A + RNA isolated from the forebrain of the lac-A and lac-CMK transgenic lines. The blot was hybridized using a lacZ specific cDNA probe and washed for 40 minutes at 68 ° C in 0.2 x SSC / 0.1% SDS and exposed for 5 hours. Histochemistry of ß-galactosidase (ß-gal). The brains were processed for histochemistry essentially as described (Forss-petter et al., 1990) . Animals were perfused with 2% paraformaldehyde / 0.2% glutaraldehyde in PBS (pH 7.3) and cryopreserved in 30% sucrose. Horizontal sections of fiftyfifth meters were prepared and stained for the β-gal activity for 4 hours at 37 ° C in 0.1 M sodium phosphate, pH 7.3 / 0.14 M NaCl / MgCl2 / 2mM / K3Fe (CN) 6 3mM / K4Fe (CN) 6 3 mM / 5-bromo-4-chloro-3-indol-β-D-galactosidase lmg / ml (X-gal). Hybridization in situ. For in situ hybridization, 20 μm coronal sections of frozen, fresh mouse fl-brains were taken. The cuts were fixed for 10 minutes in 4% paraformaldehyde and dehydrated. The slices were probed with a mixture of three oligonucleotides specific for the lacZ gene, which have been labeled by putting [a-35 S] dATP and terminal transferase at the specific end of > 1 X 109 cpm / μg. Oligonucleotide sequences: lac 1, 5'- GTGCATCTGCCAGTTTGAGGGGACGACGACAGTAT-3 '; lac 2, 5'- GCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGC-3 '; lac 3, 5 '-GTAACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCG-3'. Hybridization was overnight at 42 ° C in a solution containing 10% dextran sulfate, 50% formamide, 25 mM Hepes (pH 7.6), 600 mM NaCl, 100 mM DTT, 1 mM EDTA, sperm DNA of salmon, denatured, 200 μg / ml, 200 μg / ml of poly (A), Denhardt IX solution, 107 cpm / ml probe, the sections were washed 2X 10 'while inverted cryptase with the Kodak NTB2 emulsion, were exposed for 3 weeks, they were revealed, counteracted with tuluidine blue, and photographed under dark field illumination. Neuronal crops. For neuronal cultures, the hippocampi of the Pl-P3 mouse pups were dissected and treated for 30 minutes at 37 ° C with 0.25% trypsin (SigmaR, type XI) and then gently ground and the associated cells plated. at a concentration of 2 X 105 per I? fl) ml on covers glass objects coated with poly-D-lysine (Sigma®, 0.1 mg / ml) and laminin (Collaborative Research 10 μg / ml) as described (Rayport, et al., 1992). The cells were plated in minimal essential Eagle's medium (MEM) containing 10% fetal bovine serum, thermally inactivated (HyCLone®), 2 mM glutamine, and 0.76% glucose. On the following day, the medium was replaced with fresh SF1C medium, which includes complements B-27 (GIBCO). For immunocytochemistry, the cells were labeled as described (Craig et al., 1993). Briefly, the cells were fixed for 10 minutes at room temperature with 2% paraformaldehyde and incubated overnight at 4 ° C with MAP2 monoclonal antibody (Sigma, 5 μg / ml) and rabbit polyclonal antibody to β-gal ( Cappel®, 2 μg / ml) in PBS containing 10% goat serum. The cells were then stained or stained with the fluorescently conjugated secondary antibody (fluorescein isothiocyanate for the detection of β-gal and Cy3 for the (detection of MAP2) .In several experiments, a monoclonal antibody for β-gal (Promega®) was used. Images were obtained using a MRC-1000 laser confocal microscope (Bio-Rad®).

RESULTS The cis-action elements of the CaMKIIa ^ P gene have been isolated: one, the promoter, which controls the specific expression of the forebrain of the gene and the other, the 3 'untranslated region or 3' -UTR, which controls the localization of the dendritic mRNA. Two DNA constructs (lac-CMK and lac-A, Fig. IA) were prepared such that the lacZ reporter gene was placed in the 3 'direction of an 8.5 kb fragment of the CaMKII genomic DNA starting at 84 kb after initiation site (Sunyer et al., 1990). In one construct, the complete 3 '-UTR of the current CaMKIIa mRNA (3.2 kb) below the lacZ coding region (lac-CMK) was included to determine if the signal for the location of the genetic RNA was contained in this region . As a control, the second construct contained a 3 'polyadenylation signal provided for bovine growth hormone (lac-A). Transgenic mice were generated using the two DNA constructs. Of four founding animals obtained, two (one from each construction) expressed the lacZ gene and were analyzed in detail. To determine if the two different transgenic lines expressed the expected site mRNA, Northern blot analysis of forebrain mRNA was performed using the lacZ specific probe. The lac-A and lac-CMK lines express lacZ specific mRNAs of approximately 3.7 and 6.9 kb, respectively (Figure ^ P IB). In addition, the 6.9 kb transcript of the lac-CMK mice also hybridized to a probe specific for the 3'-UTR of CaMKII. Histochemical detection of ß-gal in sections of the brain revealed a similar pattern of expression in both lines (Figure 2). With several exceptions, this expression was limited to those regions of the forebrain that normally express fl CaMKIIa. Notably, the expression is absent in a middle layer of the cortex. Also, within the hippocampus, the expression was much stronger in the dentate circonvolution than in the CA3 and CAI regions. In this way, the CaMKIIa promoter confers the expected cell specificity in the expression of a heterologous transgene, with some variations in the level of expression. The 3'-UTR of CaMKII directs the mRNA to the Dendrites. While the presence or absence of the 3'-UTR of CaMKIIa seems to have little effect on the regional distribution of transgenic expression, in situ hybridization using the probe of the. { lacZ-specific fluorescent nucleotide revealed a different subcellular localization of lacZ mRNA between the two transgenic lines, (Figure 3A). To examine this subcellular location in greater detail, the hippocampus was examined where the neuronal and dendritic layers were also differentiated. In lac-CMK mice, the f) hybridization signal not only covered the body layers of the dentate gyrus cell and the CAI region, but also extended into the corresponding dendritic layers. In contrast, lac-A mice show strong hybridization in the cell body layer of the dentate circonvolution and a weaker signal in the bodies of the CAI cells but no signal in the corresponding dendritic layers. The ^ k hybridization signal in lac-CMK mice appears to be uniform throughout the dendritic layer and to extend into the more distant regions of the dendrite. This is parallel to the subcellular distribution of the endogenous CaMKII mRNA and differs from that of MAP2, a dendritically located mRNA found only in the proximal portion of the dendrite (Garner et al., 1988). Thus, the presence of the 3'-UTR of CaMKIIa is sufficient to localize the lacZ mRNA to the dendrites and to produce an mRNA distribution within the s dendritic layers that is indistinguishable from that of the endogenous CaMKIIa gene. ^ P In an attempt to identify a common sequence element, the nucleotide sequence of the other, dendritically located, known mRNAs [MAP2, Are, IP-3R1, BC1 (Furuichi et al., 1993; Garner et al., 1988 Lyford et al., 1995; Link et al., 1995; Tiedge et al., 1991)] was compared to that of the 3'-UTR of CaMKII. No sequence homology was found. However, the critical determinant of the cis-action element can not be reflected in its primary sequence, but in the three-dimensional structure of the folded mRNA. This seems to be the case for the RNA localization elements important in early embryonic development (Macdonald et al., 1988; Macdonald et al., 1993; Mowry et al., 1992). Alternatively, the A localization mechanism of CaMKIIa may be different from that of the other dendriticly located RNAs. The difference in the extent of dendritic transport of the MAP2 and CaMKIIa mRNAs suggests some difference in the transport mechanism (Burgin et al., 1990; Garner et al., 1988). The Dendritic Localized mRNA is Translate in an Effective Manner Does this mRNA dendritically selected as an objective translate effectively? Previous studies have produced conflicting results regarding basal protein synthesis in dendrites. (Torre et al., 1992; FGossen et al., 1992). In mice having the lacZ mRNA dendritically localized, the level of the β-gal protein in the dendrites of the pyramidal cells of the hippocampus increases with respect to the controls, in which the lacZ mRNA is restricted to the body of the cell (Figure 3B). This suggests that dendritic mRNA is fl-transductionally active in the intact animal under basal conditions. However, when expressed at high levels, ß-gal can diffuse into neuronal processes. Therefore, it is possible that some of the protein found in the dendrites actually comes from the translation in the cell body. To clearly distinguish between the protein synthesized locally within the dendrites and k those synthesized in the cell body and diffusing towards the dendrites, transgenic mice were generated in which the lacZ gene carries a nuclear localization signal (nls) so that the β-gal synthesized in the neuronal cell body will be sequestered in the nucleus, thus preventing it from spreading towards the dendrite. Transgenic mice were generated using the nls-lac-CMK construct shown in Figure IA. In this case the tet-O-promoter transgene was expressed using the tTA system (Craig et al., 1993; Mayford et al., 1995). Three mouse lines were obtained which expressed the nls-lac-CMK transgene in the hippocampus, one of which was examined in detail. In situ hybridization revealed that the lacZ mRNA from the nls-lac-CMK line of mice was transported to the dendrites (Figure 4A). The histochemical detection of ß-gal in this line revealed a pattern in which a strong tension was found in the nucleus with little or no tension in the dendrite fl) and strong tension again in the more distant dendritic layer (Figure 4B). In this way, the localization machinery is capable of transporting the ß-gal synthesized in the body and the nearby dendritic layer in the nucleus (compare Figure 3B, lac-CMK, with Figure 4B, nls-lac-CMK). In addition, this pattern of expression suggests that the ß-gal found in the distal dendrite comes from the local translation of mk mRNA from lacZ in the distant dendrite. The ß-gal is expressed non-uniformly along the dendrite. To assess whether ß-gal is expressed uniformly along the entire length of the dendrite, hippocampal neurons from nls-lac-CMK mice were cultured and double immuno-fluorescent detection of ß-gal and MAP2 was used. . The MAP2 antibody, which marks the microtubules in a specific manner along the dendrite tree, gives a smooth stress pattern. In contrast, the immuno-reactivity of ß-gal is surprisingly unequal in its distribution with localized hot spots of stress ß along the dendritic tree and within the putative dendritic spines (Figure 4C and D). This ß-gal strain pattern was observed using two different antibodies and was not detected in cultures of wild-type mice. This differential, unequal expression of ß-gal along the dendrites suggests that neurons capable of regulating transgene expression fl) locally within the dendrite. This local regulation can occur through the regulated distribution of lacZ mRNA within the dendrite or through local differences in the speed of its translation.

DISCUSSION Because neurons are cells ^ k highly polarized, a critical determinant of its function is the targeting of specific signaling molecules to their appropriate sub-cellular fate. In addition, neurons receive thousands of synaptic inputs and these can often be modulated independently in response to local differences in synaptic activity. For example, in long-term potentiation or LTP is a form dependent on the activity of synaptic plasticity that is specific to the synapse (Bliss et al., 1993). The enhancement of the synaptic concentration with LTP occurs only in those synapses that are stimulated and not in other synapses ^^ on the same cell. In this way, the LTP that induces the stimulus must produce a specific biochemical change 'to the activated synapse. A mechanism to control the local biochemistry of a synapse is by regulating the distribution and translation of the specific mRNAs at that synapse. The CaMKII gene is expressed in a fl) specific manner in forebrain neurons, plays an essential role in LTP, and is one of the few mRNAs known to target dendrites (Burgin et al., 1990; et al., 1992; Mayford et al., 1995). Therefore, the signals that control both the forebrain-specific expression and the location of the dendritic mRNA were investigated. It was discovered that a fragment of ^ k 8.5 kb of the CaMKIIa gene is able to confer specific expression to the forebrain in a heterologous lacZ transgene. Subsequently, this promoter element has been used to express several different transgenes and it has been found that in each case the expression is limited to forebrain neurons in a pattern similar to that shown in Figure 2. The ability to direct transgenic expression of specific way to forebrain neurons should prove useful in transgenic studies of neuronal function and its relationship to behavior.

While the promoter targets the forebrain CaMKIIa, the 3'-UTR locates the lacZ mRNA heterologous to the dendrites. The correlation of the dendritic selection signal from the CaMKIIa mRNA to the 3 '-UTR demonstrates that the localization process is independent of the translated protein similar to the regulation of mRNA localization in other systems (Bian et al., 1996; Sundell et al., 1990; Kleiman et al., 1993). fp The expression of a lacZ gene in which the mRNA was targeted to the dendrites was examined, but the β-gal protein itself was targeted to the nucleus. The strong stress was found for the ß-gal in the nucleus and the distant dendrites with relatively little tension in the cytoplasm of the body and the nearby dendrite. These results suggest that the nuclear localization machinery can not function efficiently in the more distant regions of the dendrites. Within the dendrite, the ß-gal has a non-uniform distribution both along the tree and in the dendritic spines. This differential expression of the gene product provides a possible mechanism for the independent modulation of the biochemistry of the individual synapses. The differential distribution could be presented either through the differential localization of the mRNA or differences in the translation of the mRNA along the dendrite.

LTP occurs only at the appropriately stimulated synapse and its late phase is blocked ^ by inhibitors of protein synthesis and mRNA (Bliss et al., 1993; Frey et al., 1998; Nguyen et al., 1994). The requirement for a gene expression value in LTP, coupled with the specificity of the synapse of the process, implies that the new gene products are targeted towards or functionally used only in flj those synapses where the LTP is induced. One mechanism by which this could occur is by the stimulus of LTP induction to convert the synapse from a transductionally inactive state to a transductionally active state. This would lead to an immediate increase in the level of gene product for those mRNA species located in this synapse. In addition, the mRNA species Recently transcribed Jfc that were transported to the dendrites will be expressed only in those transductionally active synapses that received the stimulation of LTP induction. Alternatively, an immediate increase in the translation of the mRNA in the stimulated synapse can mark that synapse with that of the newly induced gene products to maintain the LTP, it would be targeted only at those marked synapses. The poor localization of CaMKIIa mRNA, through depletion of the dendritic selection signal, can interfere with the production or maintenance of a specific late phase of Isinapsis for LTP.

Example 2; Control of the Memory Portion of the Regulated Expression of the Transsen CaMKII Abstract ^ P One of the main limitations in the use of genetically modified mice for the study of cognitive functions is the lack of regional and temporal control of gene function. To overcome these limitations, a forebrain-specific promoter was combined with the transactivator tetracycline system to achieve both regional and temporal control of transgenic expression. The expression of an activated calcium-dependent independent form of calcium-calmodulin-dependent kinase II (CaMKIIa) resulted in a loss of long-term hippocampal potentiation in response to stimulation at 10 hertz and a deficit in spatial memory, an explicit form of memory. The suppression of transgenic expression reversed both the physiological and memory deficits. When the transgene was expressed at high levels in the lateral amygdala and striatum but not in other prosencephalon substrates, there was a deficit in fear conditioning, an implicit memory task, which was also reversible. In this way, the CaMKII signaling path is critical for both explicit and implicit memory storage, a way that is independent of its potential role in development. Explicit memory, a memory for events, places and events, which requires the hippocampus and related temporal, middle, and related lobe structures (Scoville et al., 1957; Squire et al., 1992), while the implicit memory, a memory for the perceptual and motor skills, comprises a variety of anatomical systems (Schacter et al., 1994). For example, an implicit form of memory, which for the conditioned medium, comprises the amygdala (Blanchard et al., 1972; 1992). Studies with genetically modified animals have sought to relate specific genes to specific forms of explicit or implicit memory storage (Grant et al., 1992, Silva et al., 1992, Mayford et al., 1995, Bach et al., 1995; ). However, the current methodology does not allow to distinguish between a direct effect on memory or its underlying synaptic mechanisms and an indirect effect of the development of neuronal circuits from which memory storage is presented (Grant et al., 1992; Mayford et al., 1995).

In addition, the gene under study is typically overexpressed or removed throughout the brain. As a result, genetic modifications often indiscriminately affect both implicit and explicit memory as well as perceptual or motor performance. Thus, to analyze the molecular contribution of a given gene to a particular type of memory, it is essentially not only to control the synchronization of expression, but also to restrict expression to appropriate cell populations. To address these issues and to achieve transgenic expression regulated in restricted regions of the forebrain, a specific forebrain promoter was used in combination with the tetracycline trans-activator (tTA) developed by Bujard and his colleagues (Gossen et al., 1992; Furth et al., 1994). The role of CaMKII signaling in synaptic plasticity, as well as implicit and explicit memory storage, was examined. CaMKIIa is a serine-threonine protein kinase that is restricted to the forebrain (Miller et al., 1986; Burgin et al., 1990; Hanson et al., 1992). It is expressed in the neurons of the neocortex, the hippocampus, the amygdala, and the ganglia b salts. After a brief exposure to Ca2 +, CaMKII can be converted to an independent Ca2 + state through autophosphorylation in Thr286 (Miller et (flal., 1986; Hanson et al., 1992; Fong et al., 1989, Thiel et al., 1988; Waldmann et al., 1990) This ability to become persistently active in response to a transient Ca 2+ stimulus leads to the suggestion that CaMKII can be a molecular substrate of memory (Lisman, 1994). The targeted or selected interruption of the fflCaMKIIa gene causes a deficit in long-term potentiation (LTP) and severely impairs performance in hippocampal dependent memory tasks.

(Silva et al., 1992; Silva et al., 1992, pp. 201). The mutation of Thr285 to Asp in CaMKIIa mimics the effect of autophosphorylation on Thr286 and converts the enzyme to an independent form of Ca2 + (Fong et al., 1989; Walsmann et al., 1990). Transgenic expression of fl \ this dominant mutation of CaMKIIa (CaMKII -Asp286) results in a systematic change in the response to stimulation at low frequency such that long-term depression (LTD) is favored in transgenic mice (Mayford et al. al., 1995). Thus, although the collateral LTP of Schaffer in response to tetanus at 100 Hz is not altered, the LTP is eliminated in the range of 5 to 10 Hz, a frequency (theta frequency) characteristic of the endogenous oscillation in neuronal activity seen in the hippocampus of animals during space exploration (Bland, 1986). Correlated with this selective deficit in the LTP in the frequency interval ) theta is a severe defect in spatial memory (Bach et al., 1995). These phenomena have been examined with the regulated expression of the transgene CaMKI I -Asp 2 86 Regulation with doxycycline of transgenic expression. The first type of mouse generated to achieve regulated expression of CaMKII -Asp286 in prosencephalon neurons (Figure 5A) expressed the tTA gene under the control of the CaMKIIa promoter (line B), which limits the expression of the tTa transgene to forebrain neurons (Bland, et al., 1986). The CaMKII promoter consisted of 8.5 kb of genomic DNA in the 5 'direction of the transcription initiation site of the mouse CaMKIIa gene, as well as 84 ifl base pairs of the 5' non-coding exon. He Genomic DNA was isolated from a cosmid library of the C57 B16 / J mouse spleen with a rat genomic probe consisting of a 0.4 kb Ava I fragment comprising the transcription initiation region of rat CaMKIIa (Sunyer et al. , 1990) . The tTA gene of plasmid pUHD 15-1 (Gossen et al., 1992) was flanked by an artificial intron and the binding sites at the 5 'end (Choi et al., 1991) and by an SV40 polyadenylation signal at the 3 'end. The cDNA with the intron and the polyadenylation signal was placed in the 3 'direction of the 8.5 kb CaMKII promoter fragment. The cDNAs for lacZ from (AEscnericn a coli and mouse CaMKIIa were similarly flanked by the hybrid intron and the polyadenylation signal and placed in the 3 'direction of the promoter element tet-0 of plasmid pUHD 10-3 (Gossen et al., 1991). The CaMKIla gene was a full-length cDNA (4.8 kb) isolated from a C57B16 / J mouse brain cDNA library. The lacZ gene imported into the nuclear localization signal of the SV40 T antigen as well as the 3'-nontranslated region (UTR) of CaMKIIa, which targets the MRNA to the dendrites (Mayford et al., 1996). In the second mouse type, the tTA-responsive tTA promoter binds to the target gene of interest, in this case either the lacZ or CaMKII-Asp286 gene. The tTA gene expresses a eukaryotic transcription activator that binds to and activates the transcription of the tet-O promoter element; this transcription is blocked by the tetracycline analog, doxycycline (Gossen et al., 1992). When both tet-O and tTA transgenes were introduced into the same mouse, the gene linked to tet-O was activated but only in those cells expressing tTA. The regulation of the CaMKII-Asp 286 transgene was evaluated using a Southern blot (DNA) with inverted polymerase-transcriptase chain reaction (RT-PCR) (RT-PCR) was performed essentially as described (Mayford et al., nineteen ninety five) . The total RNA of the forebrain (100 ng) was used in each reaction with the oligonucleotide primers to amplify a region of the transcript that includes the mutation of Thr28S "Asp. Equal amounts of the amplified cDNA (both wild-type and mutant sequences) were separated in A 3% agarose gel was transferred to nylon membranes and hybridized with an oligonucleotide probe labeled with 3 P ^ specific for the Asp286 mutation (oligonucleotide sequence 5 'CTT CAGGCAGTCGACGTCCTCCTGTCTGTG-3'). in which only the mutant cDNA Asp286 was detected (2 '15 min., 60 ° C. 0.2 'citrate normal saline solution). A Northern blot (RNA) of the prosencephalon total mRNA revealed the expression of a shorter than expected CaMKII -Asp286 transcript (-3.4 fl kb). As shown in Figures 7A-D, this shorter CaMKII -Asp286 transcript was not localized to the dendrites, presumably as a result of the loss of the sequence element in the 3 'UTR that is necessary for targeting the mRNA to the dendrites (Mayford et al., 1996)) to detect only the mutant transcripts (Figure 5B). Mice that have any of the transgenes only show little or no expression of the CaMKII-Asp286 mRNA. When both transgenes were introduced into the same mouse, there was a greater activation of the expression of CaMKII -Asp28e. The expression of this transgene suppressed it completely when the fljratones were given doxycycline (2 mg / ml) in the drinking water for 4 weeks. Restricted expression of transgenes linked to tet-O. Expression of β-galactosidase was examined on two lacZ tet-O indicator lines from mice that also carried that tTA transgene from the CaMKIIa promoter (Figure 6A). In the first line, the expression was a report all along the forebrain, neocortex, hippocampus, amygdala, and stratum.This pattern limits the expression of the endogenous CaMKIIa gene (Burgin et al., 1990). , in the expression was observed throughout the forebrain, but surprisingly, the expression is absent in the pyramidal cell layer CA # of the hippocampus ák (Figure 6B) .Using the in situ hybridization, the expression pattern in 3 lines of double transgenic mice expressing CaMKII-Asp286 bound to tet-O was examined (lines B-13, B21 and B22) (Figure 7A-7D). In the first line (B13), the expression was evident throughout the forebrain. However, in the hippocampus, the expression was strong in the dentate gyrus and the CA2 region but was weak or absent in the CA3 region. In the second line of mice (B22), there was moderate expression in the hippocampus, subiculum, stratum, and amygdala, with little expression in the neocortex. In the hippocampus, the expression was again presented in the CAI region and was absent in the CA3 region. In the third line (B21), there was little expression in the neocortex and hippocampus, but a stronger expression in the stratum, in the anterior and lateral nuclei of the amygdala, and in the underlying olfactory tubercle. Thus, as long as the promoter is fl'CaMKII can limit expression to prosencephalon neurons in general, the expression of the transgene linked to tet-O is further limited to the particular subsets of neurons of the forebrain, presumably due to the effects dependent on the integration site. In double transgenic mice, a high level of mRNA expression of CaMKII-Asp286 tffc was obtained (Figures 5B and 7A-D). To determine the effect of this expression on enzymatic activity, the activity of CaMKII in the stratum of line B21 of mice was measured (Table 1). Table I. Effect of the expression of CaMKII-Asp286 mRNA on the capacity of the action of the enzyme. The brains were removed and the stratum was dissected and immediately homogenized in tris- 20mM (pH 7.5), EGTA, 0.5mM, EDTA, 0.5mM, leupeptin, 2mM, dithiothreitol, 0.4mM, phenylmethylsulfonyl fluoride 0.1mM, molybdate 0.4 mM, and 10 mM sodium pyrophosphate. The activity of the CaMKII enzyme was determined as described (Mayford et al., 1995). Animals B21 + Dox removal received doxycycline (1 mg / ml) plus 5% sucrose in drinking water for 3 to 5 weeks. Animals B21 + Dox removal received doxycycline (lmg / ml) for 3 to 5 weeks and then changed to normal water for 6 weeks. The number of mice is given in parentheses.

CaMKII activity In these mice, the activity of CaMKII independent of Ca2 + was increased seven times in relation to that of the wild type. However, when the mice were treated with doxycycline (1 mg / ml), the activity of CaMKII was suppressed at wild-type values. When the treatment of doxycycline was discontinued, CaMKII activity independent of Ca returned to those of the untreated transgenic mice. In this way, CaMKII -Asp286 is functionally expressed and can be regulated with doxycycline. Effects of LTP on the expression CaMKI I -Asp286 in the hippocampus. The constitutive expression of the transgene CaMKII -Asp285 in the mouse forebrain changes the stimulation frequency required for the production of LTP and LTD in the collateral pathway of ^ Schaffer of the hippocampus (Mayford et al., 1995). In wild-type mice, stimulation at 1 Hz produced LTD, whereas stimulation at 5, 10 or 100 Hz produced LTP. However, stimulation in the range of 5 to 10 Hz did not produce LTP any longer, but rather produced LTD or no change in synaptic concentration. Yes the transgen was driving in a way ^ k pre-synaptic or post-synaptic, was investigated by asking whether expression of the transgene specifically in post-synaptic CAI neurons will produce a change in the frequency threshold for LTP and LTD. Line B13 of mice, which shows a uniformly high level of expression in the CAI region was examined with little or no expression in CA3 (cross sections (400 μg thick) of the mouse hippocampus were prepared and placed in a cutting chamber of enter, perfused with artificial cerebrospinal fluid as described (Mayford et al., 1995) Post-ianeptic, excitatory field (EPSP) potentials were produced once per minute with fine electrodes of bipolar tungsten stimulation, (pulse duration 0.05-ms). Stainless steel recording electrodes were placed in the stratum radiatum. The stimulation concentration was adjusted to produce 50% of the maximum EPSP obtainable in each cut. The synaptic baseline response was collected for 20 minutes before tetanus. Tetanus at 10 Hz was distributed for 1.5 minutes at the same intensity as used in the baseline record. For treatment with doxycycline, the animals were administered doxycycline (1 mg / ml) to 5% sucrose in the drinking water for 2 to 3 weeks, and the sections were then exposed to doxycycline (1 mg / ml) in the perfusate. All animals were 2.5 to 6 months old at the time of registration). In this way when the LTP was measured with the Schaffer lateral in the B13 mice, the transgene will be expressed only in the post-synaptic neurons. Stimulation of wild-type mouse sections at 10 Hz resulted in a prolonged duration of enhancement of 123+. 9% (n = 12 cuts, 6 mice) (Figure 8). In contrast, stimulation at 10 Hz in B13 transgenic mice produced a slight depression at 89 +. 6% of baseline (n = 9 cuts, 3 mice), which was significantly different from wild-type mice [t (19) = 3.148, P <; 0.01, student's t-test] To determine if this effect was reversible, expression of the transgene was suppressed by administering doxycycline (Img / ml) for 2 to 3 weeks. Stimulation at ten-hertz then produced potentiation similar to that in wild type mice (132 ± 10%, N = 8 cuts, 4 mice) (Figure ^ P 8). In this way, suppression of transgene expression in adult mice reversed the electrophysiological genotype [t (15) 0 3.675, P < 0.005]. These results suggest that the selective expression of the CaMKII -Asp286 transgene in neurons is a post-synaptic one of the Schaffer collateral synapse is sufficient to alter the lateral frequency threshold for LTP. In addition, the change in the frequency threshold fc is due to the acute expression of the transgene, rather than the reversible defect in development (it will also be useful to suppress transgenic expression during development and then activate, the gene only in the adult animal However, it was found that treatment in wild type mice with doxycycline (1 mg / ml) during development damages adult spatial memory and memory for fear conditioning.The result follows that doxycycline itself produces a defect in neuronal development.

Transgenic suppression was used only in the adult animal in which treatment with doxycycline did not (it affected the memory.) Given the activation of the transgene throughout the development, it is possible that the LTP and the memory phenotypes observed with the active transgene in the adult animal result from a synergistic interaction between adult development and expression rather than a direct acute effect of transgenic expression in the adult animal Effect on explicit memory storage of the expression CaMKII -Asp28S in the hippocampus. The expression of the transgene CaMKII -Asp286 in the forebrain interferes with spatial memory, a form of explicit memory as measured in the circular labyrinth of Barnes (Bach et al., 1995) .The circular labyrinth of Barnes is an open disc A brightly lit with 40 holes in the perimeter (Figure 9A). Mice have an aversion to brightly lit open areas and are therefore motivated to escape from the labyrinth. This can be achieved by finding hole 1 in 40 that leads to a darkened exhaust tunnel. In the spatial version of this task, the mouse must use distant suggestions in the room to locate the hole that leads to the escape tunnel (in the circular labyrinth of Barnes (Bach et al., 1995), the mice (from 2.5 to 6). months of age) were tested once a day until they met the criteria (five out of six sessions with three or fewer errors, or until 40 days had elapsed.) The order of the holes sought is recorded by an observer who does not know the condition of the genotype and doxycycline, and from these data the number of errors was determined.The errors were defined as searches of any hole that does not have the tunnel below it.The searches ^ p included stitches with the nose and deviations of the head over the hole At the end of each session, the search strategy used was recorded by the observer.The spatial search strategy was defined operationally as the search for the escape tunnel with punctuation. both error and distance less than or equal to 3. The distance was calculated by counting the number of holes between the first hole sought within a session and the escape tunnel. An analysis of variance of one factor (ANOVA) (sex) did not reveal significant effect of sex either for transgenic or wild type mice, so that the data collapsed through this variable. For the error data the three-factor ANOVA (genotype, doxycycline, and session block) with a repeated measurement was used. For the spatial search strategy data, the two groups of B22 transgenic mice were compared with a bi-directional ANOVA (doxycycline and session block) with a repeated measurement. The expression of the transgene CaMKII -Asp 8S to Ato ° 1 ° long of the forebrain as seen in the B13 mice resulted in damage in the spatial version, but not in the suggestion of the labyrinth task of Barnes (Bach et al. ., nineteen ninety five) . To investigate those areas in the forebrain that are critical for this type of spatial memory defect, B22 transgenic mice were examined ^ which show expression in the hippocampus, subiculum, stratum and amygdala, but relatively little expression in the neocortex ( Figures 7A-D). These mice received significant damage or deterioration in spatial memory in the circular labyrinth of Barnes. None of the transgenic mice was able to acquire the task when using the spatial strategy, despite the fact that they trained for 40 days A consecutive (Figures 9B to 9D). However, this profound damage to memory was reversed by the suppression of transgenic expression. Effect on the implicit memory of the expression of CaMKII-Asp286 in the amygdala and stratum. Conditioning to fear is a simple associative form of learning, in which both a new environment and a tone meet with a foot shock on the training day. In the task of conditioned fear (Bach et al., 1995), freezing was defined as a total lack of movement with the exception of respiration and was measured by an experimenter who did not know the condition of ß ^ genotype and doxycycline. The percentage of time from freezing to context and suggestion was calculated. No significant effect of sex was observed in B22 or B21 transgenic mice or wild type mice, so that the data collapsed through this variable. The context freezing and suggestion on the day of day was analyzed by two three-factor ANOVA (genotype, line, and doxycycline) that were used to compare the transgenic and wild type B22 and B21 mice. Two unidirectional ANOVAs were used to purchase the freezing amount six weeks after the suggestion and context in B21 transgenic mice in doxycycline, transgenic B21 changed to water, and wild type. The memory was evaluated 24 hours later by measuring the amount of freezing (the response to fear) produced by either the new environment (conditioning to the context) or the tone (conditioning to the suggestion). The conditioning to fear showed components of both the implicit and explicit forms of learning. The contextual version of the task is selectively impaired by lesions of the hippocampus (Kim et al., 1992) and can thus be seen as an explicit form of learning, while both versions with suggestion and context of the task are damaged by the amygdala lesion and therefore are seen as implied. In contrast to their spatial memory deficit, line B22 of mice showed normal conditioning to the fear of both suggestion and context (Figures 10A and 10B). Thus, although B22 mice were damaged in spatial memory in the labyrinth of Barnes, they were not damaged in a second task dependent on the hippocampus (pcontextual conditioning of fear.) This dissociation has been previously observed with the constitutive expression of the CaMKII transgene. -Asp28e and to reflect the use of different synaptic mechanisms for memory storage in the two tasks (Mayford et al., 1995) .In addition, these results show that the moderate level of transgenic expression in the amygdala and stratum seen in B22 mice (Figure 3) fl is insufficient to interfere with the implicit component of fear conditioning, a high expression level of the transgene CaMKII-Asp286 in the stratum and amygdala affects implicit memory storage? .- To explore this question, B21 mice were studied which showed strong expression in the lateral amygdala and stratum but little transgenic expression in the hippocampus or neocortex (Figure 7A-7D). B21 transgenic mice exhibited severe damage in both conditioning with context and suggestion (Figures 10A and 10B). This learning damage was reversed again through the (^ administration of doxycycline for 4 weeks before training., m This deficit in fear conditioning probably arises from expression in the lateral amygdala, a structure that has been implicated in this form of learning by injury studies.

(LeDoux et al., 1990). However, because there are reciprocal connections between the stratum and the amygdala (Kita et al., 1990: Canteras et al., 1995), the possibility that the deficit results from a functional interruption can not be considered as a rule. in the stratum that secondarily alters the amygdala. Effect on memory recovery of the expression CaMKII -Asp285 in the amygdala and the stratum.

MEI removal of doxycycline after the initial period of transgene suppression resulted in a reactivation of gene expression (Table 1). We examined whether re-expression of the transgene after normal learning has occurred interferes with late stages of memory storage such as consolidation or recovery. B21 mice were trained with the deleted transgenic expression and strong fear conditioning was observed. Once the animals had learned the task, the transgenic expression was reactivated by withdrawal of doxycycline (Figure 10C). After a period of 6 weeks, the expression of the CaMKII-Asp286 transgene (^ returned to the same levels found in animals that did not receive the drug (Table 1) .These mice were then examined for the retention of the conditioning with both context and with suggestion and a significant reduction in freezing was found in comparison to the B21 mice in which the suppression of transgene f was maintained (Figure 10D) .This reduction in freezing reflects either a damage in the consolidation or revocation of the memory or a deficit in performance The evaluation of performance deficits is critical to the study of memory because it can not be inferred only that memory storage is defective once all possible defects in the memory have been excluded.

Apperception, motor performance and cognitive understanding of the task. Although it is difficult to control all the consequences of genetic manipulation in several performance components, the two most likely classes of performance variables have been examined: (i) the ability to perceive the unconditioned stimulus, and (ii) the ability to attend and freeze in response to fearful stimuli (the conditioned response). To rule out damage to the perception of the unconditioned stimulus (shock in the foot), sensitivity to shock was examined and no difference was found between the transgenic and wild-type B21 mice suggesting that the deficit of fear conditioning, observed not results from a difference in the perception of the unconditioned stimulus (simple sensitivity was measured in transgenic (n = 4) and wild type (n = 6) B21 mice.The mice were individually placed in an operant chamber, mouse with a floor of metallic grid and were given shocks on the foot of 1-s of increasing intensity (for example 1, 2, 3, mA ...). An experimenter who did not know of the genotype of the mice recorded or recorded the intensity of the foot shock required to produce each of the following three responses: jolts, vocalizations and jumps A t-test for each response revealed no significant effect of the genotype, then the possibility of a fl ue was examined. defect in the performance of the conditioned response (freezing) by measuring unconditioned freezing in response to an intruder. Unconditioned freezing in the presence of an intruder was measured in transgenic mice (n = 8) and wild type (n = 10) B21 in a plastic metabolism cage of Nalgen. The mice and the intruder were placed in upper and lower chambers respectively. The cameras were separated by a metallic grid floor. A 7-week-old male Sprague-Dawley rat served as the intruder and was placed in the lower chamber 10 minutes before the introduction of the mouse. The amount of unconditioned fl omenation that occurred during the first 120 seconds after the mouse was introduced was measured by an experimenter who did not know about the genotype.A t test revealed no significant genotype effects. the ability of B21 mice to freeze an intruder (a rat) when the transgene was expressed (Figure 10E) .Thus, B21 transgenic mice were able to attend to the fearful stimuli and express a normal freezing response. Although some hidden defect in performance could have been presented that was not detected, these control experiments support the view that the transgene does not produce its effect on the perception of the unconditioned stimulus or on the performance of the conditioned response. Results suggest that the CaMKII signaling path is important for some later effects of memory storage such as or the ability to consolidate or return the information learned. Discussion. The high levels of Ca2K-dependent CaMKII activity changed the stimulation frequency threshold for the LTP and LTD of the hippocampus to favor LTD (Mayford et al., 1995). This change in the threshold is associated with an explicit but not implicit memory damage (Bach et al., 1995). To obtain the regulated expression of this transgene in restricted regions of the forebrain, so that the underlying cellular and behavioral functions can be studied more effectively, the tTA system was used for regulated gene expression (Burgin et al., 1990; Hanson et al., 1992) It has been found that the expression of fltransgen CaMKII -Asp28G "altered synaptic plasticity in adults and memory formation in a direct way, and not by effects on neuronal development. Post-synaptic transgene expression was sufficient to alter the frequency threshold for the induction of LTP, at least at 10 Hz. Finally, high-level activation of CaMKII in the stratum and lateral amygdala fk also interfered with the forms implicit in memory, how an increase in the activity of CaMKII independent of Ca2 + can alter the stimulation frequency required to produce LTP and LTD and how this in turn can alter learning and memory storage? The results demonstrate that the effect of the CaMKII-Asp28e transgene was probably measured by changes in the post-synoptic CAI neurons of the Schaffer collateral pathway. A simple mechanism for systematically changing the frequency threshold of LTP and LTD to favor LTD will be to reduce the size of the post-synaptic Ca2 + signal produced during stimulation [(Cummings et al., 1996); however, see (Neveu et al., (1996).] This could be presented either through increased phosphorylation of the particular substrate proteins of CaMKII or by increased binding of Ca2 + - calmodulin by autophosphorylated CaMKII (Meyer et al. ., 1992) of its detailed mechanisms, however, the data indicate that activation of CaMKII alone may not be sufficient to produce the increase in synaptic concentration associated with LTP, as has been suggested (Lisman, 1994, Petit et al. ., 1992) Rather, the activation level of CaMKII regulates the stimulation conditions under which LTP and LTD occur. In this study, synaptic physiology and behavior were not measured in the same group of animals. The expression of the CaMKII -Asp286 transgene in the CAI region in the B22 mice was uneven; that is, some neurons expressed the transgene well, while in other neurons the expression is absent. This unequal expression made it impossible to evaluate LTP in this line of mice by means of field records, which sample many synapses of different neurons in a region. However, it is assumed that in these neurons where the transgene was strongly expressed in these mice, a change in the LTP / LTD frequency threshold occurred, however, the effects of CaMKII activation on behavior are probably a consequence of its effect on the frequency threshold for the induction of LTP and LTD. That the activation of CaMKII interferes with synaptic plasticity in the 5- to 10-Hz interval is particularly relevant for the spatial memory paradigm based on the explicit hippocampus. Animals that explore the space of a new environment show a rhythmic oscillation in hippocampal activity in the range of 5-10 Hz (the theta rhythm) (Bland, 1986). Changes in synaptic concentration can be produced by this endogenous activity and are thought to be necessary to store information about space. Synaptic plasticity in the frequency interval theta can regulate the cells of hippocampal placement, the pyramidal neurons (in subfields CA3 and CAI) whose activity is related to the location of the animals in the environment (O'Keefe et al. al., 1978). Several lines of evidence implicate the lateral amygdala as the site of plasticity for fear conditioning. First, the lateral amygdala is the first site of convergence of somatosensory (unconditioned stimulus) and auditory (conditioned stimulus) information on the fear conditioning pathway (Kim et al., 1992). Second, fear conditioning the responses produced by auditory neurons in the lateral amygdala (Quirk et al., 1995. Third, these neurons exhibit a strong LTP that can contribute to improved responses triggered by auditory effect (Rogan et al., 1995) Finally, lesions of the lateral amygdala block fear conditioning (Kim et al., 1992) How the expression of CaMKII -Asp286 could affect fear conditioning? - The expression of this transgene in the hippocampus. increases the frequency of stimulus required to produce the LTP (Figure 8) Where there was a similar increase in the frequency threshold to the excitatory synapses in the lateral amygdala, this increase in threshold could form the psychological basis for the atk damage observed in implicit memory storage The expression of the transgene in the stratum and the amygdala also affected the consolidation or revocation of memory. of learning generally call changes in synaptic concentration only during the initial learning process (Churchland et al., 1992). Once formed, the changes in synaptic concentration remain stable and have the real trace of memory. However, for some memories, such as explicit memories based on the hippocampus, the anatomical site of memory changes over time over a period of several weeks after initial learning (Kim et al., 1992). In addition, memory retentiveness is typically reconstructive, requiring a new recapitulation of learned experience. Both the transfer and the reconstruction of the memory may require a change dependent on the activity in the synaptic concentration. If a similar process for fear conditioning in the amygdala occurs, the defect in recovery observed in the transgenic mice could reflect a defect in synaptic plasticity caused by the expression of CaMII-ASP28e during this memory transfer or phase-shift. reconstruction. The methods for the regional and regulated transgenic expression that are described in this They present the development of an optimal technology for the genetic study of the cognitive process. To take the molecular dissection of the behavior additionally, it will be necessary to use promoters that are even more restricted in their expression pattern and adapt this technology to the regulation of the selected gene disruption. The methods described herein should prove useful and should help clarify the molecular and cellular signaling pathways important for the cognitive process.

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The use 3_ Genetic and pharmacological evidence of one? new intermediate phase of long-term potentiation (I-LTP) suppressed by calcineurin. To begin to investigate the role of phosphatases in synaptic plasticity using genetic approaches, transgenic mice were generated that overexpress a truncated form of calcineurin under the control of the CaMKIIa promoter. The mice overexpressing this transgene show increased calcium-dependent phosphatase activity in the hippocampus. Physiological studies of mice overexpressing calcineurin and parallel pharmacological studies in wild-type mice revealed a new intermediate phase of LTP (I-LTP) in the CAI region of the hippocampus. This intermediate phase differs from E-LTP in requiring multiple series for induction and to be dependent on PKA. It differs from L-LTP by not requiring new protein synthesis. These data suggest that calcineurin catua is an inhibitory limitation in I-LTP that is mitigated by PKA. This inhibitory limitation of learning acts as a gateway to regulate the synaptic induction of L-LTP.

Introduction. The long-term modification of the synaptic transmission is thought to play a role in a variety of brain functions ranging from memory storage to a fine-tuning of the synaptic connections during development. As a result, an intensive search has been carried out in both invertebrates and vertebrates to identify the molecular components of various forms of synaptic plasticity. This search has been a central focus in two types of synaptic improvement: long-term facilitation in ^ Aplysia and long-term potentiation (LTP) in the hippocampus of mammals. Both of these forms of synaptic plasticity last from minutes to days, depending on the concentration in number of stimulus inducer. A major theme emerging from these studies is that protein kinases play key roles in the long-term improvement of synaptic transmission (for review, see Roberson et iflal., 1996). In this way, inhibitors of several kinases damage the induction or maintenance of both long-term facilitation in Aplysia and LTP in the hippocampus (for review, see Roberson, et al., 1996; Huang et al., 1996b; Martin; et al., 1997). Additionally, genetically modified mice in which the genes that code for specific kinases are already overexpressed or deleted, exhibit phenotypes that in most cases are parallel to those obtained with pharmacological inhibitors (Mayford et al., 1995a, 1997; et al., 1997). Much attention has been focused on the protein-kinases in synaptic plasticity, relatively little attention has been paid to protein-phosphatases. Yet, the phosphatases will probably have signaling roles in the synaptic plasticity that is equal in importance to those of the kinases if only that is due to their inherent antagonistic relationship with the protein kinases. (JPA furthermore, most cellular learning models postulate erasure mechanisms designed to counteract the long-term synaptic improvement that is thought to be required for memory storage.) Consistent with this idea, recent experiments have shown that while a brief High frequency stimulation in the collateral pathway of Schaffer in the hippocampus leads to LTP, a prolonged low-frequency stimulus of this same pathway results in a long-term depression (LTD) of synaptic transmission and experiments with pharmacological inhibitors suggest a important role for phosphatases in LTD (Mulkey et al., 1993, 1994; O'Dell and Kandel, 1994; for review see Bear and Abraham, 1996). Despite the potential importance of phosphatases for synaptic plasticity, however, the study of phosphatases in the hippocampus has been limited by the lack of specificity of the appropriate pharmacological inhibitors (for example, see Helakar and Patrick, 1997). as well as for prolonged pre-incubation periods frequently necessary for the inhibitors to produce alterations in synaptic function (Mulkey, et al., 1993, 1994). As a result, the role of phosphatases in LTP is unclear. While several experiments suggest that the pharmacological inhibitors of phosphatases have no effect, or do not improve LTP (Blitzer, et al., 1995, Mulkey et © -il 1993, Muller, et al., 1995, Wang and Kelly , 1996), other studies report that these inhibitors block LTP (Wang and Stelzer, 1994, Lu et al., 1996a, b). To overcome these limitations and to begin to examine more directly the precise role of specific phosphatases in synaptic plasticity, we now return to a phlegmatic approach. Initial effort has been focused on calcíneurina (PP2B), because this enzyme is thought to be the first step in the phosphatases cascade initiated by the Ca2 + signal through the NMDA receptor. Consistent with the idea that the Ca2 + signal through the NMDA receptor is the initial event for both LTP and LTD in the hippocampus, the pharmacological inhibitors of calcineurin block LTD (Mulkey et al, 1994), and it has been reported by some that they improve the LTP (Wang and Kelly, 1996, but see Wang and Stelzer, 1994, Wang and Kelly, 1997, Lu et al, 1996a, b). Calcineurin is a calcium-sensitive serine / threonine- ^ fl ^ phosphatase that is present at high levels in the hippocampus, is enriched at the synapse, and is a heteromultimer that has catalytic activity (calcineurin A, is a 60 kD protein that It exists as three isoforms (a, ß and), two of which, a and ß, are present in the brain (Kuno, et al., 1992). Once activated, calcineurin ^ pacts into two types of protein directly and This mode regulates specific cellular functions, Second, it can modulate an even greater variety of substrates indirectly by its ability to dephosphorylate inhibitor-1, a key protein-phosphatase-1, (PPl). a protein of low molecular weight that, when phosphorylated, inhibits the function of PPl.The dephosphorylation of inhibitor-1 by calcineurin activates PPl and leads to the dephosphorylation of a large and independent set of obj ective proteins. One of the regulatory actions of calcineurin comes from its interactions with the protein-dependent kinase of cAMP, PKA. Calcineurin dephosphorylates and inhibits the action of inhibitor-1 by dephosphorylating the site in inhibitor-1 that is phosphorylated by PKA (Hubbard and Klee, 1991). This dephosphorylation enhances the ability of Rllβ to re-associate with and inhibit the function of the catalytic subunit of PKA (Rangel-fl / dao and Rosen, 1976). In addition, calcineurin and PKA antagonistically regulate the function of the glutamate receptor NMDA and GluR6 (Tong, et al., 1995, Raman et al., 1996, Traynelis and Wahl, 1997). Calcineurin also inhibits a new isoform of adenylyl-cyclase (Paterson et al., 1995). Finally, calcineurin can dephosphorylate the CREB transcription factor (Bito et al., 1996; Liu and Graybiel, 1996), another target of PKA, which regulates CRE-mediated transcription in this way. This modulation of CREB phosphorylation by calcineurin was probably measured at least and partially by the inhibitor-1 cascade (Bito et al., 1996). While the dephosphorylation of CREB Serl33 by calcineurin is thought to reduce ^^ CRE-mediated transcription, calcineurin-mediated dephosphorylation of CREB in another regulatory site may result in an improvement in CRE-mediated transcription (Schwaninger, et al., 1995). The interactions of PKA and calcineurin are of particular interest in the context of LTP. Based on the requirements for macromolecular synthesis, the LTP can be divided into at least two components (an early component (E-LTP) and a late component (L-LTP) .The distribution of a series of 100 Hz individual lasts a second to the CA1-collateral pyramidal cells synapse of ß ^ chaffer causes E-LTP, a relatively short and weak life improvement of synaptic transmission lasting 1-2 hours, which does not require protein and RNA synthesis, and does not it is dependent on PKA (for review see Haung, et al., 1996b, Roberson et al., 1996) In contrast, the administration of three or four 100 Hz series causes L-LTP, a stronger and more stable LTP. which lasts many hours which is dependent on the synthesis of both RNA and protein (for review see Haung, et al., 1996b, Roberson et al., 1996). In addition, L-LTP is blocked by PKA inhibitors (for review see Huang et al., 1996b), and it is dramatically damaged in mice that express a negative, dominant form of a regulatory subunit of PKA (Abel et al., 1997).

"Recent experiments with phosphatase inhibitors suggest that a role of PK in LTP in the CAI area may exist to suppress the actions of either PP" or PP2A (Blitzer et al., 1995; Thomas et al., 1996). , Blitzer et al., (1995) found that when LTP in the CAI area is induced by strong stimuli, it can be blocked by PKA inhibitors, however, its inhibition can be removed by pre-incubation of cuts with PP1 / inhibitors. PP2A This led to Blitzer, et al., Suggesting that under certain circumstances, PKA can "regulate" LTP by suppressing a phosphatase cascade.To further examine, the role of phosphatases in synaptic plasticity and in memory storage, as well as to determine more precisely the inter-role between PKA and phosphatases in the regulation of LTP, a truncated form of calcineurin Aa was overexpressed in the mouse forebrain. The overexpression of this transgene results in an increase in of about 75% ^ in phosphatase activity in the hippocampus. Using these mice, two questions have been addressed: (1) What is the role of calcineurin in the expression of the various phases of LTP? (2) Does PKA modulate the action exerted by calcineurin in each of these phases? . Both generic and pharmacological evidence is provided that is consistent with the fl "regulation" model for PKA actions in LTP (Blitzer, et al., 1995). In addition, the date presented in this article and his partner (Mansuy et al., Submitted to Cell) extends this model by showing that the "gateway" of PKA has a different temporal component representing an intermediate phase of LTP (I-LTP) . This intermediate phase is induced by multiple series and is suppressed by calcineurin. It differs from E-LTP by requiring a much stronger stimulus, the activation of PKA and the suppression of calcineurin. The intermediate phase differs from L-LTP and the suppression of calcineurin. The intermediate phase differs from L-LTP in that it does not require protein synthesis (these data also suggest that this restriction in I-LTP is not requiring protein synthesis.) The data suggest even more than this restriction in I-LTP. imposed by calcineurin can be mitigated by the activation of PKA and that this relief is required for the full expression of L-LTP.In this way, overexpression suppresses both I-LTP and L-LTP.The results of detailed behaviors in the annexed article (Mansuy, et al., presented to Cell), suggest that the function of temporarily different regulation, mediated by calcineurin, is important behaviorally and suppresses long-term memory formation.

RESULTS Generation of transgenic mice that overexpress a truncated form of calcineurin. To increase the activity of calcineurin-mediated phosphatase in the forebrain of transgenic mice, a mutant by suppression of the catalytic subunit Aa (CAM-AI) of murine calcineurin (O'Keefe et al., 1992) was expressed under the control of the promoter. CaMKIIa (CN 98 Line, Figure A, Mayford et al., 1996). The CAM-IA calcineurin mutant is a fragment of the catalytic subunit Aa that lacks the self-inhibitory domain of a portion of the calmodulin binding domain, but retains the binding domain of calcineurin B (O'Keefe et al., 1992; Persons et al., 1994). This suppression weakens the calcium requirement for the activation of calcineurin. Although this construct shows some activity independent of CA 2+ when expressed in Jurkat cells (O'Keefe et al., 1992), it finds that this mutant form requires calcium for the activation of hippocampal neurons (Figure 11C). Overexpression of Calcineurin Restricts Primarily to the Hippocampus in Mutant Mice CN 98 The CaMKIIa promoter has the advantage of activating or activating the expression of transgenes post-natally to a restricted subset of Aneurons in the central nervous system; in this way it was used to activate or activate transgenic expression selectively in neurons of forebrain structure including the hippocampus, stratum, cortex and amygdala (Mayford et al., 1995a: Kojima et al., 1997). Northern blot analysis performed on the adult CN98 mutant mouse forebrain revealed the expression of a 1.9 kb transcript corresponding to the transgene mRNA (Figure 11B). The brain distribution of the mRNA was determined by in situ hybridization using a radiolabelled oligonucleotide specific for the transgene. The mRNA was detected in the forebrain, mainly in the hippocampus in the CAI, CA2 and CA3 region as well as in the dentate gyrus (Figure 11D). No signal was detected in the wild type littermates (Figures 11D). To determine if the transgene mRNA was properly translated into a functional protein, phosphatase activity was measured in crude homogenates ^ p of the hippocampus in the presence of okadaic acid (Figure 11C). In extracts of transgenic hippocampi, there was an increase of 76% ± 12% in phosphatase activity compared to wild type extracts. In the presence of the calcium chelator EGTA, phosphatase activity in hippocampal extracts both mutant and wild-type CN98 was virtually abolished, with no significant difference in phosphatase activity between the wild type and CN98 mutant extracts (Figure 11C). In this manner, CN98 mutant mice have significantly increased levels of calcium-stimulated phosphatase activity in the hippocampus. Basal Synaptic Transmission is Not Altered in Mice Overexpressing Calcineurin. Studies with pharmacological phosphatase inhibitors have suggested that endogenous phosphatase activity can regulate the basal level of synaptic transmission in the collateral synapses of Schaffer (Firgurov et al., 1993, but see Mulkey et al., 1993; O'Dell and Kandel, 1994). However, in ^ CN98 mice, no difference was found in the synaptic, basal transmission. The stimulus response curves obtained from mutant and wild-type CN98 mice were not significantly different (Figure 12A), and the slope of a fEPSP produced by a pre-synaptic fiber discharge, given did not define dramatically between wild type and wild type mice. mutants (mean dependent ratio of fEPSP and pre-synaptic fiber discharge amplitude expressed with standard deviation was 3.1 ± 1.68 for wild type CN98 and 2.9 ± 1.3 for mutant, Figure 12B). In addition to basal transmission mediated mainly by non-NMDA ionotropic glutamate receptors, previous studies have ? demonstrated that under certain circumstances calcineurin can subtly desensitize to synaptic acts mediated by the NMDA receptor (Tong et al., 1995; Raman et al., 1996). To determine whether overexpression of calcineurin altered NMDA-mediated synaptic transmission in CN98 mice, NMDA-mediated synaptic potentials were measured in the presence of 10 μM of 6,7-dinitroquinozaline-2,3-dione (DNQX) and reduced MG2 + (50 μM). Under these conditions, the field potentials exhibited less kinetics than in the absence of DNQX, and were completely antagonized by 50 μM DL-AP5, indicating that they were mediated by receptors of the stimulus response curves generated for both mutant and wild type animals. CN98 under these conditions were not significantly different, suggesting that overexpression of calcineurin does not alter the function of the NMDA receptor (Figure 12C). Furthermore, under these conditions, the NMDA-mediated synaptic responses in cuts followed a one-second 100-Hz tetanus, in a manner qualitatively similar to the wild types (Figure 12C, insertion). Because in the CN98 mutant mice the transgene is expressed in both pyramidal CAI and CA3 cells, then an assessment of presynaptic function was made in wild type mice in CN98 mutants. We started by evaluating fl-tetanus potentiation (PTP, for review, see Zucker, 1989), in a short-term manner of the presynaptic plasticity produced by a high-frequency tetanus (1 second, 100 Hz). In the presence of DL-AP5 (50 μM) to block the NMDA receptors, administration of a single 100 Hz tetanus resulted in a transient improvement of the transmission that rapidly decayed to the baseline in the space of 2- 3 minutes as previously described (Huang et al., 1995; Abel et al., 1997). As is evident in Figure 12D, there was no difference in the peak PTP produced between the wild-type and mutant mice (160 ± 5% peak potentiation in wild, 11 cuts, 5 mice: 163 ± 11% peak potentiation in CN98 mutants, 11 cuts, 4 mice). These results suggest that the overexpression of calcineurin does not markedly affect the ability of Schaffer's co-lateral CAI synapse to respond to controlled, high-frequency simulation speeds. (f) To obtain a second measure of the presynaptic function, the paired impulse facilitation (PPF) was examined.The PPF is a transient form of the pre-synaptic plasticity in which the second of the two closely separated stimuli produces the enhanced release of transmitters due to residual calcium increases in the pre-synaptic terminal after Aprimer stimulus (for review, see Zucker, 1989).

During a range of 20-250 msec, PPF was significantly reduced in CN98 mutant mice compared to wild-type mice (15 cuts, 5 CN98 wild-type mice, 14 cuts, 5 mutant CN98 mice, for intervals of 20, 50 and 100 ms between stimuli, p <0.05 for wild type against mutant CN98; Figure 12E). In this way, the data in PTP and PPF are consistent in showing that overexpression of calcineurin does not produce enormous deficits in synaptic transmission, however, it produces a clear alteration in a form of acute pre-synaptic plasticity. ^ P Overexpression of Calcineurin Does Not Affect Expression of LTD at the CAI Collateral Synapse of Schaffer Collateral Cells. To start studying calcineurin roles in synaptic plasticity, the LTD induced 15 minutes of stimulation at 1 Hz at the synapses of the CAl-collateral cells of ifljSchaffer in adult and wild type animals CN98 was compared to mutants. It was found that the response to 15 minutes of stimulation at 1 Hz was clearly identical in wild type animals and CN98 mutants (percent slope of baseline fEPSP 30 minutes after the end of 15 minutes of stimulation at 1 Hz: wild type CN98 93 ± 6%, 7 mice, 11 cuts; mutants CN98 95 + 7%, ? A mice, 14 cuts). As previously reported (O 'Dell and Kandel, 1994; Bear and Abraham, 1996), this stimulation protocol produced little, if any, LTD in hippocampal cuts of adult animals. Therefore, these studies were repeated in the section of young mice (3-4 weeks of age) where the LTD is strongest (Bear and Abraham, 1996). As shown in Figure 12F, although the LTD was much stronger in these younger animals, there was still no detectable difference between the wild type animals and in CN98 mutants (percent dependent on baseline fEPSP 30 minutes after the end of 15 minutes of stimulation of 1 Hz: type (^^ ilves re CN98 79 ± 8%, 2 animals, 4 cuts, mutant CN98 76 ± 7%, 4 animals, 7 cuts) A consistent possibility with these data is that the Calcineurin may already be present at saturation concentrations, particularly since calcineurin is one of the most abundant proteins in the brain (Yakel, 1997) .If calcineurin is present at saturation concentrations, it will be predicted that further overexpression of Calcineurin will not affect processes such as LTD that were probably measured by phosphatase activation, however, overexpression could alter synaptic processes such as LTP where deletion of phosphatase activity is thought to be required. o Calcineurin overexpression Decreases LTP induced by multiple series at high frequency, but not an individual series. To begin to explore the roles of calcineurin in long-term synaptic improvement, LTP induced by high-frequency series (100 Hz) of a second, single or multiple, was studied in hippocampal slices of CN98 and wild type mutant mice. In the sections of both wild-type and CN98 mutant mice, administration of a single series at 100 Hz produced a transient form of LTP that was comparable to one hour post-tetanus, although after tetanus, LTP was slightly reduced in CN98 mutant (mutant CN98: 129 ± 10% baseline at 1 hour, 9 cuts, 5 mice, wild type CN98: 130 ± 6% baseline at 1 hour, 7 cuts , 4 mice, Figure 13A). In contrast, the administration of four series at 100 Hz separated by 5 minutes produced strong LTP, which does not decrease f ^ in wild type hippocampal sections, but produced a greatly reduced LTP in mutant mice (CN98 wild type: 169 ± 8% baseline at 1 hour after the stimulus, 173 ± 8% at 3 hours, 7 cuts, 7 mice, CN98 mutant: 139 ± 9% baseline at 1 hour after the stimulus, 118 ± 10% at 3 hours, 8 cuts, 7 mice, Figure 13B). This defect in CN98 mutant animals was visible immediately after the four tetanus were administered (p <0.05 at 1 minute after the last tetanus). Overexpression of Calcineurin Does not Affect Chemically Induced L-LTP. The finding that LTP induced by four series but not an individual series is reduced in CN98 mutant mice suggests that overexpression of calcineurin may suppress the late phase of LTP. Is this reduction from life to a direct effect on the components in the 3 'direction of L-LTP, or is it due to a failure to fully start the L-LTP? . To begin to explore this question, we examined the one provoked by the pharmacological activation of the PKA route, which derives the tetanic stimulation in the CAI area. In addition to being caused by multiple high-frequency tetanus to Schaffer collaterals, L-LTP in the CAI area can be induced chemically through either the application of dopamine D1 / D5 receptor agonists or direct flactivators. of adenylyl-cyclase and PKA. In wild-type sections, the application of agonists at the D1 / D5 dopamine receptors or the PKA agonist, Sp-cAMPS, will result in a slow onset enhancement of synaptic transmission that is sensitive to inhibitors of RNA synthesis and protein, and mutually occlusive with L-LTP produced by multiple high frequency series (Huang and A Kandel, 1994; Huang et al., 1994; Bolshakov et al., 1997). If the overexpression of calcineurin directly affects the machinery necessary to produce the late phase, the pharmacologically induced L-LTP, which derives the E and I-LTP, will be damaged in CN98 mutant mice, as is the case with the late phase deficit. in nude mice (anesthetized) tPA (Huang et al., 1996a). The ability of both the agonist 6 Br-APB (100 μM) the D1 / D5 receptor and the PKA activator, Sp-cAMPs (100 μM) was tested to produce a slow start enhancement at the Schaffer CAl-collateral synapse in CN98 mice. As shown in the 13C and 13D, the application of 6-Br-APB and Sp-cAMP produced a slow developmental increase in synaptic transmission in CN98 mutant mice that was indistinguishable from that seen in wild type mice (mutant CN98: 181 ± 41% of line basal to 3 hours after the application of 6-Br-APB, 5 cuts, mice; wild type CN98; 204 ± 40% line ifljbasal at 3 hours after the application of Sp-cAMPs, 6 cuts, 6 mice; wild type: 124 + 13% baseline at 3 hours after the application of SpACAMPs, 7 cuts, 6 mice). Multiple Series Produce Two Different Dependent Phases of PKA, from LTP: One Dependent and the other Independent of the Synthesis Protein In contrast to wild type hippocampal cuts where the LTP induced by an individual series is much weaker than that induced by four series, in sections of CN98 mutants, the magnitude of LTP following protocols of one series and four series were Similar. Actually, the LTP after four series in CN98 mutants is * 4 completely similar to that evident after four series in wild type hippocampal sections incubated with PKA inhibitors (for review see Huang et al., 1996b), as well as L-LTP in hippocampal slices of mouse expression , a negative, dominant form of PKA (Abel et al., 1997). This makes it seem as if the PKA system is defective or reduced in its effectiveness in the mutant mice.Although the L-LTP induced by pharmacological activation of the cAMP cascade was not damaged dramatically in the mutant mice. then do PKA and calcineurin interact? A suggestion to the possible interaction of jjpcalcineurin with the PKA system in regulating LTP comes from the work of Blitzer et al., (1995) and Abel et al. (1997), which shows that the application of Inhibitors of PPl and PP2A removes the ability of PKA in LTP in the area CAI may be to inhibit the actions of phosphatases that are activated by tetanus This suggested that PKA can serve a double function. First, you can directly activate the phase Atardía (Figure 13C, 13D). Second, PKA has an early role in inactivating an opposite phosphatase cascade. Consistent with this hypothesis, LTP generated by multiple series at 100 Hz in rat hippocampal slices (Huang et al., 1996b), as well as mouse hippocampal slices (Figure 14A) decreases more rapidly in the presence of PKA inhibitors such as Rp-cAMPs or KT5720 than in the presence of the inhibitor of protein synthesis, anisomycin, which suggests that in addition to mediating a late phase action of LTP that requires protein synthesis, PKA can mediate a second action, perhaps more above in the 5 'direction, which is (^ independently of protein synthesis (Blitzer et al., 1995; Huang et al., 1996b) .To further examine the possibility that there are two independent phases, both dependent on PKA, they became analyze the effects of anisomycin, an inhibitor of protein synthesis in LTP in mouse hippocampal slices, (now increasing the pre-incubation time to anisomycin for 30 minutes. The concentrations of anisomycin used were sufficient to completely block protein synthesis in the CAI area of the hippocampus (Stanton and Sarvey, 1984; Osten et al., 1996). However, the difference in the time course of inhibition by anisomycin and PKA inhibitors may be due Ékk to the pharmacokinetic properties of these drugs.

However, even in experiments where anisomycin (30 μM) is present in the bath for one full hour before tetanus (compared to the 20 minute pretreatment with PKA inhibitor, KT5720, 1 μM), the PKA inhibitor still produced an even more rapid decrease in LTP induced by four series at 100 Hz than anisomycin (Figure 14A, 14B). This difference in the time course between inhibitors of protein synthesis and PKA suggests that multiple series that produce L-LTP also seem to induce a new intermediate phase of LTP that requires PKA, but does not require protein synthesis. A New Intermediate Phase dependent on PKA can also be pharmacologically isolated and by varying the number of Stimulus Series. In an attempt to isolate, in some other way, the new intermediate phase in which PKA acts to suppress a phosphatase cascade, the number of tetanic stimulation series is varied. One of the characteristics that distinguishes E-LTP from L-LTP is that in weak stimuli such as a single series at 100 Hz they produce E-LTP, but not L-LTP. In contrast, to reliably induce L-LTP, 3-4 repeated sets at 100 Hz are required. Therefore, it is sought to determine if an intermediate phase of LTP could also be distinguished from these phases based on the concentration of the stimulus required. m LTP was produced with two series at 100 Hz separated by seconds This protocol produced LTP that was stronger than what was produced by a series at 100 Hz, but less maintained than that produced by four series (Figure 14C). In contrast to the LTP produced by a single series at 100 Hz that is not affected by PKA inhibitors (Huang et al., 1996), the LTP produced by two series was partially sensitive to PKA inhibitor KT5720 (no drug: 206 ± 23% baseline at 1 hour, 5 sections, 5 mice, KT5720 1 μM: 153 + 5% baseline at 1 hour, 5 sections, 4 mice, p <0.05, Figure 14D). However, different from L-LTP, the LTP produced by two series was completely insensitive to preincubation with the protein synthesis inhibitor, anisomycin, even at time points where the LTP induced by four series was reduced by anisomycin (Figure 4C).

These two types of experiments revealed that there is a new intermediate phase of LTP (I-LTP) that requires 1) a stimulus stronger than E-LTP, and 2) that of PKA. But different from L-LTP, this intermediate phase does not require protein synthesis. Genetic Evidence for an Interaction between PKA and Phosphatases in the Regulation of a New Intermediate Phase of LTP (I-LTP). To strengthen these pharmacological attempts to delineate an intermediate phase, it was changed to a genetic approach. Blitzer et al., A ^ (1995) and Thomas et al., (1996) suggested that an independent role of protein synthesis of PKA in LTP is to suppress the activity of PPl or PP2A, perhaps through phosphorylation of the inhibitor. -1. Since the phosphorylation site of inhibitor-1 is dephosphorylated by calcineurin, it would be predicted that PKA and calcineurin will antagonistically regulate PPI function and thus regulate the level of synaptic output. If this were the case, it will be predicted that in mice overexpressing calcineurin, the cAMP-dependent forms of LTP in the CAI area will be defective. Of course, as we have seen, the LTP of one series, which is independent of PKA, did not decrease in the CN98 mutant mice, whereas the four-series LTP did depend on PKA. To examine this additionally, CN98 mutant wild-type mice were compared when examining the LTP induced by two series, which have been shown to recruit the intermediate phase without significantly recruiting the late phase. (^ Consistent with the idea that the intermediate phase of LTP is antagonistically regulated by PKA and calcineurin, the LTP produced by two series in mutant mice was markedly damaged (mutant CN98: 127 ± 7% baseline at 1 hour, 12 cuts, 7 mice, wild type CN98: 182 ± 17% of baseline at 1 hour, 8 cuts, 4 mice, p <0.05; 14E). In addition, the LTP that remained in the mutant mice was insensitive to the emission of PKA, further suggesting that the PKA function in the intermediate phase is to mitigate further calcineurin actions (Figure 14F). The Overexpression of. Transference from Restricted Calcineurin to Pyramidal Cells CAI Post-synaptic is sufficient to interfere with the Intermediate Phase of LTP. The phenotype of CN98 mutant mice suggests that calcineurin suppresses an intermediate phase of LTP. However, because the calcineurin construction in these mice is expressed both in pre-synaptic CA3 cells and Kopio in post-synaptic CAI pyramidal cells, it can not be said that these experiments alone where calcineurin is producing its action . Furthermore, it is conceived that subtle alterations in the pre-synaptic function, such as those observed in PPF and PTP in these mice could contribute to the phenotype. To investigate this possibility, as well as to verify that the deficit in I-LTP seen is not due to an effect at the insertion site, two additional lines of mice expressing the calcineurin transgene were analyzed in a more spatially restricted manner. The hippocampus, the two lines tested, (Tet-CN279 and Tet-CN273), have the additional advantage that the expression of the calcineurin transgene is regulated by the transactivator system «Controlled by tetracycline (tTA) (see attached article, Mansuy et al., Presented to Cell, for details of the generation and characterization of these two lines). In contrast to the CN98 line, in which the transgene is strongly expressed both in CA3 and CAI pyramidal cells, in the Tet-CN279 and Tet-CN273 lines, the transgene is expressed much more strongly in pyramidal, post-synaptic, CAI cells. that in the CA3 pyramidal areas (Mansuy et al., presented to Cell). The effects of overexpression of the transgene in CATI pyramidal cells in LTP were first determined by comparing sections of Tet-CN273 and Tet-ß! N279 in the LTP produced by one or two series, the LTP induced by four series at 100 Hz in mice Tet-CN279. Consistent with the results in the CN98 line, it was found that overexpression of the calcineurin transgene under the Tet system had no effect on the LTP induced by an individual series, but reduced the LTP induced by two and four series (© (Figures 15A, B, C; Figure 17B) It is interesting to note that, in contrast to CN98 mice, where LTP was reduced immediately after two series at 100 Hz, both Tet-CN279 and Tet-CN273 mutant mice, which also exhibit a deficit in two 1-hour series, they showed little or no deficits immediately after tetanus, thus the phenotype of these lines was more parallel to the ^^ defec or observed after the application of PKA inhibitors to the wild type cuts that that the CN98 line does, which supports the notion that the delineation of the intermediate phase in these mutant mice is not a device with a reduced pre-synaptic function.In addition, these data imply that the site of action of the Phosphate scada is post-synaptic. The Elimination of the Intermediate Phase of LTP due to the overexpression of Calcineurin can be rescued by the application of PPI Inhibitors Similar to the results obtained in the CN98 line, no detectable differences were found in the synaptic transmission, basal, potential mediated by the NMDA receptor, and PTP in wild type animals and mutants in the Tet-CN279 and Tet-CN273 lines (Figures 16A, B, C). In contrast, the results in the CN98 line, however, did not show deficits in PPF in the line Tet-CN279 and Tet-CN273, consistent with the weak or absent expression of the transgene in a pre-synaptic manner (Figure 16D). discussed earlier, Blitzer et al., (1995) reported that pre-incubation of hippocampal cuts with PPI inhibitors removed the ability of PKA inhibitors to block LTP produced by a strong stimulus, suggesting that a role of PKA in LTP may be to suppress the activity of phosphatase in a type manner "gate" . Because PKA regulates the function of ? kPPl through phosphorylation of inhibitor-1, a site that is dephosphorylated by calcineurin, it will be predicted that pre-incubation of hippocampal slices from mice overexpressing calcineurin should rescue LTP. To test this hypothesis, sections of mutant and wild-type mice Tet-CN279 were pre-treated for 30 minutes with 750 nM caliculin A, after which the LTP was induced with two series at 100 Hz. Consistent with the hypothesis that calcineurin on expressed is suppressing LTP by regulating PPI activity, pre-treatment of cuts with caliculin A resulted in LTP in mutant mice that was indistinguishable from that in the wild type. (Figure 17A). Regulated Overexpression of the Calcineurin Transgene Suggests that the Deficit in I-LTP is Not Due to the Effects on the Development of the Transgen in the Hippocampus. Since the tTA system allows the regulation of transgenic expression, experiments were carried out to confront whether the phenotype observed in mice overexpressing calcineurin reflected a consequence of the transgene in the development of the nervous system or represented an acute effect of the transgene. in synaptic plasticity., In the absence of the inhibitor, doxycycline, the transgene is expressed in the mice Tet-CN279 (Mansuy et al., Submitted to Cell). Without However, when doxycycline (1 mg / ml) is administered in the animal's water supply, or in the ACSF (1 ng / ml) during electrophysiological experiments, expression of the transgene is suppressed (Mansuy et al., Submitted to Cell). Therefore, the LTP induced by two series in mutant and wild type Tet-CN279 mice was compared in or out of doxycycline. In wild type mice, either on or off doxycycline, stimulation with two series resulted in strong LTP that was indistinguishable from that produced in wild type mice CN 98 S 8 (Tet-CN279 weight: 195 ± 13% baseline) at 1 hour, 7 cuts, 6 mice, mutant mice Tet-CN279 in (flioxycycline: 191 ± 18% baseline at 1 hour, 12 cuts, 7 mice, Figure 17B). In Tet-CN279 mutant mice without doxycycline, the response to the two strains was significantly lower than that seen in the wild type one hour after tetanus, and was completely reversed by the pretreatment with doxycycline (mutant Tet-CN279: 147 ± 8% fbasal line at 1 hour, 15 cuts, 9 mice, Tet-CN279 on doxycycline: 184 ± 18% baseline at 1 hour, 8 cuts, 5 mice, p <0.01 for mutant Tet-CN279 against wild type Tet-CN279, Figure 17B These results clearly show that the calcineurin transgene produces its effect in the intermediate phase of LTP in a post-synaptic manner in the adult animal, and its effect can not be attributed to a consequence of the development of the transgene. .

Discussion In an attempt to develop a genetic approach to study the role of phosphatases in synaptic plasticity, it has focused on calcineurin, because it appears to work in the hippocampus as the first step in a calcium-dependent molecular signaling cascade of phosphatases. Both to limit the expression of the transgene to the forebrain, and to reduce the likelihood that the phenotype produced is a result of the presence of the transgene during the Calcineurin has been over expressed using the CaMKII promoter. To further control a role of the development of the transgene, as well as to control the effects dependent on the insertion site, two other lines of mice (Tet-CN279, Tet-CN273) in which the phenotype exhibited by CN98 mice have also been studied It can be reproduced and reversed by the suppression of the expression of the transgene using an adjustable trans-activator (see, Mansuy et al., presented to Cell.) With these lines, it was able to show that the expression of calcineurin essentially limited CAI neurons of the hippocampus selectively interfere with a new phase of LTP that was isolated independently by pharmacological means and Aal using a stimulation protocol of two series.

Furthermore, this phenotype in mice overexpressing calcineurin is due to the expression of the transgene in the adult animal.

An Intermediate Component of LTP, -LTP, Modulated by Calcineurin and PKA These experiments have revealed several important features about the role of calcineurin and PKA in synaptic function at the Schaffer CAl-collateral synapse. Convergent lines of evidence, both from pharmacological studies in wild type mice and in genetic studies with mice overexpressing calcineurin, suggest that there is an intermediate phase of LTP, and that this phase is suppressed by calcineurin. Unlike LTP, it is based on two sets of findings (Figure 18): First, E-LTP and I-LTP are distinguishable in three ways: 1) E-LTP is (dependent on PKA, whereas I-LTP is PKA-dependent 2) I-LTP, but not E-LTP, is inhibited by the over expression of calcineurin. Finally, 3) I-LTP requires a stronger stimulus for initiation than E-LTP. Second, I-LTP in turn can also be distinguished from L-LTP in two ways. First, while the I-LTP and the L-LTP are dependent on APKA, only L-LTP is dependent on macromolecular synthesis. Second, while I-LTP could not be generated in mice that over-express calcineurin, the slow-onset, pharmacologically induced potentiation, which is thought to utilize the same mechanisms as tetanus-induced L-LTP can still be generated. Although the temporal characteristics that suggest that PKA participates in an independent phase of macromolecular synthesis have not been clearly defined in previous studies, it has been implicit in several of them. Several groups have pointed out that an apparently independent component of the protein synthesis, early, of LT ^ produced by multiple series, requires PKA (Huang and Kandel, 1994, Blitzer et al., 1995). For example, Huang and Kandel (1994) have found that while LTP induced by multiple series is rapidly inhibited by PKA inhibitors, it was inhibited more slowly by inhibitors of protein synthesis. Consistent with this finding, Blitzer et al. , (1995) found that LTP is induced by three series is partially blocked by PKA inhibitors and that this blockade has a rapid time course.In addition, Thomas et al., (1996) found that the activation of ß- Adrenergic therapy by isoproterenol allows sub-threshold stimuli to produce a strong improvement of transmission A ^ synaptic in the collateral synapse of Schaffer in a PKA-dependent manner. Both effects delineated by Blitzer et al. and by Thomas et al. they were interpreted to reflect a PKA-mediated suppression of phosphatase activity. In each case, the ability of PKA inhibitors to block LTP is reduced by phosphatase inhibitors such as caliculin A and okadaic acid. While these previous studies suggested that a role of PKA in LTP will suppress phosphatase activity, these studies could not exclude an alternative explanation, that phosphatase inhibitors improved the actions of incompletely antagonized, residual ß KA. Although calcineurin was proposed to participate in the suppression of LTP, caliculin A and okadaic acid are ineffective in the inhibition of calcineurin at the concentrations used in these experiments, making it clear whether calcineurin is important in the regulation of LTP. In fact, the application of calcineurin inhibitors to hippocampal cuts has produced contradictory results, with some studies reporting no effect (Mulkey et al., 1993; Muller et al., 1995) or improvement (Wang and Kelly, 1996). ) of LTP, while other studies report the blockage of LTP (Wang and Stelzer, 1994, Wang and Kelly, 1997, Lu et al., 1996 a, b) Using genetic approaches, the opposite and proven approach has been taken ^ k that PKA suppressed a phosphatase cascade by showing that the over expression of calcineurin removes the PKA-dependent LTP component. Because this deletion is rescued by the inhibitor PP1 / PP2, caliculin A, these data are also consistent with the proposed model that calcineurin and PKA interact at the level of inhibitor-1, a molecule that controls this activity of PPl. The distinctive characteristics of I-LTP are reported here to have these previous results in the roles of phosphatases and PKA in LTP by showing that the over expression of calcineurin removes the PKA-dependent LTP constituents in the CAI area. , the findings confirm the observation by Blitzer et al. (1995) that PKA plays a regulatory role, important in LTP by suppressing phosphatase activity, and extends this idea by delineating that this role of PKA specifically comprises a competition with calcineurin, which fljbre presents a different temporal phase and that this phase has behavioral consequences (Mansuy et al., presented to Cell.) It is emphasized that although I-LTP and E-LTP differ in several different ways, I-LTP most likely also shares several mechanisms in common with E-LTP, for example, the suppression of phosphatase activity by PKA during I-LTP, a Asuppression that requires a stronger stimulus than the 100 Hz series needed to produce E-LTP, can act simply to allow a stronger utilization of the mechanisms recruited by the E-LTP. Furthermore, as long as there is a temporal distinction between I-LTP, E-LTP and L-LTP in response to repeated series at high frequency, as well as the distinction in concentration of the stimulus force required to produce these phases, these distinctions they may become confusing under other circumstances, such as during periods in which neuromodulatory influences are not recruited (Thomas et al., 1996). However, I-LTP sensitivity to stimulus intensity explains why in a previous report the over-expression of a negative, dominant form of PKA had no effect on LTP produced by two series (Abel et al., 1997). When a two-series protocol, stronger than that producing LTP of a magnitude comparable to the present data, was used, defective LTP was observed in both series in R (AB) mutant mice.

(D.G.W. and Abel, T. Personal communication). The evidence suggests that the intermediate phase of LTP is inhibited by the over expression of calcineurin. If calcineurin performs the same function, it remains to be determined. However, pharmacological experiments suggest that this may be the case (Wang and Kelly, 1996). This Amanera, in future experiments, it will be important to use other genetic manipulations, such as dominant negative constructs of calcineurin eliminations to investigate this additional intermediate phase. Interestingly, it is found that several aspects of synaptic transmission that are believed to be measured by calcineurin are not altered by the overexpression of this enzyme. For example, the over expression of calcineurin failed to modulate LTD, basal synaptic transmission, or synaptic potentials mediated by the NMDA receptor. While there are several possible explanations for these findings, one possibility is that a large excess of calcineurin exists in CAI (a calcineurin pool). Consistent with this idea, calcineurin is one of the most abundant proteins in the brain (Yagel, 1997). If this hypothesis is correct, the over expression of calcineurin will only be expected to affect the physiological actions that suppress endogenous suppression of phosphatase activity, since overexpression will create a large pool of calcineurin that can make it more difficult to completely inhibit the activity of phosphatase. Consistent with this idea, it is found that over expression of calcineurin places an inhibitory restriction on I-LTP. Ám PKA is a Regulator of Re-Feeding the Activity of Calcium-stimulated Kinase. Calcineurin has a particularly high affinity for calcium / calmodulin. For example, it is at least an order of magnitude more sensitive to calcium / calmodulin than CaMKII. It was this characteristic of calcineurin that led Lisman (1989; 1994) to propose that low-level increases in calcium, induced by low-frequency stimuli, will lead to synaptic depression through the activation of calcineurin, while that high-frequency stimuli will lead to the large increases in calcium needed to be activated in CaMKII and lead to LTP (Lisman, 1994). These aspects of the Lisman model have been supported by several studies (Malenka and Nicoll, 1993, Cummings et al., 1996). The studies provide support for an additional prediction of the model. According to the Lisman model, strong LTP requires the inactivation of phosphatases. Consistent with this idea, it is found that phosphatases do not in fact impose an inhibitory restriction on LTP, and suggest that, PKA is required to suppress phosphatase activity in a manner sufficient to completely produce LTP. Calcium-sensitive adenyl cyclases are ideally suited to increase cAMP levels and to inhibit phosphatases only when large increases in intracellular calcium occur (Lisman, 1994). In fact, activation of NMDA receptors by strong tetanization that induces LTP increases cAMP levels in CAI through a calmodulin-dependent process (Chetkovich et al., 1991, 1993). Therefore, while calcium directly regulates the balance of kinase and phosphatase activity, the generation of cAMP by NMDA receptor-dependent activation of calcium-sensitive adenyl-cyclase may further favor kinases by inducing inactivation PKA-dependent activation of fl l by calcineurin through inhibitor-1 phosphorylation. In this way, the data extend the Lisman model in the suggestion that there are four critical steps, instead of two, in the relationship between the re-exit for the LTP and LTD of the hippocampus. First, the weak stimuli that produce ^ pincrements at low level in the intracellular calcium * result in LTD, due to more complete activation of phosphatases than kinases. Second, moderate stimuli produce higher increases in intracellular calcium (such as a series at 100 Hz), but produce sub-maximal LTP (E-LTP) because, the kinases activated by the long influx of calcium are opposed by the phosphatases that are not present.

? Third, the strong stimuli (multiple series a 100 Hz, by way of example) produce the LTP (I-LTP) because the calcium-dependent activation of the kinases is now combined with a PKA-dependent suppression of the phosphatases. Finally, if the stimuli are of sufficient strength (3-4 series at 100 Hz), an additional role of PKA will be activated, which will result in the establishment of the late phase dependent on the macromolecular synthesis of LTP. This model further explains why the slow start potentiations induced by the dopamine D1 / D5 Sp-cAMPS receptor agonists are not affected by the over expression of calcineruin. These agents elicit synaptic tetanization to slow-start, induced potentiation, thereby preventing the activation of the calcium-dependent calcineurin-phosphatase cascade, and therefore need not inhibit this activated cascade.

Calcineurin can act as a deviation from fl a. L-LTP Synaptically Provoid. It is found that L-LTP induced by four series at 100 Hz is defective in CN98 mutant mice. In an effort to determine if the machinery required to induce L-LTP is intact in the CN98 mice, it is determined whether the late phase could be pharmacologically elicited., or not, in a way that derives tetanus. In this way, it is found that the application of the activators of the PKA cascade induced a slow onset enhancement of the transmission that was normal in CN98 mutant sections. This potentiation of slow onset of transmission is thought to use the same machinery as the four series at 100 Hz because they are both PKA-dependent and macromolecular synthesis, and mutually occlusive (Frey et al., 1993; Huang et al., 1996b). In contrast, both L-LTP induced by tetanus and that induced archaeologically are damaged in cases in which molecules are scrambled that are predicted to be in the 3 'direction of the macromolecular synthesis in the L-LTP synthesis . For example, in tPA "/" mice that exhibit an L-LTP caused by tetanus, defective, slow-onset potentiation if induced by the D1 / D5 agonists and Sp-cAMPS is absent, consistent with the idea that tPA mice " '"lack the machinery in the 3' direction needed to produce the late phase (Huang et al., © L996a). As discussed above, this reduction of LTP in CN98 mutant mice overexpressing calcineurin is probably due to a deviation of the kinases in the 5 'direction, important for initiating L-LTP. Actually, two recent reports are consistent with this possibility. First, Liu and Graybiel (1996) have Proven that phosphorylation of CREB and transcriptional activity in striosomes are relatively regulated by calcineurin. In addition, Bito et al. (1996) has reported that phosphorylation of CREB in hippocampal neurons in culture is also relatively regulated by calcineurin. In this way, the regulation of transcription factors is thought to be necessary for long-term synaptic modifications can prevent the formation of L-LTP in cases in which PKA is not sufficiently activated.

Multiple Inhibitory Restrictions Must Be Exceeded to Produce Synaptic Plasticity Dependent on PKA The data suggest that calcineurin acts as an inhibitory constraint on synaptic plasticity that precludes the formation of an intermediate and late phase of tetanus-induced LTP. As indicated, PKA, which contravenes the calcineurin factions, appears to act both as a disinhibitor of these phases and as a direct facilitator of the late phase.The studies on invertebrate Aplysia and Drosophila first revealed that the expression of plasticity Synaptic-related learning is restricted by several inhibitory constraints that operate in different compartments within the cell, ranging from the cell membrane to the nucleus (Yin et al., 1994, 1995; Bartsch et al., 1995). et al. (1995) found that an isoform of the transcription factor CREB (CREB-2) normally suppresses the formation of long-term facilitation by an individual serotonin boost. However, the removal of this restriction by injection of antisense antibodies or oligonucleotides directed against this transcription factor allows an impulse of serotonin, which normally only produces short-term facilitation to produce long-term facilitation. These studies imply that in order to induce the long-term enhancement of synaptic transmission, different types of inhibitory constraints are also acting on the mammalian brain plasticity.In the annexed article (Mansuy et al., 1997), it is shown that Excessive activation of this inhibitory restriction interferes with memory storage.

MATERIALS / METHODS Plasmid Construction A cDNA encoding a truncated form of the murine calcineurin sub unit Aa,? CaM-AI (provided by S. J. O'Keefe) was used to construct the expression vector for the generation of CN98 mice. CaM-AI lacks the self-inhibitory domain and a portion of the calmodulin binding domain of calcineurin Aa and was shown to be active in Jurkat T cells (O'Keefe et al., 1992). An EcoRI fragment of 1.27 kb of the? CaM-AI cDNA was made blunt-ended and subcloned into the EcoRV site of vector pNN265 (provided by N. Nakanishi). Plasmid pNN265 carries in the 5 'direction from the EcoRV site, a 230 bp hybrid intron containing an adenovirus splice donor and an immunoglobulin G splice acceptor (Choi et al., 1992) and has a polyadenylation signal SV40 in the 3 'direction from the EcoRV site. The? CaM-AI cDNA flanked by the 5 'hybrid intron and the oli (A) signal was excised in 3' of pNN265 with NotI and the resulting 2.7 kb fragment that was placed in the 3 'direction of the CaMKIIa promoter of mouse of 8.5 kb including the transcriptional initiation site (Mayford et al., 1996) to generate the CN98 mice (Figure HA) was excised from the vector by digestion with Sfil. Before the microinjection all the unions (© 3e cloning were verified by DNA sequencing.

Generation and_ Maintenance of CN98 Transgenic Mice CN98 transgenic mice were generated by microinjection of the linear constructs in fertilized eggs collected from BL6 / CBA Fl / J super-ovulated females paired with BL6 / CBA males Fl | (Jackson Laboratories; Hogan et al, 1991). Before the microinjection, the DNA fragment was gel purified then put through Elutip (Scheicher and Schuell) for additional purification. The microinjected eggs were kept overnight 37 ° C in 5% CO2 and one day later, the embryos of two females were transferred to pseudo-pregnant females BL6 / CBA Fl / J. The analysis of the founder mice for the integration of the transgene was carried out by Southern blotting and PCR. The found mouse was backcrossed to C57BL6 Fl / J mice to generate the CN98 transgenic line. The genotype of the offspring was verified by Southern blot or. The transgenic mice were maintained in the animal colony according to the normal protocol.

Northern blot analysis The forebrains of mice were harvested CN98 adult and total RNA was isolated by the guanidinium thiocyanate method (Chomczynsi and Sacchi, 1987). (Fliezy micrograms of RNA were denatured in 1 M formaldehyde, 50% formamide, 40 mM triethanolamine, 2 mM EDTA (pH 8), electrophoresed on a 1% agarose gel and transferred to a nylon membrane ( GenScreen Plus, NEN) in 0.4 N NaOH. The membrane was hybridized to an EcoRV-Notl fragment labeled with [a32P] dCTP of 1.1 kb from pNN265. Hybridization was performed overnight A42 ° C in 50% formamide, 2 X SSC, 1% SDS, 10% dextran sulfate, Denatured salmon sperm DNA 0.5 mg / ml. The membrane was washed 10 minutes at room temperature in 2 X SSC, 1% SDS then twice 15 minutes at 42 ° C in 0.2 X SSC, 1% SDS and was exposed to the movie for three days.

In Situ Hybridization The brains of adult mice were dissected and embedded rapidly in the Tissue-Tek medium on dry ice (Miles, Inc.). Sections of 15 mM cryostat were placed on gelatin-coated glass slides, dried 15 minutes ^ 55 ° C, then fixed 10 minutes in freshly prepared 4% paraformaldehyde, rinsed twice in PBS (pH 7.2) and were dehydrated through an ethanol gradient. The sections were re-hydrated, permeabilized in 0.1 M triethanolamine pH 8, 0.25% acetic anhydride, washed twice in 2 X SSC, rinsed 70% ethanol, then dried. The sections were hybridized overnight at 37 ° C to an oligonucleotide purified by OPC (5'-GCAGGATCCGCTTGGGCTGCAGTTGGACCT-3 ') derived from pNN265. The oligonucleotide was labeled by the 3 '-poly (A) end using [a35S] dATP (NEN) and terminal transferase (Boehringer Mannheim) and the hybridization was performed in a humidified chamber in 50% formamide, 10% dextran sulfate, HEPES mM (pH 7), 1 mM EDTA (pH 8), 100 mM DTT, denatured salmon sperm DNA 400 mg / ml, poly (dA) 400 mg / ml, 1 X Denhart, 600 mM NaCl and 107 cpm oligonucleotide / ml hybridization solution.

After hybridization, the slides were washed twice for 10 minutes in 2 X SSC at room temperature, twice for 60 minutes in 0.2 X SSX at 65 ° C then once 10 minutes in 2 X SSC at room temperature. The slides were dehydrated in 70% ethanol, dried and exposed to the Kodak Biomax MR film for 2-3 weeks.

Phosphatase assay Phosphatase assays were performed according to Hubbard and Klee (1991). Briefly, mice were injected with 5 ml / kg of pentobarbital and decapitated. The hippocampi were dissected, homogenized in 2 mM EDTA (pH 8), sucrose 250 Mm, ß- 0.1% and centrifuged. Supernatants were diluted in 40 mM Tris-HCl (ph 8), 0.1 M NaCl, 0.04 mg / ml bovine serum albumin, 1 mM DTT, 0.45 mM okadaic acid (Shock absorber 1) and incubated at 30 ° C for 1 minute in Shock absorber 1 containing 1 mM of the subunit [? 32P] -RII of AMP-dependent protein kinase peptide (PKA) and either 0.1 mM calmodulin (Sigma) and Ca2 + 0.66 AmM or 0.33 mM EGTA (pH 7.5). The peptide 97 [Ala] -RII (Peninsula Labs) was labeled with [? 32P] ATP (NEN) 0.3 mM using 4 mg of the catalytic subunit of PKA (Fluka). The reaction was stopped with 5% TCA in 0.1 m KH2P04 and the enzymatic activity was calculated as previously described (Klee et al., 1983) and expressed in nmol release of Pi / min / mg protein. The protein concentration was determined using the bicinchroninic acid protein assay kit (Sigma). All samples were made in triplicate.

Electrophysiology fl) Hippocampal slices were prepared, transverse as previously described (Huang and Kandel, 1994). Mice of either sex, age 7-18 weeks were used. In all the appropriate cases, the experimenter did not know about the genotype of the animal. The hippocampi were cut unilaterally rapidly on ice, and cuts were cut fl ue 400 mm in a blade of Mcllwain tissue and placed in oxygenated ACSF (NaCl, 124 mM, KCl, 4.4 mM, CaCl 2, 2.5 mM MgSO4, 1.3 mM, NaH2P04, 1 mM, glucose, 10 mM, and NaHCO3 26 mM). The slices were then transferred to a face chamber where they were sub-fused with oxygenated ACSF (1-2 ml / min) and allowed to reach equilibrium for 60-90 minutes at 28 ° C. For the extracellular records, glass electrodes filled with ACSF (1-3 MO) were placed in the radiatum stratum of the CAI area.

A bipolar nichrome stimulation electrode was also placed in the radiatum stratum for the stimulation of Schaffer collateral afferents. (duration of 0.05 ms). Unless otherwise mentioned, the test stimuli were applied at a frequency of 1 per minute (0.017 Hz), and at a stimulus intensity that produces a slope of fEPSP that was 35% of the maximum. The experiments in which a significant change in the amplitude of the fiber discharge was presented were discarded. they applied the drugs through the perfusion medium. DL-AP5, caliculin A, KT5720 and R (+) - 6-Bromo-7, 8-dihydroxy-3-allyl-1-phenyl-2, 3,4-r * tetrahydro-1H-3-benzazepine (6 -Br-APB) were compared from Research Biochemicals International, Natick, MA. The DL-AP5 was dissolved directly in ACSF before use. Caliculin A, KT5720, and 6-Br-APB were as lOOx concentrated solutions in DMSO, and were diluted in ACSF just before use. Sp-cAMPS (Biolog, La Jolla, CA) was dissolved directly in ACSF.

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Res. Comm. 214; 1000-1008; Raman, IM et al., (1996). "ß-adrenergic regulation of (|? ynaptic NMDA receptors by cAMP-dependt protein kinase," Neuron 16: 415-421; Rangel-Alsao, R et al., (1976). "Dissociation and reassociation of the phosphorylated and nonphosphorylated forms of adenosine 3 ': 5' - monophosphate-dependt protein kinase from bovine cardiac muscle, "J. Biol. Chem. 251: 3375-3380; ED et al., (1996). "A biochemist's view of long-term potentiation," Learn. and Mem. 3: 1-24; Schwaninger; M et al., (1995). "Involvement of the Ca2 + -dependet phosphatase calcineurin in gene transcription that is stimulated by cAMP response elements," J. Biol. Chem. 270: 8860-8866; Stanton, PK et al., (1984). "Blockade of long-term potentiation in rat hippocampal CAI region by jAinhibitors of protein synthesis," J. Neurosci. 4: 3080-3088; Thomas, MJ et al., (1996). "Activity-depends ß- adrenergic modulation of low frequency stimulation induced LTP in the hippocampal CAI region," Neuron 17: 475-482; Tong, G et al., (1995). "Synaptic desensitization of NMDA receptors by calcineurin," Science 267: 1510-1512; Traynelis, S.F. et al., (1997). "Control of rat GlyR6 glutamate receptor open probability by protein kinase A and calcineurin," J. Physiol. 503: 513-531; Wang, JH et al., (1996). "The balance between ^ ostsynaptic Ca2 + -dependet protein kinase and phosphatase activities controlling synaptic strength," Learn. And Mem. 3: 170-181; Wang, JH et al., (1994). "Inhibition of phosphatase 2B prevents expression of hippocampal long-term potentiation, "Neuroreport 5: 2377-2380; Yakel, JL et al., (1994)." Calcineurin regulation of function: from ion channels to transmitter relay and gene transcription, "Trends Neurosci. : 124-134; Yin, JCP et al., (1994). "Induction of a dominant negative CREB transgene speciafically blocks long-term memory in Drosophila," Cell 79: 49-58; Yin, JCP et al., (1995). "CREB as a memory modulator: induced expression of a dCREB2 activator isoform Aenhances long-term memory in Drosophila," Cell 81: 107-115; Zucker, RS (1989). "Short-term synaptic plasticity," Annu. Rev Neurosci. 12: 13-31.

Example 4; The Restricted Expression Over and. Regulated Revealed Calcineurin as a Key Component in the Transition from Short-Term Memory to Long-Term Memory. To investigate whether phosphatases play a role in memory storage, hippocampal dependent memory was evaluated in transgenic mice by expression, mainly in the of a truncated form of calcineurin. These mice have short-term, normal memory, but they have a long-term memory defect that is evident both in a spatial task (the spatial version of Barnes' labyrinth) and in a visual recognition task, thus providing evidence genetics for the role of the lipocampopo of the rodent in storage in spatial as well as non-spatial memory. In addition to Barnes' labyrinth, the long-term memory defect could be completely overcome by increasing the number of training trials. These results suggest that transgenic mice that overexpress calcineurin have the capacity for long-term memory that prevents storage Ade long-term memory. Using the tTA system, the transgenic mice overexpressing calcineurin in a regulated manner were analyzed and found that the observed memory defect was reversible and therefore is more likely due to the transgene and not to the developmental abnormality. Along with the electrophysiological findings that mice overexpressing calcineurin have a defect in an intermediate phase of long-term potentiation (I-LTP), the behavioral results suggest that calcineurin has a role in the transition to short-term or long-term memory and that there is a correlation between this transition in memory storage and a new intermediate phase of LTP.

Introduction Mice overexpressing a truncated form of phosphatase calcineurin in the hippocampus are described (lines CN98, Tet-CN98m Tet- © CN279 and Tet-CN 273). These mice exhibit a specific defect in an intermediate phase of long-term potentiation (I-LTP). Now there is growing evidence that LTP can contribute to the storage of declarative forms of memory (Bliss and Collingridge, 1993; Eichenbaum, 1995; Mayford et al., 1996; Tsien et al., 1996). As in the temporal phases of memory, the LTP also Ano is unitary but has at least two main phases: an early phase (E-LTP) produced by a weak stimulus (a series of ls at 100 Hz) and which is independent of PKA and protein synthesis, and a late phase ( L-LTP) induced by strong stimuli (4 series of IS 100 Hz) that requires PKA and protein synthesis (Huang and Kandel, 1994, Huang et al., 1996). In addition to its role in the late phase of LTP, PKA is thought to be a component of a gate that regulates the initiation of LTP by opposing the actions of the PPl and PP2A phosphatases (Blitzer et al., 1995; Thomas et al. ., nineteen ninety six) . The electrophysiological results with mice expressing a truncated form of calcineurin are consistent with this idea and suggest that this gate has a temporal, distinct component and forms a new intermediate phase of LTP. (I-LTP) that can be suppressed by calcineurin and that has three definitive characteristics: (1) it requires strong stimulation (a minimum of 2 series of one ls to 100 Hz) (2) depends on (3) does not require protein synthesis . In the present study, hippocampal dependent memory was evaluated in mice expressing a truncated form of calcineurin. It was found that mutant mice have normal short-term memory but exhibit a deep and specific defect in long-term memory in both the spatial version of Barnes' labyrinth and in a task that requires the ^ visual recognition of a new object. To determine whether the mutant mice have long-term memory capacity, the training protocol was intensified in the spatial version of Barnes maze by increasing the number of daily training trials and the memory defect was found to be reversed. complete manner, indicating that these mice were able to form long-term memory. This rescue experiment suggests that mice overexpressing calcineurin have damaged long-term memory possibly due to a specific defect in the transition between short-term memory and long flazo which may reflect a weakening of an intermediate component of memory. Finally, it is shown that the observed memory defect was not the result of an abnormality of development due to genetic manipulation. In mice in which the expression of the calcineurin transgene is regulated by the transactivator system controlled by tetracycline (tTA), the spatial memory defect was reversed when the expression of the transgene was repressed by doxycycline.

Results Mice overexpressing calcineurin are deficient in the spatial version of ABarnes maze with a one-day trial. A physiological analysis of transgenic mice overexpressing calcineurin mainly in the hippocampus (CN98 line) is described. This analysis revealed that the CN98 mutant mice lacked an intermediate phase of LTP between the early phase independent of protein synthesis and PKA and the late phase dependent protein synthesis of PKA. As a first step in the analysis of the memory capacity of these mice, the mice were tested in a hippocampal-dependent memory task. The spatial version of Barnes' labyrinth (Barnes, 1979, Bach et al., > 995). The labyrinth of Barnes is a circular maze that has 40 holes in the perimeter and a hidden escape tunnel placed under one of the holes. The mouse is placed in the center of the labyrinth and is motivated to find the tunnel to escape to the brilliantly lit labyrinth, and an unfriendly timbre. To locate the tunnel the mouse needs to remember and use the relationships between distant suggestions in the environment. To achieve the learning criterion in this task, the mouse must make three or fewer errors through five of six consecutive trials. Errors are defined as the search for any hole that does not have the tunnel below it. Previous research has established that performance in this task depends on the hippocampus (Barnes et al., 1979). CN98 mice were tested in the Barnes labyrinth once each day (one attempt per day, 24-hour interval between attempts) until the learning criterion was met or until 40 consecutive days have elapsed. Despite the fact that they were tested for 40 consecutive days, only 25% of the CN98 mutant mice met the learning criterion compared to 88% of the wild type littermates (Figure 19A). An analysis of the mean errors made through (five blocks of five attempts by mutant and wild type mice revealed that the mutant mice have significantly more errors than the wild type mice through the last two blocks of attempts (genotype effect Main F [1.30] 4.63 mp, 0.05 m Figure 19B) Damage in the spatial version of the labyrinth in CN98 mutant mice could be due to a deficit in spatial memory or a deficit in performance such as motor damage In order to exclude a performance deficit, another group of CN98 mice was tested in a version with suggestion of Barnes labyrinth, a task that does not require the hippocampus.The version with suggestion has similar contingencies and (requirements of response as the spatial version, except that the position of the escape tunnel becomes visible to mice by putting a suggestion behind the hole where it is placed. In this way, to locate the escape tunnel, the mice simply need to associate the suggestion with the tunnel. The CN98 mutant mice acquired the task in a manner similar to that of their wild type littermates (Figure 19A) and made a similar number of errors across all try blocks (genotype of main effect F [l, 18] = 2.44, p <; 0.05; Figure 19C). These data indicate that the CN98 mutant mice exhibit normal motivation and do not have any motor, motivational or visual damage.

The deficit in spatial memory can be completely rescued by repeated training trials. The results of the experiments in the spatial version of Barnes' labyrinth, which is a hippocampal-dependent task, indicates that CN98 mutant mice have a defect in long-term spatial memory.The mutant mice have totally lost their ability to form long-term memory? Or do these mice have a block in the transition from short-term memory to long-term memory? Can kratons store long-term memory when they train with a more intensive protocol? Electrophysiological experiments indicated that L-LTP was reduced in CN98 mutant mice (Winder et al., 1997), however, potentiation similar to L-LTP could be induced by pharmacological agents that activate the PKA pathway. that the machinery for the expression of L-LTP is intact in CN98 mutant mice and that the damage seen presides in an intermediate phase, between the early and late phase, which is n necessary for the production of the late phase (winder et al., 1997). (Since L-LTP is thought to be parallel to long-term memory (Abel et al., 1997), these results suggest that CN98 mutant mice may actually have the capacity to form long-term memory but may be deficient in an early phase of memory essential for long memory storage To test whether CN98 mutant mice have long-term memory capacity, the Barnes Maze protocol was modified by increasing the number of daily attempts of one to four per day, attempts were separated by an interval between attempts of 1.5 minutes, when trained with four attempts per day, 100% of the (^ CN98 mutant mice were able to learn the spatial version of the Barnes maze as was 100% of the wild mice (Figure 20A) A comparison of the average attempts and days to the criteria through the protocols of attempts Individuals against repeated, rebelled that a similar number of attempts were required for wild type mice to learn the task if they were given an individual or repeated attempt each day (Figure 20B) .However, the number of days needed for the acquisition of the task was much smaller with the ß S four attempts per day than with only one attempt per day (Figure 20B) .An analysis of the average of rrores revealed that the mutant mice were similar to the wild type mice through all the test blocks (main effect genotype F [l, 8] = 0.5191, P> 0.05) (Figure 20C) These results demonstrate that CN98 mutant mice have a long-term memory, damaged in a spatial version of Barnes' labyrinth when they were tested with one trial per day (24-hour test interval), but they have a long-term memory, normal when tested with four trials per day (intervals between trials 1.5 min) suggesting that CN98 mutant mice have the capacity for long-term memory but have a deficiency in long-term memory storage.

Memory a. Short term is normal in mice that overexpress calcineurin. The demonstration that CN98 mutant mice have the capacity for long-term memory, dependent on the hippocampus, when trained with repeated tests formulated the question: Why mutant mice have a defective spatial memory when training with one trial per day ? Is short-term memory damaged? If so, can the long-term memory defect be explained by a defect in short-term memory? . Since spatial tasks such as Barnes spatial persistence do not easily lend themselves to exploring short-term memory, CN98 mutant mice were assessed for short-term memory using a recognition task for new objects. Spontaneous exploratory activity in rodents can be used as a measure of memory and in particular, can be assessed to determine the recognition of a new object against a familiar one, in a task of object recognition (Aggleton, 1985; Ennaceur and Dalacour, 1998). In humans, the hippocampal region has been shown to play a role in the detection of new visual stimuli (Tulving et al., 1996).

Patients with lesions in the hippocampus exhibit damaged responses to new stimuli (Knight et al., ßk l 996; Reed and Squire, 1997). Monkeys and rodents with lesions in the hippocampus are similarly defective in a task that requires the recognition of new objects (Myhrer, 1988a, b, Phillips et al., 1988, Mumby et al., 1995). In the task of recognition for new * objects, the mice were trained to be placed in a new environment that contained two new objects and were allowed to explore the objects for 15 minutes. During the testing phase, after the different retention intervals, the mice were placed back in the environment, but one of the two familiar objects was replaced with a third new object. The mice with a normal object recognition memory showed an increase in the exploration of the third new object. This increase in exploration indicates that the information regarding the familiar object was stored during the training and that further exploration of this object will no longer be necessary. k The exploration was assessed using the phase of -training by examining the amount of time spent exploring both new objects and not observing any difference between the mutant and wild-type mice (main effect of genotype F [l, 67] = 1.48, p = 0.228). Then, the exploration of the new object was assessed following different retention intervals: short term (30 minutes), intermediate term (2 hours), and long term (24 hours) . For this analysis, a preference index (Pl) was determined by calculating the relationship between the amount of time spent exploring a new object and the amount of time spent exploring both the new object and the familiar during the first 5 minutes of the phase of test (the preference index was normalized and expressed as a percentage with Pl = 100% indicating no preference and Pl greater than 100% indicating preference for the new object). A significant difference in the exploration of the new object between the wild type mutant mice was observed (genotype of main effect F [l, 67] = 4.03, p = 0.049). Post-hoc analysis using the Student's t-test was performed for each retention interval and revealed that the mutant mice exhibited an increase in exploration towards the new object comparable to the wild type at 30 minutes (t = 0.449, p > 0.05) (Figure 21). This indicates that the components of short-term memory are intact in the mutant mice. When the mutant mice were tested in the 2 hour retention interval, they exhibited a slight memory defect compared to the wild type, although this difference was not significant (t = 1.114, p> 0.05) (Figure 21). However, when tested in the 24-hour retention interval, the mutant mice showed a statistically significant long-term memory deficit. While the wild-type mice exhibited a significant preference for the new object, the mutant mice explored both objects equally (t = 2.061, p <0.05) (Figure 21). These results provide independent evidence for a long-term memory deficit in CN98 mutant mice and suggest that the early components of short-term memory are intact. These results support the findings of the single versus repeat assay protocol in the Barnes maze by showing that Patones that overexpress calcineurin have normal short-term memory and capacity for long-term memory that is reinforced with repetition (protocol four trials) and allows long-term memory to be stored.

Overexpression of calcineurin can be regulated ^ p by the tTA system. To verify that the memory damage observed in CN98 mutant mice is not due to a developmental defect caused by the increase in calcineurin activity during post-natal development or to an effect at the transgene insertion site, spatial memory was assessed in mice expressing the calcineurin transgene in a regulated manner under the control of the tTA system (Tet-CN279 and Tet-CN273 lines, Figure 22A). To obtain regulated expression of the calcineurin transgene, mice expressing the tTA gene under the control of the CaMKIIa promoter (line B, Mayford et al., 1996) were crossed with mice carrying the terato-sensitive promoter fused to a cDNA encoding for the truncated CaM-AI form of calcineurin (lines CN279 and CN273) (Figure 22A). Northern blot analysis revealed a 1.9 kb transcript corresponding to the transgene mRNA in Tet-CN279 and Tet-CN273 mutant mice (Figure 22B). In addition, an RT-PCR revealed the (flj) pressure of the transgene mRNA in Tet-CN279 and Tet-CN273 mutant mice that decreased dramatically when the mutant mice were administered doxycycline for at least one week (Figure 22B). with phosphatase revealed an increase of 112% ± 9% and 114% ± 5% in Ca2 + -dependent calcineurin activity, respectively in the Tet-CN279 and Tet-CN273 mice when compared to the wild type (Figure 22C). Increase in phosphatase activity in Tet-CN279 and Tet-CN273 mutant mice was slightly higher than that detected in CN98 mutant mice (76% ± 12%, see Winder et al., 1997) In Tet-CN279 and Tet mutant mice -CN273, the phosphatase activity was suppressed at wild-type levels in the administration of doxycycline for at least one week (Figure 22C) .The spatial distribution of the transgene transcript was examined by in situ hybridization in the brain of adults in Tet mice -CN279 and Tet-CN273. The mRNA of the transgene was detected mainly in the hippocampus and stratum, almost no expression was detected in the neocortex. In the hippocampus, it was found mainly in the CAI area and the dentate gyrus with relatively little expression in the CA3 area (Figure 23). In contrast, no signal was detected in the mutant mice administered with 1 mg / ml doxycycline for at least one week or in wild type mice (Figure 23).

The defect in memory can be reversed by the repression of the calcineurin transgene by djQX-Lcicliii. To assess whether the memory defect can be reversed by repressing the calcineurin transgene with doxycycline in adult mice, Tet-CN279 and Tet-CN273 mice or the spatial version of Barnes labyrinth were tested. When the spatial version of Barnes' labyrinth is performed, the mice progress normally through three search strategies. Random, serial and spatial (Barnes, 1979, Bach et al., 1995) (Figure 24A). The search strategy is defined operationally as a localized, random search for the holes separated by central crosses that result in a large number of errors. In the serial search strategy, it is operationally defined as a systematic search for consecutive holes in a clockwise or counterclockwise manner and the use of the strategy results in less errors that for the random search strategy (Figure 24A). During the first five trials, the CN98 and Tet-CN279 mutant mice and their respective wild-type mice either on or off doxycycline (Figures 24B and 24C) mainly used the randomized strategy and both a similar decrease in use across the remaining test blocks (CN98: genotype of main effect by time (F [3.28] = 0.5, p> 0.05; Tet-CN279: genotype of main effect F [1.54] 1.63, p> 0.05). The use of the randomized strategy is paralleled by an increase in the use of the search strategy in flJBerie in mutant and wild type mice CN98m Tet-CN279.The serial strategy was used significantly more frequently by CN98 and Tet mutant mice. CN279 during the last two test blocks (Figures 24D and 24E) (CN98: Main effect genotype for always F [3,28] = 5.22, p <0.01; Tet- CN279: Main effect genotype for doxycycline F [1, 54] = 6.12, p < 0.05). In contrast, during Mine two last test blocks, the wild-type mice CN98, the Tet-CN279 mutant mice in doxycycline and the wild-type mice used mainly the spatial search strategy (Figures 24F and 24G) CN98: Main effect genotype for time F [3,28] = 5.4, p < 0.005; Tet- CN279: Genotype of main effect F [1, 54] = 4.64, p < 0.05). These results show that the mutant CN98 and Tet-CN279 mice have a similar defect in spatial memory since they do not use the spatial search strategy. When the expression of the calcineurin transgene was repressed by in Tet-CN279 mutant mice, this defect was inverted. The ability to reverse memory loss suggests that the observed defect is probably not development but rather due to the expression of the calcineurin transgene and the resulting increase in calcineurin and its interference with memory storage in the Juvenile adult Discussion Calcineurin plays a role in the transition from hippocampal-dependent memory to short-term long-term memory. Mice expressing a truncated form of calcineurin exhibit an afflicting memory defect in the spatial version of the labyrinth of Barnes, a task dependent on the hippocampus. No defect was observed in the version with suggestion of the task, which is independent of the hippocampus, indicating that the defect observed in the labyrinth of Barnes was in the spatial memory and was not a defect of motivation or sensory-motor. In addition, the defect in spatial memory was reversible in adult mice overexpressing calcineurin in a regulated manner with the tTA system. These results provide the first genetic evidence that a phosphatase, and specifically calcineurin, has a role in memory storage based on the hippocampus. Although the present data do not allow to determine if a specific phase of the memory is damaged by overexpression of the calcineurin transgene, allow to delineate the components of the memory that are affected and identify the memory components that are not damaged. The results indicate that by increasing the number of daily trials in the spatial version of the Barnes labyrinth, the long-term memory defect observed in CN98 mutant mice was completely rescued. This shows that although it exhibits an apparent defect in long-term, spatial memory, the mutant mice actually still have the ability to store long-term memory. The finding that the memory deficit observed with a one day trial can be rescued with repeated training suggests that the mutant mice have a defect in some processes in the 5 'direction required for long-term memory storage. These results therefore suggest that the short-term memory trace generated by an individual daily trial disintegrates before the transition in long-term memory is complete. When training is intensified so that the short-term, defective stroke is reinforced, long-term memory can be achieved. Genetic evidence supports the notion that the hippocampus stores some aspects of short-term as well as long-term memory for non-spatial spatial tasks. The results of the Barnes labyrinth support those obtained in the task of recognizing new objects. In this task, the mutant mice have normal short-term memory, m ^ 0 minutes, but have a significant long-term memory defect at 24 hours. The combined results in the spatial version of the labyrinth of Barnes and the task of recognizing new objects further strengthens the hypothesis that the defect that leads to damage in long-term memory storage is a defect in the process or stages, so that short-term memory becomes ? long-term memory. Since the calcineurin transgene is expressed mainly in the hippocampus, this defect in the transition probably lies in the hippocampus. While additional genetic manipulations will be required to establish this idea more firmly, the present results strengthen the important idea, well documented in humans and primates (Scoville and Milner, 1957; Mishkin, 1978; Zola-Morgan and Squire, 1985; Overman et al., 1990), that the hippocampus is comprised not only in the storage of long-term memory, but also in some aspects of the storage of short-term memory in the 3 'direction of working memory. As a corollary, the experiments provide independent evidence that the rodent hippocampus is related to the storage of information other than space.In addition to forming a defect in spatial memory, genetic interference with I-LTP is restricted ^^ 1 hippocampus It also interferes with the recognition of the new object.These findings support the idea (Squire et al., 1992) that the rodent's hippocampus is similar to that of humans by supporting a variety of memories that require the complex association of suggestions in all sensory modalities.

The defect in the transition from short-term memory to. The long-term correlates with a defect in I-LTP The behavioral and electrophysiological results suggest that an increase in calcineurin activity in the hippocampus leads to a defect in a transition phase of spatial memory between short-term and long-term memory term as well as a defect in the new intermediate phase E-LTP between the early and late phase (Winder et al., 1997). Since short-term memory and E-LTP on the one hand, long-term memory and L-LTP on the other hand have common properties since short-term memory and E-LTP do not require protein synthesis, while that in long-term memory and L-LTP. depend on PKA and the synthesis of new proteins, the results that show a similarity in behavioral and electrophysiological phenotypes suggest a correlation between the transition of short and long-term memory and the intermediate phase of LTP The data also suggest a possible correlation between short-term memory and E-LTP since they are intact in mice Finally, the results further extend the suggested correlation between long-term memory storage and L-LTP. -LTP (Abel et al., 1997) First, both long-term memory and L-LTP are damaged in mice.

The defects in both long-term memory and L-LTP were rescued when the electrophysiological and behavioral protocols were systematically manipulated. Rescue of long-term memory defect behavior by repeated training is not seen in CREB and CaMKIII -Asp285 mutant mice. Repeated training experiments similar to those carried out here have been performed on other genetically modified mice. In mice with elimination of CREB, the deficit in the 3 long-term spatial memory observed in Morris's water maze tasks was attenuated, but (fl was completely rescued by increasing the number of daily trials from 1 to 12 with intervals between 1-minute trials, or from 1 to 2 with a 10-minute interval between trials (Bourtchouladze et al., 1994; Kogan et al., 1996). However, when the interval between the daily trials (2 trials per day) was increased to 60 minutes, the performance of mutant mice was improved (Kogan et al., 1996). In addition, mice overexpressing a constitutively active form of CaMKII (CaMKII-Asp286) were shown to have a spatial memory defect in the Barnes labyrinth with one assay per day. In these mice, no improvement in spatial memory was observed when the number of trials increased to trials per day with a 1-minute interval between trials and, in addition, there was no improvement in performance within a day across the 10 trials (Mayford et al., 1995, Bach, M. unpublished results). These results suggest that CaMKII-Asp286 mutant and CREB-deleted mice may have spatial memory defects other than the defect observed in mice overexpressing calcineurin (a comparison of performance in the Morris and Barnes water maze is possible since both tasks involve similar cognitive processes). Specifically, the CREB mutant mice have a long-term memory defect although the mutant CaMKII -Asp286 mice may have a defect In turn, the behavioral deficits observed in mice overexpressing calcineurin and in CREB elimination mice provides an interesting comparison with mice expressing a negative, dominant form of the subunit. regulator of PKA, R (AB) (Abel et al., 1997) In mice overexpressing calcineurin and in R (AB) mutant mice, the PKA pathway is modified.In mice overexpressing calcineurin, the PKA pathway is affected indirectly through an increase in calcineurin activity that is suggested to suppress the PKA pathway (Winder et al., 1997) whereas the R (AB) mice, the PKA pathway is directly affected by genetic manipulation I bet that PKA activity itself is decreased.

In CREB elimination mice, the defect appears to be additionally in the 3 'direction of PKA since CREB has been implicated in the activation of gene transcription (Brindle and Monminy, 1992; Lee and Masson, 1993). Consistent with these three genetic manipulations that act in complementary sites, all three types of mice have a similar phenotype: short-term memory and E-LTP are normal but L-LTP and long-term memory are damaged.

Experimental Procedures Barnes Circular Maze fP The Barnes Maze experiments were performed as previously described with animals housed individually for at least three days before the first day of the experiment (Bach et al., 1995). Thirty-four CN98 mice (mutant: n = 17, wild type: n = 17), 58 tet-CN279 (mutant: n = 14, in doxycycline n = 20, wild type: b = 13, fl ^ n doxycycline n = 11) were tested in the spatial version of Barnes labyrinth. Thirteen CN98 mice (mutant: n = 7, wild type: n = 6) were tested in the labyrinth-clamped version. Briefly, the labyrinth of Barnes is a circular platform with forty holes in the periphery with an escape tunnel placed under one of the holes. On the first day of the test, each mouse was placed on the (Tune and left there for 1 minute.) The first session started 1 minute after the training trial.At the beginning of each session, each mouse was placed in a start chamber in the center of the labyrinth for 10 seconds and a buzzer was lit The start camera was then lifted and the mouse was allowed to explore the labyrinth.The session ended when the mouse entered the tunnel or after 5 minutes had elapsed.The buzzer was then turned off and the mouse was left in the tunnel for 1 In the spatial version of the labyrinth, the tunnel was always placed under the same hole that was determined randomly for each mouse, (fljbando was tested with 4 tests per day, after it was removed in the escape tunnel, The mouse was placed in a start chamber in the labyrinth for 30 seconds, in this way, each test was separated by an interval between trials of 90 seconds (60 seconds in the escape term of 30 seconds in the In the version with suggestion of (fllaberinto, mice are tested once a day until they met the criteria of three errors or less in 5 of 6 consecutive days or until 40 days have elapsed. An error was defined as the search for a hole that does not have the tunnel due to it. The order of the holes sought and the search strategy used were recorded manually by an experimenter who did not know about the Afenotipo. For both the 'spatial' test versions, with suggestion and repeated, within the CN98 line, a two-factor ANOVA (genotype and a repeated measurement) was used. For the Tet-CN279 line, a three-factor ANOVA (genotype, doxycycline, a repeated measurement) was used.

New Obesity Recognition Task Seventy three mice of the CN98 line (mutant: 30 min n = 9, 2 hours n = 12, 24 hours n = ss 15, wild type: 30 min n = 9, 2 hours n = 11; 24 hours n = 17) were assessed individually in the (flfeirea of recognition of the new object.) Three mutant and three wild type mice were excluded because they exhibited a strong preference (preference index <60) to the family object during training and testing. During the training trial, the mice were placed in a new, square environment (20 inches long by flfe inches high) constructed of laminated wood and painted with white with epoxy paint. Two plastic toys (of three possible) (between 2.5 and 3 inches) that varied in color, shape and texture were placed in specific locations in the environment 14 inches apart. Two different combinations of object pairs were counterbalanced through the retention and genotype intervals. The flratons were able to freely explore the environment and objects for 15 minutes and then put them back in their individual, homemade cages. After several retention intervals (30 minutes, 2 hours to 24 hours), the mice were placed back in the environment with two objects in the same location but now one of the familiar objects was replaced with a third new object. The mice were then allowed to freely explore both objects again for 15 minutes. The objects were thoroughly cleaned with a mild detergent (Roccal diluted 1:50 in water) before each experiment to avoid instinctive evasion by (^ tLor due to the mouse smell of familiar objects.) During both the training and test phases An experimenter who does not know about the genotype recorded the number of seconds elapsed by scanning each individual object during each minute through 15 minutes.A mouse was considered to be scanning the object when its head is giving the object a distance of one inch or less or when any part of your body except the tail is touching the object.For the purpose of data analysis, the total number of seconds elapsed by scanning each object during the first 5 minutes during the test phase was added and an index of preference (Pl). The amount of time spent exploring the new object is (divided by the amount of time spent exploring new and familiar objects.) The resulting value was divided by 0.5 representing no preference for any object and that result was then multiplied by 100. A Pl greater than 100 indicates preference for the new object during the A Pl equal to 100 does not indicate preference while a Pl less than 100 indicates a preference for the familiar object, a second ANOVA of two factors (genotype and a repeated measurement) and the individual Student t tests for each interval of Retention was explored to assess the effect of the genotype on the Pl at the different retention intervals.

Construction of the Plasmid The construction of the plasmid used to generate the CN98 mice is described in Winder et al., 1997. For the generation of the Tet-CN279 and Tet-CN273 mice, a plasmid was constructed with a cDNA coding for a form Truncated and active flJta subunit Aa catalytic murine calcineurin, CaM-AI (provided by S.J. O'Keefe). CaM-AI lacks the self-inhibitory domain and a portion of the calmodulin-binding domain of calcineurin Aa and was shown to be constitutively active in Jurkat T cells (O'Keefe et al., 1992). An EcoRI 1.27 kb fragment of the CaM-AI cDNA was blunt-ended and subcloned into the site ABCORV of vector pNN265 (provided by N.

Nakanishi). Plasmid pNN265 carries in the 5 'direction from the EcoRV site a 230 bp hybrid intron containing an adenovirus splice donor and an immunoglobulin G splice acceptor (Choi et al., 1991) and has a SV40 polyadenylation signal in the 3 'direction from the EcoRV site. The CaM-AI cDNA flanked by the 5 'intron hybrid and the 3' poly (A) signal was excised from pNN265 with Notl and the resulting 2.7 kb fragment was placed in the 3 'direction of the teto promoter of the plasmid pUHD10 -3 (Gossen and Bujard, 1992) to generate the CN279 and CN273 mice (Figure 22A). The final tetO-CaM-AI fragment of 3.1 (© b (Figure 22A) was excised from the vector by digestion with Notl. Prior to microinjection, all cloning linkages were verified by DNA sequencing.

Generation and_ Maintenance of Tet-CN279 and Transgenic Mice. Tet-CN273 (flJ The transgenic mice Tet-CN279 and Tet-CN273 were generated by microinjection of the linear construct as previously described (Hogan et al., 1994; Windor et al., 1997). Transgen integration was done by transfer Southern and PCR. The founder mice were backcrossed to C57BL6 Fl / J mice to generate the transgenic cells Tet-CN279 and Tet-CN273. To generate the Tet-CN279 and Tet-CN273 mice, CN279 and CN273 Fl mice were crossed with mice from the CaMKIIa tTA promoter (line B, Mayford et al., 1996) (Figure 22A). Offspring was verified by Southern or PCT transfer. The transgenic mice were maintained in the animal colony according to the normal protocol. The Tet-CN279 and Tet-CN273 mice were administered either water or 1 mg / ml doxycycline (in 5% sucrose) in the drinking water at least one week before they were used. fl) Northern blotting Northern blot analysis was performed as described in Winder et al., 1997. Briefly, the forebrains of Tet-CN279 and Tet-CN273 mice, adults administered with water or doxycycline were harvested and isolated. Total RNA by the method of guanidinium thiocyanate and Sacchi, 1987). 10 micrograms of RNA were denatured, subjected to electrophoresis on a 1% agarose gel and transferred to a nylon membrane in 0.4 N NaOH. The membrane was hybridized overnight at 42 ° C to an EcoRV-Notl fragment. 1.1 kb radiolabelled from pNN265, washed and exposed to the film for three days.

A RT-PCR For RT-PCR, the total RNA of the forebrain was amplified according to the manufacturer's protocol (Gibco BRL). Briefly, cDNA was synthesized from 3 μg of total RNA with Superscript II RT in 20 μl of reaction. Amplification was performed with Taq-polymerase (Boehringer Mannheim) for 25 cycles as follows: 94 ° C for 30 seconds, 50 ° C for 30 seconds and 72 ° C for 1 minute. The following oligonucleotides were used as primers: 5 '-CCTGCAGCACAATAATTTGTTATC-3' and 5'-TAGGTGACACTATAGAATAGGGCCO-3 '. They produced a 478 bp fragment containing 406 bp of the cDNA of and 75 bp of the as sequences of pNN265. The samples were run on a 2% agarose gel, then transferred onto a nylon membrane. The membrane was hybridized to the probe labeled with [a32P] dCTP specific for the pNN265 sequences in the PCR product. Hybridization was performed overnight at 42 ° C in 50% formamide, 2 X SSX, 1% SDS, (10% dextran fljulfate, 0.5 mg / ml denatured salmon sperm DNA. minutes at room temperature in 2 X SSC, 1% SDS, twice 15 minutes at 42 ° C in 2 X SSC, 1% SDS, 0.2 X SSX and exposed to the film.

In situ hybridization In situ hybridization was performed as described in Winder et al., 1997. Briefly, the brains of adult Tet-CN279 and Tet-CN273 mice were cut and sectioned, either on or off doxycycline. Sections were fixed for 10 minutes in 4% paraformaldehyde, rinsed in PBS and dehydrated. The sections were re-hydrated, permeabrated, washed and rinsed before they hybridized overnight at 37 ° C to an oligonucleotide labeled with [a35 S] ATP (5 'GCAGGATCCGCTTGGGCTGCAGTTGGACCT-3') specific for transgenes. After hybridization, the slices were washed, dehydrated then exposed to the Kodak Biomax MR film for 2 to 3 weeks.

Phosph assay assay Phosphatase assays were performed as described in Winder et al., 1997. Briefly, mice were injected with 5 ml / kg pentobarbital and decapitated. The hippocampi were cut, they were glycogenized in 2mM EDTA (pH 8), 250mM sucrose, 0.1% β-mercaptoethanol. The supernatants were incubated at 30 ° C for 1 minute in the presence of the [Ala97] -RII peptide labeled with [a32P] and either calmodulin 0.01 mM and Ca2 + 0.66 mM or 0.33 mM EGTA. The reaction was stopped and the enzyme activity previously calculated as described (Klee et al., 1983; 1987). The activity was expressed in nmol Pi ^ cubed / min / mg of protein. The concentration of the protein was determined using the bicinchroninic acid protein assay kit (Sigma). All samples were made in triplicate.

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Example 5; Memory and. behavior; a second generation of genetically modified mice. [Figures corresponding to the legends of the figures at the end of this example can be found in Mayford et al., Current Biology 1997; R580-R589.] Introduction One of the understandings of modern cognitive neural science is that memory is not unitary, but that it has at least two forms; implicit (or non-declarative) and explicit (or declarative) (Squire et al., 1996). Explicit memory refers to the conscious gathering of information about events and events that includes places, people, objects. Implicit memory refers to the unconscious use of information that refers to various habits and perceptual and motor strategies, and to memories for simple forms of learning in Aplysia and Drosophila has provided some understanding of the molecular mechanisms that contribute to the Implicit memory storage (Carew, 1996, Martin et al., 1996, Tully et al., 1996) In contrast, although now there have been four decades since Scoville and Milner (Scoville et al., 1957) first established that The explicit forms of memory require the temporal lobe system, the brain's own, much less is known about the mechanisms that contribute to these forms of memory storage.The studies of the medical temporal lobe system have been impeded by their complexity ( Figures 1, 2) In humans, this system consists of several interconnected cortical structures, including multimodal association areas in the M neocortex, the peririnal and entorinal cortices, the dentate gyrus, the hippocampus and the entorinal cortexes, the dentate gyrus, the hippocampus and the subicular complex, each of which is thought to be important for the aspects of explicit memory storage. To study the function of these individual regions in humans, many patients with very specific brain lesions will be required. Fortunately, recent anatomical and behavioral studies indicate that, although there are some differences in detail, there is a remarkable similarity between the organization of the central temporal lobe system, humans, nonhuman primates, and simpler mammals such as. rats and mice (figure 1) [Burwell et al. , nineteen ninety six] . Furthermore, a simple mammal is just as the mouse requires the temporary, central system for storing memory about places and objects, and this type of memory has several of the characteristics, the integration not simply of a single variable of a multiplicity of distant suggestions. The study of an explicit form of memory in mice has the advantage that makes this cognitive process accessible to a genetic approach. - * Of the various structures of the medical temporal lobe in the mouse, the hippocampus is proven to be the most appropriate, and the most accessible goal for a rigorous genetic analysis of the To aspects of explicit memory storage.

Each of the three main synaptic pathways within the hippocampus (Figure 2) is well defined anatomically and is capable of understanding long-term potentiation (LTP), a dependent form of plasticity activity that is thought to be important for memory storage (Bliss et al., 1973; Bliss et al., 1993). Hippocampal lesions interfere with the formation of new spatial memories, memory for places, which is particularly well studied in rodents (Morris et al., 1982). In the animal that behaves freely, the pyramidal cells of the lipocampole, the cells that cause LTP, code for spatial locations in their potential activation activation patterns. Therefore, pyramidal cells are "place cells" that are activated only when an animal occupies a particular location in its environment (O'Keefe et al., 1971). These findings formulate a series of questions that has been the central point of studies of spatial memory during the last years. What are the molecular mechanisms of LTP? Is LTP important for spatial memory storage? If so, how does LTP moderate the properties of the site cell to cause spatial memory storage? Does it do so when acting during the initial formation or subsequent stabilization of the local Acampos? In the review, he limited himself to two areas. First, it was looked at as' new techniques to produce temporally regulated and anatomically restricted genetic modification in the mouse have been used to examine the LTP mechanisms of the role it plays in spatial memory. Second, it is considered a complementary set of studies in genetically modified mice that use the individual unit record of the place cell in the hippocampus. Here, the intent is to examine the relationship of LTP to the cognitive map of space in the hippocampus. In this section of the review, we will determine if the LTP is required for the formation of place fields and for their stability over time, if so, how these properties of the local cells are related to the acquisition and maintenance of spatial memory ?.

A first generation of genetically modified mice With the development of genetically modified mice, it becomes possible to ask how the perturbation of an individual gene affects LTP, on the one hand, and the complete behavior of the animal, on the other hand. Initial studies of spatial memory in genetically modified animals (Silva et al., 1992, p.201, 206; Grant et al., 1992) take as their starting point several important and well-documented findings from pharmacological studies, Previous studies on the sequence of steps involved in LTP induction (Bliss et al., 1993) These studies focused on one of the key pathways in the hippocampus, the Schaffer collateral pathway among the mice of the pyramidal cells of the CA3 region and the post-synaptic target cells in the CAI region The previous search has shown that, in this route, the session step for LP comprises the release of glutamate from the pre-synaptic terminals of CA3 neurons. This leads to the activation of m > & N-methyl-D-aspartate (NMDA) receptors in post-synaptic CAI pyramidal cells, which result in an influx of Ca2 + in the post-synaptic neuron. The Ca2 + signal, in turn, acts a number of second messenger-kinases, including Ca2 + / calmodulin-dependent protein kinase II (CaMKII), protein C, protein kinase A and one or more ßirosine kinases. Based on these pharmacological findings, homologous recombination in embryonic mast cells was used to detect in mice the genes encoding the CaMKII subunit (CaMKII) and Fyn tyrosine (Silva et al., 1992, p.201, 206; et al., 1992. In each case, the deletion of the target gene led to a defect in LP and to a damage in the explicit spatial memory.In this way, the initial genetic studies did not support only the previous pharmacological work when showing that both CaMKII and Fyn seem to be included in the important signal transduction path for LP, but also showed that it interferes with LP that affects memory.

A second generation approach Although these results illustrated the potential usefulness of genetic approaches to analyze synaptic plasticity and to relate it to explicit memory, it was also (I clarify that there were limitations that need to be overcome, for example, to understand the role of storage of LP memory within a given component of the central temporal lobe system, such as the hippocampus, the generic change must be restricted to that specific of the anatomical component.Genetic change also needs to be (ß regulated in a temporary manner, to exclude possible developmental defects and to ensure that the physiological or behavioral phenotype reflects a change in the adult brain questioning To understand the effects of genetically induced alterations in memory LP, changes in LP in mutant mice should be related to changes in the neuroma activity in ^ animals that behave freely. Within the last year, substantial progress has been made in each of these three areas.

The CaMKII promoter Genetically modified mice come in two main varieties, called "elimination" and "transgenic." In elimination mice, the endogenous gene of interest is specifically suppressed by homologous recombination in mast cells, embryos. Therefore, the gene of interest is deleted in all cells of the body and is absent during the entire life of the animal. In this way, those of conventional elimination lack both anatomical and temporal regulation restrictions.In transgenic mice, an additional gene, the transgene, is added to the mouse genome by microinjection of DNA into the oocyte. be the wild-type version and a gene, in which case the gene product is overexpressed, or it may be a mutant version of the gene, designed to improve or suppress the function, the transgene carries with it an appropriate promoter element that directs the anatomical and temporal pattern of its expression.When selecting the appropriate promoter, the anatomical and temporal expression of genetic change can be controlled, at least partially, since many molecules will probably be used during development and learning.

(Martin et al., 1996), the promoter will activate transgene expression in temporal, central, critical lobe structures, with the onset of expression occurring later in brain development. Otherwise, you can not be sure that you are examining the. learning and memory in a specific way without interfering with the development. As a first step in these directions, the promoter of the CaMKII gene (Mayford et al., 1996) was isolated, which triggers or drives the expression of a transgene specifically in the structures of the forebrain, especially the hippocampus. The active only in the neurons and not in the glial cells (figure 3a), and the beginning of the expression occurs in a relatively late stage of development, usually the first or second post natal week (Kojima et al., 1997) . As discussed below, the anatomical sites at which gene expression occurs are uniquely restricted when the CaMKII promoter is combined with other regulatory elements such as Crerecombinase or the tetracycline transactivator (tTA) (Mayford et al., 1996; Taien et al., 1996).

These characteristics of the CaMKII promoter have been crucial in the development of the second generation of genetic approaches to behavior in mice and illustrate, as will be discussed later, that future efforts will require the isolation of other promoters that are specific to each of the components of the temporal, central lobe.

Regional restriction: the CaMKII promoter y_ Crerecombinasa The most dramatic evidence for the anatomical restriction of the CaMKII promoter arose from the experiments in collaboration with Susumu Tonegawa and his colleagues in which the Cre-loxP system was applied to the brain, a system developed by the group by Klaus Rajewski for specific gene suppression of B cells (Gu et al., 1994; Lakso ket al., 1992). This system uses Cre recombinase, a sequence-specific recombinase or site derived from bacteriophage Pl that catalyzes recombination between loxP recognition sequences of 34 base pairs (Abremski et al., 1984; Sauer et al., 1988). . When the two appropriately oriented loxP sites flank a piece of DNA, (? Cre-mediated recombination leads to deletion of DNA between the loxP sites.) Two different types of mice are required to obtain gene suppression mediated by creloxP. first it is the transgenic mouse in which a promoter, in this case a CaMKII promoter, is used to activate or boost the expression of Cre-recombinase in a specific subset of neurons in the brain (without effect, since the sites target loxP are absent from the genome.) In the second type of mouse, loxP sites are introduced by homologous recombination into the endogenous gene of interest (the gene that is "deleted") such that they flank an exon critical to the function of the gene. The loxP sites are placed in introns so that they do not alter the normal function of the gene and do not produce a phenotype in the absence of Cre recombinase.When through mating, if the Cre-recom transgene Binasa and the endogenous gene that is flanked by loxP are introduced into the same mouse, the portion of the endogenous gene between the loxP sites will be suppressed by the recombinase. This deletion will lead to an elimination of the gene flanked by loxP only in neurons expressing Cre recombinase. In cells that do not express Cre-recombinase, the gene flanked by the loop remains intact and functional (Figures 4a). Surprisingly, when the mk Cre-recombinase expression was driven by the CaMKII promoter (Tsien et al., 1996), Cre-mediated suppression was restricted to only CAI neurons of the hippocampus in three of the five lines (Figures 4b, c) The molecular basis for the CAI restriction of Cre-mediated recombination is not yet clear. The neurons' of the forebrain outside CAI also expressed that cre-recombinase, although at a high level, but this pressure did not lead to the effective suppression of the Tsien et al., 1996 gene. This suggests that a high level of Cre expression is required to achieve recombination, and in many mouse lines this High level of expression threshold is achieved only in CAI neurons. In the CaMKII-Cre-loxP systems, they were then used by the Tonegawa laboratory to eliminate the NMDA receptor 1 gene (Ri) in a definitive manner by CAI, (Tsien et al., 1996, p.1327). Mutant mice have a complete loss of LPT in CAI as well as a deep defect in spatial memory, showing that transmission by the NMDA receptor in neurons is one of the hippocampus is critical for the formation of explicit memory and the strengthening of the idea that LP Schaffer's collateral route is important in memory formation. These results are complementary to previous findings that selective interference with mossy fiber L.P. granule cells and CA3 cells have no effect on spatial memory (Huang et al., 1995). The CaMKII -Cre-loop approach solves one of the anatomical restrictions, but there is still the possibility that the damage in the spatial memory observed in these mice results from some abnormality in the development caused by the Prolonged absence of the NMDA Ri gene. Although this is unlikely, given the late onset of expression of the CaMKII promoter, it turns to a technology to obtain the transient regulation of transgene expression.

Temporary Restriction: the promoter CaMKII y. tTA In a parallel series of excrement is designed to obtain temporal as well as anatomical control over the expression of a transgene, the tTA system arranged by tetracycline # * developed by the Herman Bujard 'system (Gossen et al., 1992; Furth et al., 1994). The repressor of jfl ^ tetracycline (tetR) is a protein from the tetracycline resistance operon TnlO of Escherichia coli that recognizes and binds teto, a specific DNA sequence in the operon. The interaction of tetR with its target teto DNA breaks down under the antibiotic tetracycline and its derivatives. By fusing the tetR protein to the activation and transcription domain of VP16, a herpes simplex virus protein, Bujard produced an eukaryotic, regulatable transcription factor called the tetracycline transactivator (tTA). When the teto sequences, together with a minimal eukaryotic promoter element, are placed close to the target being of interest, the transcription factor of tTA can activate the expression of the teto-linked gene in eukaryotic cells. When exposed to low levels of the tetracycline analog, doxycycline, however, the binding of tTA to teto is prevented and the transcription of the teto-linked gene is deactivated. To obtain expression of the transgene that can be regulated by doxycycline, two different types of transgenic mice are required (Figure 5a). In the first line of mice, the CaMKII promoter is used to activate expression of the tTA gene of forebrain neurons (no effect, since there are no endogenous teto sites). The second line of transgenic mice has a site bound to the particular gene of interest, in this case a constitutively active form of CaMKII (CaMKII-Asp286), whose properties are subsequently considered (Mayford et al., 1996). Both transgenes are introduced into an individual mouse through pairing, the transgene is expressed CaMKII-Asp286 is expressed only in proencephalon neurons expressing tTA In the strong expression of the CaMKII- gene (fljAsp286 can now be suppressed when doxycycline is administered in mice in their drinking water CaMKII-Asp286 was selected as a transgene for the study because, as discussed above, early pharmacological and genetic studies require CaMKII as a key molecular mediator of synaptic plasticity and memory formation. Pharmacological blocking of α CaMKII prevents LTP (Malinow et al., 1989; Miller et al., 1988), and deletion of the CaMKII gene in mice e a loss of LTP in spatial memory (Silva et al., 1992, p. 201; Silva et al., 1992, p. 206). In addition, previous biochemical studies revealed several interesting features of this kinase (Miller et al., 19986). In the absence of Ca2 + / calmodulin, CaMKII shows little or no enzymatic activity. After a brief boost of Ca2 + / calmodulin, the complete enzymatic activity is induced. When it falls in Ca2 + levels, however, it rather returns to the low basal level of activity as seen before the boost Ca 2+ the enzyme remains substantially active even in the complete absence of Ca 2+ This persistent change of a Ca 2+ dependent state and an independent Ca 2+ state represents a form of biochemical memory for the Ca 2+ signal. Because LTP is, in essence, a long-lasting biochemical alteration resulting from a brief Ca2 + signal, ßpisman (1994) suggested that the change of CaMKII from the Ca2 + dependent state to the Ca2 + independent state represents the biochemical mechanism of the LTP. To test the Lisman model, it became important to ask whether the activation of CaMKII was sufficient, by itself, to produce the LTP. The conversion of CaMKII from the dependent state of Ca 2 + to the independent state of Ca requires the ^ • phosphorylation of an individual amino acid residue, threonine 286 (Schworer et al., 1988; Miller et al., 1988; Thiel et al., 1988). The mutation of this residue to aspartate limits the effects of autophosphorylation and produces an enzyme independent of Ca2 + (Fong et al., 1989, Waldmann et al., 1990). The mutant CaMKII-Asp286 kinase now provides a molecular genetic means to increase the activity of the baseline CaMKII. In early studies, the CaMKII promoter is used to express CaMKII-Asp286 and to examine the effect of LTP and memory (Mayford et al., 1995, Bach et al., 1995). It was found that the activation of ([^ tMKII alone was not enough to change the LTP, on the other hand, CaMKII seems to act as a regulator of the frequency of the synaptic activity at which LTP or long-term depression will occur (LTP When the CaMKII-independent CaMKII levels were elevated in transgenic CaMKII-Asp286 mice at levels higher than those produced during LTP, the stimulation frequencies necessary to produce LTP or LTD were altered, in wild type animals, the stimulation at 1 Hz produces LTP, while 5 Hz or 10 Hz produces a modest amount of LTP and 100 Hz produces maximum LTP.In transgenic mice CaMKII -Asp286, LTP at 100 Hz, while in the range of 5-10 Hz of stimulation the LTD is favored over the LTP. M The change from LTP in wild types to LTD in mutants in the 5-10 Hz frequency range is particularly interesting because there is an oscillation of endogenous 5-10 Hz in neuronal activity ("theta-rhythm") in the hippocampus of rodents. It has been suggested that the pattern neuronal activity in the theta frequency range represents the endogenous mechanism to induce LTP in the hippocampus during spatial learning (Staubli et al., 1987, Huerta et al, 1995). If this idea is correct, then mice lacking LTP in the theta frequency could show damaged spatial memory. Consistent with this idea, the analysis of the transgenic mice CaMKII -Asp286 showed that they do not have a severe deficit in the function of spatial memory. These experiments suffer, however, from both problems discussed above; possible abnormalities of development and lack of precise anatomically restricted expression. To address development problems, the tTA system was used to express CaMKII -Asp286 (Mayford et al., 1996). When the transgenic expression was suppressed by doxycycline, the damage in memory, evident in the spatial memory task described in Figure 5b, was reversed completely, the transgenic mice also performed like the wild mice. In parallel, the suppression of transgenic expression Ata also reversed the deficit in LTP at theta frequency observed in the hippocampus. These experiments with regulated gene expression therefore showed that the behavioral and electrophysiological effects of the transgenic CaMKII-Asp286 are the direct consequence of the acute elevation in CaMKII activity, and are not the effect of an abnormality on the whole neuronal circuits caused by the expression of the transgene during development.

Combination of temporal and regional restriction In the course of this work, it was found that, in a line of mice in which the CaMKII promoter was combined with tTA, there was little or no expression of the CaMKII -Asp286 transection in the neocortex (Figure 3a) . The expression was limited to certain deep structures of the forebrain, in subiculum, stratum, amygdala and hippocampus. Within the hippocampus, expression of the transgene was strong in the (B-CAI region, which contains the post-synaptic cells of the Schaffer collateral pathway, but was not expressed in the CA3 region, which contains the pre-synaptic neurons of this route (Figure 3b) This line of mice could therefore be used to ask whether the transgenic CaMKII -Asp286 has to be expressed in pre-synaptic CA3 neurons to produce the deficit in LTP at 5-10 Hz, A \ or if it is sufficient to restrict the expression to post-synaptic CAI neurons. Since transgenic expression was temporarily regulatable, one can also ask whether its expression in CAI neurons varied LPT and memory by interfering directly with normal plasticity in the adult brain, or if it is through an interruption of neuronal development . It was found that the reversible expression of the CaMKII-Asp286 transgene, limited to CAI neurons, was sufficient to reversibly damage LTP at the theta frequency of 5-10 Hz. In addition, expression of the transgene in the deep structures of the QP > Sencephalon was enough to reversibly damage spatial memory. Although the regional restriction achieved by combining the CaMKII promoter with Tta is not as limited as that achieved by combining the CaMKII promoter with Cre, the above is only informative, especially in physiological terms where it has been able to examine the relative contribution of the pre-element. - and post-synaptic of a mono-synaptic connection. In addition, this restriction, although limited, continues with the great benefit of also being adjustable, ensuring that the phenotype is due to direct effects in the adult brain and is not due to developmental abnormalities.

A Spatial memory in the adult mouse: the mouse of the LTP and_ the fields of place The study of the neuronal mechanisms of explicit memory requires not only the production of highly defined molecular lesions in the brain, but also an analysis of the properties Physiological and plastic neurons in animals that behave freely when stimulated by learning and remembering the brief information. Are the changes in concentration caused by induced connection-by LTP occurring naturally in an intact animal that makes a spatial memory test? If so, how is it? (They reflect these modifications in the activation properties of neurons within the network that control the behavior under study.) As was first shown by O'Keefe and Dostrovsky (1971), pyramidal cells of the hippocampus that were stimulated artificially during LTP experiments are in rats (free-range, "place cells" that code for spatial location in their action potential activation patterns.) A given place cell will only be activated when an animal occupies a particular location in When the animal moves to a different location in the same environment, other place cells are activated.If the animal enters a new environment, the selection changes «From the place cells among the pyramidal cells. These new place cells are formed within a matter of minutes and remain stable for weeks (Bostock et al., 1991). These results have formulated the idea that the hippocampus contains a map-like representation of the animal's current environment, and that 'the activation of the cells of place in the region of CAI and Ca3 indicates the moment-to-moment location of the animal within the environment. This map is interesting because it is the best example in the brain of an internal, complex representation, a true cognitive map. It differs in various ways from the classical maps found, for example, in visual or somatosensory systems. Different from sensory maps, the space map is not topographic since the neighboring cells in the hippocampus do not represent the neighboring regions in the environment. In addition, the activation of the cells of place may persist after the outgoing suggestions are removed and even in the dark. In this way, although the activity of a place cell can be modulated by sensory input, in contrast to neurons in the sensory system, activity is not controlled by this sensory input (Muller, 1996). If the hippocampal neurons code for an internal representation of space that is used to solve memory problems, how is this Spatial Aapa when LTP interferes with genetic management ?. To address this question, positional activation properties were examined where pyramidal neurons in the hippocampus of mice express the CaMKII -Asp286 transgene (Rotenberg et al., 1996). The experimental arrangement for studying the cells of place is shown in Figure 6. A mouse is equipped with a registration or recording electrode implanted in the hippocampus. The potential activation of the action of a single hippocampal neuron can be reliably recorded from the electrode over a period of several weeks. The mouse is placed in a cylindrical sand and left to settle for 16 minutes while the location of the animal and the activation of the hippocampal neuron are recorded simultaneously As shown in Figure 6b, the activation speed of the neuron When the mouse is in each location in the cylinder it can be plotted, these studies show that different cells have fields in parts of the apparatus and that the fields of place are with approximately equal density anywhere in the apparatus, reinforcing the idea of the place cell with map elements (Muller et al., 1987). Sequential records in the cell of place in wild type mice in a familiar environment show that their fields are stable (Figure TO ) . Activation fields are also formed in transgenic CaMKII-Asp286 mice, indicating that LTP in the 5-10 Hz range is not required for hippocampal pyramidal cells to transform sensory information into spatial information. However, the place cells of the transgenic CaMKII-Asp286 mice have several deficits. First, the activation fields are less well defined, they seem more confusing, with the boundaries between the regions of high and low activation speed less distinct. Second, the rates of cell activation of place in the transgenic mice is reduced. This effect could be a direct generation of abnormal LTD instead of LTP in response to stimulation in the range of 5-10 Hz. Third, place cells in transgenic mice is unstable. When a place cell of a wild-type mouse is registered, and the mouse is then removed from the environment of the recording or recording for a period of time mk then it is retested, the field of activation of the cells of place remains remarkably stable (Figure 7). In this way, when the animal is repeatedly exposed to the same environment, as in a paradigm of solving the spatial problem, the information gained about that environment remains stable. However, when a similar experiment is performed on a CaMKII-Asp286 mouse, the ^ Field of place is unstable and in a different location during different sessions (Figure 7). What defects account for the deficit in spatial memory ?. By themselves, the less precise activation fields and the lower activation rates of the cells in place in the transgenic CaMKII-Asp286 mice can account for the deficits in spatial memory by providing the animal with a less accurate representation in this ambient. However, given hundreds of thousands of place cells, it is not clear that the environment map degrades to be unstable to support normal navigation. However, a deficit in the affability of the place cells will damage or severely impair the animal's ability to learn special tasks, information gained in a given training session will be lost, and in a subsequent training session it will be like the animal showed up with homework for the first time. If the place cells are the building blocks of flJfi cognitive map, the instability of the place cells will suggest that the map itself is unstable and therefore not suitable for the sufficient calculation of the navigation routes. In fact, the deficit at the diagonal level is very similar to the memory deficits seen in human patients with lesions in the temporal, central lobe. A classic example is the H.M. patient, for whom explicit information each session of a multi-session learning test is like the first; he does not remember that the experiment takes place previously, or even recognizes the psychologist who gave him the test. In a parallel set to the study, Wilson et al. (1996) investigated the positional activation properties of CAI pyramidal cells in mice with a specific CAI elimination of the NMDA Rl subunit. These cells have stable activation fields, but the fields are larger than those in wild type mice and instead of having an individual peak, the activation fields have multiple peaks. In addition, the cells of the Q-CAI with overlapping activation fields do not tend to be activated together at the same time (in covariance of significant temporal activation). In this way, until now, the properties of the cells have been examined in two types of mice with the genetically altered LTP in the collateral pathway of Schaffer CA3-CA1. The results ^ ndic n that LTP is not required for the transformation of afferent information into place fields. In contrast, LTP is needed for the fine tuning of high-order properties of the field such as stability and psychic activation. These characteristics of place fields seem to be necessary for spatial memory.

General view The study of explicit memory storage has clearly benefited from the use of new technologies to produce genetically modified mice. First, by using the CaMKII promoter, it has been possible to activate transgenic expression in the temporal, central and, in particular, hippocampal neurons. Second, combining the CaMKII promoter with Cre-recombinase has helped to be able to restrict expression to the CAI region of the hippocampus and suppress the genes in this region. Third, combining the CaMKII promoter and the transcription factor tTA with tetracycline has helped to get out and put the transgenes in limited groups in neurons in the brain. Finally, the analysis of the activation properties of the cell in the place of the modified mice adds a new dimension to the understanding of the molecular and cellular bases of memory. For example, a change in individual amino acid causes CaMKII, an important enzyme in the translation of Ca2 + signals, to become constitutively active. This increase in the affinity of CaMKII leads to a deficit in the response in LTP to a stimulation at 5-10 Hz, presumably by reducing the ability to store information at the synapse between cells that signal the spatial location of an animal. This ^ Loss of storage capacity in the spatial map can destabilize positional activation patterns of place cells and cause severe deficits in performance in spatial memory tasks in transgenic CaMKII-Asp286 mice. The combination of new genetic techniques with in vitro synaptic function analysis and in vivo neuronal activation patterns provides a powerful set of tools for studying the behavior of mammals, from the level of an individual molecule to memory in the animal full. However, as indicated by the lightness of the circuitry of the temporal lobe, central system (Figure 2), when studying the storage of explicit memory, one is only in the hills of a mountain range of great mountains. The next step is to further encourage these methodologies. It is necessary to be able to evaluate the contribution to memory storage of each © aa of the main regions of the hippocampus (Figure 2). Do these regions store different types of information or process the same type of information, but differ in their role in memory per se? Some regions specialize in coding, consolidation or storage, while others specialize in recovery ? The answers to these questions will require The now additional generation of genetically modified mice using promoters to restrict expression to the various individual regions of the temporal, central lobe. In addition, attempts are under way to extend the tTA system to make it more useful in fostering genetic analysis of behavior. For example, it may be possible to produce step changes in the level of transgene expression by administering lower levels of doxycycline to the animals. Also, the Bujard group '(Gossen et al., 1995) has generated a mutant of the tTA molecule that does an inverted response to doxycycline. This inverted tTA allows the expression of a transgene outside during development, then activated rapidly by the administration of doxycycline to the adult mouse (Kistner et al., 1996 and unpublished observations of the author). The use of this inducible system combined with Cre recombinase should provide a way to inducibly remove genes in the brain. Other systems to regulate gene expression in gene suppression are also being explored (Feil et al., 1996, No et al., 1996). With appropriate promoters, these technologies should generally prove useful for the selective genetic modification of precisely defined neural circuits that control behavior. In addition, this approach can allow to explore not only To the individual genes, but also the important genetic routes for LTP.

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Figure 1. A comparison of the central temporal lobe system in rodents, primates and humans. The upper part is shown on the lateral surface of the rat brain, the central surface of the brain (l monkey brain and the central surface of the human brain.) Subsequently, each of these views of the brain is a two-dimensional unfolded map of the cortex. entorinal, the peririnal cortex and the postrinal cortices for the hippocampus. illustrate these comparisons, rodents, primates and humans have an organization of their central temporal lobe structures observed "Greatly among mammals (Burwell et al., nineteen ninety six). Abbreviations: R, rostral; C, flow; D, dorsal; V, ventral. Figure 2. The flow of information in the central temporal memory system in primates (Burwell et al., 1996). This extended view of the central temporal lobe system emphasizes the importance of projects direct from layer II and III of the entorinal cortex (EC) to the CA3, CAI and subicular regions. Figure 3. Regional specificity in transgenic expression with the CaMKII promoter (Mayford et al., 1996). Regional distribution of the transgenic mRNA CaMKII-Asp286 when expressed under the CaMKII promoter alone (a) or in combination with the tTA (b) system. In (a), the CAI, CA2 and CA3 areas and the dentate circonvolution (DG) in the hippocampus, and in the neocortex (Ctx), stratum (Str), amygdala (Amy), and subiculum (Sub). In (b), the expression is found only in CAI and dentate gyrus in the hippocampus, and in the stratum and amygdala. fl igura 4. Regional specificity in gene elimination with the CaMKII promoter in combination with the Cre-loxP system. (a) strategy used to obtain gene elimination restricted by CAI. Two independent lines of transgenic mice were generated: mouse 1 has the CaMKII promoter fused to the Cre transgene; in mouse 2, the gene of interest is flanked by two loxP sequences. The transgene is introduced into the mouse with the gene flanked by loxP through pairing. In mice that have both genetic modifications (mouse 1 + 2), the sequence flanked by loxP is suppressed only in CAI neurons. (b, c,) recombination pattern in an "indicator" mouse having the CaMKII -transgene Cre promoter and a lacZ gene that is interrupted by a stop sequence flanked by two loxP sequences. In this indicator mouse, recombination by Cre removes the arrest sequence that prevents lacZ expression. (b) Sagittal section of the 28-day mouse brain showing blue staining (X-gal) in CAI pyramidal cells in the (^ ipoc mpo indicating recombination in these cells (IMM, MM, and ERK, unpublished results). (c) View at high magnification of the hippocampus Figure 5. Temporal and regional expression of the CaMKII-Asp286 transgene with the tTA system. (a) Strategy used to obtain the transgenic expression specific to the forebrain regulated by doxycycline (j | pRaton 1 carries the CaMKII promoter fused in tTA transgene, mouse 2 carries the teto promoter fused to the CaMKII-Asp286 transgene. an individual mouse through mating (b) The memory task impeded by the mice, the spatial version of the Barnes labyrinth. 40 holes in the perimeter and an escape tunnel «\ Hidden under one of the holes. The mouse is placed in the center of the labyrinth and is motivated to escape by bright lights and a disgusting timbre. To find the tunnel, the mouse tends to remember and use the relationships between distant suggestions in the environment. To achieve the learning criterion in this period, the mouse must make three or fewer errors through five of six consecutive trials. Errors are defined as the search for any hole that does not have a tunnel under it.

Figure 6. Mouse place cell records (modified from Rotenberg et al., 1996). (a) The registration process: A mouse trained to run on the floor of a cylinder 49 cm in diameter.The record is made simultaneously of the activity of nailing one or more pyramidal cells and the position of the head The experiment is done with a TV camera on high whose signal is fed to a detector that digitizes the position of a light in the head of a mouse. (b) The positional activation patterns of three cells of the place sequentially recorded for 16 minutes, each one of a wild type mouse, the circles are seen on the head of the cylinder, and the color represents the activation speed in each small square region (pixel). is the colored region of ML dark. When the head of the animal is in this region, the cell activates approximately 10 spikes / second. Outside the activation field, the download speed is virtually as indicated by the yellow pixels. In this way, the positional signal is extremely strong. It is noted that the activation fields of the example cells are in different places in the environment. If many cells were shown, it will be clear that the fields cover the surface of the cylinder. Figure 7. Individual place cells subtracted repetitively from wild type mice and transgenic mice CaMKII-Asp286 (Rotenberg et al., ^ .996). The four 16-minute registration sessions in the top row for the wild type mouse were made in two pairs. Sessions 1 and 2 were made within 3 minutes of each other, without removing the table and cylinder. Similarly, sessions 3 and 4 were given in the space of 2 minutes of each other, again with the mouse continuously present in the ßpcilindro. However, within sessions 2 and 3, the mouse was removed from the cylinder for 1 hour before repositioning. Point out that the position of the activation field is constant at approximately 10:30 o'clock through the four sessions. When the same time sequence of four recording or recording sessions is repeated the transgenic mouse CaMKII -Asp286, the activation field moves from To the position to the position between sessions. In this example, the change in field position was greater after the mouse was removed from and replaced in the apparatus than for the session pairs made at two minute intervals. With respect to many cells, however, the instability was approximately the same for session pairs separated by minutes or by one hour.

Claims (20)

1. CLAIMS 1. A recombinant nucleic acid molecule comprising a calcium-calmodulin-dependent kinase promoter region operably linked to a gene of interest.
2. 2. The recombinant nucleic acid molecule according to claim 1, wherein the region comprises a nucleic acid sequence of 8.5 kilobases corresponding to the nucleic acid sequence of Accession Number ATCC 98582.
3. 3. The recombinant nucleic acid molecule according to claim 1, wherein the gene of interest comprises a gene of acalcinurin, a gene comprised in brain function, a gene of the growth factor, a gene of the ion channel, a kinase gene, a neurotransmitter gene, a gene of the neurotrophic factor, a phosphatase gene, a recombinase gene, an indicator gene, a receptor gene, a gene of the transactivator transcription factor, a gene of the transcription factor.
4. 4. The recombinant nucleic acid molecule of claim 3, wherein the neurotrophic factor comprises the ciliary neurotrophic factor; the nerve growth factor; the neurotrophic factor 4/5; neurotrophic factor derived from the brain; neurotrophic factor derived from glial cells.
5. 5. The recombinant nucleic acid molecule according to claim 3, wherein the neurotransmitter gene comprises a gene of (srotonin, a dopamine gene, and an epinephrine gene) 6. A human cell line that has been stably transformed by a recombinant nucleic acid molecule, comprising a gene of interest operably linked to a nucleic acid encoding for a kinase promoter region Ila (Calcium-calmodulin dependent) having a nucleotide sequence corresponding to ATCC accession sequence number 98582. 7. The human cell line according to claim 6, wherein the gene of interest comprises a gene of acalcinurin, a gene comprised in brain function, a gene of growth factor, an ion channel gene, a gene of Acinase, a neurotransmitter gene, a neurotrophic factor gene, a phosphatase gene, a recombinase gene, a reporter gene, a receptor gene, a transactivator transcription factor gene, a transcription factor gene. 8. The cell line according to claim 6, wherein the cell line is a line of neuronal, human cells. 9. A non-human, transgenic mammal whose germ or somatic cells contain a nucleic acid molecule that codes for a gene of interest under the control of a CaMKIIa promoter (accession number ATCC 98583), introduced into the mammal, or a flank of the same, in an embryonic stage. The non-human, transgenic mammal according to claim 9, wherein the gene of interest comprises an aklincinurin gene, a gene comprised in brain function, a growth factor gene, an ion channel gene, a kinase gene , a neurotransmitter gene, a "neurotrophic factor" gene, a phosphatase gene, a recombinase gene, a reporter gene, a receptor gene, a transactivator transcription factor gene, a transcription factor gene. The non-human, transgenic mammal of claim 9, wherein the nucleic acid molecule contains an appropriate piece of the DNA of the mammalian genomic clone designed by homologous recommendation 12. A method for treating a neurological disorder in a subject which comprises administering to the subject an effective amount of the recombinant nucleic acid of claim 1 to express the gene of interest in the subject and thereby treat the neurological disorder. The method according to claim 13, wherein the neurological disorder is amnesia, Alzheimer's disease, amyotrophic lateral sclerosis, a brain injury, cerebral senility, chronic peripheral neuropathy, a cognitive disability, a degenerative disorder associated with learning, Down's syndrome, dyslexia , amnesia or amnesia induced by electric shock, Guillain-Barre syndrome, head trauma, Huntington's disease, a learning disability, a memory deficiency, memory loss, mental illness, mental retardation, cognitive or memory dysfunction, dementia due to several infarcts and senile dementia, myasthenia flgravis, a neuromuscular disorder, Parkinson's, Picks disease, a reduction in space memory retention, senility, or Turret syndrome. 14. A method for evaluating whether a compound is effective in the treatment of the symptoms of a neurological disorder in a subject, which comprises: A (a) administering the compound to the non-human, transgenic mammal in claim 9, and ) comparing the neurological function of the mammal in step i [a) with the neurological function of the transgenic mammal in the absence of the compound, thereby determining whether the compound is effective in the treatment of the symptoms of the neurological disorder in a subject. 15. The method according to claim 14, wherein the neurological disorder is amnesia, Alzheimer's disease, amyotrophic lateral sclerosis, a brain injury, cerebral senility, chronic peripheral neuropathy, a cognitive disability, a degenerative disorder associated with learning, Down, dyslexia, amnesia or amnesia induced by electric shock, Guillain-Barre syndrome, head trauma, Huntington's disease, a learning disability, a memory deficit, memory loss, mental illness, mental retardation, cognitive dysfunction or of memory, dementia fl or several infarcts and senile dementia, myasthenia gravis, a neuromuscular disorder, Parkinson's disease, Picks disease, a reduction in the retention of space memory senility, or Turret syndrome. The method according to claim 14, wherein the compound is an organic compound, a nucleic acid, a small molecule, an inorganic compound A, a liquid or a synthetic compound. 17. The method according to claim 14, wherein the mammal is a mouse, a sheep, a bovine, a canine, a porcine or a primate. 18. The method according to claim 14, wherein the subject is a human. The method according to claim 14, wherein the administration comprises intralesional, intraperitoneal, intramuscular, intravenous injection; infusion; distribution mediated by liposomes; Gene bombardment; topical, nasal, oral, anal, ocular or otic distribution. 20. A method for evaluating whether a compound Fes effective in the treatment of the symptoms of a neurological disorder in a subject, which comprises: (a) contacting a human neuronal cell of the human neuronal cell line in claim 6 with the compound, and (b) comparing the function of the neuronal cell in step (a) with the function of the neuronal cell in the absence of the compound, thereby determining whether the compound is effective in treating the symptoms of the neuronal cell. neurological disorder. SUMMARY OF THE INVENTION The present invention provides a recombinant nucleic acid molecule comprising a region, a calcium-calmodulin dependent kinase promoter linked operably to a gene of interest. The calcium-calmodulin-dependent kinase promoter region can comprise a nucleic acid sequence of 8.5 kilobases corresponding to the nucleic acid sequence with accession number ATCC 98582, designated pMM 281. The present invention also provides a human cell line that has been transformed in stable form by a recombinant nucleic acid molecule comprising a gene of interest operatively linked to a calcium dependent kinase-calmodulin dependent kinase promoter region, having a nucleotide sequence corresponding to the B sequence with accession number ATCC 98581, designated pMM 281. The present invention also provides a non-human transgenic mammal whose somatic or germ cells contain a nucleic acid molecule encoding a gene of interest under the control of the CaMKIIa promoter (accession number ATCC 98583), introduced in the mammal or in an ancestor of the same, in an embryonic stage. Another embodiment of this invention is a method for evaluating whether a compound is effective in treating the symptoms of a neurological disorder in a subject, making use of the transgenic mammal as a test model in vivo.