WO2004035001A2 - Calcium channel agonists for psychotherapeutic use - Google Patents

Abstract

The invention provides a pharmaceutical composition comprising a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist and a pharmaceutically acceptable carrier. The invention further provides methods of enhancing inhibitory learning in a subject, ameliorating schizophrenic symptoms in a subject, ameliorating symptoms of autism in a subject, reducing addiction in a subject, and ameliorating symptoms of anxiety disorder in a subject. The method comprises administering to the subject a therapeutically effective amount of an LVGCC agonist. In a typical embodiment, the inhibitory learning comprises extinction, latent inhibition, novel object recognition, negative contingency learning and/or inhibition of delay.

Description

CALCIUM CHANNEL AGONISTS FOR PSYCHOTHERAPEUTIC USE

This application claims priority to United States provisional patent application number 60/417,995, filed October 14, 2002, the entire contents of which are incorporated herein by reference.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

The neural mechanisms underlying learning behavior have been intensively studied in the last century. Learning allows us to adapt to our environments and affects nearly every aspect of our lives. A significant amount of learned behavior involves the formation of excitatory associations between stimuli and responses. Equally important, however, is the ability to suppress, or inhibit, maladaptive responses. Though a great deal of progress has been made in understanding the neural mechanisms underlying the acquisition of new behavior, research regarding the mechanisms of inhibitory learning have lagged far behind. This is unfortunate, as nearly all therapies for psychological disorders are critically dependent on the formation of inhibitory memories to suppress maladaptive responses. There remains a need to facilitate inhibitory learning with new drugs and enhance the treatment of disorders ranging from anxiety and addiction to schizophrenia and autism.

SUMMARY OF THE INVENTION

The invention provides metiiods of enhancing inhibitory learning in a subject, ameliorating schizophrenic symptoms in a subject, ameliorating symptoms of autism in a subject, reducing addiction in a subject, and ameliorating symptoms of anxiety disorder in a subject. The method comprises adrninistering to the subject a dierapeutically effective amount of an LNGCC agonist. In a typical embodiment, the inhibitory learning comprises extinction, latent inhibition, novel object recognition, negative contingency learning and/ or inhibition of delay. The invention further provides a pharmaceutical composition comprising a therapeutically effective amount of an L-type voltage-gated calcium channels (LNGCC) agonist and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A.-F. Nifedipine does not affect cue fear acquisition. Fig. 1A, Experimental design (7-8 mice/group); open circles: vehicle, filled circles: nifedipine. Fig. IB, Freezing during the five cues that preceded each shock for mice injected with nifedipine (40 mg/kg dose shown, 20 min. pretreatmeni) or vehicle. Fig. IC, Freezing 24 hours later during a 2- minute, drug-free CS test. Fig. D, Freezing during the five cues that preceded each shock for mice injected with nifedipine (40 mg/kg dose shown, 50 min. pretreatmeni) or vehicle. Also included is freezing during the 2-minute stimulus free period following the last CS-US pairing (ctxt) Fig. IE, Freezing 24 hours later during a 2-minute acclimation period (Pre-CS) followed by a 2-minute, drug-free CS test. Fig. F, Freezing during a 2- minute, drug-free CS test, 24 hours after injections (50 min. pretreatmeni) and a single cue- shock pairing. ^*p < 0.05 vs. vehicle.

Figures 2A-G. Nimodipine does not affect cue fear acquisition, though recall appears to be state-dependent. Fig. 2A, Experimental design (8 mice/group). Fig. 2B, Freezing during the five cues that preceded each shock for mice injected with nimodipine (15 mg/kg, 20 min. pretreatment) or vehicle. Fig. 2C, Freezing 24 hours later during a 2- rninute, drug-free CS test. Fig. 2D, Freezing during a second state-dependence test (3 hrs after test 1) following re-injections of drug or vehicle (20 min. pretreatment). Fig. 2E, Fig. 2F, Fig. 2G, Repeat of the above experiment (Fig. 2A-C) using a weaker training protocol (two cue-shock pairings), 'p < 0.05 vs. vehicle.

Figures 3Λ-C. Recall of fear is partially state-dependent with nimodipine. Fig. 3A, Experimental design (8 mice/group; between-subjects design). Fig. 3B, Freezing during the two cues that preceded each shock for mice injected with nimodipine (16 mg/kg, 20 min. pretreatment, n=16) or vehicle (n=16). Fig. 3C, Freezing 24 hours later during a 2- minute CS test, 20 minutes after drug or vehicle injections. Half the mice from each of the previous day's treatment groups received nimodipine (16 mg/kg) or vehicle. ^*p < 0.05 vs. 0-0 group, ⁺ρ < 0.05 vs. 16-0 group.

Figures 4Λ-E. LNGCC inhibitors do not prevent acquisition of context fear. Fig. 4A, Experimental design (7-8 mice/group). Fig. 4B, Freezing shown in 2-minute blocks during the entire 12-minute session for mice injected with nifedipine (40 mg/kg, 50- minute pretreatmeni) or vehicle. Unsignaled footshocks occurred at the 2^nd, 4^th, 6^th, 8^th and 10^th minutes. Fig. 4C, Freezing 24 hours later during a 5-minute drug-free context exposure. Fig. 4D & Fig. 4E, Identical experiment conducted with nimodipine (16 mg/kg, 20 minute pretreatmeni).

Figures 5A-E. Nifedipine blocks extinction, but not expression, of cued fear. Fig. 5A, Experimental design. Extinction sessions began 24 hours after cue fear acquisition (five cue-shock pairings). Fig. 5B, Freezing during the 2-minute acclimation period and first 15 CS presentations, after injections of nifedipine (40 mg/kg dose shown, 20 min. pretreatmeni) or vehicle. Fig. 5C, Freezing 24 hours later during a 2-minute acclimation period (Pre-CS) followed ^'by a 2-minute, drug-free CS test (n's = 8 for extinction groups). Retention control mice (retention control, n = 16) were injected with vehicle and placed in the extinction chamber on day 2, but not exposed to any CS. Fig. 5D & Fig. 5E, Identical experiment with a longer pretreatment (50 min) of a single nifedipine dose (40 mg/kg) or vehicle (all n's = 8). ^*p<0.05 vs. retention control, ⁺p<0.05 vs. vehicle/extinction.

Figures 6A-FL. Nimodipine blocks extinction, but not expression of cued fear. Fig. 6A, Experimental design (12 mice/group). Extinction sessions began 24 hours after cue fear acquisition (five cue-shock pairings). Fig. 6B, Freezing during the first 15 CS presentations, after injections of nimodipine (15 mg/kg, 20 min. pretreatment) or vehicle. Fig. 6C, Freezing 24 hours later during a 2-minute, drug-free CS test. Retention control mice were injected with vehicle and placed in the extinction chamber on day 2, but not exposed to any CS. Fig. 6D & Fig. 6E, Identical experiment with several doses of nimodipine. Only die 16 mg/kg dose is shown for acute extinction. ^*p<0.05 vs. retention control, ⁺p<0.05 vs. vehicle/extinction.

Figures 7A.-B. Extinction with LNGCC blockers is not state-dependent. Fig. 7A, Experimental design (7-8 ice/gnoxφ). The 3-day experiment was identical to earlier experiments (Figure 3) with the exception that drugs and vehicle were injected 20 minutes prior to botii day-2 extinction and die day-3 test. Fig. 7B, Freezing during a 2- minute CS test, 24 hours after extinction. ^'fp<0.05 vs. retention control, ⁺p<0.05 vs. vehicle/extinction.

Figures 8Λ-C. LNGCC inliibitors block extinction, but not expression, of context fear. Fig. 8A, Experimental design (16 mice/group). Extinction was conducted 24 hours after context fear acquisition (5 unsignaled footshocks, 2 minute ITI). Fig. 8B, Freezing in 5-minute blocks for die first 30 minutes of context exposure (120 total). Mice were injected with nifedipine (40 mg/kg, 50 minute pretreatment), nimodipine (16 mg/kg, 20 minute pretreatmeni) or vehicle (all n's = 6). Retention control mice were injected with vehicle and placed in dissimilar chambers for 120 minutes. Fig. 8C, Freezing 24 hours later during a 5 minute drug-free context fear test (data were lost for one mouse in the nimodipine group due to a camera failure).

Figures 9Λ-B. Effects of nifedipine and nimodipine on spontaneous locomotor activity in an open field. Fig. 9A, Experimental design (6 mice/group). Fig. 9B left panel, Total distance traveled during a 60 minute session in a novel chamber (arbitrary units). Mice were injected 20 minutes before the session with nifedipine (40 mg/kg), nimodipine (16 mg/kg) or vehicle. agbt panel, An identical test of the same mice 24 hours later in the drug-free state. ^*p<0.05 vs. vehicle.

Figure 10. Nifedipine blocks LI. Upper scheme shows experimental design. The graphs show that animals receiving pre-exposure to the CS prior to CS-US pairing show reduced freezing to the CS (LI), that injection of Nifedipine prior to pre-exposure abolishes LI, and d at injection of Nifedipine alone without pre-exposure to the CS does not affect conditional fear.

Figure 11. Test of state-dependence. Upper scheme shows experimental design. The graphs show that blockade of LI by Nifedipine shown in die pre-exposure context is not state-dependent (context A), and that injection of nifedipine prior to pre-exposure, acquisition and test fails to restore LI. However, if nifedipine is given before pre- exposure and acquisition, reduced freezing to the CS is evident in the training context (context B).

Figure 12. Diltiazem also blocks LI. These graphs show that diltiazem blocks LI when injected prior to pre-exposure. No state-dependent effect is evident when diltiazem is administered prior to pre-exposure, acquisition and testing.

Figures 13-14. LI: Evidence for 3 processes. These graphs show that LI appears to consist of 3 processes which result in: 1) Reduced freezing to CS presentations during CS-US pairing; 2) Transient reduction in fear to CS presentations in the training context (context B) which is apparent on day 3 but disappears by day 5; and 3) Long- lasting reduction of fear to CS presentations in pre-exposure context (context A).

Figure 15. Nifedipine blocks NOR. These graphs show that, when pre-exposed to A, the vehicle group shows a preference for novel object B during the test. Animals that receive nifedipine prior to pre-exposure show no preference for either object and explore the familiar object A significantly more than d e vehicle group .

Figure 16. These graphs show tiiat inhibition of delay is also impaired by L-NGCC blockade.

Figure 17. These graphs show that nifedipine impairs negative contingency learning to context during cue fear acquisition.

Figures 18A-B. These graphs show that nifedipine does not prevent the affect of reducing CS-US contingency during acquisition. Figures 19A-C. These graphs show that, acutely, nifedipine impairs negative contingency learning to unpaired conditional stimuli.

Figures 20Λ-C. These graphs show that, when tested the next day, nifedipine-treated mice freeze less to d e paired CS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that LVGCCs are necessary for several forms of inhibitory learning, including extinction, latent inhibition, novel object recognition, negative contingency (or safety) learning, and inhibition of delay (or timing). Accordingly, pharmacological agents designed to enhance the activity of LVGCCs can be used to facilitate inhibitory learning and aid in die treatment of disorders caused by failure of inhibition.

Rodent models and pharmacological agents have been used in the studies described herein to elucidate the induction mechanisms of several forms of inhibitory learning. Memory induction likely requires influx of calcium into neurons, which triggers enzymatic cascades resulting in plasticity of neuro transmission. Neural calcium influx is achieved by the opening of calcium channels. There are a wide variety of known calcium channels, one type of which includes the L-type voltage-gated calcium channels (LVGCCs).

To demonstrate d e utility of LVGCC agonists in enhancing inhibitory learning, antagonists, which block the passage of calcium through these channels, were employed for three reasons. First, a necessary step in implicating a molecule in a learning process is to demonstrate that blocking its activity prevents learning. Second, it is typically difficult to enhance learning in unimpaired animals. Because inhibitory learning deficits affect a small percentage of the human and rodent populations, blocking learning proves to be a more productive strategy than attempting to facilitate it. Finally, while there are many effective LVGCC antagonists available for research, only a few agonists have been developed. As demonstrated in the Examples herein, LVGCCs are required for several rodent forms of inhibitory learning. This has been shown using behavioral assays of fear extinction, latent inhibition, discrimination and inhibition of delay.

These findings are significant because LVGCCs now define a general mechanism particular to inhibitory learning. These types of learning are crucial in multiple aspects of human behavior. Briefly, humans acquire many patterns of behavior, i.e. skills and habits, through their lifetime. Inhibition is crucial for the capacity to express the correct pattern in a given context. For example, writing depends on the ability to express the proper pattern of letters in the context of sentence, "there" in one sentence, and "their" in another. An F may be the proper note in the context of one musical passage, while an F# may be appropriate in anotiier. A humorous, even off-color joke may be appropriate in one context, but totally inappropriate in a board meeting. The list of such context driven choices is endless, and it is clear that the ability to make such choices, and suppress inappropriate ones, develops independentiy of the ability to learn the patterns in the first place. That development is called "maturity" in general life, and its delayed development is the reason that we do not assign children and adolescents the same responsibility for their actions as we do adults; why people can't vote until they are 21 and can't be president until they are 35.

Failures of inhibition cause numerous human disorders. And all of these disorders will be important targets of drugs that potentiate the LVGCC system, including agonists that act at the channels themselves. These include anxiety disorders, including obsessive compulsive disorder, trichotillomania (hair pulling and skin picking), panic disorder, post- traumatic stress disorder, generalized anxiety disorder; autism (autistic patients characteristically show extremes of perseverative repetitive behaviors); schizophrenia (acute schizophrenics show a failure of latent inhibition); impulse control disorders (including eating disorders like anorexia nervosa and bulimia nervosa, compulsive gambling and, perhaps, mania); addictive disorders including all forms of alcohol and drug dependence; disorders of sustained attention, such as adult and childhood attention- deficit hyperactivity disorder; and some learning and conduct disorders in children. Furthermore, psychotherapy in general depends on processes similar to extinction. Thus facilitators of the LVGCC system are likely to be useful adjuncts to all forms of psychotherapy, including psychoanalysis.

Beyond these defined disorders, LVGCC system facilitators are likely to be effective performance enhancers in almost every realm of learning. For example, extinction is the basis of all error correction and should allow easier learning of skills like new languages, musical performance, typing, driving, sports performance etc. In addition, they may improve performance in a variety of skills requiring sustained attention.

Definitions

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have die meanings specified.

As used herein, "agonist" of an LVGCC refers to any agent that enhances the activity of an LVGCC.

As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or (c) salts formed with an organic cation formed from N,N'- dibenzyletiiylenediamine or etiiylenediamine; or (d) combinations of (a) and (b) or (c), e.g., a zinc tannate salt; and the like. The preferred acid addition salts are the trifluoroacetate salt and the acetate salt. As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.

Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton PA 18042, USA).

Methods

The invention provides methods of enhancing inhibitory learning in a subject, ameliorating schizophrenic symptoms in a subject, ameliorating symptoms of autism in a subject, reducing addiction in a subject, and ameliorating symptoms of anxiety disorder in a subject. The method comprises adrninistering to die subject a therapeutically effective amount of an LVGCC agonist. In a typical embodiment, the inhibitory learning comprises extinction, latent inhibition, novel object recognition, negative contingency learning and/or inhibition of delay.

LVGCC agonists for use in accordance with the invention are typically selected based on such factors as suitability for pharmaceutical delivery, minimization of side effects, duration and potency of action, and other factors that will vary with the patient, the severity and nature of the disorder or other treatment objectives. To avoid impracticalities of direct delivery to the brain, consideration is given to a particular agonist's ability to cross the blood-brain barrier. To avoid unintended action on related channels in the heart and/ or musculature, one can select an agonist that is relatively selective for subtypes of LVGCC channels that are expressed in die brain. In some embodiments, the agonist is selected to facilitate opening of the channels in response to a signal, such as a physiological signal, rather than acting constituitively. Examples of LVGCC agonists include, but are not limited to, BayK 8644, PCA 50941, FPL 64 76, and CGP-28392. Agonists can act by a variety of mechanisms, so long as they have the effect of enhancing die function of LVGCCs. BayK 8644, for example, acts by binding the dihydropyridine binding site of LVGCCs and increases the mean open time of the channel in response to depolarization. Other agents that can enhance the function of LVGCCs include CB1 agonists, such as BAY 38-7271 and DALN, and agonists of the small conductance potassium SK channels.

Compositions

The invention provides compositions which are useful for treating psychiatric disorders. In one embodiment, d e composition is a pharmaceutical composition. The composition can comprise a therapeutically or prophylactically effective amount of a LVGCC agonist or pharmaceutically acceptable salt thereof of the invention, as described above. The composition can optionally include a carrier, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular metiiod used to administer die composition. Accordingly, diere is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Methods of Aά_τιinistration

Treatment includes prophylaxis and therapy. Prophylaxis or treatment can be accomplished by a single direct injection at a single time point or multiple time points. Adiiiinistration can also be nearly simultaneous to multiple sites.

Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals.

Compositions are typically administered in vivo via parenteral (e.g. intravenous, subcutaneous, and intramuscular) or other traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or directly into neural tissue. Non-parenteral routes are discussed further in WO 96/20732.

The cells or vectors are administered in any suitable manner, often with pharmaceutically acceptable carriers. Suitable methods of administering cells in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular cell composition, a particular route can often provide a more immediate and more effective reaction than another route.

The dose of agonist aά__ιinistrated to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time, or to inhibit progression of the disorder. Thus, agonist is administered to a patient in an amount sufficient to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disorder. An amount adequate to accomplish this is defined as a "therapeutically effective dose."

The dose will be determined by the activity of the agonist and the condition of the patient, as well as the body weight or surface areas of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular agonist in a particular patient. Administration by many of the routes of administration described herein or otherwise known in the art may be accomplished simply by direct administration using a needle, catheter or related device, at a single time point or at multiple time points.

EXAMPLES

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in malting and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

Example 1: LVGCC antagonists prevent the extinction of conditional fear. Acquisition of Pavlovian conditional fear in rodents is a widely-used model of human associative learning and anxiogenesis. The procedure involves presenting d e rodent with a neutral stimulus, such as a tone (conditional stimulus, CS), that is paired in time with an aversive stimulus, such as a footshock (unconditional stimulus, US). Following training, mice and humans exhibit fear behaviors when presented with the CS. Thus, a previously neutral stimulus becomes associated with the aversive event and triggers fear. Extinction procedures can reduce fear to the CS by inducing an inhibitory memory that acts to suppress expression of the acquisition memory. The extinction procedure involves repeatedly presenting the animal with the CS (tone) in the absence of the US (footshock). With enough CS presentations, fear behavior can be eliminated completely. Extinction of conditional fear is the prototypical rodent model of inhibitory learning and is also the explicit model for behavioral therapy of anxiety disorders in humans. Human therapy for anxiety disorders often involves exposure to fear-eliciting cues in a safe environment and is one of the most effective ways to reduce pathological fear in the clinical setting. Additionally, resistance to extinction is one of the hallmarks of anxiety disorders.

Using systemic injections of LVGCC antagonists (nifedipine and nimodipine), the requirement of LVGCCs in rodent extinction memory formation was investigated. When the drugs were injected prior to die CS exposure session, extinction memory formation was completely blocked. Despite being presented with up to 60 unpaired CS presentations, mice exhibited robust fear reactions to the CSs both during the exposures and a day later. The blockade of extinction with LVGCC antagonists was also dose- dependent. These data strongly suggest that LVGCC activity is required for the induction of inhibitory extinction memory formation. The findings also raise the possibility that pharmacological agents designed to enhance the function of LVGCCs may facilitate the formation of inhibitory extinction memories.

L-type Noltage-gated Calcium Channels are Required for Extinction, but not for Acquisition or Expression, of Conditional Fear in Mice.

Fear conditioning is an important form of behavioral plasticity that has been correlated to changes in synaptic strength in the amygdala (Rogan and LeDoux, 1995; Rogan et al., 1997). Both die acquisition and extinction of conditional fear are forms of active learning. The acquisition of conditional fear requires the establishment of a novel association by pairing an initially neutral conditional stimulus (CS), like a tone, with an intrinsically aversive unconditional stimulus (US), typically a mild footshock. While extinction, the reduction of conditional responding after repeated exposures to the CS alone, might initially appear to be a passive decay or erasure of this association, many observations indicate that extinction is new inhibitory learning, which leaves the original memory intact. Conditional fear responding shows: spontaneous recovery with time (Baum, 1988), reinstatement after unpaired US presentations (Rescorla and Heth, 1975), and renewal with context change (Bouton and King, 1983). These observations indicate that the original association is not lost during extinction, but rather suppressed by new, context-dependent learning, which is likely due to plasticity at separate synapses from those mediating acquisition.

Many recent studies have investigated the molecular basis of fear conditioning and its extinction. Accumulating evidence indicates that fear acquisition and expression require ΝMDA-type glutamate receptor (ΝMDAR) activity in the amygdala (Miserendino et al, 1990; Kim et al., 1991; Falls et al., 1992; Kim et al., 1992; Tang et al., 1999; Rodrigues et al., 2001). There is also evidence that extinction of fear depends on NMDARs (Falls et al., 1992; Baker and Azorlosa, 1996; Tang et al., 1999). However, the NMDAR inhibitors used in these experiments may also have altered basal synaptic transmission in the amygdala, and thus, fear expression, which may be required for extinction (Li et al., 1995; Maren et al., 1996; Lee and Kim, 1998). A recent experiment demonstrated that

NMDAR activity is necessary for the long-term retention of extinction memories, but is not required for the generation of extinction acutely (Santini et al., 2001). Thus, while it seems clear that NMDAR activity is required for the formation of fear acquisition memories, considerably less is known about the molecules that mediate the induction of fear extinction.

Recently, a form of NMDAR-independent long-term potentiation (LTP) has been described in synapses between thalamic afferents and neurons in the lateral amygdala (Weisskopf et al., 1999). This LTP depends, instead, on L-type voltage-gated calcium channels (LNGCCs). Since these synapses have also been implicated in auditory fear conditioning (Rogan and LeDoux, 1995), amygdaloid LNGCC-LTP is an important candidate for a mechanism that may underlie some aspect of conditional fear (Blair et al., 2001; Bauer et al., 2002). In tiiis paper, we tested the specific hypotheses that LVGCC activity is required for the (1) acquisition, (2) expression and (3) extinction of conditional fear in mice. To do so, we used systemic injections of two dihydropyridine LVGCC antagonists with good penetration through the blood brain barrier, nifedipine and nimodipine. Our results indicate that LVGCCs are necessary for die extinction of conditional fear, but are not required for its acquisition or expression.

MATERIALS AND METHODS

Subjects. Naϊve 12-20 week old C57/B16 male mice (Laconic, Germantown, NY) were housed 4/ cage, maintained on a 12:12 light/ dark schedule and allowed free access to food and water. All testing was conducted during the light phase in iUu ninated testing rooms following protocols approved by UCLA's Institutional Animal Care and Use Committee. Ωrugs. The LVGCC antagonists nifedipine (1.25 - 80 mg/kg) and nimodipine (4 - 16 mg/kg, Sigma, St. Louis) were sonicated into 100% Cremophor EL (BASF, Mt. Olive, NJ). PBS was added to make the final vehicle 10% Cremophor/90% PBS. The highest nifedipine doses were pardy suspensions and care was taken to thoroughly mix the drugs prior to injecting them. Mice were injected subcutaneously 20 or 50 minutes prior to behavioral testing (10 ml/kg). Drug pretreatment times were chosen based on pilot studies and previous reports of systemic aiiministration in rodents (Janicki et al., 1988; Larkin et aL, 1992).

Conditioning Apparatus. Two contexts (A and B), in separate rooms, were used for all behavioral fear testing. Shuttle box compartments (Med Associates, St. Albans, VT) measuring 20.3 x 15.9 x 21.3 cm served as context A, and conditioning boxes (Med Associates) measuring 30.5 x 24.1 x 21 cm served as context B. Both contexts had two transparent walls and stainless steel grid floors (3.2 mm diameter, 8 mm centers), however, the grid floors in context B were covered with flat white acrylic inserts in order to minimize context generalization. Context A was wiped down before testing with 10% ethanol and context B with 10% mefhanol. Individual video cameras were mounted in the ceiling of each chamber and connected via a quad processor to a standard VCR and monitor for video-taping and scoring of freezing. Grid floors were connected to a scrambled shock source (Med Associates). Auditory stimuli (Med Associates) were delivered via a speaker in the chamber wall. Delivery of stimuli was controlled with a PC and Med-PC software through a SmartCTL Interface System (DIG-716, Med Associates). Background white noise was maintained at 62 dB throughout behavioral testing.

Open field. Spontaneous locomotor activity was monitored by placing mice in one of four chambers (40 cm x 40 cm x 40 cm) and allowing them to freely explore for 60 minutes. Chambers had white floors and two white walls; the remaining two walls were transparent. A video camera was mounted above the chambers and total distance traveled was tracked with a PC and software (Poly-Track Video Tracking System, Chromotrack v 4.02, San Diego Instruments, San Diego, CA) and expressed in arbitrary units.

Conditional Fear Testing.

Cue fear acquisition. Experiments investigating the effects of LVGCC antagonists on the acquisition of cue fear consisted of two phases: fear acquisition (context A), and testing (context B). Following injections, conditional fear was induced by presenting audible cues (CS: white noise, 2 min., 80 dB) that co-terminated with mild footshocks (US: shock, 2s, 0.7 mA). Two-minute stimulus free periods preceded, separated and followed the pairings. Most experiments employed five CS-US pairings, however, experiments using one (Figure IF) or two (Figures 2E-G & 3B-C) pairings were also conducted to ensure that LVGCC blockers did not impair fear acquisition with weaker training protocols. After allowing one day for memory consolidation, cue fear was tested by presenting one, continuous, 2-minute CS after a 2-minute acclimation. In two cases where reductions in fear acquisition were observed, a second test was conducted three hours later to determine whether the reductions reflected state-dependent memory retrieval (Figures 2D & G). For these state-dependence tests, mice were re-injected and subjected to an identical test of cue fear. In a separate test of state-dependence, using a 2 x 2 inter-subject design, mice were injected with vehicle or nimodipine (16 mg/kg, Figure 3) 20 minutes before both ttaining and testing for fear expression 24 hours later.

Cue fear extinction. Experiments investigating die effects of LVGCC antagonists on cue fear extinction consisted of three phases: fear acquisition (context A), fear extinction (context B), and testing (context B), each separated by one day to allow for memory consolidation. In all experiments cue fear was induced in non-drugged, naϊve mice with the 5-pairing protocol described above. Mice were matched into equivalent treatment groups based on freezing during the third training CS. One day later, following injections, mice were placed in context B and allowed to acclimate for 2 minutes. Extinction was induced with 60 2-minute CS presentations (5s ITI). Additionally, non- extinguished (retention control) mice were injected with vehicle and placed in context B for an equivalent period of time, but not exposed to any CS presentations. One day following extinction, all mice were returned to context B in the drug-free state. After a 2-minute acclimation, freezing was assessed during a 2-minute, continuous CS presentation. In a subsequent experiment (Figure 7) examining the potential for state- dependent recall of extinction memories, fear acquisition, extinction and testing were conducted as described above, however, mice were re-injected 20 minutes prior to the final test with drug or vehicle.

Context fear acquisition: Experiments investigating the effects of LVGCC antagonists on context fear acquisition consisted of two phases, both conducted in Context B with the white inserts removed. Following injections, mice were placed in the chambers where they received five 0.7 mA x 2s unsignaled footshocks. Two-minute stimulus free periods preceded, separated and followed the footshocks. 24 hours later mice were returned to the same chambers for a five-minute test of context fear.

Context fear extinction: The experiment investigating the effects of LVGCC antagonists on context fear extinction consisted of tiiree phases, all conducted in Context B with the white inserts removed. Context fear was induced in naϊve, untreated, mice with the 5- shock protocol described above. Mice were then matched into equivalent treatment groups based on freezing during the 2-minute period following the fifth shock. One day later, mice were injected and returned the conditioning chambers for a 120-rninute shock-free session. Non-extinguished retention control mice were injected with vehicle and placed in different chambers (see Open Field) for 120 minutes. One day after extinction, all mice were returned to the Context B chambers for a 5-minute drug-free test session.

Statistical analyses. Behavioral freezing, the absence of all non-respiratory movements, was rated during all phases by a blinded, experienced investigator using a 5s instantaneous time sampling technique. Percent freezing scores were calculated for each mouse and data represent mean freezing percentages (± SEM) for groups of mice during specified time bins. Total session means and individual CS exposures were analyzed with one-way ANOVA and planned post-hoc Dunnett's Test comparisons. Student's t-tests were used to analyze experiments with only two treatment groups. Multiple trial data were analyzed with matched two-way ANOVA and Bonferroni post-tests to compare individual timepoints. Differences were considered significant if p < 0.05.

RESULTS

LNGCC inhibitors do not prevent the acquisition or retention of conditional cue fear. To test whether LNGCC activity is required for cue fear acquisition, we injected nifedipine 20 minutes before a moderate ttaining protocol (5 CS-US pairings). We generated a dose-response curve for nifedipine (5-80 mg/kg), and assessed freezing during acquisition and 24 hours later during a drug-free retention test. Acute acquisition of freezing was unaffected by nifedipine administration; mice injected with vehicle or nifedipine (40 mg/kg) froze identically during the CS preceding each shock (F ,65 = 3.22, p = 0.08 for drug; Fι,65 = 1-05, p = 0.39 for drug x trial interaction; Figure IB). After allowing 24 hours for consolidation of learning, the mice were presented with the CS in a novel context. None of the drug doses reduced freezing during this test (Fs, 2 = 1.64, p = 0.17, Figure IC). In fact, mice previously injected with 10 mg/kg of nifedipine froze slightiy, but significantly, more than mice previously injected with vehicle (p < 0.05).

Since the rate of absorption and brain penetration of systemically administered nifedipine varies depending on its vehicle (Larkin et al., 1992), we also generated an abbreviated dose-response curve with a 50 minute pretreatment (2.5-40 mg/kg). Again we saw no impairment of fear acquisition or retention with nifedipine administration (F.,35 = 0.88, p = 0.36 for 40 mg/kg nifedipine during acquisition; F ,35 = 0.57, p = 0.69 for the drug x trial interaction, Figure ID; and F₃,28 = 0.48, p = 0.70 for die retention test, Figure IE). To confirm that confounding effects of context conditioning or generalization were not somehow obscuring a blockade of acquisition, we also scored context freezing in this experiment, both in the last 2-minute stimulus-free period of training, and during the 2- minute pre-CS period of testing on day 2 in context B. There was no evidence that nifedipine impaired conditioning to the training context during CS-US pairings. In fact, nifedipine-treated mice froze nearly twice as much as control mice during the final 2- minute stimulus free period of ttaining (p < 0.05 vs. vehicle; Figure ID, Ctxt). Since nifedipine does not induce freezing or potentiate context conditioning when the shocks are unsignaled (see Figure 4), these data suggest that nifedipine may retard negative contingency learning (i.e. that the CS, not the context, predicts US delivery). During the retention test, all groups showed a small amount of context generalization. However, freezing before the CS delivery was low and statistically undistinguishable for all groups F3,28 = 1.30, p = 0.29; Figure IE, Pre-CS) and is unlikely to have obscured a blockade of long-term cue fear acquisition. Lastly, to eliminate the possibility that our lack of effect with nifedipine was due to an overly strong ttaining protocol, we injected the drug (2.5- 40 mg/kg) 50 minutes prior to a single CS-US pairing. This protocol does not allow the measurement of short-term acquisition, but again we detected no significant reduction in retained conditional fear 24 hours later (F₃₎ 28 = 1.19, p = 0.33; Figure IF).

To ensure that our failure to block acquisition was not specific to nifedipine, we tested nimodipine (15 mg/kg, 20 min. pretreatment), another LVGCC antagonist. Nimodipine had no effect on the acute acquisition of fear measured by freezing during the CS that preceded each of the five shocks (Fι,3_ = 0.07, p = 0.79 for drug; F_4;35 = 0.65, p = 0.63 for drug x trial interaction; Figure 2B). When tested for retention of conditional fear on the next day in a drug-free state, animals treated witii nimodipine froze slighdy less than vehicle-treated controls, however, this effect was not significant (t_ = 1.35, p = 0.20; Figure 2C). State-dependent effects on learning have been reported for a number of pharmacological agents (Connelly et al., 1975; Jackson et al., 1992; Bloldand et al, 1998), suggesting that such drugs provide a salient internal context that contributes to the learned cue association. We therefore assayed the same mice for freezing again, three hours after d e first test, 20 minutes after re-injections. Nimodipine-treated animals now showed the same levels of freezing as vehicle-treated animals (tι = 0.58, p = 0.57; Figure 2D). Thus, nimodipine did not interfere with the process of fear conditioning, although our data indicates that it made the recall of fear partially state-dependent. Again, to rule out the possibility that the five-shock ttaining protocol represented overttaining that might obscure a subde role for LVGCC in the acquisition of conditional fear, we also tested the role of nimodipine on a weaker ttaining protocol, using only two CS-US pairings. There were no differences in freezing during the CS that preceded each shock (Fι,i4 = 0. , p = 0.74 for drug; Fι,ι = 0.84, p = 0.38 for drug x trial interaction; Figure 2E), and nimodipine again decreased freezing slightly, though this time significandy, when assayed a day after ttaining in the drug-free state (tι₄ = 2.32, p < 0.05; Figure 2F). Again, the difference disappeared when the animals were re-injected before a second fear assay (t_i4 = 1.69, p = 0.11; Figure 2G).

We had not anticipated state-dependent recall of cue fear with nimodipine, and the initial experiments were not designed to test this. Therefore, we next assessed state- dependence direcdy in a between-subjects design, using two CS-US pairings for training followed after a day by a test CS presentation in a novel context. Eight mice per group received an injection prior to both ttaining and testing, in a two by two design: either nimodipine (16 mg/kg) before ttaining, then nimodipine before testing, or vehicle both days, or vehicle first followed by nimodipine, or vice versa. As seen before, nimodipine had no effect on freezing during the tones that preceded each shock during acquisition (Fι,3o = 0.03, p = 0.86 for drug; Fι₎₃o = 0.01, p = 0.93 for drug x ttial interaction; Figure 3B). Again, nimodipine treatment prior to acquisition reduced freezing 24 hours later compared to vehicle treated mice (0-0 vs. 16-0, p < 0.01; Figure 3C). However, freezing in mice that received nimodipine prior to both the acquisition and test sessions was indistinguishable from vehicle treated mice (0-0 vs. 16-16, p > 0.05) supporting the hypothesis that nimodipine makes cue fear recall partially state-dependent. Importantly, nimodipine injections prior to testing did not increase freezing in mice that were trained with vehicle (0-0 vs. 0-16, p > 0.05). Thus while recall of fear is state-dependent with nimodipine, expression of fear is unaltered. LNGCC inhibitors do not prevent the acquisition or retention of conditional context feat. We also examined the effects of nifedipine (40 mg/kg) and nimodipine (16 m/kg) on acquisition of context fear in separate experiments (Figure 4). Acutely, mice injected with either nifedipine or vehicle acquired context fear (Fs,78 = 10.12, p < 0.01 for time), though nifedipine-tteated mice appeared to learn at a slightly slower rate, the effect of drug treatment was statistically significant (Fι, 8 = 8.33, p < 0.01; Figure 4B). However, the effect was small as freezing by nifedipine-tteated mice was never statistically different at any single timepoint (p's > 0.05) and the group x time interaction was statistically insignificant (Fs s = 0.87, p = 0.51). Νimodipine-treated mice were indistinguishable from vehicle-treated mice in the acquisition of context fear (Fs,84 =

17.27, p < 0.01 for time; Fι,₈4 = 2.46, p = 0.12 for drug, and F₅,84 = 0.83, p = 0.54 for the drug x time interaction; Figure 4D). When tested drug-free 24-hours later, both nifedipiαe- and nimodipine-teeated mice froze the same as vehicle-treated mice indicating that retention of context fear was unimpaired by LNGCC blockade (tι₃ = 0.74, p = 0.47 and tw = 0.29, p = 0.78 respectively; Figures 4C & E).

LNGCC inhibitors block extinction but not expression of conditional cue fear.

Using five CS-US pairings for training groups of naϊve mice, we next tested the effect of LNGCC inhibitors on the expression and extinction of cue fear. One day after ttaining, mice were injected with drug or vehicle, placed in a novel context and exposed to 60 2- minute CS (5s ITI). Retention control mice were injected with vehicle and placed in the extinction chambers for an equivalent period of time, but were not exposed to any CS. Expression of conditional fear was assessed by measuring freezing during the first CS exposure of the day 2 session, before any extinction could occur. Acute extinction was assessed during the first 15 CS exposures, and retained extinction was assessed during a single 2-minute CS exposure in the same context one day later. The 60 CS protocol generated substantial persistent extinction in vehicle-treated mice compared to retention controls (p's < 0.05; Figures 5C,E & 6C,E). Neither inhibitor affected the expression of conditional fear during the first 2-minute exposure to the cue (p's > 0.05 compared to vehicle; Figures 5B,D & 6B,D). Furthermore, nifedipine did not affect pre-CS freezing to the novel context before cue exposure began (tι₄ = 1.1, p = 0.28; Figure 5D, Pre), nor pre-CS freezing on the test of extinction (F_2j2i = 1.64, p = 0.22; Figure 5E, Pre-CS). However, while vehicle-treated mice showed a progressive decline in freezing with repeated CS exposures, freezing by mice treated with LNGCC blockers remained elevated in all four experiments. Although mice treated with 40 mg/kg nifedipine (20 min. pretreatment) appear to begin to extinguish, they rapidly return to initial freezing levels while controls show continuing declines (Fι,ιo5 = 39, p < 0.01 for drug; Fι₄₎ιo5 = 2.27, p < 0.01 for drug x ttial interaction; Figure 5B). Furthermore, freezing during each of the CS was never significandy less than freezing during the first CS (Fι_4>ιo5 = 1.61, p = 0.09). In conttast, vehicle controls show a significant reduction in freezing by the seventh CS presentation (Fι ,ιo5 — 4.71, p < 0.01). Nevertheless, a trend towards some early extinction suggested that nifedipine may be more efficient witii a longer pretreatment, and we repeated both our extinction experiment and our acquisition experiments (see above) with longer pretteatments. When injected with nifedipine (40 mg/kg) 50 minutes before extinction began there was no hint of acute extinction in the nifedipine-tteated group (Fι,ιo5 = 228, p < 0.01 for drug; Fι _ιιos = 2.05, p < 0.05 for drug x ttial; Figure 5D). To verify that the blockade of extinction was not particular to nifedipine, we also tested nimodipine (15 mg/kg), and it, too, blocked acute extinction entirely (Fι_;i65 = 220, p < 0.01 for drug; Fι₄,i65 — 5.05, p < 0.01 for drug x trial; Figure 6B). A dose-response curve was then generated for nimodipine (4-16 mg/kg) and an identical result was also obtained for the 16 mg/kg dose (Fi.iβs = 179, p < 0.01 for drug; Fu,i65 = 1.72, p = 0.06 for drug x trial; Figure 6D); acute extinction was blocked entirely.

Consistent with the acute results, when the same mice were tested drug-free after a day for consolidation, both LNGCC antagonists had completely blocked the extinction seen in vehicle-treated mice (p < 0.01 for 40 mg/kg nifedipine, 20-minute pretreatment; p < 0.01 for 40 mg/kg nifedipine, 50-minute pretreatment; p's < 0.05 for 15 & 16 mg/kg nimodipine, 20-minute pretreatment). Additionally, freezing in these groups was statistically equivalent to non-extinguished retention controls (p > 0.05 for 20, 40 and 80 mg/kg of nifedipine, 20-minute pretreatment; p > 0.05 for 40 mg/kg nifedipine, 50- minute pretreatment; and p's > 0.05 for nimodipine 4, 8, 15 & 16 mg/kg, Figures 5C,E & 6C,E).

It has previously been shown that extinction generated in the presence of benzodiazepines can be state-dependent (Bouton et al., 1990). Thus, although no persistent extinction is seen when rats extinguished in the presence of benzodiazepines are tested in the absence of drugs, extinction can be uncovered by aά_ταinistering benzodiazepines again before the extinction test. We therefore tested whetiier such state-dependent extinction might occur with LNGCC-antagonist treatment, by both extinguishing and testing after drug injections. There was no evidence of state- dependent extinction; mice given extinction ttaining in the presence of nifedipine or nimodipine and then re-injected with drug before testing showed freezing no lower than retention controls (p's > 0.05), and froze significantly more than mice extinguished and tested in the presence of vehicle (p's < 0.05, Figure 7). Nifedipine-tteated animals showed a trend to more freezing than retention controls in the final drugged test, however this effect was not statistically significant (p > 0.05).

LNGCC inhibitors block extinction but not expression of conditional context feat.

To further explore the effects of LVGCC blockade, we also tested the effects of nifedipine (40 mg/kg) and nimodipine (16 mg/kg) directly on the expression and extinction of context fear (Figure 8). Neither inhibitor altered the expression of freezing as measured during the first 5-minute block of die extinction session ( p's > 0.05 vs. vehicle; Figure 8B). Freezing was assessed during die first 30 minutes of the 120-minute extinction session. As with cue fear, both LVGCC inhibitors blocked the acute extinction of context fear evident in veliicle-tteated mice (nifedipine: Fι,₉o = 45.7, p

<0.01 for group, Fs^o = 10.9, p < 0.01 for time, and Fs,9o = 8.3, p < 0.01 for the group x time interaction; nimodipine: Fι,90 = 54.2, p < 0.01 for group, Fs,9o =19.6, p < 0.01 for time, and Fs_>9o = 5.7, p < 0.01 for the group x time interaction; Figure 8B). Likewise, vehicle-treated mice showed significant long-term extinction compared to retention conttol mice when tested 24 hours later (Fi.iso = 20.7, p < 0.01 for group; Figure 8C). Treatment during context exposure with nifedipine or nimodipine completely blocked long-term extinction of context fear; freezing in these groups was indistinguishable from non-extinguished retention control mice (nifedipine vs vehicle: Fι,i5o = 33.1, p < 0.01; nimodipine vs vehicle: Fι,ι₄5 = 22.1, p < 0.01; nifedipine vs. retention control: Fi.iso = 0.24, p = 0.62; nimodipine vs. retention control: Fι,i45 = 0.27, p = 0.60). All mice showed extinction during this final 5-minute drug-free test (main effect for time: F_4j295 = 15.9, p < 0.01), however there was no indication that the groups extinguished at different rates during this test (main effect for group x time interaction: Fi2,_95 = 0.31, p = 0.99).

Spontaneous locomotor activity and LVGCC blockers.

To rule out the possibility that our behavioral effects with the LVGCC inliibitors were due to gross reductions in movement we next assessed the effects of nifedipine and nimodipine on spontaneous locomotor activity in a novel open field. Mice were injected with vehicle, nifedipine (40 mg/kg) or nimodipine (16 mg/kg) 20 minutes prior to a 60- minute session. One day later, mice were returned to the same chambers and allowed to freely explore again for 60 minutes, though now in the drug-free state. Total distance traveled (arbitrary units) was recorded in each session. The distance traveled by nifedipine-tteated mice was no different than vehicle-treated mice in either session (p's > 0.05, Figure 9). Nimodipine-tteated mice showed suppressed locomotion acutely (Test 1: p < 0.01) but normal locomotion the next day (Test 2: p > 0.05).

DISCUSSION

We have presented data that point to a distinction in the molecular mechanisms underlying acquisition and extinction of conditional fear in mice. Blockade of LVGCCs effectively prevents extinction in a dose-related manner (Figures 5-8), both acutely (during extinction exposures) and persistently (a day after exposures). These effects are not state-dependent; no hidden extinction is uncovered by treating mice with the same drugs at testing as during extinction exposures (Figure 7). LVGCC activity thus appears to be essential to induce conditional fear extinction. This is the first intervention reported to completely block the induction of extinction. Since it has been shown recently that NMDAR activity is not required for extinction induction (Santini et al., 2001), this work provides a candidate induction mechanism: calcium entry through LVGCCs may initiate plasticity underlying extinction memories.

On the other hand, doses of LVGCC inhibitors that completely block extinction fail to prevent conditional fear acquisition (Figures 1-4). For cue fear, drug-injected animals showed a pattern of increasing freezing with ttaining trials identical to that of vehicle- treated controls, indicating that acute fear learning is independent of LVGCC activity. Across a wide range of nifedipine doses and two pretteatment durations, conditional fear one day later was at least as great as that of controls. These data indicate diat long-term retention of fear learning does not require LVGCC activity, and that the failure to block acquisition was not due to delayed absorption or early metabolism of the drug. While drug-free freezing was sometimes significantly lower in the long-term retention test one day later in animals treated witii nimodipine, this was clearly a state-dependent effect on recall (Figures 2 & 3). When nimodipine was re-injected (Figure 2) or injected before both ttaining and testing (Figure 3), mice showed conditional freezing identical to that expressed by animals doubly injected witii vehicle. Normal fear acquisition occurred with both moderate and weak ttaining protocols, indicating that it was not overttaining tiiat prevented detection of a role for LVGCCs in fear acquisition.

LVGCC inhibitors also failed to block context fear acquisition (Figures 1 & 4). The rate of acute context fear acquisition in nimodipine-teeated mice was indistinguishable from vehicle-treated mice. Nifedipine-tteated mice appear to acquire context fear more slowly in the absence of auditory CS, but the difference in acquistion rate was not significant, and animals achieved the same final freezing levels. The 24-hour retention test was unambiguous; neither LVGCC inhibitor prevented the long-term acquisition of context fear (Figure 4), and nifedipine caused no significant increase in context generalization of fear (Figure 1). This last finding may be especially relevant to the present studies, as extinction expression is context-dependent (Bouton and Bolles, 1979). Our context experiments indicate that LVGCC inhibitors do not impair context-dependent learning, and that their blockade of extinction is probably not by preventing associations with the context of extinction.

Several of our findings make it clear that LVGCCs are also not required for the expression of conditional fear. Vehicle- and drug-treated mice acquire freezing to the tone at the same rate (Figures 1-4). Furthermore, initial freezing levels during the extinction sessions were equivalent for drug-injected and vehicle-injected mice (Figures 5,6 & 8). Finally, in the state-dependence test of acquisition, animals injected first with vehicle and then with nimodipine show no difference in freezing from those injected with vehicle twice (Figure 3). These findings argue strongly that the drugs interfere neither with the detection of the CS or US, nor with expression of conditional fear (freezing).

We also tested whether non-specific effects of the drugs on locomotion could account for our results. In an open field test, nimodipine, but not nifedipine, acutely decreased total distance traveled, but neither affected locomotion the next day (Figure 9). Thus, affects on locomotion cannot account for the blockade of extinction in our drug-free tests. It is also unlikely that our blockade of acute extinction is a result of reduced locomotion since nifedipine blocks extinction acutely but does not reduce locomotion. In addition, nimodipine-injected animals were never scored with increased freezing acutely, compared to vehicle-injected animals (Figures 2,3,4,6 & 8). These results confirm that freezing is behaviorally distinct from reduced locomotion, and that the acute effects of nimodipine on locomotion neither confounded our freezing scores nor accounted for the persistent blockade of extinction.

Forms of LVGCC-dependent synaptic modification have been described in a number of synapses in the brain (Johnston et al., 1992; Huang and Malenka, 1993; Christie and Abraham, 1994; Huber et al, 1995; Zhuo and Hawkins, 1995; Kurotani et al., 1996; Izumi and Zorumsld, 1998; Kapur et al, 1998; Morgan and Teyler, 2001; Zaldiarenko et al., 2001). Usually, these synapses also show NMDAR-dependent LTP. However, LTP at synapses between thalamic afferents and neurons in the lateral amygdala is NMDAR- independent (Weisskopf et al., 1 99). This LTP depends, instead, on L-type voltage- gated calcium channels (LVGCCs), since it can be blocked by nifedipine. Consistent with this, few previous reports indicate a clear dependence of learning on LVGCCs (Lee and Lin, 1991; Deyo et al., 1992; Borroni et al., 2000). To the contrary, many studies indicate that LVGCC blockade promotes learning, rather than blocking it (Disterhoft et al., 1993; Vetulani et al., 1993; Fulga and Sttoescu, 1997; Quevedo et al., 1998; Quartermain et al, 2001). We also show a significantly increased acquisition of fear with 10 mg/kg of nifedipine (Figure IC) and trends towards better learning at other doses in acquisition and in extinction (Figures IC and 4C). Previous investigators hypothesize various explanations for the paradoxical enhancement of learning by LVGCC blockade, including compensatory cellular changes (Quevedo et al., 1998), low concentrations of antagonists acting to hold channels open rather than closing them (Fulga and Sttoescu, 1997), or non-specific vasodilatory effects (Vetulani et al., 1993). Importantly, this learning enhancement has repeatedly been observed to disappear as the dose of LVGCC inhibitors increases, suggesting that it results from modulation rather than complete blockade of the channels.

However, in the present studies we did not pursue the noottopic effects of low doses of LVGCC inhibitors, because we wanted to determine when LVGCC-dependent plasticity was required in fear learning. We, therefore, chose high drug doses in order to maximally inhibit LVGCCs. The results demonstrate a robust blockade of one type of inhibitory learning (extinction) with no effect on a type of excitatory learning (acquisition). The fact that LVGCCs are implicated in extinction but not in acquisition of conditional fear, while NMDARs are implicated in bodi (Falls et al, 1992; Baker and Azorlosa, 1996; Tang et al., 1999), raises questions about the need for this extra molecule in extinction learning.- We hypothesize that LVGCCs are needed in extinction but not in acquisition, because no CS-US pairing occurs during extinction. LVGCCs may allow plasticity to occur after presentation of CS alone, a hypothesis we hope to test using other forms of CS-alone learning, such as latent inhibition (Lubow, 1973).

Although systemic injections cannot support any anatomical hypothesis about the sites where these inhibitors have their effect on extinction, other evidence suggests that the amygdala may be the relevant location. First, while long-lasting extinction may depend on areas of prefrontal cortex (Morgan et al., 1993), the induction of extinction proceeds normally in animals with frontal lesions (Quirk et al., 2000). Similarly, extinction induction occurs normaEy in animals with hippocampal lesions (Frohardt et al., 2000). The amygdala, on the other hand, clearly plays a role in extinction, as inttaparenchymal infusions of NMDAR or MAP kinase inhibitors there block extinction (Falls et al., 1992; Lu et al., 2001). Furthermore, the identification of LVGCC-dependent, but NMDAR- independent, LTP in the thalamo-amygdala pathway (Weisskopf et al., 1999) has led to the hypothesis that this LTP is crucial for fear conditioning (Blair et al., 2001), and a very recent paper from this group shows an attenuation of cue fear acquisition with the LVGCC blocket verapamil (Bauer et al., 2002). We cannot account entirely for the inconsistency of these results with our own. Our data argue strongly against the importance of LVGCC-dependent LTP, whether in amygdala or elsewhere in the brain, in the acquisition of conditional fear. Two potential confounds may account for the inconsistency. First, it is difficult to compare inttaparenchymal infusions of verapamil to our systemic administrations, and, second, no test of state-dependent recall was performed in Bauer et al. Since our data clearly indicate that LVGCCs participate in die extinction of conditional fear at doses that don't affect acquisition, we expect that intta- amygdala infusions of LVGCC inhibitors will also block extinction of conditional fear at doses tiiat fail to block acquisition. We are currently testing this hypothesis with inttaparenchymal aά-riinisttations, and performing state-dependence controls, in order to resolve the apparent inconsistency. However, it should be noted that LVGCCs are ubiquitous in the brain, and LVGCC-dependent plasticity outside the amygdala may well make the relevant contribution to extinction memory formation. The demonstration that LVGCCs are required for extinction but not acquisition of conditional fear suggests that it may be possible to identify cells, synapses or molecular pathways specific to extinction. Because extinction is the explicit model for behavior' therapy (Wolpe, 1969), die most efficacious treatment for human anxiety disorders, this discovery also holds out hope for the development of new drugs that can make such therapy easier and more effective by selectively facilitating the extinction of fear.

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Example 2: LVGCC antagonists prevent latent inhibition of fear.

Latent inhibition is another well-accepted model of inhibitory learning. Latent inhibition procedures are similar to extinction procedures, except that repeated exposure to die CS occurs before fear acquisition. Repeated presentation of a stimulus before it has any emotional significance leads rodents and humans to respond less to subsequent conditioning with the stimulus. Thus, prior exposure to a CS reduces fearful responding when that CS is later paired witii an aversive event. Latent inhibition has also been used to model attention, since repeated exposure to the irrelevant stimulus causes animals to place less emphasis on the stimulus during future experiences. One well-defined characteristic of schizophrenia is an impairment in latent inliibition. Additionally, procedures akin to latent inhibition are commonly employed in the ttaining of policemen, firemen and soldiers in order to protect against fear acquisition during the traumatic events they encounter in the field. Mice were injected with LVGCC antagonists prior to the CS exposure session. In a subsequent session, mice were subjected to fear acquisition (pairing of the CS and US). Veliicle-tteated mice showed a substantial reduction in acquisition of fear compared to mice that never received CS exposure. However, mice treated with LVGCC antagonists acquired fear as if they had never received CS exposure. Like extinction, latent inhibition was completely blocked by the LVGCC antagonists. These data show that latent inhibition also requires LVGCC activity, and LVGCC activity may be a general requirement for inhibitory learning, not just the suppression of previously acquired memories.

These data are further elaborated in Figures 10-15.

Example 3: LVGCC antagonists prevent ciiscrimination.

The environment we experience is a composite of countless stimuli. When we experience a significant event we tend to attribute the event to the occurrence of specific stimuh that precede it. The remaining stimuli are usually discarded as irrelevant. In other words, certain stimuli predict the occurrence of certain events and we discriminate between the predictive cues and the irrelevant ones.

This process is alternatively described as a calculation of contingency. Events that are contingent upon the occurrence a certain stimulus lead to the formation of an association between the two. The formation of associations between stimuli that occur in the absence of the event is suppressed.

The ability to discriminate is critically dependent on inhibitory learning and is modeled by the Pavlovian procedure of discrimination. An inability to discriminate is also a feature of many anxiety disorders in humans. In these cases, individuals will exhibit fear to stimuli present during the traumatic event that had nothing to do with the occurrence of the event.

Discrimination procedures involve die presentation of at least two neutral stimuli. One is paired in time with the US (called the CS+), while the others occur without the US (called CS-). Initially, animals will show some responding to all the stimuli. However, with further ttaining, responding to the unpaired stimuli is inhibited, and responding becomes specific to the predictive stimulus. The animals discriminate between the contingent stimulus and the non-contingent stimuli. To test the role of LVGCCs in discrimination learning, mice were injected with vehicle or an LVGCC antagonist and place in a novel context. In this context, mice were presented with tones that always ended witii a footshock. Control mice initially displayed fear responses to both the tone and die context itself (in the intervals between the tones). However, with repeated ttaining, fear responding became selective to the tone presentation. In other words, since the tone had a highly contingent relationship with the shocks, but the context did not, responding was inhibited to the context alone. Mice treated with the LVGCC antagonist were unable to make this discrimination. These mice responded to the tone and context equally, showing again that LVGCC activity is necessary for inhibitory learning.

Example 4: LVGCC antagonists prevent inhibition of delay.

Inhibitory learning also plays a role in conditional responses involving a precise temporal relationship between the CS and the US. With extensive ttaining with invariant CS-US pairings, animals will begin to predict the occurrence of the US with the appropriate timing and adjust their behavior accordingly. For instance, if a brief 2s footshock always occurs at the end of a 3-minute tone presentation, animals will show little fear at the beginning of the tone presentation but considerable fear towards the end of the tone. This does not happen initially. During early ttaining trials animals will show fear responses throughout the tone presentation. However, with extensive ttaining an inhibitory memory is formed and responding is suppressed at the beginning of the tone CS. They will inhibit responding in the delay between the CS and US onsets. This phenomenon is known as inhibition of delay.

To examine the role of LVGCCs in inhibition of delay, mice were injected with an LVGCC antagonist or vehicle prior to 20 pairings of a 3-minute tone with coterminating 2s footshocks. During the latter ttaining trials, and the CS alone test session the next day, vehicle-tteated mice showed suppression of fear responding during the early portion of the CS presentations. Mice treated with the LVGCC antagonist showed fear during the entire CS presentation. This finding shows that LVGCC blockade prevented the formation of the inhibitory memory and further demonstrates that LVGCC activity is required for inhibitory learning.

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From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of enhancing inhibitory learning in a subject, comprising administering to the subject a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist.

2. The method of claim 1, wherein the inhibitory learning comprises extinction, latent inhibition, novel object recognition, negative contingency learning and/or inhibition of delay.

3. A method of ameliorating schizophrenic symptoms in a subject, comprising administering to the subject a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist.

4. A method of ameliorating symptoms of anxiety disorder in a subject, comprising administering to the subject a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist.

5. A metiiod of ameliorating symptoms of autism in a subject, comprising administering to the subject a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist.

6. A method of reducing addiction in a subject, comprising administering to the subject a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist.

7. A pharmaceutical composition comprising a therapeutically effective amount of an L-type voltage-gated calcium channels (LVGCC) agonist and a pharmaceutically acceptable carrier.