EP1774318A2 - Models and methods for nociception, pain transduction, and screening for analgesic compounds - Google Patents

Abstract

The alpha-2-delta-1 subunit of a voltage-gated calcium channel is preferentially over-expressed in a non-human transgenic animal model for neuropathic pain. Such transgenic animals advantageously exhibit non-injurious tactile allodynia and/or thermal hyperalgesia while retaining normal pain reaction to tissue injury and inflammatory pain. Thus, and in significant contrast to heretofore known animal models for neuropathic pain that require injury to precipitate the neuropathic pain, response behavior of the animal to a stimulus can be clearly attributed to the over-expression of the alpha-2-delta-1 subunit.

Description

MODELS AND METHODS FOR NOCICEPTION, PAIN TRANSDUCTION, AND SCREENING FOR ANALGESIC COMPOUNDS

This application claims the benefit of our U.S. provisional applications with the serial numbers 60/587,883 and 60/606,315, filed July 13, 2004 and August 31, 2004, respectively, both of which are incorporated by reference herein.

Government Support

This invention was made with government support under grants NS40135 and DE 14545 from the National Institutes of Health/National Institute of Neurological Disorders and Stroke and National Institute of Dental and Craniofacial Research, respectively. The government may have certain rights in the invention.

Background of the Invention

Classic animal models and various cell-based models to determine nociception, pain transduction, and pain threshold are well known in the art, and an overview over numerous methods is provided, for example, in "Pain Research: Methods and Protocols" (Methods in Molecular Medicine) by Z. David Luo (Humana Press 2004, ISBN: 1588291030).

Further known methods include those focusing on certain components (e.g., pain- specific receptors) that are expressed in cell lines in which a receptor-activation or other response can be observed. For example, alpha- 1 adrenergic receptors (G-protein coupled transmembrane receptors) are thought to mediate various actions of the sympathetic nervous system through binding of catecholamines, epinephrine, and/or norepinephrine, and selected cell systems and uses are described in U.S. Pat. No. 6,733,982, and U.S. Pat. App. No. 20040132115. Similarly, recombinant expression of an isoform of human VGSC β IA subunit (preferentially expressed in tissues associated with neurological function) is reported in U.S. Pat. App. No. 20040002439 in a cell-based model for screening of analgesic compounds.

Voltage-gated calcium channels (VGCCs) within sensory neurones are also believed to perform an important role in neuropathic pain. Among other things, it has been observed that induction of diabetic neuropathy is associated with significant changes in the expression of various VGCC mRNAs (typically an increase in alpha(2)delta(l), alpha(2)delta(2), and alpha(2)delta(3) mRNA levels; see e.g., Yusaf et al. Biochem Biophys Res Commun. 2001 Nov 30;289(2):402-6). On the other hand, downregulation of certain VGCC components was reported by Kim et al (Brain Res MoI Brain Res. 2001 Nov 30;96(l-2):151-6). For example, expression of the VGCC alpha(l) gene in the dorsal root ganglion (DRG) following chronic constriction injury (CCI) and axotomy of the rat sciatic nerve resulted in decreased alpha(lC), alpha(lD), alpha(lH), and alpha(ll) mRNA expression at 7 days in the ipsilateral DRG, to approximately 34-50% of the contralateral side, hi still further published studies, novel splice isoforms of the Ca V2.2. alpha subunit of N-type calcium channels (preferentially expressed in a subset of nociceptive neurons) containing exon 37a were implicated in neuropathic pain as described in U.S. Pat. App. No. 20040214238.

Neuropathic pain diagnostics has therefore looked at a variety of target molecules and an exemplary molecular diagnostic test is described in U.S. Pat. App. No. 20030216341, in which expression of various sequence tags (representing genes differentially expressed under neuropathic pain in the models of Bennett, Seltzer and Kim & Chung (Bennett & Xie 1988 Pain 33:87-107, Seltzer et al. 1990 Pain 43:205-218; Kim & Chung 1992 Pain 50:355-363)) is quantitatively monitored. Similarly, Brooksbank et al. describe in U.S. Pat. App. No.

20030138803 a system in which sequences associated with streptozocin-induced diabetes and implicated with pain are analyzed and used for various models.

While the above and other models provide at least some insight into molecular and systemic mechanisms of pain perception and transduction, all or almost all of the known models suffer from one or more disadvantages. Among other things, many cell-based systems often fail to accurately predict the biological effects of analgesics and/or fail to provide information about pharmacokinetic and pharmacodynamic of tested compounds. On the other hand, systemic animal models tend to have a relatively narrow dynamic range and therefore often provide less than desirable accuracy in data. In addition, classic animal models are generated using surgery, irritants, and/or stimulations, which often introduce pathological changes and complications such as trauma, tissue injury, inflammation, and motor dysfunction that are not directly related to pain transduction. These complications may affect or cover up the responses of animal models to nociceptive stimuli, and analgesic compound screening.

Consequently, while numerous models for analysis of neuropathic pain are known in art, all or almost all of them suffer from one or more disadvantages. Therefore, there is still a need to provide improved models and methods for nociception, pain transduction, and screening for analgesic compounds.

Summary of the Invention

The inventor has discovered that overexpression of voltage-gated calcium channel alpha-2-delta-l subunit in neural tissue correlates in vivo with pain responses in transgenic animals otherwise observed with nerve injury-induced neuropathic pain. Significantly, such transgenic animals can be employed in animal models for nerve injury-induced neuropathic pain without inflicting nerve injury, which often interferes with test results. Similarly, the inventor discovered that neural cells obtained from transgenic animals or cell transformed to overexpress the alpha-2-delta-l subunit exhibit physiological parameters remarkably similar to those of neural tissue obtained from animals thought to have nerve injury-induced neuropathic pain.

Therefore, in one aspect of the inventive subject matter, the inventor contemplates a model for peripheral nerve injury-induced neuropathic pain in which a transgenic non-human animal preferentially expresses the alpha-2-delta-l subunit of a voltage gated calcium channel in a neuronal tissue in an amount sufficient to produce non-injurious tactile allodynia and/or thermal hyperalgesia, preferably while retaining normal pain reaction to inflammatory pain (as compared to a control animal). Typically, the alpha-2-delta-l subunit is expressed in the hippocampus, the cortex, the spinal cord, and the dorsal root ganglion, and is substantially not expressed in the heart, skeletal muscles, the lung, kidneys, the spleen, and/or the intestines. The expression may be facilitated using various manners, however, it is generally preferred that expression is driven from the Thy- 1.2 gene promoter. Among other suitable choices, preferred transgenic animals include mammals, and most preferably mice.

Consequently, a method of testing a pharmaceutically active compound for use in treatment of peripheral nerve injury-induced neuropathic pain will preferably include a step in which the compounds is administered in vivo to the transgenic animal (or recombinant cell, which may or may not be isolated from the transgenic animal). In some contemplated aspects of the inventive subject matter, administration is parenteral administration of the compound at a dosage effective to reduce the non-injurious tactile allodynia and/or thermal hyperalgesia, and most typically intrathecal inj ection. Suitable compounds for reduction of neuropathic pain will include those that reduce expression and/or activity of the alpha-2-delta-l subunit or a component that is functionally associated with the alpha-2-delta-l subunit. Among other advantages, it should be recognized that contemplated animal models will allow the investigator to determine metabolic effects of a drug in vivo (e.g., the animal metabolizes the compound to thereby generate an active metabolite that is effective to reduce the allodynia and/or hyperalgesia). Most typically, and especially where mice are the transgenic animals, contemplated methods will include a step of measuring paw withdrawal threshold and a measurement of thermal escape latency.

Various objects, features, aspects and advantages of the present invention will become more apparent from the accompanying drawing and the following detailed description of preferred embodiments of the invention.

Brief Description of the Drawings

Figures IA and IB depict a Western blot and a graph illustrating Ca_Va₂O₁ expression in neuronal tissues from transgenic mice according to the inventive subject matter.

Figures 2A-2F are graphs depicting specific pain behaviors under defined conditions in transgenic and wild type mice.

Figure 3 is a set of micrographs depicting Ca_V(X₂O₁ immunohistostaining in spinal dorsal horn (DH) sections taken from lumbar spinal cord of adult transgenic mice (TG) and wild-type (WT) littermates.

Figures 4A and 4B are graphs depicting gabapentin dose-dependent reversal of tactile allodynia in TG mice without significant alteration of paw sensitivity to mechanical stimulation in WT mice.

Figures 5A-5C are graphs depicting results for various parameters of whole-cell voltage-clamp experiments in acutely isolated DRG neurons from TG and WT mice.

Figures 6A and 6B are graphs depicting significant increased activation rates of voltage gated calcium channels in cells with Ca_Va₂O₁ over-expression.

Figures 7A and 7B are graphs depicting voltage-dependent changes in deactivation rates of voltage gated calcium channels. Figures 8A and 8B are graphs depicting gabapentin dose-dependent attenuation of high threshold voltage-gated barium currents in TG sensory neurons.

Detailed Description

The inventor has discovered that expression of voltage-gated calcium channel alpha- 2-delta-l subunits is implicated in various aspects of neuropathic pain. Notably, the inventors have now discovered that increased expression of the voltage-gated calcium channel alphas- delta- 1 subunit, and especially increased expression in spinal cord and dorsal root ganglia in transgenic mice correlates in vivo with typical nerve injury-induced nociceptive responses to innocuous mechanical and thermal stimulation (tactile allodynia and thermal hyperalgesia). Remarkably, pain perception due to tissue injury and/or inflammation was not affected to a measurable degree in such transgenic animals. Thus, contemplated animal models advantageously lend themselves to investigate the specific role of the voltage-gated calcium channel alpha-2-delta-l subunits in various aspects of treatment and research of neuropathic pain. Using such model, responses to certain stimuli attributable to prior injury of the animals (which was needed in previously known models to generate the neuropathic pain model) can now be excluded.

hi one particularly contemplated aspect of the inventive subject matter, the inventors have observed that in transgenic mice over-expressing the calcium channel alpha-2-delta-l subunit, the abnormal sensations that mimic the pain states after nerve injury indicate that the alpha-2-delta subunit is a molecular determinant of abnormal sensations. This observation was supported by the finding that abnormal sensations in such transgenic mice can be normalized in a dose-dependent manner by gabapentin, a drug binds to the alpha-2-delta subunit, but not by ketorolac, a non-steroidal anti-inflammatory drug.

Remarkably, the prepared transgenic mice overexpressing the calcium channel alpha- 2-delta-l subunit in neurons (see experimental section) have hypersensitivity to mechanical and thermal stimuli, mimicking pain states after injuries such as nerve injuries, neuropathies and inflammation involving neural tissue. Consequently, the presently contemplated injury- free transgenic mouse model provides an excellent tool for screening compounds related to the development and evaluation of pain medications (e.g., anti-hyperalgesic agents, general analgesic agents, etc.), and for mechanistic studies of pain transduction mechanisms. Based on the above findings, the inventors contemplate numerous systems and uses, and exemplary systems and uses are described below.

Animal Models

In one aspect of the inventive subject matter, the inventors contemplate that transgenic animals may be made in which the gene for the voltage-gated calcium channel alpha-2-delta-l subunit is constitutively overexpressed. However, in further contemplated aspects, inducible, repressible, and/or temporal overexpression is also deemed suitable. Consequently, suitable overexpression may be performed using viral transfection, chemical transfection, ballistic gene transfer, electroporation, cell fusion, and transgenic animals. There are numerous manners of temporarily or permanently overexpressing genes in an animal, and all of the known manners are deemed suitable for use herein. Similarly, it is contemplated that the amount of overexpressed alpha-2-delta-l subunit maybe either variable, or constant. Nucleic acid constructs for regulated expression are well known in the art and all of them are deemed suitable for use herein.

It is particularly preferred that in animals overexpressing the alpha-2-delta-l subunit gene, the expression is driven from a promoter and/or other regulatory element that is tissue specific to neuronal tissue, and/or that transgenic expression is inducible/controllable by exogenously added agents. Most preferably, the alpha-2-delta-l subunit gene is expressed in the hippocampus, cortex, spinal cord, and/or dorsal root ganglion. However, other locations are also deemed suitable and will predominantly depend on the choice of animal, recombinant DNA construct, and promoter. Still further, it is generally preferred that suitable alphas- delta- 1 subunit genes are expressed from a cDNA construct. However, in alternative aspects, expression constructs may also include one or more introns and/or other non-coding (preferably regulatory) elements. Once more, there are numerous arrangements and promoter sequences known in the art for transgenic expression, and all of the known sequences and elements are considered suitable for use herein.

Furthermore, it is generally preferred (but not necessarily so) that the alpha-2-delta-l subunit gene is homologously expressed in the animal (e.g., murine gene in transgenic mouse), and that the gene is the wildtype form for that particular animal. For example, where the animal is a mouse, particularly suitable sequence is identical with the sequence accessible at the Genbank accession number U73484. However, in alternative aspects of the inventive subject matter, heterologous expression may also be advantageous (e.g., for immunological distinction over wildtype, or for kinetic analyses of the human subunit expressed in a rodent). In still further contemplated aspects, it should be recognized that the overexpressed alpha-2- delta- 1 subunit gene may be modified to provide one or more advantages over the unmodified form. For example, modified forms may include affinity tags for labeling and/or isolation, or may have deletions or alterations to remove or alter a particular biochemical parameter (e.g., degree of glycosylation, degree of interaction with components up- or downstream in signal transduction, drug and/or ligand binding, or association with other subunits). In other examples, modifications may also include base substitutions to modify the primary sequence of the overexpressed protein to obtain desired physical properties. Therefore, all at least partially functional, and even non- functional homologues of the alpha-2-delta-l subunit gene are specifically contemplated herein.

In particularly preferred models, suitable animals include those in which the voltage- gated calcium channel alpha-2-delta-l subunit is naturally expressed as a component in nociception and/or pain transduction. Therefore, contemplated animals include numerous non-human mammals, and preferably rodents for which suitable test protocols are already well established. Alternatively, it is also contemplated that suitable animals are chosen from a group that does not naturally express the voltage-gated calcium channel alpha-2-delta-l subunit as a component in nociception and/or pain transduction. Among other phyla, contemplated animals may be selected from invertebrates (e.g., cnidaria or nematoda, where relatively simple neuronal systems are desired) or from arthropods (e.g., where large progeny in relatively short period are desired). Such animals may provide particularly desirable advantages as a particular signal transduction chain that may be engineered into such animals. For example, activation of the voltage gated calcium channel may be coupled with beta 2 (β2) adrenergic receptors (Davare et al, 2001), which may be detected by numerous methods well known in the art. Additionally, or alternatively, expression of the alpha-2-delta-l subunit may also be performed in yeast. For example, where it is desirable to investigate the role of the alpha-2-delta-l subunit in signal transduction, or cooperation with other components, expression systems may use Pichia, Saccharomyces, or other suitable species. Such animal models may be useful in numerous experimental settings, and especially preferred uses include those in which the animal is a transgenic animal for in vivo study of nociception, and especially of neuropathic pain. However, it should be recognized that such animals may also be useful for numerous other abnormal and/or pathologic conditions in which the alpha-2-delta-l subunit is overexpressed relative to a normal and/or non-pathologic condition. For example, contemplated models may be useful for investigation of various aspects related to ischemic stroke, epilepsy, neoplastic diseases, etc. (see below).

Consequently, and in one preferred aspect of the inventive subject matter, the inventor contemplates a model for peripheral nerve injury-induced neuropathic pain that comprises a transgenic non-human animal that preferentially expresses (i.e., at least two times, more typically at least three times, and most typically at least five times more than in non-neuronal tissue) an alpha-2-delta-l subunit of a voltage gated calcium channel in a neuronal tissue and in an amount sufficient to produce at least one of non-injurious tactile allodynia and non- injurious thermal hyperalgesia while the animal retains normal pain reaction to tissue injury and inflammatory pain. Most preferably, the expression is driven by a tissue specific promoter or other construct such that the preferential expression is observed in the hippocampus, the cortex, the spinal cord, and/or the dorsal root ganglion. Viewed from another perspective, preferential expression will provide substantially no recombinant expression (i.e., less than 10% of neuronal tissue) in the heart, skeletal muscles, the lung, kidneys, the spleen, and/or the intestine. While various nucleic acid constructs may be suitable, it is generally preferred that the alpha-2-delta-l subunit is expressed under the control of a Thy- 1.2 gene promoter as exemplified below.

Thus, it should be recognize that contemplated animal models may be provided for research and development, as well as a commercial product together with an information to use the animal for testing of a compound for use in treatment of pain, and especially peripheral nerve injury-induced neuropathic pain. Most typically, the compound to be tested is administered in vivo. Such models will therefore allow not only to study the direct interaction of certain pharmaceutical compounds in their role as modifiers of the alphas- delta- 1 subunit mediated pain, but also allow for pharmacokinetic and pharmacodynamic studies in which the role of the animal's metabolism is observed. Consequently, suitable models also allow identification of active metabolites, studies of prodrug activation, passage of the drug to the target cells, and clearance of the drug and/or metabolite from the animal's system. For example, administration of the compound maybe parenterally (intravenously, intraperitoneally, intrathecally, etc.), orally, or any reasonable combination thereof. Suitable compounds will typically include those that bind (reversibly or irreversibly) to the alpha-2- delta- 1 subunit, those that reduce activity of the alpha-2-delta-l subunit or a component functionally associated with the alpha-2-delta-l subunit, and/or those that interfere with expression of the alpha-2-delta-l subunit (see below).

Cell-Based Models

In further contemplated models, it should be appreciated that where identification of binding agents that interfere with the biological function of the alpha-2-delta-l subunit is desired, recombinant eukaryotic cells or microorganisms may be employed. For example, where various lead compounds have been identified based on their activity in contemplated animal models, refinement of the compound (e.g., to increase affinity to the subunit, to reduce expression of the subunit) may be performed in a cell-based format. Among other appropriate cells, particularly suitable cells include eukaryotic cells (e.g., SF9 cells, Vero cells, HeLa cells, etc.) while exemplary microorganisms include various yeasts (e.g., Pichia spec). In such cell-based systems, calcium flux may be directly observed by change in fluorescence (e.g., using fluorescent calcium indicator Fura-2) or other physico-chemical parameter. Among other advantages of cell-based expression, the quantity of recombinant subunit may be significantly increased. Furthermore, heterologous expression of the alpha-2-delta-l subunit in numerous non-neuronal cells may be especially advantageous as no intrinsic background expression is expected.

Consequently, the inventors also contemplate a method of testing a pharmaceutical compound in which in one step a pharmaceutical compound is administered to a tissue and/or a cell of a transgenic animal in vitro. Alternatively, the compound may also be administered to a recombinant cell over-expressing the alpha-2-delta-l subunit. Where the cell or tissue is isolated from the transgenic animal, suitable tissues especially include hippocampus, cortex, spinal cord, and dorsal root ganglion or cells therefrom. As already discussed, the transgenic animal or recombinant cell may or may not naturally express the alpha-2-delta-l subunit. Exemplary Uses of Contemplated Models

In still other contemplated aspects, it should be appreciated that natural expression of the alpha-2-delta-l subunit may be modulated (and most typically down-regulated or even abrogated) in animals, and particularly human suffering from neuropathic pain. For example, using the above animal and cell-based models, it is contemplated that therapeutically active small molecules can be identified that interfere with the biological function of the alphas- delta- 1 subunit. Alternatively, antibodies (or fragments thereof) maybe at least temporarily used to reduce biological activity of alpha-2-delta-l subunit in a patient. Alternatively, or additionally, and especially where the alpha-2-delta-l subunit is overexpressed in a patient, it is contemplated that anti-sense RNA and/or siRNA may be employed to reduce or even completely abolish functional expression of the alpha-2-delta-l subunit gene in corresponding cells. There are numerous procedures for in vivo use of anti-sense RNA and/or siRNA known in the art, and all of such uses are deemed suitable for use herein.

Furthermore, it should also be recognized that analysis of the expression level of alpha-2-delta-l subunit may be employed in an analytic and/or diagnostic manner in which the subunit is detected in vivo or in vitro using methods well known in the art. For example, peptide expression may be monitored using immunoscintygraphy (e.g., using technetium- labeled antibodies) in vivo. In another example, peptide expression may be quantified ex vivo from biopsy specimen using western blot or other immunographic methods. Similarly, where nucleic acid detection is desired, all known hybridization and/or amplification methods are deemed appropriate. Contemplated diagnostic applications also include a determination of the sequence (nucleic acid and/or peptide) of the alpha-2-delta-l subunit to identify and/or characterize mutations that may be present.

In still further contemplated uses, it should be appreciated that components in pain transduction may be identified that functionally cooperate with the alpha-2-delta-l subunit. Such components may then be employed as molecular targets for further therapeutic use, and most preferably for reduction in pain perception of an individual.

Based on the inventors discovery of the implication of the alpha-2-delta-l subunit in pain, and particularly neuropathic pain, it should be appreciated that numerous compounds and compositions may be prepared that have therapeutic use in treatment and/or prevention of neuropathic pain. For example, using the models described above, it is contemplated that inhibitors of the alpha-2-delta-l subunit maybe identified that act as modulating agents, reduce expression of the alpha-2-delta-l subunit or functionally associated component, and/or reduce pain perception of an individual.

Experiments

Animal subjects were used in the project under protocols in conformance with approved IACUC protocol #2003-2470.

Transgenic Mice

Transgenic mice (TG) over-expressing the voltage-gated calcium channel alpha-2- delta-1 subunit (Ca_vα₂δi) gene were generated substantially following a protocol as described previously ¹³ . A transgene vector was used containing the mouse brain Ca_Va₂O₁ cDNA (Genbank accession number U73484) that was cloned into the vector down-stream of a 6.5 kb murine thy- 1.2 gene extending from the promoter region to the intron after exon 4 without exon 3 and its flanking introns. Deletion of exon 3 and its flanking introns has been shown to abolish expression in non-neuronal cells ¹⁴. Only adult male transgenic mice and wild-type littermates (WT) with the same genetic background (129SV/C57BL6) and age were used for the experiments. All the mice appeared normal with respect to grooming, social interactions and feeding, and showed no signs of abnormality or any obvious motor defects, tremor, seizure, or ataxia. Thus, it was impossible to distinguish the transgenic mice from the wild- type mice visually. All animal care and experiments were performed according to protocols approved by the Institutional Animal Care Committee of the University of California, Irvine.

Western Blot

Frozen tissues were pulverized and extracted with lysis buffer (50 mM Tris-HCl buffer, pH 7.5, containing 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA) containing protease inhibitors. Equal amounts of total protein extracts in gel loading buffer from each sample were applied to electrophoresis in NuPAGE Tris-acetate gels under reducing conditions (0.05 M dithiothreitol), then electrophoretically transferred to the nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The membranes were incubated with monoclonal antibodies against the Ca_Va₂O₁ subunit in phosphate-buffered saline containing 0.1% Tween 20 for 1 hr at room temperature or overnight at 4⁰C after blocking nonspecific binding with 5% non-fat milk for 1 hr at room temperature. The antigen-antibody complexes were detected by incubating the membranes with horseradish peroxidase (HRP) labeled secondary antibody for 1 h at room temperature followed by washing and addition of HRP substrate. Under reducing conditions, the 6-peptide separates from the α₂ subunit so the positive bands detected by the primary antibody reflect the α₂ subunit only. For internal control of equal loading, the blots were stripped and re-blotted with primary antibodies against house-keeping protein glyceraldehyde-3 -phosphate dehydrogenase (GAPDH, Ambion, Austin, TX) that was not changed due to Ca_Va₂S₁ over-expression and other manipulations on the mice.

Behavioral Assays

Behavioral response to mechanical stimulation was tested as described previously with minor modifications: Mice were placed in a clear plastic cage with a wire mesh bottom and allowed to acclimatize for 1 hr. The 50% paw withdrawal thresholds (PWT) to calibrated von Frey filament (Stoelting, Wood Dale, IL) stimulation were determined using a modified up-down method of Dixon ³⁷. A series of filaments with buckling weight between 0.04 and 2.0 gm were applied with a bending force to plantar surface of the hindpaw, starting with a filament with a 0.4 gm buckling weight. Paw lifting within 5 s was considered a positive response and led to the use of the next weaker filament. Absence of a paw lifting after 5 s led to the use of the next filament with increasing weight. The 50% paw withdrawal threshold was calculated from the resulting scores of six measurements starting from the one prior to the first positive response as described previously \ or until four consecutive positive (assigned a score of 0.01 gm), or three consecutive negative (assigned a score of 3.0 gm) responses had occurred. Unless indicated, averaged paw withdrawal threshold from both sides of hindpaws was used for comparing sensitivities between WT and transgenic mice, or before and after systemic and intrathecal drug treatments.

The escape latencies of hindpaw withdrawal from a thermal stimulation in mice were measured in a modified Hargreaves-type thermal testing device ³⁸. Briefly, mice were placed into individual enclosures on the glass surface of the hot box maintained at 30 °C and allowed to acclimatize for at least 30 min. After acclimation, the escape latencies to a thermal stimulation were measured in both hindpaws of the WT and TG mice. The source of radiant thermal stimulus underneath the glass surface was aligned to the planter surface of the hindpaw. Activation of the light source activated a timer, and paw withdrawal from the light source or 20 s of light stimulation turned off the light bulb and timer. Unless indicated, averaged escape latency (in seconds) from both sides of hindpaws was used for comparing sensitivities between WT and TG mice, or before and after systemic and intrathecal drug treatments.

For measuring behavioral responses to the formalin test ³⁹, mice were placed into individual transparent cylinders for acclimation at least 1 hr before the formalin injection. To induce nociceptive behaviors, 20 μl of 2% formalin was injected subcutaneously into the plantar surface of right hindpaw of WT and TG mice. This induced two distinct phases of spontaneous behaviors: flinching and lifting/licking. The flinching responses were recorded automatically in an automated nociception analyzer (University of California, San Diego) and lifting/licking was recorded by a digital camcorder and counted manually. After formalin injection (time 0), the number of flinching/lifting and licking was recorded from 0 to 60 min. For analyzing the nociceptive behaviors in each phase, the numbers of flinching/min or lifting/licking per a 5 min interval were compared between groups.

For measurement of carrageenan-induced inflammation and mechanical hyperalgesia, mice were placed into a clear plastic cage with a wire mesh bottom for acclimation at least 1 hr before carrageenan injections. Behavioral base lines were determined immediately before carrageenan injections. To induce inflammation, 20 μl of 1% (w/v) carrageenan was injected intraplantarly into the right hindpaw of WT and TG mice. The paw withdrawal thresholds to mechanical stimulation (von Frey filaments) were measured at designated time points post injection. The degree of inflammation induced by carrageenan was determined by measuring the thickness of right hindpaws before and at designated time points post injection with a caliper.

Drug Administration

Working solutions of gabapentin, ketorolac and morphine were prepared in sterile saline before intrathecal injection between lumbar regions 5 and 6 (in 5 μl) or intraperitoneal administration, respectively. Behavioral testing started 1 hr post drug treatment unless indicated. Tissue Preparation and Sectioning

Tissue preparations and sectioning from TG and WT mice were performed simultaneously to minimize experimental variations. Mice were deeply anesthetized with 3% — 4% isofluorane and the L5/L6 level spinal cord segments were dissected and fixed in 4% paraformaldehyde. Paraffin-embedded spinal cord samples were sectioned (5 μM) using a microtome and mounted onto Superplus precleaned slides (Fisher Scientific, Pittsburg, PA) and kept at room temperature.

Immunohistostaining

Immunohistological staining was used to localize Ca_Va₂O₁ expression in paraffin sections of spinal cord from the WT and TG mice. Briefly, tissue sections were treated with a citrate buffer (pH 6.0) followed by 3% H₂O₂ in PBS, washed and blocked with 1% bovine serum albumin and 10% normal goat serum, then incubated with the monoclonal antibodies against the Ca_Va₂O₁ subunit (Sigma, Saint Louis, MO) overnight at 4 °C. After thorough rinsing, sections were incubated with biotinylated secondary antibody conjugated to horseradish peroxidase and then with avidin-biotin complex solution (Vectastain Elite ABC kit, Vector Laboratories). After several rinses, sections were developed in diaminobenzidine- H₂O₂ solution, washed, and mounted on slides, air-dried, dehydrated and coverslipped with Permount. The positive staining in sections was examined and images were taken under a microscope.

Electrophysiological Recordings

Sensory neurons were obtained from the lumbar dorsal root ganglia of adult mice. Ganglia were enzymatically treated and mechanically dispersed as described ²¹, except that the ganglia were bubbled in carbogen (5% CO₂, 95% O₂) during the 20 minute collagenase treatment. DRG neurons were plated onto laminin/ornithine-coated glass coverslips and incubated for 2 hrs in MEM containing 10% fetal bovine serum at 37°C, 90% humidity, and 3% CO₂. Neurons were then transferred to an L- 15 based medium containing 10 % fetal bovine serum, and stored at room temperature before recording. AU recording was performed within 8 hours of harvesting ganglia. Voltage-clamp recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) in the whole-cell patch configuration as described. Data were filtered with a 4-pole Bessel filter and digitized. Series resistance <12 MD was compensated (>80%) by using amplifier circuitry. Only data obtained from neurons in which uncompensated series resistance resulted in voltage-clamp errors of less than 5 mV were used. A P/4 protocol was used for leak subtraction.

Ba²⁺ was used as the charge carrier. The bath solution contained 130 mM choline chloride, 5 mM BaCl₂, 0.6 mM MgCl₂, 10 mM Hepes, and 10 mM glucose (pH was adjusted to 7.4 with Tris base and osmolality was adjusted with sucrose to 325 milliosmolar). The electrode solution contained 110 mM Cs-Methansulfonate, 30 mM TEA-Cl, 1 mM CaCl₂, 5 mM MgCl₂, 11 mM EGTA, 10 mM Hepes, 2 mM Mg-ATP, and 1 mM Li-GTP (pH was adjusted to 7.2 with Tris base and osmolality was adjusted with sucrose to 310 milliosmolar). Patch pipettes filled with electrode solution had resistances of 1.5-3 MΩ .

Ba²⁺ currents were evoked from a holding potential of -70 mV. Conductance- voltage curves were constructed for each neuron from I-V curves generated by assessing peak current evoked with 40-ms voltage steps between -80 and +60 mV taken at every 5 mV. Reversal potential was determined by interpolating between inward and outward currents. Conductance was determined by dividing peak current by driving force. In order to estimate maximal conductance, conductance voltage-curves were fitted with a modified Boltzmann equation of the form: Conductance (g) = gMax/(l+exp(-( Vm- V ^₂)Zk)); where gMax is maximal conductance, Vm is membrane potential, Vy₂ is the potential at which conductance is half of maximal and k is a slope factor. Dose-response data were fitted with a modified Hill equation of the form: Percent inhibition = maximal inhibition / (drug^ + EC₅₀^); where drug is the concentration of gabapentin, EC₅₀ is the concentration of gabapentin inhibiting 50% of the maximal inhibition and N is a Hill coefficient.

Statistical Analyses

Data were reported as means ± S.E.M. Unpaired Student's t tests were performed where significance was indicated by two-tailed p values: *p < 0.05. Results

Neuronal Overexpression

Transgenic mice overexpressing the mouse Ca_VO₂O₁ cDNA were generated under the control of a mutant thy-1 promoter. Thy-1 is a member of the immunoglobulin superfamily that is expressed in both neuronal and non-neuronal tissues, including thymocytes ¹⁵. The mutant used in the present study involved the deletion of a particular intron in the thy-1 gene, which produces the neuronal specificity of this promoter to drive down-stream target gene expression ¹³'¹⁴. Data from Western blot analysis indicated that Ca_Va₂O₁ protein levels were elevated in forebrain, cortex, hippocampus, cerebellum, spinal cord, and DRG of the TG mice compared with their WT littermates.

Figures IA and IB depict enhanced Ca_Va₂O₁ expression in neuronal tissues from the transgenic mice. Figure IA illustrates representative Western blot data from three independent determinations showing selective increases of Ca_vα₂δi subunit expression in neuronal tissue samples of adult transgenic mice. 1 - wild-type mice, 2 - transgenic mice. Figure IB illustrates summarized Western blot data presented as the mean ± SEM from three independent determinations. WT — wild-type mice, TG — transgenic mice.

Similar increases in neuronal Ca_vα₂δ_! expression were observed in different lines but not in non-neuronal tissues, although relative Ca_vα₂δ_! expression levels varied among different transgenic lines and tissues (data not shown). Endogenous Ca_Va₂O₁ proteins from non-DRG tissues of the WT mice differed from that in the DRG as indicated by the faster migration of the former in Western blot, consistent with findings in rat tissues ¹⁶. Interestingly, heterogeneous Ca₇O₂O₁ expression was observed in some tissues, and most prominently in DRG from the TG mice where two bands with similar densities were observed. These Ca_Va₂O₁ species were likely derived from post-translational modifications since the transgene did not contain introns for alternative splicing, hi transgenic spinal cord samples, the increases in Ca_VO₂O₁ proteins were mainly in the faster migrating band with a similar migration rate to that of endogenous spinal Ca₇O₂O₁ subunit. However, a minor band with a similar migration rate as the endogenous DRG Ca_vα₂δ[ proteins was also observed.

Since this slowly migrating band can be eliminated by dorsal rhizotomy , it is likely representative of Ca_vα₂δ_! proteins at the pre-synaptic central terminals of DRG neurons. Expression of other VGCC subunits, such as the channel forming Ca_vαi_b and intracellular Ca_v/3₃ subunits, were not changed in the TG mice.

Elevated Ca^aJ) 7 Expression In TG Mice Results In Abnormal Nociceptive Responses

Hindpaw withdrawal thresholds to mechanical stimulation were significantly reduced (tactile allodynia). Importantly, this reduction in mechanical threshold was similar to that seen in spinal nerve injured WT mice as can be seen from Figure 2A. Paw withdrawal latencies to noxious thermal stimulation were also significantly reduced (thermal hyperalgesia) in TG mice compared with WT littermates as can be seen from Figure 2B. The tactile allodynia state lasted for six months, the longest time tested (see Figure 2C).

Here, specific pain behaviors are induced by elevated Ca_Va₂O₁ subunit in the transgenic mice that can be blocked by specific drug treatment. Figure 2 A shows that Ca_Va₂O₁ overexpression in the TG mice induced tactile allodynia (shown as reduced paw withdrawal thresholds to mechanical stimulation). The tactile allodynia state in the transgenic mice was similar to that observed in mice with one- week L5 spinal nerve ligation injury, and reversed by systemic gabapentin, but not ketorolac, treatment. N = 8 each for the TG and WT mice, and n = 10 for the nerve ligated mice. Ca_Va₂O₁ overexpression in the TG mice also induced thermal hyperalgesia shown in Figure 2B as reduced paw withdrawal latency to thermal stimulation that could be blocked by systemic gabapentin, but not ketorolac, treatment. Gabap.-gabapentin (50 mg/kg, i.p.). Ket.-ketorolac (7.5 mg/kg, i.p.). N = 16 each for the TG and WT mice. The tactile allodynia state in the transgenic mice lasted at least six months as illustrated in Figure 2C, the longest duration tested. Evidence against the possibility that altered nociceptive processing in the TG mice reflected disruption of a host gene by random transgene incorporation comes from the observation that a similar behavioral phenotype was observed in another transgenic line with similarly enhanced Ca_vα₂δi expression (data not shown).

While enhanced Ca_vα₂δ_! expression resulted in dramatic changes in the response to acute stimuli, there were no detectable changes in response to inflammatory stimuli: the behavioral responses to intradermal formalin and carrageenan and the inflammatory response to carrageenan injection were not significantly altered in the TG mice compared with their WT littermates as shown in Figures 2D-2F, respectively. Interestingly, the carrageenan-, but not formalin-, induced hyperalgesia state is additive to that induced by elevated Ca_Va₂S₁ expression in the transgenic mice. In Figure 2D, the TG and WT mice showed similar flinching as well as lifting/licking (data not shown) responses in the formalin test. The TG mice and WT littermates showed similar onset and duration of carrageenan-induced tactile allodynia (Figure 2E) and inflammatory response (Figure 2F). All data are presented as the mean ± SEM. *p < 0.05 compared with WT or contralateral side of spinal nerve ligated mice.

Spinal Ca_vaJ>i Subunit Contributes To Tlie Abnormal Sensations

To evaluate the specific contribution of elevated spinal cord Ca_Va₂O₁ to abnormal sensations in the TG mice, the localization of elevated Ca_Va₂O₁ proteins was assessed in the spinal cord along with the influence of intrathecal gabapentin, an antihyperalgesic drug that binds to the Ca_vθ₂δi subunit ¹⁷'¹⁸ on tactile allodynia in the TG mice. Compared with the WT samples, Ca_Va₂O₁ immunoreactivity in transgenic spinal cords was high in superficial and deep dorsal horn neurons, axons and synapse-like structures as can be seen in Figure 3, in addition to ventral horn neurons and associated structures (data not shown). Figure 3 depicts images Of Ca_Va₂O₁ immunohistostaining in spinal dorsal horn (DH) sections taken from lumbar spinal cord of adult transgenic mice (TG) and wild-type (WT) littermates. Scale bar = 20 μM. Double arrows: neurons; Single arrow: axons; Arrow head: synapse-like structures.

Intrathecal gabapentin dose-dependently reversed the tactile allodynia in the TG mice without significantly altering the paw sensitivity to mechanical stimulation in the WT mice as shown in Figure 4A. Here, intrathecal gabapentin dose-dependently reversed the tactile allodynia state shown as reduced paw withdrawal thresholds to von Frey filament stimulation in the transgenic mice (TG) without affecting the paw withdrawal thresholds significantly in the wild-type littermates (WT). Furthermore, as illustrated in Figure 4B, systemically administered gabapentin (Gab) had better efficacy than morphine (Mor) in allodynia reversal in the transgenic mice (TG) while both drugs did not affect significantly the response thresholds to mechanical stimulation in the wild-type littermates (WT). All data are presented as the mean ± SEM. * p < 0.05 compared with WT littermates.

These data indicate that the gabapentin-sensitive allodynic state in the TG mice is mediated by a mechanism, similar to that observed in spinal nerve-injured animals ^lj8'¹⁹. Thus, elevated Ca_vQ!₂δi subunit expression at the levels of spinal cord and associated dorsal root ganglia is highly likely a critical contributor to the abnormal sensations in the TG mice.

Pharmacological Profiles Of The Abnormal Sensations In The Transgenic Mice Mimic That

Of Neuropathic Pain

To test the sensitivity of abnormal sensations in the TG animals to different classes of compounds known to have anti-hyperalgesic effects on human patients and experimental animals, the inventor compared the efficacies of gabapentin, morphine, and ketorolac on the allodynic and thermal hyperalgesic states of the TG mice. Intraperitoneal administration of gabapentin (50 mg/kg) reversed the tactile allodynia (see e.g., Figure 2A) and thermal hyperalgesia (see e.g., Figure 2B) in the TG mice. The effects of gabapentin were dose- dependent with an estimated EC50 value -13 mg/kg (see Figure 4B). Intraperitoneal ketorolac (cyclooxygenase inhibitor used to treat inflammation and related pain conditions), at a dose that has been shown to exhibit analgesic efficacy (7.5 mg/kg) ²⁰, failed to reverse the tactile allodynia and thermal hyperalgesia states in the TG mice (see e.g., Figures 2A and B). Intraperitoneal morphine could only partially reverse tactile allodynia at the dose of 1 mg/kg, and failed to further reduce allodynia at a higher dose (5 mg/kg) (Figure 4B). Interestingly, the tested drugs did not affect significantly the baseline tactile responses in the WT animals. Thus, the sensitivity of TG mice to tactile stimulation has a pharmacological profile similar to that observed following nerve injury with the efficacies of gabapentin > morphine > cyclooxygenase inhibitors.

Increased Ca_vci₂§i Expression Resulted In An Increased Current Density And Altered Gating Properties Of High Threshold VGCC In DRG Neurons

To assess the impact of increased Ca_Va₂O₁ on current density and other biophysical properties of VGCC, we employed whole-cell voltage-clamp techniques to study voltage- gated barium currents (Iβ_a) in acutely isolated DRG neurons from the TG and WT mice. Electrode and bath solutions were formulated so as to eliminate voltage-gated sodium, potassium currents as well as calcium activated potassium and chloride currents. Barium (5 mM) was used as a charge carrier in place of calcium in order to limit run-down associated with the use of calcium as a charge carrier ²¹. Iβ_a was evoked from a holding potential of -70 mV following a 100 ms pre-pulse to -100 mV as shown in Figure 5 A. Here, whole cell current evoked from a 15.6 pF neuron obtained from a TG mouse. Five mV depolarizing voltage steps (40 ms duration) following a 100 ms pre-pulse to -100 mV and ranging between -80 mV and +60 mV were used to evoke barium currents. Only traces to -10 mV (peak inward current) are shown for clarity. Activation and deactivation rates were determined with a 40 ms voltage step while that of inactivation was determined with a 400 ms voltage step (data not shown). The current-voltage relationship was used to assess peak inward current for each cell, which was evoked between -20 and 0 mV. To validate conductance measurements based on estimates of current reversal potential (i.e., conductance (g) = I/(Vt_est-V_rev)₅ where I is membrane current, V_test, is the potential at which current was evoked and V_rev is the current reversal potential), derived conductance- voltage relationship was compared to that based on tail currents (i.e., an instantaneous G-V); both methods yielded similar results (data not shown).

Given that there is a rough correlation between DRG cell body size and afferent function with nociceptive afferents generally arising from small DRG neurons, the impact of enhanced Ca_Va₂O₁ expression was assessed on barium current in small diameter neurons (i.e., < 27 μm). Both the peak inward current density (Figure 5B, left) and maximal conductance (Figure 5B, right) were significantly increased in small DRG neurons from TG mice compared to that in small DRG neurons from WT mice. This increase in conductance was associated with a 5 mV hyperpolarizing shift in the potential for half activation (V₀.₅ = -20 ± 1 mV and -25 ± 1 mV for WT and TG neurons, respectively. See Figure 5C; p < 0.01. All data are presented as the mean ± SEM). This shift in Vo.₅ was largely due to an increase in the voltage-dependence of activation as it was associated with a decrease in the slope of the conductance-voltage curve (inset Figure 5C) with little change in thresholds.

The increased Ca_Va₂O₁ subunit also caused a significant increase in the activation rate as can be seen from Figure 6A (assessed with an exponential fit of the increasing phase of voltage-gated Iβ_a following membrane depolarization), compared with that in Iβ_a from WT neurons, at voltages > -20 mV. Here, current activation was fitted with a single exponential equation in order to determine the activation time constant. Both traces were normalized to the peak inward current obtained during a 40 ms test pulse to -10 mV. Barium currents in TG neurons activated more rapidly than those in WT neurons. Interestingly, fea activation rate became voltage dependent in TG neurons in contrast to the minimal voltage-dependence observed in fea from WT neurons as shown in Figure 6B. Here, the activation time constant for currents evoked at potentials between -25 mV and +10 mV were determined for each neuron in each group and pooled. Pooled data illustrate that there is a voltage-dependence to the activation rate of currents from TG neurons (n = 11), while there is little voltage- dependence to the current evoked from WT neurons (n = 12). All data are presented as the mean ± SEM.

While there is no dramatic apparent difference between WT and TG mice in deactivation rates of high threshold Iβ_a (assessed with an exponential fit of the decay phase of the tail current; se Figure 7A), the voltage-dependent changes in these rates did differ between the two. In Figure 7A, tail currents, evoked following membrane repolarization to - 70 mV, following 40 ms depolarizing voltage-steps to potentials ranging between -25 and +40 mV, were fitted with a single exponential equation in order to determine the deactivation time constant. The first 35 ms of the current trace has been omitted for clarity.

The deactivation rate was slowed by increased membrane depolarization in Iβ_a evoked in TG neurons, but increased with membrane depolarization in Iβ_a evoked from WT neurons as can be seen from Figure 7B. Iβ_a inactivation was similar in DRG neurons from TG and WT mice (data not shown). Here, time constant data pooled from TG (n = 11) and WT (n = 12) neurons indicated that voltage-dependent changes in deactivation time constants varied between TG and WT neurons. AU data are presented as the mean ± SEM. The increases in current and maximal conductance are consistent with results from heterologous expression systems where the Ca_Va₂S₁ subunit has been shown to increase membrane expression of Ca_Va₁ subunits ²² as well as increase open channel probability ²³.

Increased Ca_va₂δj Expression Mediates Gabapentin Inhibition Of High Threshold VGCC Current In DRG Neurons

To assess the impact of increased Ca_vα₂δi expression on the efficacy of gabapentin directly, the effects of gabapentin on voltage-gated Iβ_a were examined in WT and TG DRG neurons. The results for this experiment are depicted in Figures 8A and 8B. While the inhibitory effects of gabapentin on high threshold voltage-gated Iβ_a in WT DRG neurons were minimal, gabapentin significantly inhibited peak inward Iβ_a in a dose-dependent fashion with an EC₅₀ ~2 μM in TG neurons. As can be seen, gabapentin dose-dependently attenuates high threshold voltage-gated barium currents in TG sensory neurons while it has little effects on that in WT neurons. Figure 8A depicts raw current evoked at -5 mV from a TG neuron before (control) and after application of increasing concentrations of gabapentin. Figure 8B depicts the above data converted to % inhibition ([current in presence of gabapentin / current in absence of gabapentin] x 100) and plotted as function of gabapentin concentrations. The data were fitted by a modified Hill equation resulting in a maximal inhibition of 42%, and EC_5O of 2.0 μM and a Hill coefficient of 0.8. The curve was generated with data from four independent neurons except the data point of 10 μM (10^~5 M) that was from 21 neurons. N = 6 -10 for WT neurons. All data are presented as the mean ± SEM.

Thus, it should be noted that the transgenic mice were generally indistinguishable from their wild-type littermates and had normal motor functions, appearance, growth rate, and fertility. However, compared with the WT littermates, the TG mice exhibited hypersensitivity to light touch (tactile allodynia) and thermal stimulation (thermal hyperalgesia). The pharmacological sensitivity of the nociceptive changes was similar to that for neuropathic pain states. In contrast, inflammation-induced nociception and its pharmacological sensitivity were similar between WT and TG mice. Intrathecal administration of gabapentin reversed dose-dependently the tactile allodynia state in the transgenic mice. Furthermore, VGCC in DRG neurons from TG mice displayed altered kinetics, voltage-dependence of activation, increased densities of high threshold VGCC and gabapentin sensitivity. Together, these data support the notion that modulation of VGCC activity at the spinal level by elevated calcium channel Ca_Va₂O₁ subunit is the molecular mechanism underlying the development and maintenance of certain types of abnormal sensations.

Two lines of evidence support the suggestion that an increase in Ca_Va₂O₁ subunit mediates specifically neuropathic, but not inflammatory, pain. First, overexpression of the Ca_vα₂δi subunit results in behavioral changes that are similar to those observed following nerve injury. Furthermore, Ca_Va₂O₁ overexpression has no influence on formalin-induced behavior and has only additive effects to carrageenan-induced hyperalgesia. Second, behavioral changes associated with Ca_Va₂O₁ overexpression in the TG mice have a pharmacological sensitivity similar to that of neuropathic pain with Ca_Va₂O₁ induction post injury, rather than that of inflammatory pain. Gabapentin, a compound with efficacy in neuropathic pain treatment ²⁴"²⁶, can attenuate tactile allodynia in the TG mice without affecting the tactile threshold in normal animals, while other analgesics such as morphine and ketorolac have less or little efficacy in allodynia reversal ²⁷>²⁸.

These findings in the TG mice support that elevated Ca_Va₂O₁ at the spinal level can contribute to neuropathic pain-like sensations without the influence of other nerve injury factors. While these data suggest that expression of other VGCC subunits is not changed in the TG mice, it cannot be ruled out, however, that other compensatory changes may have occurred during the development of the TG mice. But it is unlikely that these changes, if any, are similar to those associated with nerve injury. In addition, a difference between the TG mice and nerve injury models is that the former has overexpressed Ca_vce₂δ_! through out the nervous system while the latter only has that induced at the DRG and spinal level. Thus, the abnormal sensations in the TG mice could be mediated by changes in different areas of the central nervous system. However, the dose-dependent allodynia reversal by spinal gabapentin in the TG mice suggests that spinal site of action mediated by elevated Ca_vα₂δi is sufficient to confer the behavioral changes.

While the exact mechanism underlying elevated Ca_Va₂O₁ in sensory hypersensitivity remain elusive, the inhibitory effects of gabapentin on VGCC of transgenic DRG neurons suggest that changes in primary afferent neurons are likely a contributing factor. The increase in activation rate, sensitivity to membrane potential changes, current density and amplitudes of VGCC associated with elevated Ca^₂O₁ subunit as shown herein suggests that overexpression of the subunit may influence both neuronal excitability and transmitter release at locations critical to sensory information processing. Assuming that Ca_Va₂O₁ subunit has similar effects on different types of VGCC, which are differentially distributed at different tissues and even different locations within a neuron ²⁹, a Ca_Va₂O₁ subunit-mediated increase in low threshold T-type currents may contribute to bursting activity in DRG and spinal neurons following nerve injury as T-type currents have been shown to mediate depolarizing after- potential in DRG neurons ³⁰. Similarly, an increase in high threshold VGCC currents and excitability at the pre-synaptic terminals may contribute to an increase in transmitter release ³¹'³². Alternatively, an increase in inward calcium currents through high threshold VGCC in inhibitory dorsal horn neurons may make the inhibitory neurons less excitable by activating calcium-activated potassium channels ³³, thus contributing to neuropathic pain-like behaviors. These pathological changes may not be mutually exclusive, and their roles in the abnormal sensations remain to be determined.

The observed gabapentin efficacy in the TG mice is consistent with in vitro binding data suggesting that the Ca_vθ!₂δi is the target site for the drug actions. In this regard, a density threshold of elevated Ca_vα₂δi appears to be necessary for the gabapentin effects since the drug does not affect tactile threshold and DRG neuron VGCC in WT mice. However, the inventor cannot exclude the possibility that elevated Ca_Va₂S₁ increases a subpopulation of VGCC that are more sensitive than the endogenous VGCC to gabapentin. Nevertheless, the data support the notion that modulating the effects of elevated Ca_Va₂O₁ on VGCC by gabapentin accounts for at least partially the mechanism of its actions and explain why gabapentin is more efficacious in neuropathic pain, especially that associated with elevated Ca_vα₂δi ⁷, than other pain states.

Together, the data presented herein strongly support that elevated Ca_Va₂O₁ subunit is a molecular determinant of certain types of neuropathic pain. Emerging data indicate that VGCC expressed centrally have different structural, biophysical and pharmacological properties than those of VGCC expressed peripherally ³⁴'³ . These differences have imposed an important challenge to the design of novel therapeutic interventions targeting specific VGCC that may contribute to injury-induced nociception. However, results of the present study suggest that blocking pathways related to Ca_Va₂O₁ subunit induction post injury or the effects of elevated Ca_Va₂S₁ subunit on VGCC may enable the development of compounds that act on both peripheral and central VGCC specifically involved in neuropathic pain expression.

Thus, specific embodiments and applications of models and methods for nociception, pain transduction, and screening for analgesic compounds have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the present disclosure. Moreover, in interpreting the specification, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non¬ exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

References

All references cited throughout the specification are incorporated by reference herein to their appropriate extent.

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Claims

CLAIMSWhat is claimed is:

1. A model for peripheral nerve injury-induced neuropathic pain comprising a transgenic non-human animal preferentially expressing an alpha-2-delta-l subunit of a voltage gated calcium channel in a neuronal tissue and in an amount sufficient to produce at least one of non-injurious tactile allodynia and non-injurious thermal hyperalgesia while retaining normal pain reaction to tissue injury and inflammatory pain.

2. The transgenic animal of claim 1 wherein the alpha-2-delta-l subunit is expressed in a tissue selected from the group consisting of hippocampus, cortex, spinal cord, and dorsal root ganglion, and substantially not expressed in a tissue selected from the group consisting of heart, skeletal muscle, lung, kidney, spleen, and intestine.

3. The transgenic animal of claim 2 wherein the alpha-2-delta-l subunit is expressed under control of a Thy- 1.2 gene promoter.

4. The transgenic animal of claim 1 wherein the alpha-2-delta-l subunit is expressed in an amount sufficient to produce non-injurious tactile allodynia.

5. The transgenic animal of claim 2 wherein the animal is a mouse.

6. A method of providing an animal model, comprising:

providing the transgenic animal according to claim 1; and providing an information to use the animal for testing of a compound for use in treatment of peripheral nerve injury-induced neuropathic pain, wherein the compound is administered in vivo.

7. The method of claim 6 wherein the administration comprises parenteral administration of the compound at a dosage effective to reduce at least one of non-injurious tactile allodynia and non-injurious thermal hyperalgesia.

8. The method of claim 7 wherein the parenteral administration comprises intrathecal injection.

9. The method of claim 6 wherein the compound reduces expression of the alpha-2- delta-1 subunit or a component functionally associated with the alpha-2-delta-l subunit.

10. The method of claim 6 wherein the compound reduces activity of the subunit or the component functionally associated with the subunit.

11. The method of claim 6 wherein the animal metabolizes the compound to thereby generate an active metabolite that is effective to reduce the at least one of the non- injurious tactile allodynia and the non-injurious thermal hyperalgesia.

12. The method of claim 6 further comprising a step of measuring response to a stimulus using at least one of a measurement of paw withdrawal threshold and a measurement of thermal escape latency.

13. A method of testing a pharmaceutical compound comprising a step of administering in vitro the pharmaceutical compound to at least one of a tissue and a cell of an animal according to claim 1.

14. The method of claim 13 wherein the tissue is a cell from a tissue selected from the group consisting of hippocampus, cortex, spinal cord, and dorsal root ganglion.

15. The method of claim 13 wherein the animal is an animal that does not naturally express the alpha-2-delta-l subunit.