Introduction

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Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid, Pfizer Global R & D] was developed as a brain penetrant 3-alkyl-substituted analog of -amino-butyric acid (GABA) (reviewed by Bryans and Wustrow, 1999). Gabapentin is approved for clinical use in the treatment of refractory partial seizures and secondary generalized tonic-clonic seizures and is being investigated as treatment for a number of disorders including bipolar, social phobias, neuropathic pain, dental pain, osteoarthritis, and migraine. Gabapentin has been reported to bind with nanomolar affinity to the auxiliary ₂ subunit of voltage-dependent calcium channels (VD-CCs) (Gee et al., 1996). However, no direct functional correlation to this binding has been reported to date, and it is unknown whether this accounts for the anticonvulsant, antihyperalgesic, and antinociceptive actions of gabapentin (Taylor et al., 1998).

The principal physiological role of GABA in the neural axis is synaptic inhibition. Ionotropic GABA_A multisubunit chloride channel receptors mediate the fast synaptic inhibitory actions of GABA, whereas metabotropic GABA_B G protein-coupled receptors mediate the slower, longer lasting synaptic inhibitory actions implicated in hippocampal long-term potentiation, slow-wave sleep, absence epilepsy, muscle relaxation, and antinociception (see Bowery and Enna, 2000 and references therein). Recent studies have suggested that the functional and high-affinity agonist binding neuronal GABA_B receptor is a heterodimer of individually inactive gb1 and gb2 seven-transmembrane spanning subunits (reviewed by Bowery and Enna, 2000). Three molecularly and pharmacologically distinct human GABA_B receptor subtypes termed gb1a-gb2, gb1b-gb2, and gb1c-gb2 have been identified that could account at least in part for the diverse biological functions of GABA_B receptors (Ng et al., 2001). Many of the physiological roles of GABA_B receptors can be attributed to the modulation of P/Q- (₂, ₁, _1A subunits) and N-type (₂, ₁, _1B subunits) VD-CCs by presynaptic receptors and modulation of inwardly rectifying K⁺ channels (GIRKs) by postsynaptic GABA_B receptors (Bowery and Enna, 2000 and references therein). GABA_B receptor regulation of VD-CC function is thought to be mediated by G protein subunits via a membrane-delimited mechanism (Herlitze et al., 1996; Ikeda, 1996) resulting in the inhibition of membrane Ca²⁺ conductance and a decrease in neurotransmitter release (Doze et al., 1995; Wu and Saggau, 1997). Activation of presynaptic GABA_B receptors negatively coupled to VD-CCs is likely the mechanism underlying the antinociceptive effects of GABA and the prototypic nonselective GABA_B agonist baclofen, which have been reported to inhibit the release of pain transmitters such as calcitonin gene-related peptide and substance P in spinal cord slices (Malcangio and Bowery, 1993, 1996). Baclofen has also been reported to be efficacious when given intrathecally for the treatment of central pain following stroke or spinal cord injury (Loubser and Akman, 1996), however its wider clinical use has been limited because doses (p.o.) needed for efficacy are associated with flaccidity and hypotonia. Compelling evidence that GABA_B receptors are attractive therapeutic targets has been lacking because of the absence of efficacious and selective GABA_B ligands with few side effects.

Gabapentin has been reported to inhibit K⁺-evoked Ca²⁺ rises in neocortical synaptosomes via inhibition of VD-CCs (Fink et al., 2000) and reduce K⁺-evoked glutamate release from neocortical and hippocampal slices (Dooley et al., 2000). Moreover, gabapentin has been reported to inhibit excitatory neurotransmitter release in the spinal cord dorsal horn (Patel et al., 2000; Shimoyama et al., 2000). We have recently reported that gabapentin is a selective agonist at the recombinant gb1a-gb2 heterodimer and neuronal GABA_B receptor coupled to GIRKs with no partial agonist or antagonist activity at gb1b-gb2 or gb1c-gb2 subtypes (Ng et al., 2001). This led us to the hypothesis that the inhibitory pharmacological effects of gabapentin on excitatory neurotransmitter release in the studies referenced above were attributed to selective activation of neuronal GABA_B receptors coupled to VD-CCs. In this study, we show for the first time that gabapentin is an agonist at endogenously expressed GABA_B gb1a-gb2 heteromers coupled to inhibition of VD-CCs in mouse pituitary intermediate melanotrope clonal mIL-tsA58 (mIL) cells and hippocampal neurons in situ. We propose that gabapentin activation of neuronal GABA_B gb1a-gb2 heteromers and inhibition of voltage-dependent calcium channels may represent a novel mechanism accounting for the anticonvulsant, antihyperalgesic, and antinociceptive properties of gabapentin.

	Experimental Procedures

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Materials. (R)-Baclofen and CGP55845 were purchased from Sigma (St. Louis, MO) and Tocris Cookson (Ballwin, MO), respectively. Gabapentin was obtained commercially (Sigma), stored at 80°C, and freshly prepared and used immediately in the functional assays. Indo-1/AM, indo-1 pentapotassium salt, carboxy SNARF-1/AM, carboxy SNARF-1, Pluronic F-127, and Ca²⁺ calibration kits were purchased from Molecular Probes, Inc. (Eugene, OR). Pertussis toxin, penicillin/streptomycin, and dimethyl sulfoxide were obtained from Sigma. The DMEM and the Gibco BRL trypsin-free buffer were purchased from Life Technologies (Grand Island, NY). Other chemicals and reagents were purchased from Fisher Scientific (St. Louis, MO).

mIL-tsA58 Cells and Culture Conditions. The mIL-tsA58 cells were isolated from a mouse intermediate lobe tumor (M. J. Low, manuscript in preparation). Briefly, a strain of transgenic mice was generated that developed pituitary tumors because of the melanotrope-specific expression of a pro-opiomelanocortin promoter-simian virus 40 large T antigen (temperature-sensitive A58 mutant) fusion gene. The phenotype of these mice was similar to those described previously (Low et al., 1993) despite the substitution of the tsA58 mutant T antigen for wild type. The mIL-tsA58 cells were isolated from a single tumor using procedures analogous to those reported for another melanotrope cell line (Hnasko et al., 1997). They express the POMC gene and dopamine D₂ receptors. Growth rate and morphology of the cells were similar at 33°C, the permissive temperature for tsA58, and at 37°C. These cells display a normal mouse karyotype after more than 60 passages. They either grow as free floating spheres of tightly associated cells or can be coaxed to adhere to plastic, although they remain clustered under this condition as well. Few individual cells are found in cultures, and the clusters are difficult to dissociate enzymatically without destroying the cells.

Measurement of Intracellular Calcium [Ca²⁺]_i Kinetics. [Ca²⁺]_i and intracellular pH (pH_i) were measured simultaneously using a custom-built ultra low light multi-imaging video microscope as described previously (Beatty et al., 1993; Morris et al., 1994). Cells grown on number 00 coverslips (Corning, Corning, NY) were simultaneously loaded with 5 µM indo-1/AM and 5 µM SNARF-1/AM (cell permeant acetoxymethyl (AM) esters), in DMEM, 12.5% dimethyl sulfoxide, and 0.04% (w/w) Pluronic F-127 for 30 min at 37°C in a humidified incubator under 10% CO₂. After incubation, the cells were washed and left in complete medium at 37°C under 10% CO₂ for a 30-min recovery period to allow the esterase to cleave the dyes to their active, impermeant forms. Cells were examined within 90 min following the recovery period.

Antisense Oligodeoxynucleotide Synthesis and Administration. Two antisense deoxynucleotides (ADNs) directed against either GABA_BR1a or GABA_BR1b isoforms of the receptor were designed and synthesized. The gb1a probe is antisense to bases 5'-CAC CAG CAG CAG CAG CAG-3' of GABA_BR1a (bases 4-22, GenBank accession number AJ102185) and the gb1b probe is antisense to bases 5'-ACA GGG TCC CCC CGG GCC-3' of GABA_BR1b (bases 4-22, GenBank accession number AJ02186). Using the National Institutes of Health BLAST search engine (http://www.ncbi.nlm.nih.gov/BLAST/), these antisense sequences showed extensive overlaps with GABA_BR1a and GABA_BR1b receptor sequences from other species but had no significant overlaps with sequences of other cDNAs as of December, 2000. A missense probe containing the same nucleotide base content as the gb1a ADN, but with bases randomly assigned, was used as a control. This missense probe, 5'-CCA GCA GAC ACG CAG CAG-3' has eight overlaps with the gb1a antisense probe and no known complementarity with other sequences. All nucleotide sequences were synthesized by Integrated DNA Technologies (Coralville, IA) as phosphorothioated derivatives.

Data Analysis. Results were analyzed either in real time or from video tape recordings. Twenty-five consecutive frames were averaged in real time, then Ca²⁺ and pH ratio images of the microscope field (uncorrected for background or shading error) displayed on the computer monitor display at one image per second. At the same time, the integrated gray levels of up to eight regions of interest were extracted from the 25-frame average image, and the data were stored on an ASCII file for further analysis. In addition, the uncorrected ratio values for Ca²⁺ and pH were plotted on the computer monitor screen, permitting immediate evaluation of cell viability and the effects of treatments on [Ca²⁺]_i and pH_i. The experiments were also recorded on 3/4-inch U-matic video tape as a backup and to allow analysis of other cells in the video field since the same data display and data extraction procedures could be applied offline. Correction of [Ca²⁺]_i using the prevailing intracellular pH, standardization, data reduction analysis, and statistical methods has been previously described (Morris et al., 1994).

Electrophysiology and Calcium Imaging of Hippocampal Neurons in Brain Slices. Experiments were performed on CA1 pyramidal neurons in 300-µm-thick hippocampal slices from 25- to 28-day-old male Sprague-Dawley rats (Nurse and Lacaille, 1999). Slices were allowed to recover for at least 1 h before use. The recording chamber was continuously perfused with oxygenated (95% O₂, 5% CO₂) artificial cerebrospinal fluid containing 124 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 2 mM MgSO₄, and 10 mM glucose, pH 7.35 to 7.4. Experiments were conducted in the presence of 0.5 µM tetrodotoxin (TTX) to block voltage-dependent Na⁺ channels. To block K⁺ channels in the recorded neuron, patch pipettes (4-8 M) were filled with a cesium-based solution containing 140 mM CsMeSO₃, 1 mM MgCl₂, 5 mM NaCl, 2 mM ATP, 0.4 mM GTP, 10 mM HEPES, and 100 µM of the Ca²⁺ indicator Oregon Green BAPTA-I (Molecular Probes, Inc.) titrated with CsOH to pH 7.25 to 7.28. Combined whole cell current clamp recordings and confocal calcium imaging were performed from CA1 pyramidal neurons using an Axopatch 200B amplifier (Axon Instrument, Foster City, CA) and a multiphoton confocal laser scanning microscope LSM 510 (Carl Zeiss, Kirkland, QC) equipped with a 40× long-range water-immersion objective (numerical aperture 0.8).

Statistical Analysis. The level of significance for differences between means was measured by Fisher's test or analysis of variance followed by the Bonferroni post-test (InStat, GraphPad Software, Inc., San Diego, CA).

	Results

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Gabapentin Inhibition of Calcium Mobilization via Selective Activation of Endogenously Expressed GABA_B gb1-gb2 Heterodimers in mIL Cell Lines. It is well known that pituitary intermediate lobe melanotropes release adrenocorticotropic hormone, -melanocyte-stimulating hormone, and -endorphin by exocytosis. This process requires Ca²⁺ (Thomas et al., 1990) and is inhibited by GABA_B agonists (Taraskevich and Douglas, 1990; Morris et al., 1998). We have recently reported that immortalized pituitary melanotrope mIL cells express endogenous functional GABA_B gb1a-gb2 but not gb1b-gb2 heteromers coupled negatively to VD-CCs (Chronwall et al., 2001). Since presynaptic GABA_B receptors inhibit VD-CCs, mIL cells represented a suitable model system to study the pharmacology of the wild-type brain gb1a-gb2 subtype and to test whether gabapentin inhibition of VD-CCs in isolated neurons (Stefani et al., 1998; Dooley et al., 2000; Fink et al., 2000) was mediated by selective activation of GABA_B receptors.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Pharmacological actions of GABAB ligands on high K+-evoked activation of VD-CCs in mIL cells endogenously expressing gb1a-gb2 heteromers. A, there is a sharp increase in [Ca2+] followed by a slower second peak or shoulder in response to depolarization by high extracellular K+ concentrations. B, baclofen (1 µM) reduces the primary response. C, baclofen effects (1 µM) are completely reversed by addition of 3 µM CGP55845. CPG55845 (3 µM) has no significant effect on the response of mIL cells to depolarization (D). E, 1 µM gabapentin reduces the primary Ca2+ responses of cells in a manner similar to 1 µM baclofen (top, middle) and that it is also blocked by 1 µM CGP55845 (F). In each panel, the individual Ca2+ responses of six to eight cells are shown.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Dose-response study of the ability of gabapentin to inhibit influx of Ca2+ after high K+ depolarization. mIL cells were treated 1 to 5 min with the indicated doses of gabapentin and then depolarized with high K+ as described under Experimental Procedures. Bars represent the standard error for the number of cells indicated. The EC50 was calculated after fitting the data using GraphPad software.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Inhibition of K+-evoked calcium mobilization by 1 µM gabapentin is blocked in a dose-dependent manner with 30 nM to 3 µM CGP55845. CGP, CGP55845; GBP, gabapentin. Each error bar represents 1 S.E.M. The number of cells analyzed is noted in brackets. *p  0.05; **p  0.01; ***p  0.001; compared with control, as calculated by analysis of variance followed by the Bonferroni test between means.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Antisense knockdown of the endogenous GABAB gb1a subunit in mIL cells results in the block of gabapentin-induced inhibition of the primary increase in [Ca2+]i following K+ depolarization and activation of VD-CCs. There was no significant difference among any of the control values and the values for the various ADN treatments when compared by analysis of variance; therefore the results presented are normalized to the value for high K+ depolarization (column A) set to 100%. Both 10 and 30 µM gabapentin inhibited the depolarization-induced rise in intracellular [Ca2+] (compare A to B and C). None of the deoxynucleotide treatments in themselves affected the depolarization-induced rise in intracellular [Ca2+] (compare A, D, G, and J). Gb1a ADN treatment completely blocked the ability of gabapentin to inhibit the rise in [Ca2+]i (compare B and C to D, E, and F), whereas gb1b ADN at the two concentrations tested had no such effect on gabapentin, being indistinguishable from control values (compare A, B, and C versus G, H, and I). Treatment with the gb1a missense probe was also without effect when compared with control values (compare A, B, and C versus J, K, and L) and, like the gb1b ADN treatments (columns H and I), had no significant effect on gabapentin inhibition of the Ca2+ channels. Each error bar represents the S.E.M. The number of cells analyzed is noted in brackets. *p  0.05; **p  0.01; ***p  0.001; compared with control.

Gabapentin Inhibition of VD-CCs via Neuronal GABA_B Receptors in Rat Hippocampal Neurons in Situ. To confirm stimulation by gabapentin at neuronal GABA_B receptors coupled to VD-CCs in situ, we combined whole cell current clamp recordings of pyramidal cells with multiphoton confocal calcium imaging (Fig. 5, A1 and A2) and examined the effects of gabapentin on calcium responses evoked by somatic current injections in CA1 pyramidal neurons of rat hippocampal slices. In the presence of TTX and a K⁺ channel blocker (intracellular cesium), positive current pulses were applied to the pyramidal cell soma via the recording electrode, and the evoked calcium responses were recorded electrophysiologically (membrane potential) and optically (fluorescence) (Fig. 5A2). The amplitude of the current pulse was varied to elicit Ca²⁺ responses by subthreshold stimulation (Fig. 5, B1 and C1) and Ca²⁺ spikes by suprathreshold stimulations (Fig. 5, B2 and C2). Subthreshold current injections induced Ca²⁺ responses of small amplitude and short duration (Fig. 5, B1 and C1), whereas suprathreshold current injections triggered larger and longer lasting Ca²⁺ responses (Fig. 5, B2 and C2) at the cell soma. In our experimental conditions, these responses induced by somatic current injection were solely mediated by Ca²⁺ since they were totally blocked in Ca²⁺-free artificial cerebrospinal fluid (n = 2 cells, data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Gabapentin and baclofen inhibition of somatic Ca2+ responses in hippocampal neurons. A, two-photon confocal image of a pyramidal neuron with the line position for linescan (A1). Calibration bar, 10 µm. In the presence of TTX (0.5 µM) (A2), somatic current injection (I) evoked a Ca2+ spike in the current clamp recording (membrane potential, Vm) and a Ca2+ rise (line scan, LS; and %F/F). B, 1 mM Gabapentin reduced both sub- (B1) and suprathreshold (B2)-evoked somatic Ca2+ responses (traces 2 versus 1) but did not prevent the generation of Ca2+ spikes when somatic current injection was increased (traces 3). C, baclofen similarly reduced Ca2+ responses.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Gabapentin inhibits Ca2+ responses in a dose-dependent manner. A and B, dose-response histograms of gabapentin (100 µM to 1 mM) actions on both membrane depolarizations and Ca2+ responses for sub- (A) and suprathreshold (B) current injections. In the presence of 1 mM gabapentin, cells were still able to generate Ca2+ spikes when somatic current injection was increased. In these conditions, both membrane depolarizations and Ca2+ responses were not significantly different from control. C and D, summary histograms of baclofen (40 µM) effects on sub- (C) and suprathreshold (D) responses. Bars on histograms represent S.E.M.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Gabapentin and baclofen inhibition of Ca2+ responses via GABAB receptor activation. A and B, in the presence of the GABAB receptor antagonist CGP55845 (4 µM), gabapentin (2 mM; A) and baclofen (40 µM; B) failed to depress responses evoked by either sub- (A1 and B1) or suprathreshold (A2 and B2) current injection. C and D, summary histograms of gabapentin (2 mM) and baclofen (40 µM) effects on responses evoked by sub- (C) and suprathreshold (D) stimulation in the absence and presence of CGP55845.

	Discussion

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It has been proposed that the auxiliary ₂ subunit of voltage-dependent calcium channels may be a molecular target for gabapentin, conceivably altering VD-CC function, but direct experimental proof is still lacking to link this with the anticonvulsant, antihyperalgesic, and antinociceptive actions of this drug (Gee et al., 1996; Taylor et al., 1998). Gabapentin has been reported to have no effect on VD-CCs in cultured rodent neurons (Rock et al., 1993) and in acutely dissociated human dentate gyrus granule cells from patients with temporal lobe epilepsy (Schumacher et al., 1998). Yet gabapentin has been reported to inhibit predominantly L-type calcium currents in isolated rat neocortical, striatal, and pallidal neurons (Stefani et al., 1998). More recently, gabapentin has been found to inhibit K⁺-evoked glutamate release from rat neocortical and hippocampal slices (Dooley et al., 2000; Fink et al., 2000). However, the mechanisms underlying these gabapentin actions were not elucidated.

We have reported recently that gabapentin is a selective agonist for the recombinant and neuronal GABA_B gb1a-gb2 heteromer subtype coupled to GIRKs and that it is not a partial agonist and does not block GABA activity at gb1b-gb2 and gb1c-gb2 heteromers (Ng et al., 2001). Selective gabapentin activation of GABA_B receptors negatively coupled to VD-CCs may account for gabapentin actions in the aforementioned K⁺-evoked Ca²⁺-dependent responses, and this notion is also consistent with the depressant action of gabapentin on voltage-sensitive calcium currents in some central neurons (Stefani et al., 1998). Indeed we show herein that gabapentin is an agonist at brain GABA_B gb1a-gb2 heteromer receptors endogenously expressed in mIL cells mediating robust dose-dependent inhibition, similar to that of the prototypical GABA_B agonist baclofen, of VD-CC function. This was attributed to selective activity at the GABA_B receptor since it could be blocked with GABA_B antagonists and following selective antisense knockdown of the gb1 subunit in agreement with the selective activity reported at the recombinant GABA_B receptors (Ng et al., 2001). Recombinant GABA_B heteromers have been also shown to couple to calcium channels in cultured NG108-15 cells and sympathetic neurons (Easter and Spruce, 2000; Filippov et al., 2000), and activation of native receptors in rat pituitary melanotropes and dorsal root sensory neurons leads to inhibition of calcium currents (Morris et al., 1998; Chronwall et al., 2001; Hand et al., 2000). Our results also indicate that, in hippocampal neurons in situ, gabapentin activates GABA_B receptors negatively coupled to N- and/or P/Q-type VD-CCs. But our data do not support a predominant action of gabapentin on L-type Ca²⁺ channels since gabapentin inhibited subthreshold Ca²⁺ responses but did not prevent Ca²⁺ action potentials in the present experiments. Gabapentin actions on neuronal GABA_B receptors coupled to VD-CCs is consistent with the previously reported actions of baclofen in hippocampal neurons (Scholz and Miller, 1991; Lambert and Wilson, 1996). These studies underscore that VD-CCs represent a major and physiologically important effector for neuronal GABA_B receptors.

Our results further indicate that gabapentin may have multiple anticonvulsant actions linked to GABA_B receptors. In addition to its selective activation of gb1a-gb2 GABA_B receptors coupled to GIRKs that produce postsynaptic hyperpolarization (Ng et al. 2001), gabapentin may inhibit Ca²⁺ influx during burst discharges or seizures via its activation of postsynaptic gb1a-gb2 receptors negatively coupled to VD-CCs. It is interesting to note that gabapentin actions on hippocampal neurons are therefore dictated not only by its selective activity at the gb1a-gb2 heteromer subtype but also by the cellular domain where these receptors are found in the cell. Gabapentin-sensitive GABA_B receptors present in the soma and dendritic regions couple to VD-CCs (Fig. 3) and GIRKs (Ng et al., 2001). In contrast, the GABA_B gb1b/c-gb2 subtypes, which are likely present in glutamate and GABA axon terminals of hippocampal neurons and also are negatively coupled to VD-CCs, are insensitive to gabapentin. This is in agreement with the subtype-selective agonist activity defined using recombinant receptors and the lack of presynaptic effect of gabapentin on synaptic transmission in hippocampus (Ng et al., 2001).

Gabapentin has been recently reported to depress excitatory amino acid neurotransmission in spinal cord dorsal horn (Patel et al., 2000; Shimoyama et al., 2000), and the effect of the agonist gabapentin on GABA_B receptors coupled to VD-CCs could account for these effects since a well established physiological role of presynaptic neuronal GABA_B receptors is inhibition of P/Q- and N-type VD-CCs and transmitter release (Menon-Johansson et al., 1993; Wu and Saggau, 1997; Bowery and Enna, 2000). This conclusion is also consistent with the anatomical localization of the gb1a-gb2 heteromer to some presynaptic elements in the neural axis (Benke et al., 1999; Billinton et al., 1999; Towers et al., 2000). GABA_B distribution studies in the lumbar spinal cord and dorsal root ganglia showed that the gb1a mRNA is the predominant species (accounting for ~90%) of the total gb1 mRNA in the afferent fiber cell body. This suggests that gb1a subunits together with gb2 subunits, which exhibit equivalent density to gb1a, comprise presynaptic GABA_B receptors on primary afferent terminals (Towers et al., 2000). Indeed in this report, immunocytochemical analysis showed denser labeling of gb1a in the superficial dorsal horn and presence in neuropil, whereas gb1b was more associated with cell bodies in this region.

The predominant expression of GABA_B gb1a-gb2 receptors in the superficial laminae where nociceptive primary afferent fibers terminate, together with studies that suggest the antinociceptive effects of baclofen (Henry, 1982; Hammond and Drower, 1984; Sawynok and Dickson, 1985) and gabapentin (Xiao and Bennet, 1997; Patel et al., 2000; Shimoyama et al., 2000) are mediated presynaptically, lead us to suggest that, at least in part, the antihyperalgesic, antiallodynic, and antinociceptive effects of gabapentin can be attributed to selective activation of presynaptic GABA_B gb1a-gb2 receptors coupled to VD-CCs in the spinal cord dorsal horn.