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Localization of [3H]gabapentin to a novel site in rat brain: autoradiographic studies
European Journal of Pharmacology - Molecular Pharmacology Section, 244 (1993) 303-309 © 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/$06.00
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EJPMOL 90409
Localization of [3H]gabapentin to a novel site in rat brain: autoradiographic studies David R. Hill, N i r m a l a S u m a n - C h a u h a n and G e o f f r e y N. W o o d r u f f Parke-Dacis Neuroscience Research Centre, Addenbrookes Hospital Site, Cambridge CB2 2QB, UK Received 25 September 1992, accepted 3 November 1992
The autoradiographical distribution of [~H]gabapentin, the tritiated analogue of the novel anticonvulsant gabapentin (1-(aminomethyl)cyclohexaneacetic acid) was measured in rat brain. Binding to sections was uniformly inhibited by non-radioactive gabapentin and 3-isobutyl-y-aminobutyric acid (3-isobutyl-GABA). Specific gabapentin binding sites were unevenly distributed throughout the brain with the highest level being found in the outer layers of the cerebral cortex (38 _+7 fmol/mm2; n = 3) and the lowest amounts in the white matter. In the hippocampus, the distribution of the binding site paralleled the excitatory neuronal input with the highest levels of binding being measured in the outer layers of the dentate gyrus and in the dendritic regions of the CA1 pyramidal cell layer. The binding site appeared absent from the cell body region of granule and pyramidal cells. Lesions performed unilaterally in the striatum using quinolinic acid resulted in a marked loss of [3H]gabapentin binding sites as compared with sham-lesioned animals, suggesting the binding site was localized on neuronal cell bodies. These data complement and extend the results of experiments using [3H]gabapentin with homogenates of rat brain and show the discrete localization of this novel binding site in regions associated with excitatory amino acid input. The data do not support previous indications of an association of the gabapentin binding site and NMDA/glycine receptor complex. Gabapentin; Autoradiography; GABA (y-aminobutyric acid); NMDA (N-methyl-D-aspartate); Glycine; Hippocampus; Anticonvulsants; Excitatory amino acids
1. Introduction
In the accompanying report (Suman-Chauhan et al., 1993) we described the presence, in rat brain homogenates, of a high affinity binding site for a tritiated analogue of the novel anticonvulsant gabapentin (1(aminomethyl)cyclohexaneacetic acid). The binding site was present in high concentrations in brain membranes, saturable and apparently unique to the central nervous system. Thus, using conditions identical to those that permit labelling of the brain, little or no specific [3H]gabapentin binding could be detected in a variety of peripheral tissues such as the liver, lung, kidney, spleen and pancreas. The pharmacological profile of the binding site also appeared to be novel with a large number of putative neurotransmitters, including y-aminobutyric acid (GABA) and other neuroactive compounds failing to influence the binding of [3H]gabapentin. By contrast
Correspondence to: David R. Hill, Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK. Tel. (223) 210929; Fax (223) 249106.
analogues of gabapentin and several 3-substituted GABA derivatives, that also possess anticonvulsant properties (Silverman et al., 1991), were active and inhibited specific binding of [3H]gabapentin with nanomolar affinity, suggesting that the binding site may be a pharmacologically relevant entity. Numerous pharmacological experiments in rodents have shown gabapentin to be an effective anticonvulsant, inhibiting seizures produced by chemical and electrical means (Bartoszyk et al., 1986; Dooley et al., 1986) and it has antiepileptic properties in man (Chadwick, 1992). Despite its close structural similarity to the major inhibitory neurotransmitter GABA, it appears that gabapentin does not influence either GABA A or GABA B receptor binding (Bartoszyk and Reimann, 1985; Suman-Chauhan et al., 1993) or the uptake of this neurotransmitter (Dooley et al., 1986). Accordingly, gabapentin does not affect the response to iontophoretic GABA in cultured spinal cord neurones (Taylor et al., 1988) or influence inhibitions due to activation of chloride or potassium currents in the hippocampus (Haas and Wieser, 1986) as would be expected of a direct action at GABA receptors. An indirect action at the GABA A receptor via an al-
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losteric modulatory site is also unlikely as gabapentin does not show any affinity for the benzodiazepine site associated with the GABA A receptor (Dooley et al., 1986). More recently, electrophysiological and pharmacological experiments (Singh et al., 1990; Sprosen, 1990) performed in our laboratory suggested that in cultured rat striatal neurones, gabapentin may act as a partial agonist at the glycine co-agonist site associated with the N-methyl-D-aspartate (NMDA) receptor complex (Johnson and Ascher, 1987); potentiating the response to (NMDA) and antagonising the facilitatory effects of glycine in the same preparation (Sprosen, 1990). If exerted in vivo such effects could explain the anticonvulsant effect of gabapentin (see Singh et al., 1990). However, other data, also obtained in our laboratories, were not consistent with the electrophysiological data. For example in homogenate binding assays gabapentin did not affect strychnine-insensitive glycine binding, nor did it influence basal or glycine-stimulated binding of [3H]dizocilpine ([3H]MK801) as would be expected of a glycine partial agonist. Nonetheless, in binding studies performed with [3H]gabapentin in rat brain homogenates, the polyamines spermine and spermidine in addition to magnesium ions did show some interaction with the binding site, each inhibiting specific binding by 50%. This observation may be of significance as both the polyamines and magnesium ions have been shown to interact with the NMDA receptor complex, possibly by an action at a common site (Mayer et al., 1984; Ransom and Stec, 1988; Sacaan and Johnson, 1990). The development of the binding assay allowed us to investigate the localization of the binding site in sections of brain by autoradiography. Such an approach has yielded valuable information in a variety of other systems and particularly in the case of the glycine/ NMDA complex, where the distribution of [3H]glycine and NMDA-sensitive [3H]glutamate binding has been shown to be almost identical (McDonald et al., 1990). In the present report we describe the autoradiographic localization of [3H]gabapentin binding to sections of rat brain and compare it to that of the NMDA/glycine receptor complex described in the literature.
quired. On the day of the experiment, sections were thawed and dried and then prewashed in 10 mM HEPES buffer (pH 7.4, sodium free) at room temperature for 30 rain. Adjacent sections were incubated in HEPES buffer containing 40 nM [3H]gabapentin (73 Ci/mmoi, Amersham International, custom synthesis) at 20°C in the presence and absence of 100 #M nonradioactive gabapentin or 10/~M 3-isobutyl GABA to define non-specific binding. After 30 min, the radiolabelled sections were subjected to four sequential washes of 1 min each in ice-cold 100 mM NaC1, dipped in deionised water to remove excess salt and dried under a stream of cold air. The dried sections were exposed to tritium-sensitive film (Amersham) for 3-4 weeks together with brain paste standards (Clark and Hall, 1986). Quantitative analysis of autoradiographs was performed using a Quantimet 920 image analysis system with radioligand bound per unit area being measured. Following the development of the autoradiograph some sections were then stained using cresyl violet. The stained sections were used in conjunction with the autoradiographs to determine the anatomical localization of binding. 2.2. Lesions Male rats (250 g) were anaesthetized with sodium pentobarbitone (Sagatal, 40-50 mg/kg) and placed in a Kopf stereotaxic apparatus. Unilateral lesions of the striatum were made by injecting 200 nmol of quinolinic acid dissolved in 1 Izl of saline at a rate of 0.5 izl/min (coordinates: anterior 0.-5 mm, lateral 2.6 mm and ventral 4.4 mm, with Bregma and the brain surface as reference points). Sham-lesioned animals received the same surgical procedures but saline was infused rather than the excitotoxic amino acid. This treatment has been shown to produce extensive loss of cell bodies in the injected region (Coyle and Schwarcz, 1976; Foster et al., 1988). Fourteen days after surgery the animals were killed and the brains removed to be rapidly frozen. Sections were then cut and treated as described above.
3. Results 2. Materials and methods
3.1. Distribution of [ 3H]gabapentin binding 2.1. Autoradiography Rats (approximately 250 g) were stunned, decapitated and the brains removed. These were rapidly frozen and sections (10 izm) were cut using a cryostat. The sections were cold-mounted onto gelatin-subbed slides, thawed, dried and stored at -20°C until re-
Preliminary studies indicated that four sequential washes of 1 min each in ice-cold NaC1 resulted in optimal levels of specific binding. Non-specific binding represented about 40-50% of total, was identical for gabapentin and 3-isobutyl GABA and was uniform throughout the section.
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The autoradiographic localization of [3H]gabapentin to a horizontal section of rat brain is shown in fig. 1. [3H]Gabapentin binding was unevenly distributed in the brain with the highest levels of binding (38 ___7 fmol/mm2; n = 3) being found in the outer layers of the frontal, parietal, occipital and entorhinal cortex and the lowest levels being measured in the white matter. Fig. 1 also shows that within the cerebral cortex labelling was particularly pronounced in layers I and II of the cerebral cortex. Specific [3H]gabapentin labelling was also discretely localized in the molecular layer of the dentate gyrus and cerebellum as well as the CA1 region of the hippocampus. Within the hippocampus binding was not uniformly distributed but was highest in the CA1 region and molecular layer of the dentate gyrus. The pyramidal cell layer in the hippocampus and the granule cell layer in the dentate gyrus, where binding was almost absent, could be clearly distinguished against the surrounding densely labelled regions. The cerebellum also contained different levels of binding according to layers. Binding was absent in the white matter and highest in the molecular layer, whilst the granule cell layer showed intermediate levels of binding.
3.2. Comparison of [3H]gabapentin binding with the distribution of the NMDA glycine receptor complex The distribution of the N-methyl-D-aspartate (NMDA) receptor and its associated glycine modulatory site has been described extensively by others (Bristow et al., 1986; Bowery et al., 1988; Maragos et al., 1988; McDonald et al., 1990; Young and Fagg, 1991). Table 1 presents a summary of the distribution of [3H]glycine (McDonald et al., 1990) and NMDAsensitive glutamate (Monaghan et al., 1985) binding and compares it with the density of [3H]gabapentin binding in the same region. In order to make compar-
isons with the published data, [3H]gabapentin binding throughout the brain was normalised with respect to that measured in the CA1 region of the hippocampus (26 +_4 fmol/mm2; n = 4). Although [3H]gabapentin binding was present in all the same regions as the NMDA/glycine receptor complex, the relative densities differed. Within the regions of the hippocampus the relative levels of gabapentin, NMDA and glycine binding were roughly equal (table 1). In contrast, the high degree of [3H]gabapentin binding measured in the outer layers of the cerebral cortex was not paralleled by a similar level of NMDA or glycine binding. Moreover, in regions such as the substantia nigra and molecular layer of the cerebellum, where [3H]glycine binding was low, relatively high amounts of [3H]gabapentin binding were measured. In fact, within the cerebellum there was a reciprocal distribution of [3H]gabapentin and [3H]glycine binding in the granule and molecular layers. When the data for [3H]gabapentin and [3H]glycine binding summarized in table 1 were analyzed by regression analysis, a correlation coefficient of only 0.48 was obtained indicating a lack of any significant correlation between the relative density of the two sites within the brain areas measured.
3.3. The effect of quinolinic acid lesions on [3H]gabapentin binding As a means of determining whether the gabapentin binding site was located on neuronal cell bodies, the striatum was lesioned unilaterally using the excitotoxin quinolinic acid. The results of these experiments are summarized in fig. 2. In control animals total binding in the left striatum measured 23.0 + 1.2 fmol/mm 2 and non-specific binding was 2.0 + 0.3 fmol/mm 2 (n = 4) and was essentially the same on the sham-lesioned right side which received a saline infusion (total bind-
TABLE 1 Relative distribution of [3H]gabapentin, [3H]glycine and NMDA-sensitive [3H]glutamate binding in rat brain. Data for binding to the gabapentin site are the m e a n from three separate experiments in which 2 - 4 separate sections were analyzed for each region. Data were then normalised with respect to the level of binding in the stratum radiatum. Brain region
[3H]Gabapentin
[3H]Glycine (McDonald et al., 1990)
[3H]Glutamate (Monaghan and Cotman, 1985)
Hippocampus CA1 stratum radiatum Hippocampus CA1 stratum oriens Dentate gyrus molecular layer Parietal cortex I - I I Parietal cortex I V - V I Substantia nigra Nucleus accumbens Globus pallidus Cerebellum molecular layer Cerebellum granule layer
100 68 108 146 99 68 84 33 78 28
100 81 89 61 43 11 43 27 4 30
100 78 65 40 8 61 8 -
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307 40
3O
SHAM
LESIONED
"o E
2O o~ i0
LHS RHS LHS RHS Fig. 2. The effect of quinolinic acid lesions on [3H]gabapentin binding in rat striatum. Anaesthetized male rats received a unilateral injection of either saline or 200 nmol of quinolinic acid into the striatum. Fourteen days later, the animals were killed and the brains removed for sectioning. Adjacent sections from sham-lesioned and lesioned animals were incubated in parallel with 40 nM [3H]gabapentin with or without 100/xM non-radioactive gabapentin to define non-specific binding. After incubation the sections were washed and dried and apposed to tritium-sensitive film in order to generate the autoradiographs. These were analyzed densitometrically using brain paste standards as a calibration. Total (open bars) and non-specific binding (hatched bars) to the left and right side striatum in sham-lesioned and lesioned animals is shown. Data are the mean values from measurements made using sections taken from four separate animals. Total and non-specific binding were equal in the left and right striatum of sham-lesioned animals and the left (unlesioned) striatum of animals which had received quinolinic acid. By contrast total binding to the lesioned striatum was reduced by more than 65% without any effect on non-specific binding.
ing = 21.6 + 1.9 f m o l / m m 2 ; non-specific binding = 1.5 _+ 0.2 f m o l / m m 2 ) . In the lesioned animals binding to the left striatum was the same as in the control animals (total binding = 22.4 _+ 3.0 f m o l / m m 2 ; non-specific binding = 1.9 +_ 0.9 f m o l / m m 2, n = 4). In contrast, total binding to the striatum that had b e e n treated with quinolinic acid was reduced by 66% to 7.6_+ 1.0 f m o l / m m 2. Non-specific binding was unaffected (1.1 _+ 0.6 f m o l / m m 2 ) .
4. Discussion G a b a p e n t i n is a novel anticonvulsant showing efficacy in rodent models of epilepsy and in man (Bartoszyk et at., 1986; Dooley et al., 1986; Chadwick, 1992). Despite this c o m p o u n d being originally synthesized as a lipophilic G A B A - m i m e t i e , it does not a p p e a r to interact with either G A B A A or G A B A B receptors
either in binding assays or in more functional tests (Bartoszyk and Reimann, 1985; S u m a n - C h a u h a n et al., 1993) and its mechanism of action remains to be resolved, Many anticonvulsants exert their therapeutic effects by modifying the physiological actions of one of the two main classes of amino acid transmitter. For example, barbiturates and benzodiazepines e n h a n c e the actions of the inhibitory neurotransmitter G A B A at G A B A A receptors (see M e l d r u m and Braestrup, 1984). By contrast, the anticonvulsant effects of dizocilpine (MK-801; Clineschmidt et al., 1982), HA-966 and 7chlorokynurinic acid (7-C1-Kyn) are the result of a reduction in the activity of the N M D A receptor complex; MK-801 blocks the N M D A - a s s o c i a t e d channel directly (Kemp et al., 1986)whilst HA-966 and 7-C1-Kyn reduce the facilitatory effect of glycine at the co-agonist site (Kemp et al., 1988; Foster and K e m p 1989). M o r e recently, the n o n - N M D A glutamate antagonist N B Q X has been shown to exert anticonvulsant actions in animal models of reflex epilepsy, presumably by antagonising the excitatory effects of glutamate at the quisqualate ( A M P A ) a n d / o r the kainate receptor (Smith et al., 1991). A n action at the glycine modulatory site has recently been p r o p o s e d for gabapentin. Thus, Sprosen (1990) reported that gabapentin ( 1 - 1 0 /~M) could e n h a n c e the effects of N M D A in cultured striatal neurones whilst higher concentrations antagonised the effects of glycine itself, and suggested that gabapentin was a partial agonist at the glycine co-agonist site. Some support for this came from the work of Oles and colleagues (1990) who d e m o n s t r a t e d that the anticonvulsant effects of gabapentin were antagonised by Dserine, which is an agonist at the glycine site. Despite observations such as these suggesting an action at the glycine r e c e p t o r , g a b a p e n t i n d o e s n o t inhibit [3H]glycine binding to cortical membranes, nor does it modify glycine stimulated [3H]MK-801 binding (D.R. Hill, unpublished observations). A u t o r a d i o g r a p h i c studies which reveal the anatomical localization of ligand binding sites in the brain have helped our understanding of the way such ligands may influence the activity of neuronal systems. A good example comes from the field of excitatory amino acids where the close parallel between the distribution of [3H]glycine and NMDA-sensitive glutamate sites un-
Fig. 1. Localization of [3H]gabapentin binding to horizontal sections of rat brain (upper panel) and its displacement by non-radioactive gabapentin (lower panel). Adjacent sections of rat brain were incubated for 30 min with 40 nM [3H]gabapentin in the presence and absence of non-radioactive gabapentin (100 p,M). After washing and drying the sections were apposed to tritium-sensitive film for the generation of the autoradiographs. On the photographs shown here high levels of [3H]gabapentin binding are represented as white grains. Key: Layers I and II of the cerebral cortex (1,2), fields Cal, Ca2 and Ca3 of Ammon's horn of the hippocampus, dentate gyrus (dg), central gray (cg), granule (gr) and molecular (mol) layers of the cerebellum, lateral septum (Is), white matter (w).
308 derlies the dependence on glycine for activation of the NMDA receptor complex (Bristow et al., 1986; Johnson and Ascher, 1987; Bowery et al., 1988; Maragos et al., 1988; McDonald et al., 1990; Young and Fagg, 1991). Moreover, the uneven distribution of excitatory amino acid receptors is in good agreement with the projection areas of the major excitatory amino acid pathways (see Young and Fagg, 1991). The qualitative distribution of [3H]gabapentin binding sites measured throughout the brain was similar to that of excitatory amino acid receptors including the NMDA receptor as determined by NMDA-sensitive glutamate binding. Furthermore, high levels of [3H]gabapentin binding were found in regions having a major excitatory input such as the striatum, hippocampus and cerebellum. Apart from the outer layers of the cerebral cortex, the highest levels of gabapentin binding sites were found within the hippocampus and dentate gyrus, an area believed to be associated with seizure activity because of its low threshold to stimulation. Moreover, within the hippocampus the level of binding paralleled the terminal fields of the excitatory circuitry. Thus, binding was highest in the area dentata where a major excitatory pathway, arising in the entorhinal cortex (the perforant path), terminates on the dendrites of granule cells (Walaas, 1983). These granule ceils project their excitatory input to the CA3 region of the hippocampus and these cells in turn feed forward to terminate in the CA1 region where binding was again pronounced. Clearly, this pattern of binding suggests the possibility of influencing the excitatory input to this structure. Nonetheless, in extracellular recordings from the CA1 region of hippocampal slices, gabapentin did not influence the development of longterm potentiation (Taylor et al., 1988) which is believed to be due to activation of NMDA receptors (Harris et al., 1984) and reductions in synaptic transmission in the hippocampus were observed only at high (1 raM) concentrations of gabapentin (Haas and Weiser, 1986). Within the cerebellum, binding was also differentiated in a way that could be taken to suggest an association with excitatory fibres. Thus the molecular layer, where the granule cells terminate upon Purkinje dendrites, contained a greater level of binding than the granule cell layer which receives the major mossy fibre input to the structure. Nonetheless, significant levels of binding were still measured in the granule layer. Despite a similarity in the pattern of [3H]gabapentin binding and the NMDA/glycine complex, in terms of their relative quantitative distribution they were quite different. In relative terms, the density of the N M D A / glycine receptor complex has been found to be highest in the CA1 region of the stratum radiatum whereas the highest density of the gabapentin site was found in the outer cortical layers. Proportionately higher levels of [3H]gabapentin binding were present in structures such
as the inner cerebral cortex, the substantia nigra and nucleus accumbens than has been reported for [3H]glycine binding. Moreover, within the cerebellum the distribution of the gabapentin site was the reverse of that reported for [3H]glycine. The difference in relative density was reflected in an almost complete lack of correlation between the relative binding density of the two binding sites as determined from regression analysis, suggesting that despite their qualitatively similar distributions the two sites were not related. Indeed, the distribution of the gabapentin site agreed more closely to that of non-NMDA glutamate receptors, particularly to that of the AMPA site (Nielsen et al., 1990) and a more detailed comparison of the distribution of the two sites is currently in progress. The precise cellular localization of the binding site remains to be determined, but within the hippocampus the distribution would suggest a presence on the dendritic tree of granule and pyramidal neurones. A localization on glial elements cannot be ruled out, although the results from the quinolinic acid lesions suggest that the binding site may be present on the cell bodies of striatal neurones. Lesions produced by this neurotoxin have been shown to selectively destroy neuronal cell bodies whist sparing axons (Foster et al., 1988). Moreover, following the lesion there is a proliferation of glial elements which is reflected in an increased level of binding of [3H]PKll195 to the omega-3 benzodiazepine site (Dubois et al., 1988). One may deduce therefore that the reduction in [3H]gabapentin binding seen following the lesion is more consistent with a localization on neuronal rather than glial elements, although this needs to be determined more directly. In conclusion, we have described the presence of a novel binding site for [3H]gabapentin which is discretely localised in regions of the brain thought to be involved in the genesis of epileptiform activity. The results of the present study demonstrate that there is some similarity between the distribution of the gabapentin binding site and the NMDA receptor but also that quantitatively, the pattern of localization is sufficiently different to suggest that [3H]gabapentin does not label the NMDA/glycine receptor complex. Thus an interaction with this excitatory amino acid receptor may not be responsible for the anticonvulsant effects of gabapentin as has previously been suggested. The mechanism whereby.gabapentin does exert its therapeutic effect and the molecular nature of the gabapentin binding site still remain to be elucidated.
Note added in proof
Since submitting this and our companion paper (Suman-Chauhan et al., 1993) we have found that a number of neutral L-amino acids such as L-leucine (ICs0 =
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77 nM), L-isoleucine (IC50 = 72 nM), L-methionine (IC50 = 50 nM) and also the system-L inhibitor BCH (2-(-)-endoamino-bicycloheptane-2-carboxylic acid: IC50 = 40 nM) are potent inhibitors of [3H]gabapentin binding. This raises the possibility that [3H]gabapentin labels a site in brain that closely resembles the large neutral amino acid transporter (system-L) that has been characterised elsewhere.
References Bartoszyk, G.D. and W. Reimann, 1985, Preclinical characterization of the anticonvulsant gabapentin, in: 16th Epilepsy International Congress, Abstracts (Ciba-Geigy, Basel) p. 1. Bartoszyk, G.D., N. Meyerson, W. Reimann, D. Satzinger and A. von Hodenberg, 1986, Gabapentin, in: Current Problems in Epilepsy, Vol. 4, New Anticonvulsant Drugs, eds. B.S. Porter and R.J. Porter (John Libbey, London) p. 147. Bowery, N.G., E.F.G. Wong and A.L. Hudson, 1988, Quantitative autoradiography of [3H]MK-801 binding sites in mammalian brain, Br. J. Pharmacol. 93, 944. Bristow, D.R., N.G. Bowery and G.N. Woodruff, 1986, Light microscopic autoradiography of [3H]glycine and [3H]strychnine binding sites in rat brain, Eur. J. Pharmacol. 126, 303. Chadwick, D., 1992, Gabapentin, in: Recent Advances in Epilepsy, Vol. 5, eds. T.A. Pedley and B.S. Meldrum (Churchill-Livingstone, Edinburgh) p. 211. Clark, C.R. and M.D. Hall, 1986, Hormone receptor autoradiography: recent developments, Trends Biochem. Sci. 11, 195. Clineschmidt, B.V., G.E. Martin and P.R. Bunting, 1982, Anticonvulsant activity of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-immine (MK-801), a substance with potent anticonvulsant, central sympathomimetic and apparent anxiolytic properties, Drug Dev. Res. 2, 123. Coyle, J.T. and R. Schwarcz, 1976, Lesions of striatal neurones with kainic acid provides a model for Huntington's chorea, Nature 262, 244. Dooley, D.J., G.D. Bartoszyk, J. Hartenstein, W. Reimann, D.M. Rock and G. Satzinger, 1986, Preclinical pharmacology of gabapentin, in: Golden Jubilee Conference and Northern European Epilepsy Meeting (University of York, York) Abstract 8. Dubois, A., J. Benavides, B. Peny, D. Duverger, D. Fage, B. Gotti, E.T. MacKenzie and B. Scatton, 1988, Imaging of primary and remote ischaemic and excitotoxic brain lesions. An autoradiographic study of peripheral type benzodiazepine binding sites in the rat and cat, Brain Res. 445, 77. Foster, A.C. and J.A. Kemp, 1989, HA-966 antagonizes N-methylD-aspartate receptors through a selective action with the glycine modulatory site, J. Neurosci. 9, 2191. Foster, A.C., R. Gill and G.N. Woodruff, 1988, Neuroprotective effects of MK-801 in vivo: selectivity and evidence for delayed degeneration mediated by NMDA receptor activation, J. Neurosci. 8, 4745. Haas, H.L. and H.-G. Wieser, 1986, Gabapentin: action on hippocampal slices of the rat and effects in human epileptics, in: Golden Jubilee Conference and Northern European Epilepsy Meeting (University of York, York) Abstract 9. Harris, E.W., A.H. Ganong and C.W. Cotman, 1984, Long term potentiation in the hippocampus involves activation of N-methylD-aspartate receptors, Brain Res. 323, 132. Johnson, J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse brain neurones, Nature 325, 529.
Kemp, J.A., T. Priestly and G.N. Woodruff, 1986, MK-801, a novel orally active anticonvulsant is a potent non-competitive N-methylD-aspartate receptor antagonist, Br. J. Pharmacol. 89, 535. Kemp, J.A., A.C. Foster, P.D. Leeson, T. Priestly, R. Tridgett, L.L. Iversen and G.N. Woodruff, 1988, 7-Chlorokynurinic acid is a selective antagonist at the N-methyI-D-aspartate receptor complex, Proc. Natl. Acad. Sci. USA 85, 6547. Maragos, W.F., J.B. Penney and A.B. Young, 1988, Anatomic correlation of NMDA and [3H]TCP-labelled receptors in rat brain, J. Neurosci. 8, 493. Mayer, M.L., G.L. Westbrook and P.B. Guthrie, 1984, Voltage dependent block by Mg 2+ of NMDA responses in spinal cord neurones, Nature 309, 261. McDonald, J.W., J.B. Penny, M.V. Johnston and A.B. Young, 1990, Characterization and regional distribution of strychnine-insensitive [3H]glycine binding sites in rat brain by quantitative receptor autoradiography, Neuroscience 35, 653. Meldrum, B. and C. Braestrup, 1984, GABA and the anticonvulsant action of benzodiazepines and related drugs, in: Actions and Interactions of GABA and Benzodiazepines, ed. N.G. Bowery (Raven Press, New York) p. 133. Monagham D.T., D. Yao and C.W. Cotman, 1985, [3H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis, Brain Res. 340, 378. Nielsen, E.O., J. Drejer, J.J. Cha, A.B. Young and T. Honor6, 1990, Autoradiographic characterization and localization of quisqualate binding sites in rat brain using the antagonist [3H]6-cyano-7nitroquinoxaline-2,3-dione: comparison with (R,S)-[3H]a-amino 3-hydroxy-5-methyl-4-isoxolepropionic acid binding sites, J. Neurochem. 54, 686. Oles, R.J., L. Singh, J. Hughes and G.N. Woodruff, 1990, The anticonvulsant action of gabapentin involves the glycine/NMDA receptor, Soc. Neurosci. Abstr. 16, 783, 321.6. Ransom, R.W. and N.L. Stec, 1988, Cooperative modulation of [3H]MK-801 binding to the N-methyl-D-aspartate receptor ion channel complex by L-glutamate, glycine and polyamines, J. Neurochem. 51,830. Sacaan, A.I. and K.M. Johnson, 1990 Competitive inhibition of magnesium induced [3H]-(-[thienyl]cyclohexyl)piperidine binding by arcaine: evidence for a shared spermidine-magnesium binding site, Mol. Pharmacol. 38, 7/)5. Silverman, R.B., R. Andruszkiewicz, R. Nanavati, C. Taylor and MG. Vartanian, 1991, 3-Alkyl-4-aminobutyric acids: the first class of anticonvulsant agents that activates L-glutamic acid decarboxylase, J. Med. Chem. 34, 2295. Singh, L., R. Oles and M.D. Tricklebank, 1990, Modulation of seizure susceptibility in the mouse by the strychnine-insensitive glycine recognition site of the NMDA receptor/ion channel complex, Br. J. Pharmacol. 99, 285. Smith, S.E., N. DfirmiJller and B.S. Meldrum, 1991, The non-Nmethyl-D-aspartate receptor antagonists, GYKI 52466 and NBQX are anticonvulsant in two animal models of reflex epilepsy, Eur. J. Pharmacol. 201, 179. Sprosen, T., 1991). Allosteric Modulation of the NMDA-Receptor Complex, Ph.D. Thesis (Cambridge University, Cambridge). Suman-Chauhan, N., L. Webdale, D.R. Hill and G.N. Woodruff, 1993, Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies, Eur. J. Pharmacol. Mol. Pharmacol. 244, 293. Taylor. C.P., D.M. Rock, R.J. Weinkauf and A.H. Ganong, 1988, In vitro and in vivo effects of the anticonvulsant gabapentin, Soc. Neurosci. Abstr. 14, 866. Walaas, I., 1983, in: Chemical Neuroanatomy, ed. P.C. Emson (Raven Press, New York) p. 337. Young, A.B. and G.E. Fagg, 1991, Excitatory amino acid receptors in the brain: membrane binding and receptor autoradiographic approaches, Trends Pharmacol. Sci. Special Report, 18.
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Localization of [3H]gabapentin to a novel site in rat brain: autoradiographic studies David R. Hill, N i r m a l a S u m a n - C h a u h a n and G e o f f r e y N. W o o d r u f f Parke-Dacis Neuroscience Research Centre, Addenbrookes Hospital Site, Cambridge CB2 2QB, UK Received 25 September 1992, accepted 3 November 1992
The autoradiographical distribution of [~H]gabapentin, the tritiated analogue of the novel anticonvulsant gabapentin (1-(aminomethyl)cyclohexaneacetic acid) was measured in rat brain. Binding to sections was uniformly inhibited by non-radioactive gabapentin and 3-isobutyl-y-aminobutyric acid (3-isobutyl-GABA). Specific gabapentin binding sites were unevenly distributed throughout the brain with the highest level being found in the outer layers of the cerebral cortex (38 _+7 fmol/mm2; n = 3) and the lowest amounts in the white matter. In the hippocampus, the distribution of the binding site paralleled the excitatory neuronal input with the highest levels of binding being measured in the outer layers of the dentate gyrus and in the dendritic regions of the CA1 pyramidal cell layer. The binding site appeared absent from the cell body region of granule and pyramidal cells. Lesions performed unilaterally in the striatum using quinolinic acid resulted in a marked loss of [3H]gabapentin binding sites as compared with sham-lesioned animals, suggesting the binding site was localized on neuronal cell bodies. These data complement and extend the results of experiments using [3H]gabapentin with homogenates of rat brain and show the discrete localization of this novel binding site in regions associated with excitatory amino acid input. The data do not support previous indications of an association of the gabapentin binding site and NMDA/glycine receptor complex. Gabapentin; Autoradiography; GABA (y-aminobutyric acid); NMDA (N-methyl-D-aspartate); Glycine; Hippocampus; Anticonvulsants; Excitatory amino acids
1. Introduction
In the accompanying report (Suman-Chauhan et al., 1993) we described the presence, in rat brain homogenates, of a high affinity binding site for a tritiated analogue of the novel anticonvulsant gabapentin (1(aminomethyl)cyclohexaneacetic acid). The binding site was present in high concentrations in brain membranes, saturable and apparently unique to the central nervous system. Thus, using conditions identical to those that permit labelling of the brain, little or no specific [3H]gabapentin binding could be detected in a variety of peripheral tissues such as the liver, lung, kidney, spleen and pancreas. The pharmacological profile of the binding site also appeared to be novel with a large number of putative neurotransmitters, including y-aminobutyric acid (GABA) and other neuroactive compounds failing to influence the binding of [3H]gabapentin. By contrast
Correspondence to: David R. Hill, Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK. Tel. (223) 210929; Fax (223) 249106.
analogues of gabapentin and several 3-substituted GABA derivatives, that also possess anticonvulsant properties (Silverman et al., 1991), were active and inhibited specific binding of [3H]gabapentin with nanomolar affinity, suggesting that the binding site may be a pharmacologically relevant entity. Numerous pharmacological experiments in rodents have shown gabapentin to be an effective anticonvulsant, inhibiting seizures produced by chemical and electrical means (Bartoszyk et al., 1986; Dooley et al., 1986) and it has antiepileptic properties in man (Chadwick, 1992). Despite its close structural similarity to the major inhibitory neurotransmitter GABA, it appears that gabapentin does not influence either GABA A or GABA B receptor binding (Bartoszyk and Reimann, 1985; Suman-Chauhan et al., 1993) or the uptake of this neurotransmitter (Dooley et al., 1986). Accordingly, gabapentin does not affect the response to iontophoretic GABA in cultured spinal cord neurones (Taylor et al., 1988) or influence inhibitions due to activation of chloride or potassium currents in the hippocampus (Haas and Wieser, 1986) as would be expected of a direct action at GABA receptors. An indirect action at the GABA A receptor via an al-
304
losteric modulatory site is also unlikely as gabapentin does not show any affinity for the benzodiazepine site associated with the GABA A receptor (Dooley et al., 1986). More recently, electrophysiological and pharmacological experiments (Singh et al., 1990; Sprosen, 1990) performed in our laboratory suggested that in cultured rat striatal neurones, gabapentin may act as a partial agonist at the glycine co-agonist site associated with the N-methyl-D-aspartate (NMDA) receptor complex (Johnson and Ascher, 1987); potentiating the response to (NMDA) and antagonising the facilitatory effects of glycine in the same preparation (Sprosen, 1990). If exerted in vivo such effects could explain the anticonvulsant effect of gabapentin (see Singh et al., 1990). However, other data, also obtained in our laboratories, were not consistent with the electrophysiological data. For example in homogenate binding assays gabapentin did not affect strychnine-insensitive glycine binding, nor did it influence basal or glycine-stimulated binding of [3H]dizocilpine ([3H]MK801) as would be expected of a glycine partial agonist. Nonetheless, in binding studies performed with [3H]gabapentin in rat brain homogenates, the polyamines spermine and spermidine in addition to magnesium ions did show some interaction with the binding site, each inhibiting specific binding by 50%. This observation may be of significance as both the polyamines and magnesium ions have been shown to interact with the NMDA receptor complex, possibly by an action at a common site (Mayer et al., 1984; Ransom and Stec, 1988; Sacaan and Johnson, 1990). The development of the binding assay allowed us to investigate the localization of the binding site in sections of brain by autoradiography. Such an approach has yielded valuable information in a variety of other systems and particularly in the case of the glycine/ NMDA complex, where the distribution of [3H]glycine and NMDA-sensitive [3H]glutamate binding has been shown to be almost identical (McDonald et al., 1990). In the present report we describe the autoradiographic localization of [3H]gabapentin binding to sections of rat brain and compare it to that of the NMDA/glycine receptor complex described in the literature.
quired. On the day of the experiment, sections were thawed and dried and then prewashed in 10 mM HEPES buffer (pH 7.4, sodium free) at room temperature for 30 rain. Adjacent sections were incubated in HEPES buffer containing 40 nM [3H]gabapentin (73 Ci/mmoi, Amersham International, custom synthesis) at 20°C in the presence and absence of 100 #M nonradioactive gabapentin or 10/~M 3-isobutyl GABA to define non-specific binding. After 30 min, the radiolabelled sections were subjected to four sequential washes of 1 min each in ice-cold 100 mM NaC1, dipped in deionised water to remove excess salt and dried under a stream of cold air. The dried sections were exposed to tritium-sensitive film (Amersham) for 3-4 weeks together with brain paste standards (Clark and Hall, 1986). Quantitative analysis of autoradiographs was performed using a Quantimet 920 image analysis system with radioligand bound per unit area being measured. Following the development of the autoradiograph some sections were then stained using cresyl violet. The stained sections were used in conjunction with the autoradiographs to determine the anatomical localization of binding. 2.2. Lesions Male rats (250 g) were anaesthetized with sodium pentobarbitone (Sagatal, 40-50 mg/kg) and placed in a Kopf stereotaxic apparatus. Unilateral lesions of the striatum were made by injecting 200 nmol of quinolinic acid dissolved in 1 Izl of saline at a rate of 0.5 izl/min (coordinates: anterior 0.-5 mm, lateral 2.6 mm and ventral 4.4 mm, with Bregma and the brain surface as reference points). Sham-lesioned animals received the same surgical procedures but saline was infused rather than the excitotoxic amino acid. This treatment has been shown to produce extensive loss of cell bodies in the injected region (Coyle and Schwarcz, 1976; Foster et al., 1988). Fourteen days after surgery the animals were killed and the brains removed to be rapidly frozen. Sections were then cut and treated as described above.
3. Results 2. Materials and methods
3.1. Distribution of [ 3H]gabapentin binding 2.1. Autoradiography Rats (approximately 250 g) were stunned, decapitated and the brains removed. These were rapidly frozen and sections (10 izm) were cut using a cryostat. The sections were cold-mounted onto gelatin-subbed slides, thawed, dried and stored at -20°C until re-
Preliminary studies indicated that four sequential washes of 1 min each in ice-cold NaC1 resulted in optimal levels of specific binding. Non-specific binding represented about 40-50% of total, was identical for gabapentin and 3-isobutyl GABA and was uniform throughout the section.
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The autoradiographic localization of [3H]gabapentin to a horizontal section of rat brain is shown in fig. 1. [3H]Gabapentin binding was unevenly distributed in the brain with the highest levels of binding (38 ___7 fmol/mm2; n = 3) being found in the outer layers of the frontal, parietal, occipital and entorhinal cortex and the lowest levels being measured in the white matter. Fig. 1 also shows that within the cerebral cortex labelling was particularly pronounced in layers I and II of the cerebral cortex. Specific [3H]gabapentin labelling was also discretely localized in the molecular layer of the dentate gyrus and cerebellum as well as the CA1 region of the hippocampus. Within the hippocampus binding was not uniformly distributed but was highest in the CA1 region and molecular layer of the dentate gyrus. The pyramidal cell layer in the hippocampus and the granule cell layer in the dentate gyrus, where binding was almost absent, could be clearly distinguished against the surrounding densely labelled regions. The cerebellum also contained different levels of binding according to layers. Binding was absent in the white matter and highest in the molecular layer, whilst the granule cell layer showed intermediate levels of binding.
3.2. Comparison of [3H]gabapentin binding with the distribution of the NMDA glycine receptor complex The distribution of the N-methyl-D-aspartate (NMDA) receptor and its associated glycine modulatory site has been described extensively by others (Bristow et al., 1986; Bowery et al., 1988; Maragos et al., 1988; McDonald et al., 1990; Young and Fagg, 1991). Table 1 presents a summary of the distribution of [3H]glycine (McDonald et al., 1990) and NMDAsensitive glutamate (Monaghan et al., 1985) binding and compares it with the density of [3H]gabapentin binding in the same region. In order to make compar-
isons with the published data, [3H]gabapentin binding throughout the brain was normalised with respect to that measured in the CA1 region of the hippocampus (26 +_4 fmol/mm2; n = 4). Although [3H]gabapentin binding was present in all the same regions as the NMDA/glycine receptor complex, the relative densities differed. Within the regions of the hippocampus the relative levels of gabapentin, NMDA and glycine binding were roughly equal (table 1). In contrast, the high degree of [3H]gabapentin binding measured in the outer layers of the cerebral cortex was not paralleled by a similar level of NMDA or glycine binding. Moreover, in regions such as the substantia nigra and molecular layer of the cerebellum, where [3H]glycine binding was low, relatively high amounts of [3H]gabapentin binding were measured. In fact, within the cerebellum there was a reciprocal distribution of [3H]gabapentin and [3H]glycine binding in the granule and molecular layers. When the data for [3H]gabapentin and [3H]glycine binding summarized in table 1 were analyzed by regression analysis, a correlation coefficient of only 0.48 was obtained indicating a lack of any significant correlation between the relative density of the two sites within the brain areas measured.
3.3. The effect of quinolinic acid lesions on [3H]gabapentin binding As a means of determining whether the gabapentin binding site was located on neuronal cell bodies, the striatum was lesioned unilaterally using the excitotoxin quinolinic acid. The results of these experiments are summarized in fig. 2. In control animals total binding in the left striatum measured 23.0 + 1.2 fmol/mm 2 and non-specific binding was 2.0 + 0.3 fmol/mm 2 (n = 4) and was essentially the same on the sham-lesioned right side which received a saline infusion (total bind-
TABLE 1 Relative distribution of [3H]gabapentin, [3H]glycine and NMDA-sensitive [3H]glutamate binding in rat brain. Data for binding to the gabapentin site are the m e a n from three separate experiments in which 2 - 4 separate sections were analyzed for each region. Data were then normalised with respect to the level of binding in the stratum radiatum. Brain region
[3H]Gabapentin
[3H]Glycine (McDonald et al., 1990)
[3H]Glutamate (Monaghan and Cotman, 1985)
Hippocampus CA1 stratum radiatum Hippocampus CA1 stratum oriens Dentate gyrus molecular layer Parietal cortex I - I I Parietal cortex I V - V I Substantia nigra Nucleus accumbens Globus pallidus Cerebellum molecular layer Cerebellum granule layer
100 68 108 146 99 68 84 33 78 28
100 81 89 61 43 11 43 27 4 30
100 78 65 40 8 61 8 -
306
307 40
3O
SHAM
LESIONED
"o E
2O o~ i0
LHS RHS LHS RHS Fig. 2. The effect of quinolinic acid lesions on [3H]gabapentin binding in rat striatum. Anaesthetized male rats received a unilateral injection of either saline or 200 nmol of quinolinic acid into the striatum. Fourteen days later, the animals were killed and the brains removed for sectioning. Adjacent sections from sham-lesioned and lesioned animals were incubated in parallel with 40 nM [3H]gabapentin with or without 100/xM non-radioactive gabapentin to define non-specific binding. After incubation the sections were washed and dried and apposed to tritium-sensitive film in order to generate the autoradiographs. These were analyzed densitometrically using brain paste standards as a calibration. Total (open bars) and non-specific binding (hatched bars) to the left and right side striatum in sham-lesioned and lesioned animals is shown. Data are the mean values from measurements made using sections taken from four separate animals. Total and non-specific binding were equal in the left and right striatum of sham-lesioned animals and the left (unlesioned) striatum of animals which had received quinolinic acid. By contrast total binding to the lesioned striatum was reduced by more than 65% without any effect on non-specific binding.
ing = 21.6 + 1.9 f m o l / m m 2 ; non-specific binding = 1.5 _+ 0.2 f m o l / m m 2 ) . In the lesioned animals binding to the left striatum was the same as in the control animals (total binding = 22.4 _+ 3.0 f m o l / m m 2 ; non-specific binding = 1.9 +_ 0.9 f m o l / m m 2, n = 4). In contrast, total binding to the striatum that had b e e n treated with quinolinic acid was reduced by 66% to 7.6_+ 1.0 f m o l / m m 2. Non-specific binding was unaffected (1.1 _+ 0.6 f m o l / m m 2 ) .
4. Discussion G a b a p e n t i n is a novel anticonvulsant showing efficacy in rodent models of epilepsy and in man (Bartoszyk et at., 1986; Dooley et al., 1986; Chadwick, 1992). Despite this c o m p o u n d being originally synthesized as a lipophilic G A B A - m i m e t i e , it does not a p p e a r to interact with either G A B A A or G A B A B receptors
either in binding assays or in more functional tests (Bartoszyk and Reimann, 1985; S u m a n - C h a u h a n et al., 1993) and its mechanism of action remains to be resolved, Many anticonvulsants exert their therapeutic effects by modifying the physiological actions of one of the two main classes of amino acid transmitter. For example, barbiturates and benzodiazepines e n h a n c e the actions of the inhibitory neurotransmitter G A B A at G A B A A receptors (see M e l d r u m and Braestrup, 1984). By contrast, the anticonvulsant effects of dizocilpine (MK-801; Clineschmidt et al., 1982), HA-966 and 7chlorokynurinic acid (7-C1-Kyn) are the result of a reduction in the activity of the N M D A receptor complex; MK-801 blocks the N M D A - a s s o c i a t e d channel directly (Kemp et al., 1986)whilst HA-966 and 7-C1-Kyn reduce the facilitatory effect of glycine at the co-agonist site (Kemp et al., 1988; Foster and K e m p 1989). M o r e recently, the n o n - N M D A glutamate antagonist N B Q X has been shown to exert anticonvulsant actions in animal models of reflex epilepsy, presumably by antagonising the excitatory effects of glutamate at the quisqualate ( A M P A ) a n d / o r the kainate receptor (Smith et al., 1991). A n action at the glycine modulatory site has recently been p r o p o s e d for gabapentin. Thus, Sprosen (1990) reported that gabapentin ( 1 - 1 0 /~M) could e n h a n c e the effects of N M D A in cultured striatal neurones whilst higher concentrations antagonised the effects of glycine itself, and suggested that gabapentin was a partial agonist at the glycine co-agonist site. Some support for this came from the work of Oles and colleagues (1990) who d e m o n s t r a t e d that the anticonvulsant effects of gabapentin were antagonised by Dserine, which is an agonist at the glycine site. Despite observations such as these suggesting an action at the glycine r e c e p t o r , g a b a p e n t i n d o e s n o t inhibit [3H]glycine binding to cortical membranes, nor does it modify glycine stimulated [3H]MK-801 binding (D.R. Hill, unpublished observations). A u t o r a d i o g r a p h i c studies which reveal the anatomical localization of ligand binding sites in the brain have helped our understanding of the way such ligands may influence the activity of neuronal systems. A good example comes from the field of excitatory amino acids where the close parallel between the distribution of [3H]glycine and NMDA-sensitive glutamate sites un-
Fig. 1. Localization of [3H]gabapentin binding to horizontal sections of rat brain (upper panel) and its displacement by non-radioactive gabapentin (lower panel). Adjacent sections of rat brain were incubated for 30 min with 40 nM [3H]gabapentin in the presence and absence of non-radioactive gabapentin (100 p,M). After washing and drying the sections were apposed to tritium-sensitive film for the generation of the autoradiographs. On the photographs shown here high levels of [3H]gabapentin binding are represented as white grains. Key: Layers I and II of the cerebral cortex (1,2), fields Cal, Ca2 and Ca3 of Ammon's horn of the hippocampus, dentate gyrus (dg), central gray (cg), granule (gr) and molecular (mol) layers of the cerebellum, lateral septum (Is), white matter (w).
308 derlies the dependence on glycine for activation of the NMDA receptor complex (Bristow et al., 1986; Johnson and Ascher, 1987; Bowery et al., 1988; Maragos et al., 1988; McDonald et al., 1990; Young and Fagg, 1991). Moreover, the uneven distribution of excitatory amino acid receptors is in good agreement with the projection areas of the major excitatory amino acid pathways (see Young and Fagg, 1991). The qualitative distribution of [3H]gabapentin binding sites measured throughout the brain was similar to that of excitatory amino acid receptors including the NMDA receptor as determined by NMDA-sensitive glutamate binding. Furthermore, high levels of [3H]gabapentin binding were found in regions having a major excitatory input such as the striatum, hippocampus and cerebellum. Apart from the outer layers of the cerebral cortex, the highest levels of gabapentin binding sites were found within the hippocampus and dentate gyrus, an area believed to be associated with seizure activity because of its low threshold to stimulation. Moreover, within the hippocampus the level of binding paralleled the terminal fields of the excitatory circuitry. Thus, binding was highest in the area dentata where a major excitatory pathway, arising in the entorhinal cortex (the perforant path), terminates on the dendrites of granule cells (Walaas, 1983). These granule ceils project their excitatory input to the CA3 region of the hippocampus and these cells in turn feed forward to terminate in the CA1 region where binding was again pronounced. Clearly, this pattern of binding suggests the possibility of influencing the excitatory input to this structure. Nonetheless, in extracellular recordings from the CA1 region of hippocampal slices, gabapentin did not influence the development of longterm potentiation (Taylor et al., 1988) which is believed to be due to activation of NMDA receptors (Harris et al., 1984) and reductions in synaptic transmission in the hippocampus were observed only at high (1 raM) concentrations of gabapentin (Haas and Weiser, 1986). Within the cerebellum, binding was also differentiated in a way that could be taken to suggest an association with excitatory fibres. Thus the molecular layer, where the granule cells terminate upon Purkinje dendrites, contained a greater level of binding than the granule cell layer which receives the major mossy fibre input to the structure. Nonetheless, significant levels of binding were still measured in the granule layer. Despite a similarity in the pattern of [3H]gabapentin binding and the NMDA/glycine complex, in terms of their relative quantitative distribution they were quite different. In relative terms, the density of the N M D A / glycine receptor complex has been found to be highest in the CA1 region of the stratum radiatum whereas the highest density of the gabapentin site was found in the outer cortical layers. Proportionately higher levels of [3H]gabapentin binding were present in structures such
as the inner cerebral cortex, the substantia nigra and nucleus accumbens than has been reported for [3H]glycine binding. Moreover, within the cerebellum the distribution of the gabapentin site was the reverse of that reported for [3H]glycine. The difference in relative density was reflected in an almost complete lack of correlation between the relative binding density of the two binding sites as determined from regression analysis, suggesting that despite their qualitatively similar distributions the two sites were not related. Indeed, the distribution of the gabapentin site agreed more closely to that of non-NMDA glutamate receptors, particularly to that of the AMPA site (Nielsen et al., 1990) and a more detailed comparison of the distribution of the two sites is currently in progress. The precise cellular localization of the binding site remains to be determined, but within the hippocampus the distribution would suggest a presence on the dendritic tree of granule and pyramidal neurones. A localization on glial elements cannot be ruled out, although the results from the quinolinic acid lesions suggest that the binding site may be present on the cell bodies of striatal neurones. Lesions produced by this neurotoxin have been shown to selectively destroy neuronal cell bodies whist sparing axons (Foster et al., 1988). Moreover, following the lesion there is a proliferation of glial elements which is reflected in an increased level of binding of [3H]PKll195 to the omega-3 benzodiazepine site (Dubois et al., 1988). One may deduce therefore that the reduction in [3H]gabapentin binding seen following the lesion is more consistent with a localization on neuronal rather than glial elements, although this needs to be determined more directly. In conclusion, we have described the presence of a novel binding site for [3H]gabapentin which is discretely localised in regions of the brain thought to be involved in the genesis of epileptiform activity. The results of the present study demonstrate that there is some similarity between the distribution of the gabapentin binding site and the NMDA receptor but also that quantitatively, the pattern of localization is sufficiently different to suggest that [3H]gabapentin does not label the NMDA/glycine receptor complex. Thus an interaction with this excitatory amino acid receptor may not be responsible for the anticonvulsant effects of gabapentin as has previously been suggested. The mechanism whereby.gabapentin does exert its therapeutic effect and the molecular nature of the gabapentin binding site still remain to be elucidated.
Note added in proof
Since submitting this and our companion paper (Suman-Chauhan et al., 1993) we have found that a number of neutral L-amino acids such as L-leucine (ICs0 =
309
77 nM), L-isoleucine (IC50 = 72 nM), L-methionine (IC50 = 50 nM) and also the system-L inhibitor BCH (2-(-)-endoamino-bicycloheptane-2-carboxylic acid: IC50 = 40 nM) are potent inhibitors of [3H]gabapentin binding. This raises the possibility that [3H]gabapentin labels a site in brain that closely resembles the large neutral amino acid transporter (system-L) that has been characterised elsewhere.
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