Quantitative autoradiographic characterization of the binding of [3H]tiagabine (NNC 05-328) to the GABA uptake carrier

BRAIN RESEARCH ELSEVIER

Brain Research 647 (1994) 231-241

Research Report

Quantitative autoradiographic characterization of the binding of [3H]tiagabine (NNC 05-328) to the GABA uptake carrier P.D. Suzdak a.,, C. Foged b, K.E. A n d e r s e n c Departments of a Receptor Neuroehemistry, b Isotope Chemistry and c Medicinal Chemistry, Novo Nordisk A / S, Pharmaceuticals Research, Novo Nordisk Park, DK-2760 M~lOv, Denmark Accepted 8 February 1994

Abstract

The kinetic properties and regional distribution of [3H]tiagabine ([3,4-3H]N-[4,4-bis(3-methyl-2-thienyl)but-3-en-l-yl]nipecotic acid) binding to the central GABA uptake carrier was examined in the rat brain using quantitative receptor autoradiography. In slide mounted sections of frontal cortex, the binding of [3H]tiagabine was saturable, reversible and sodium dependent. The kinetics of association and dissociation of [3H]tiagabine were monophasic, and Scatchard transformation of saturation isotherms resulted in a linear plot with a K 0 = 58 + 7 nM and a Bmax = 58.9 _ 0.9 pmol/mg protein. The autoradiogaphic distribution of [3H]tiagabine binding sites in rat brain was heterogenously distributed. The highest density of [3H]tiagabine binding sites was present in the cerebral cortex, mammillary body, globus pallidus, substantia nigra pars reticulata, hippocampus, dorsal raph6, superior colliculus (outer layer), and cerebellum. The distribution of GABA uptake sites, as measured by [3H]tiagabine binding, in the rat brain ~s highly consistant with the organization of GABAergic terminals and cell bodies. The present investigation characterized the use of [3H]tiagabine as a novel radioligand for the GABA uptake carrier using quantitative receptor autoradiography. [3H]Tiagabine has several major advantages over the currently utilized radioligand for the GABA uptake carrier [3H]nipecotic acid, in that [3H]tiagabine has an increased affinity, specificity, and is not transported intracellularly via the GABA uptake carrier. These data suggest that [3H]tiagabine represents a novel and highly useful ligand for studying the GABA uptake carrier using quantitative receptor autoradiography. Key words: [3H]Tiagabine; GABA uptake carrier; Autoradiography; Receptor

I. Introduction

y-Aminobutyric acid (GABA) is a major inhibitory amino acid neurotransmitter in the central nervous system (CNS) [9,10,57,60]. It has been estimated that approximately 60-75% of all synapses within the CNS are GABAergic [11]. A decrease in GABAergic neurotransmission may be evoked in the etiology of several neurological disorders including anxiety, epilepsy, pain, depression and Huntington's disease [10,33,50,55]. While numerous investigations have focused on studying changes in the post-synaptic G A B A uptake corn-

* Corresponding author. Novo Nordisk A / S CNS Division Novo Nordisk Park DK-2760 M~10v Denmark. Fax: (45) (44) 66 39 39. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 2 3 3 - 3

plex in tissue from either human brain or from animal models of these disorders, alterations in pre-synaptic GABAergic neurons have not been extensively studied. Although several biochemical markers exist for presynaptic GABAergic neurons (e.g. GABA, glutamic acid decarboxylase (GAD), and the G A B A uptake carrier), methodological problems have greatly limited their usefulness in studying changes in pre-synaptic GABAergic neurons from human tissue or in various animal models of neurological disorders. The activity of GAD, the biosynthetic enzyme for GABA, while stable in human brain for many hours after death [61], is greatly affected by the general health of the patient preceding death. Brain G A B A concentrations, on the other hand, rise rapidly immediately after death [61]. The measurement of G A B A uptake in synaptosomes

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from h u m a n or a n i m a l b r a i n requires freshly p r e p a r e d tissue and is not suitable for studying small b r a i n nuclei. Recently, the b i n d i n g of the G A B A u p t a k e inhibitor, [3H]nipecotic acid, to the G A B A u p t a k e carrier has b e e n proposed as a m a r k e r to study presynaptic G A B A e r g i c n e u r o n s [7,37]. Nipecotic acid is a weak inhibitor of G A B A u p t a k e in synaptosomes (IC50 = 2000 n M ) [5], a n d b i n d s with low affinity to the G A B A u p t a k e carrier ( K a = 2600 n M ) [37]. [3H]Nipecotic acid b i n d i n g has b e e n shown to be altered in h u m a n postm o r t u m b r a i n tissue from p a t i e n t s with A l z h e i m e r ' s disease [58] or H u n t i n g t o n ' s d i s e a s e [7]. However, several methodological p r o b l e m s limit the usefulness of [3H]nipecotic acid. Initial studies with [3H]nipecotic acid b i n d i n g used filtration to harvest the receptor isotope complex [37] which may have greatly u n d e r e s t i m a t e d the n u m b e r of G A B A u p t a k e sites due to the low affinity of [3H]nipecotic acid for the G A B A u p t a k e carrier ( K a = 2600 nM). Recently, C z u d e k a n d Reynolds [7] i n t r o d u c e d a c e n t r i f u g a t i o n step for harvesting the G A B A u p t a k e isotope complex, thus increasing m e a s u r a b l e binding. However, nipecotic acid, in a d d i t i o n to b i n d i n g to the G A B A u p t a k e carrier, is also a substrate for the G A B A u p t a k e carrier, b e i n g t a k e n up by and s u b s e q u e n t l y released by G A B A e r g i c n e u r o n s [33], thus complicating the i n t e r p r e t a t i o n of the results o b t a i n e d with [3H]nipecotic acid. T h e p r e s e n t r e p o r t c h a r a c t e r i z e s the use of [3H]tiagabine, a p o t e n t a n d selective G A B A uptake inhibitor, as a novel ligand to study presynaptic G A B A e r g i c n e u r o n s . U n l i k e [ 3 H ] n i p e c o t i c acid, [3H]tiagabine b i n d s with high affinity ( K a = 18 n M ) to the G A B A u p t a k e carrier [5], a n d is not a substrate for the G A B A u p t a k e carrier [5], thus m a k i n g [3H]tiagabine a suitable ligand for the G A B A u p t a k e carrier.

dried with cold air. The sections were then incubated for 90 min at 25°C in 50 mM Tris-citrate buffer containing: 1 M NaCl (pH 7.4), varying concentrations (1 to 200 nM for Scatchard analysis, or 5 nM for single point determinations) of [3H]tiagabine, and buffer or nipecotic acid (300 mM) to define non-specific binding. The sections were then washed three times with 4 ml of Tris-citrate buffer at 0°C. For association experiments, sections were incubated with 5 nM [3H]tiagabine for up to 175 min. For dissociation experiments, sections were incubated with 5 nM [3H]tiagabine for 90 rain at 25°C. Dissociation was initiated by the addition of 300 mM nipecotic acid. The slices were then blown dry and placed in an X-ray cassette along with radioactive standards and exposed to tritium-sensitive film for 7 days. The optical densities of the resulting film images were determined using an Amersham RAS-R1000 image analyzer. The radioacitivity was determined by a computer-generated polynomial regression analysis which compared film densities produced by the tissue sections to those produced by the radioactive standards. All data presented were analyzed densitometrically from autoradiographic images. Photographs of autoradiograms were prepared from images digitized on the RAS-R1000 video processing system. KD, Bm~X, and K i values were generated by the Kinetic, EBDA, LIGAND, and Lowry program (Elsevier-Biosoft). Under the incubation conditions described above, < 2% of [3H]tiagabine was subject to degradation. Following the termination of the incubation, acetonitrile was added, the mixture centrifuged, and both the organic and aqueous phase of the supernatant was injected onto a HPLC column to determine radiochemical purity. The recovery of radioactivity in this assay was > 95%. HPLC analysis was performed using a Merck L-6200 pump with a rheodyne injector and a Merck UV-detector (operating at 214 nM). Separations were accomplished with a C-18 column (250×4.6, 5 p.M) using an eluent of A: 0.1% TFA/acetonitrile 90/10, B: 0.1% TFA/ acetonitrile 10/90. A gradient system A/B: 80/20 to A/B: 40/60 in 20 min was used at a flow rate of 1.0 ml/min. Radioactivity in the column effluent was monitored with a Radiomatic/Canberra Flo-One beta detector A-200, using a 500 /,1 liquid flow cell. Tba ratio of column effluent to liquid scintillator (Pico-aqua, Packard) was 1:2. Data collection was done by Flo-One data software on a PC-XT computer.

3. Results 3.1. Binding characteristics

2. Methods 2.1. Materials

[3H]Tiagabine [[3,4-3H]1N-(4,4-bis(3-methyl-2-thienyl)but-3-en-1yl)nipecotic acid (specific activity 44 Ci/mmol) was purchased from Amersham (UK) [5,64]. 2.2. Tissue preparations

Male Wistar rats (150-180 g) were decapitated, and the brains quickly removed, mounted with Tissue-Tek embeding matrix on a specimen stage, and frozen under powdered dry ice. The frozen brains were cut into 20/,m horizontal sections at - 15°C on a Leitz crystal and thaw mounted onto chrome alum/gelatin-subbed slides. Sections were stored for less than 24 h at -20 °. 2.3. Autoradiography

For binding studies, tissue sections were preincubated for 30 min at 25°C in 50 mM Tris-citrate buffer (pH 7.4), and subsequently

P r e l i m i n a r y b i n d i n g studies were carried out to det e r m i n e optimal buffer, p H a n d the effects of salts, t e m p e r a t u r e , a n d i n c u b a t i o n time. Initial e x p e r i m e n t s carried out in slide m o u n t e d sections of frontal cortex revealed that the best b i n d i n g was p r o d u c e d in 50 m M tris citrate buffer ( p H 7.4), c o n t a i n i n g 1 M NaCl, i n c u b a t e d at 25°C for 90 min. T h e kinetic a n d steadystate characteristics of [3H]tiagabine b i n d i n g was det e r m i n e d using s l i d e - m o u n t e d sections of frontal cortex (kinetic a n d steady-state characterization of [3H]tiagab i n e b i n d i n g was also d e t e r m i n e d in the s u b s t a n t i a nigra, pars reticular a n d inferior colliculus, yielding s i m i l a r results). F o l l o w i n g tissue p r e i n c u b a t i o n , [3H]tiagabine b i n d i n g increased with increasing incub a t i o n times (see Fig. 1). Steady-state was r e a c h e d by 75 min of i n c u b a t i o n at 25°C. Specific b i n d i n g was stable at i n c u b t i o n times up to 175 m i n (the longest

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Fig. 2. Association curve of 5 nM [3H]tiagabine to slide-mounted sections of frontal cortex. Total and non-specific binding were determined at each point. The data are expressed as the percentage of total specific binding at equilibrium. B e is the binding equilibrium, and Kob S was determined from the slope of In (B e / B e - B ) versus time. The data represent the mean of 5 experiments carried out in triplicate.

3.3. Cerebral cortex

time point measured). Association and dissociation experiments were carried out using 5 nM [3H]tiagabine (see Figs. 2 and 3). The best fit to the experimental values was monoexponential on-kinetics with Kobs of 0.009 m i n - 1. This binding was fully reversable (see Fig. 3) and the off-kinetics were apparently monoexponential with K.~ of 0.008 m i n - l . Calculation of K~ to 0.0002 M-~ min-1 gave a K o --- 40 nM. [3H]tiagabine labeled high-affinity binding sites in the rat frontal cortex in a saturable manner (Fig. 4A). Scatchard analysis of saturation data (1 to 200 nM) indicated that [3H]tiagabine bound to a single population of binding sites (see Fig. 4B) with a K D = 58 + 7 nM and a Bmax = 58.9 ___0.9 p m o l / m g protein. Nonspecific binding (determined in the presence of 300 mM nipecotic acid) was linear over the range of [3H]tiagabine concentrations used and was 15 to 25% of the total binding at 5 nM. Similar results were obtained in parallel experiments using 30/xM tiagabine to define non-specific binding.

The binding of [3H]tiagabine in the cerebral cortex was evenly distributed. A high density of [3H]tiagabine binding sites was seen in the dorsal prefrontal cortex, ventral prefrontal cortex, motor cortex, sensory cortex, pyriform cortex, parietal cortex, auditory cortex, striate cortex 17, striate cortex 18, entorhinal cortex, frontal motor cortex, temporal cortex, retrospenial cortex, frontal somatosensory cortex, posterior cingulate cortex and cingulate cortex. A medium density of [3H]tiagabine receptors was seen in the anterior cingu-

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TIME (MINUTES) The distribution of [3H]tiagabine binding in the rat central nervous system is shown in Table 1 and Fig. 5. In the descriptions that follow, specific terms are used to describe the following ranges of binding densities: high ( > 3 5 0 f m o l / m g protein); moderate (251-350 f m o l / m g protein); low ( < 225 f m o l / m g protein).

Fig. 3. Dissociation curve of 5 nM [3H]tiagabine to slide-mounted sections of frontal cortex. Total and non-specific binding were determined at each point. The data are expressed as the percentage of total specific binding at equilibrium, and K_ l was determined from the slope of In ( B / B o ) , where Bo is the amount bound at equilibrium. The data represent the mean of 5 experiments carried out in triplicate.

P.D. Suzdak et al. / Brain Research 647 (1994) 231-241

234

late cortex, olfactory cortex, medial prefrontal cortex, and retrospinal cortex. 3.4. Limbic system A high density of [3H]tiagabine binding sites was seen in the mammillary body, olfactory bulb, hippocampus dentate, hippocampus dentate molecular layer, hippocampus dendritic zone, CA 3 of the hippocampus, and medial amygdala. A medium density of [3H]tiagabine binding sites was seen in the CA1 of the hippocampus and lateral amygdala. A low density of [3H]tiagabine binding sites was seen in the nucleus accumbens, CA4 of the hippocampus, fornix and central amygdala.

3. 7. Monoaminergic nuclei A high density of [3H]tiagabine binding sites was seen in the substantia nigra pars reticulata, ventra tegmentum, and dorsal raphe. A medium density of [3H]tiagabine binding sites was seen in the substantia nigra pars compacta, median raphe, and locus coeruleus.

3.8. Medial basal cholinergic nuclei A medium density of [3H]tiagabine binding sites was seen in the medial septum and diagonal band.

3. 9. Other diencephalic structures 3.5. Extrapyramidal regions A high density of [3H]tiagabine binding sites was seen in the globus pallidus, and a low density of [3H]tiagabine binding sites was seen in the caudate. 3.6. Thalamus A high density of [3H]tiagabine binding sites was seen in the paraventricular thalamic nucleus anterior and posterior regions. A medium density of [3H]tiagabine binding sites was seen in the lateral habenula, medial habnula, anteroventral nucleus, central medial thalamic nucleus, and lateral posterior thalamic nucleus. A low density of [3H]tiagabine binding sites was seen in the medial geniculate, ventrolateral nucleus, lateral ventroposterior nucleus, dorsal lateral geniculate, ventral lateral geniculate, and ventroposterior thalamic nucleus.

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3.10. Brainstem A high density of [3H]tiagabine binding sites was seen in the interpendicular nucleus, periaqueductal gray, and dorsal tegmental nucleus. A medium density of [3H]tiagabine binding sites was seen in the vestibular nucleus and ventral tegmental area. A low density of [3H]tiagabine binding sites was seen in the raphe pontis, raphe magnis, and cochlear nucleus.

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A high density of [3H]tiagabine binding sites was seen in the medial hypothalamus and superior colliculus outer layer. A medium density of [3H]tiagabine binding sites was seen in the medial preoptic area, lateral preoptic area, lateral hypothalamus, superior colliculus inner layer, and inferior colliculus. A low density of [3H]tiagabine binding sites was seen in the corpus callosum and lateral septum.

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P.D. Suzdak et al. / Brain Research 647 (1994) 231-241 Table 1 Quantitative autoradiographic determination of the anatomical distribution of [3H]tiagabine sites Distribution of [3H]tiagabine binding sites in the rat brain Brain region

Cerebral cortex Anterior cingulate cortex Olfactory cortex Dorsal prefrontal cortex Ventral prefrontal cortex Medial prefrontal cortex Motor cortex Sensory cortex Pyriform cortex Parietal cortex Auditory cortex Striate cortex 17 Striate cortex 18 Entorhinal cortex Retrospenial cortex Temporal cortex Frontal motor cortex Frontal somatosensory cortex Posterior cingulate cortex Cingulate cortex Frontal cortex Limbic system Mamillary body N accumbens Olfactory tubercle Hippocampus dentate Hippocampus dentate mol lyr Hippocampus CAI, pyr lyr Hippocampus CA3, pyr lyr Hippocampus CA4, pyr lyr Fornix Medial amygdala Central amygdala Lateral amygdala Extrapyramidal regions Caudate putamen Globus pallidus Thalamus Lateral habenula Medial habenula Medial geniculate Anteroventral nucleus Central medial thalamus nucleus Ventrolateral nucleus Lateral ventroposterior nucleus Lateral posterior nucleus Dorsal lateral geniculate Ventral lateral geniculate Paraventricular thalamic nucleus, anterior Paraventricular thalamic nucleus, posterior Centroposterior thalamic nucleus, medial Lateral dorsal thalamic nucleus

[3H]Tiagabine bound (fmol/mg protein) 346 ± 23 323 ± 25 393 ± 49 364 ± 21 329 ± 35 388 ± 55 373 ± 48 374 ± 39 352± 6 399 ± 10 395 ± 18 381 _+35 365 ± 17 329 ± 38 394 ± 26 378 ± 19 402 ± 25 352 ± 16 354 ± 34 320 ± 12 396 _+79 184 ± 57 352 ± 67 352 ± 41 352 ± 25 329 ± 27 414 ± 55 224 ± 8 166 ± 20 411 ± 11 282 ± 46 329 ± 24 181 ± 16 280 ± 19 326 ± 41 313 ± 66 198 ± 66 228 ± 19 301 ± 90 209 ± 30 156 ± 35 327 ± 33 212 ± 38 200 ± 20 367 ± 58

235

Table 1 (continued) Distribution of [3H]tiagabine binding sites in the rat brain Brain region

[3H]Tiagabine bound (fmol/mg protein)

Monoaminergic nuclei Substantia nigra, pars compacta Ventral tegmentum Dorsal raphe Medial raphe Locus coeruleus

310 ± 30 456 + 25 429 ± 53 414± 12 282 ± 35 369 ± 51

Medial basal cholinergic nuclei Medial septum Diagonal band

283 ± 20 388 ± 99

Other diencephalic structures Medial preoptic area Lateral preoptic area Corpus callosum Lateral septum Lateral hypothalamus Medial hypothalamus Superior colliculus, inner layer outer layer Inferior colliculus

322 ± 60 335 ± 28 28± 2 164 ± 50 323 ± 59 360 ± 36 285 ± 41 441 ± 50 266 ± 28

Brainstem Interpendicular nucleus Dorsal tegmental nucleus Raphe pontis Raphe magnis Ventral tegmental area

402 ± 95 429 ± 11 249 ± 30 93 _+43 332 ± 87

Spinal cord Dorsal horn Ventral horn

192 ± 10 41 ± 31

Cerebellum Granule cell layer Molecular layer

155 ± 11 355 ± 27

[3H]Tiagabine binding was determined as described in section 2. Data represent the mean +_S.E.M. (n = 6).

3.11. Spinal cord A low density of [3H]tiagabine binding sites was seen in the dorsal and ventral horn.

3.12. Cerebellum

374 + 44 166± 7 225 ± 12

A high density of [3H]tiagabine binding sites was seen in molecular cell layer, while a low density of [3H]tiagabine binding sites was seen in the granule cell layer.

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P.D. Suzdak et al. / Brain Research 647 (1994) 231-241

4. Discussion

Tiagabine has been previously shown to potently inhibit GABA uptake in synaptosomes, neurons and glial cells in cell culture [5]. Tiagabine also lacks a significant affinity for other neurotransmitter receptor or uptake sites [5]. Unlike previously described GABA uptake inhibitors, such as nipecotic acid, tiagabine readily crosses the blood brain barrier and produces potent anticonvulsant effects [43]. We have previously characterized the use of [3H]tiagabine as an in-vitro ligand for the GABA uptake carrier in synaptosomes [5] and in in-vivo binding experiments [64]. The present report describes the use of [3H]tiagabine as a ligand for receptor autoradiographic studies of the GABA carrier. 4.1. Binding characterization

The binding of [3H]tiagabine to synaptosomes has been previously demonstrated to be saturable, reversible and sodium-dependent [5]. The binding of [3H]tiagabine to synaptosomes has also been shown to be of high affinity (K D = 18 nM) and of large capacity (Bmax = 669 p m o l / g tissue) [5]. In the present study, the binding of [3H]tiagabine to slide-mounted brain sections was characterized. The binding of [3H]tiagabine was dependent upon both the temperature of incubation and the concentration of sodium present. Maximal binding was obtained in the presence of 1 M NaC1 at 25°C. The association and dissociation experiments confirm the high affinity and demonstrate that [3H]tiagabine binding is reversable. [3H]Tiagabine bound to slide-mounted sections of frontal cortex with high affinity (K d = 58 + 7 nM) and high capacity (Bmax = 58.9 p m o l / m g protein). 4.2. Regional distribution

The distribution pattern of [3H]tiagabine binding sites in the rat brain in the present study is highly consistent with the organization of GABAergic terminals and cell bodies as previously identified by a variety of methods including the immunohistochemical localization of GAD [41], the immunohistochemical localizaion of the GABA transporter [49], the distribution of m R N A encoding for GAD [42], and the autoradiographic distribution of GABA A and GABA B receptor

237

binding sites [3]. However, in areas such as he hippocampus, dentate gyrus, substantia nigra pars reticulate and substantia nigra pars compacta, which appear to contain a medium to high density of the GABA uptake carrier [49], previous studies examining the localization of GAD [41,42] have suggested that only a minority of the neurons contain GAD. This difference in the ratio between GAD and the GABA transporter may be expected due to regional differences in GABA turnover and GABA release [62]. In addition, the GABA uptake carrier is also present on glial cell bodies which do not contain GAD [49]. The autoradiographic distribution of [3H]tiagabine binding sites in the rat brain was heterogeneously distributed. The highest density of the GABA uptake carrier was present in the cerebral cortex, mammillary body, globus pallidus, substantia nigra pars reticulate, hippocampus, ventral tegmental area, dorsal raphe, superior colliculus (outer layer), interpendicular nucleus and cerebellum (molecular layer). 4.3. Cerebral cortex

The cerebral cortex contained a high density of [3H]tiagabine binding sites. These data are consistent with the immunohistochemical localization of GAD [41], the GABA transporter [49], and the distribution of messenger RNA encoding GAD [42]. The cerebral cortex contains high levels of GABA A and GABA B receptors [3], benzodiazepine receptors and TBPS binding sites [69]. Previously we have demonstrated a high concentration of [3H]tiagabine binding sites in the frontal cortex, motor cortex, parietal cortex and entorhinal cortex in-vitro in rat brain synaptosomes [5], or using [3H]tiagabine in-vivo binding [64]. lmmunocytochemical data have also demonstrated a high level of GABAergic inter-neurons within the cerebral cortex [25]. GABA has been shown to play a major role in the control of neuronal activity in the cortex. GABA has been shown to mediate inhibition of spontaneous cortical neuronal activity produced by electrical stimulation of adjacent cortical areas [31,32]. In addition, GABA B receptor activation appears to regulate neurotransmitter release in the cerebral cortex [2,65]. Within discrete areas of the cerebral cortex, GABA has been suggested to be involved in the anxiolytic a n d / o r anticonsulsant action of benzodiazepine agonists [8,9,23,60]. This is consistant with the potent anticonvulsant effects pro-

Fig. 5. Autoradiographicdistributionof [3H]tiagabinebindingsites in the adult rat CNS. Imageswere generated by computerizedsubtraction of autoradiogramsobtained from sectionsin the presence, or absence, of 300/~M nipecotic acid. ENT, entorhinal cortex; CPU, caudate putamen; CA1, field CA1 of the hippocampus; FrPS, frontal somatosensorycortex; CC, corpus callosum;LD, laterodorsal thalamicnucleus; DLG, dorsal lateral geniculate; IC, inferior colliculus;SNR, substantia nigra pars reticular; Acb, accumbens nucleus; Fr, frontal cortex; FrPm, frontal motor cortex; SUG, superior colliculus;Str 18, striate cortex, area 18.

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duced by GABA uptake inhibitors such as tiagabine [43].

4.4. Limbic system The limbic system contained areas of high, medium and low density of [3H]tiagabine receptors. The hippocampus and dentate gyrus showed medium to high levels of [3H]tiagabine sites. The immunohistochemical localization [41] and messenger RNA localization of GAD also demonstrate the presence of GAD in the hippocampus and dentate gyrus [42]. However, these studies suggest that only a minority of the neurons contains GAD in the hippocampus and dentate gyms. Immunohistochemical localization of the GABA transporter suggests a high density in the hippocampus and dentate gyrus [49]. Differences in the ratios of GAD and the GABA uptake carrier may be expected due to regional differences in GABA turnover and GABA release [62]. In addition, the GABA uptake carrier is also present on glial cell bodies which do not contain GAD [49]. The hippocampus contains high levels of benzodiazepine, TBPS and GABA receptors [69]. In addition, high levels of [3H]tiagabine binding sites have been demonstrated in the hippocampus in both in-vitro synaptosomes [5] and in-vivo binding [64] experiments. The hippocampus appears to be involved in anxiety, memory, and anxiolytic drug action [21] mediated in part through GABAergic neurotransmission. The high density of [3H]tiagabine binding sites present in thc hippocampus may explain the memory impairment seen in rodents with high doses of GABA uptake inhibitors [3(I]. The molecular layer of the hippocampus and dentate gyrus are rich in GABA, benzodiazepine and GABA uptake sites [69]. The clear laminar structure of the hippocampus with its known neuronal circuits and at least one established GABAergic inhibitory interneuron, the basket cell, which inhibits the hippocampal output-forming pyramidal neurons [62]. The recurrent inhibition of dentate granular cells presumably is also mediated by GABAergic basket cells [1]. The medium to high levels of [3H]tiagabine binding sites present in the hippocampus and dentate gyrus are consistant with the blockage of the cerebral ischemiainduced loss of hippocampal CAI pyramidal cells produced by systemic administration of tiagabine [29]. In addition, tiagabine has been shown to increase the amplitude and duration of response to exogenous GABA in pyramidal neurons of the CA1 region in rat hippocampal slices [5 i].

4.5. Extrapyramidal regions The extrapyramidal regions examined contained a high to low density of [3H]tiagabine binding sites. These data are in agreement with the localization of messen-

ger RNA for GAD [42] and the binding of [3H]nipecotic acid [37]. The globus pallidus contains a high density of benzodiazepine GABA A and GABA B receptors [69]. The globus pallidus also contains a high concentration of GABA [61] and of [3H]nipecotic acid binding sites [7], which may result from the dense GABAergic input from the striatum and nucleus accumbens [56].

4.6. Monoaminergic nuclei Monoaminergic nuclei contain a moderate to high level of [3H]tiagabine binding sites. A higher density of [3H]tiagabine sites was found in the substantia nigra, pars reticulata as compared to substantia nigra, pars compacta. These data correlate well with the immunocytochemical localization of the GABA uptake carrier [41], the presence of mRNA for G A D [42], and the distribution of GABA A and GABA B receptors [3,69]. However, the absolute difference in [3H]tiagabine binding sites between the substantia nigra pars reticulata and substantia nigra pars compacta does not correlate with the three fold difference seen when measuring GAD. The substantia nigra contains a high level of benzodiazepine [54], GABA A and GABA B [3,69] receptors, as well as the presence of extensive GABAergic nerve terminals and cell bodies [53,45]. Several afferent inputs to the substantia nigra from the striaturn and nucleus accumbens have been shown to be GABAergic in nature [15]. GABA has been shown to inhibit the nigral projections from the substantia nigra pars reticulata [34,46,47]. It has been suggested [17] that GABA afferents to the substantia nigra inhibit efferent neurons that normally permit or facilitate the generalization of seizures. The substantia nigra does not appear to a site for initiation of seizures, but rather a gating mechanism for seizure spread. This is consistent with the lack of seizure production following intra nigral injection of GABA antagonists, or depletion of GABA in the substantia nigra [40] and the supression of seizures following the application of GABA, GABA agonists or the elevation of extracellular GABA in the substantia nigra [16,19,20,26,39,67]. Thus, the net result of increasing GABAergic activity in the substantia nigra is disinhibition of target neurons in the thalamus, tectum and tegmentum [17]. The GABA uptake carrier may play a critical role in regulating the overall GABAergic tone within the substantia nigra, and this may be the reason why GABA uptake inhibitors such as tiagabine are highly efficacous anticonvulsants. The dorsal raph6 nucleus, which contains serotonergic cell bodies, has widespread projections in the limbic forebrain, and locus coeruleus [21,27,52], and has been implicated in anxiety and stress. The dorsal raph6 nucleus contains an intermediate density of benzodiazepine, GABA A and GABA B receptors [3,66,69].

P.D. Suzdak et al. / Brain Research 647 (1994) 231-241

Within the dorsal raph6 nucleus G A B A may regulate the output of serotonin. G A B A has been shown to exert an inhibiting influence on central serotonergic neurons emanating from the raph~ nucleus resulting in a decrease in serotonin synthesis, release and metabolism, as well as, a decrease in the firing rate of raph(~ neurons [18,44]. 4. 7. O t h e r diencephalic structures

The inferior colliculus contained a moderate level of [3H]tiagabine binding sites. A similar moderate level of G A D m R N A [42] and G A B A A and G A B A n receptors [3] was found in the inferior colliculus. The inferior colliculus contains a high level of benzodiazepine [3,69], TBPS and G A B A receptors [69]. The inferior colliculus has been implicated in the mechanism leading to audiogenic seizures in genetic epilepsy prone rats [68]. G A B A has also been proposed as the neurotransmitter involved in the sound-induced inhibition of ceils within the inferior colliculus [12-14], and this may underlie the epileptogenesis in this genetic form of epilepsy [12]. Tiagabine has been shown to potently inhibit sound-induced convulsions in the genetic epilepsy prone rat following microinjection into the inferior colliculus [14]. 4.8. Cerebellum

Within the cerebellum, the granule cell layer contained a low level and the the molecular layer contained a high level of [3H]tiagabine binding sites. This is not surprising considering the extensive GABAergic innervation in the cerebellum. Four out of the five major cell types within the cerebellum are GABAergic. A high level of G A D [41], G A D m R N A [42] and G A B A A and G A B A B receptors [3] are also found in the granule and molecular layer of the cerebellum. The cerebellar cortex has a well defined neuronal circuit with three GABA-releasing inhibitory interneurons (basket, stellate and golgi ceils) and the purkinje cells as the cerebellar output neurons which by themselves release G A B A on deep cerebellar nuclei and the lateral vestibular nucleus. The molecular layer of the cerebellum has been shown to contain a high density of benzodiazepine binding sites [3,69] and G A B A receptors [3,69].

5. Conclusions The present study characterized the distribution of the G A B A u p t a k e carrier in the CNS, using [3H]tiagabine receptor autoradiography. The binding of [3H]tiagabine was heterogeneously distributed within the CNS. [3H]Tiagabine may be a useful ligand for studying changes in the G A B A uptake carrier in dis-

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ease states such as epilepsy where changes in the functioning of the neuronal G A B A uptake carrier may be involved. Recently, three G A B A uptake carriers have been cloned from rat brain [6,22], and it appears that nipecotic acid-based compounds, like tiagabine, may be selective for the GAT-1 transporter. The present data demonstrate that [3H]tiagabine is a high-affinity ligand for the central G A B A uptake carrier using receptor autoradiographic techniques. [3H]Tiagabine autoradiography may be a useful technique for studying changes in GABAergic function, which appears to offer several advantages over [3H]nipecotic acid (including affinity, selectivity, and the inability to be transported intracellularly through the G A B A uptake carrier). Thus, [3H]tiagabine is a novel and highly useful ligand for studying the G A B A uptake carrier using receptor autoradiography techniques.

Acknowledgements The technical assistance of Lisbeth Eriksen, Marianne L. Jacobsen and Jette Platou is highly appreciated. Tine K. Hansen is also thanked for help in the preparation of the manuscript.

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