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An in situ hybridization study of the distribution of the GABAB2 protein mRNA in the rat CNS
Molecular Brain Research 71 Ž1999. 185–200 www.elsevier.comrlocaterbres
Research report
An in situ hybridization study of the distribution of the GABA B2 protein mRNA in the rat CNS Margaret M. Durkin ) , Caryn A. Gunwaldsen, Beth Borowsky, Kenneth A. Jones, Theresa A. Branchek Department of Pharmacology, Synaptic Pharmaceutical Corporation, 215 College Road, Paramus, NJ 07652, USA Accepted 25 May 1999
Abstract g-Aminobutyric acid ŽGABA. is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA exerts its actions through two classes of receptors: GABA A , multimeric ligand-gated Cly ion channels Ža class which has been proposed to include the homomeric variant previously called GABA C , to be designated GABA A0r .; and GABA B , G-protein coupled receptors which regulate Ca2q and Kq channels. Currently, within the GABA B receptor family two proteins have been identified through molecular cloning techniques and designated GABA B1 and GABA B2 . Two N-terminal variants of GABA B1 were isolated and designated GABA B1a and GABA B1b . The distribution of neurons in the rat CNS expressing the mRNA for the GABA B1 isoforms have been previously described by in situ hybridization histochemistry. The recent isolation and identification of the GABA B2 protein by homology cloning has enabled the use of radiolabeled oligonucleotides to detect the distribution of the expression of GABA B2 mRNA in the rat CNS. The expression of GABA B2 mRNA was observed to be primarily related to neuronal profiles. The highest levels of GABA B2 mRNA expression were detected in the piriform cortex, hippocampus, and medial habenula. GABA B2 mRNA was abundant in all layers of the cerebral cortex, the thalamus and in cerebellar Purkinje cells. Moderate expression was observed in several hypothalamic and brainstem nuclei. In contrast to the distribution of GABA B1 mRNA, only a weak hybridization signal for GABA B2 was detected over cells of the basal ganglia, including the caudate–putamen, nucleus accumbens, olfactory tubercle and throughout most of the hypothalamus. Moderate-to-heavy GABA B2 mRNA expression was also seen over dorsal root and trigeminal ganglion cells. In general, the pattern of GABA B2 mRNA expression in the rat brain overlaps considerably with the distributions described for both GABA B1 mRNAs, and is concordant with the distribution described for GABA B receptor binding sites. However, differences between GABA B2 expression levels and GABA B binding sites were observed in the basal ganglia. q 1999 Elsevier Science B.V. All rights reserved. Keywords: GABA; GABA B2 protein; In situ hybridization; Rat CNS
1. Introduction g-Aminobutyric acid ŽGABA. is the main inhibitory neurotransmitter in the mammalian CNS. GABA exerts its actions through two classes of receptors: GABA A , multimeric ligand-gated Cly ion channels Ža class which has been proposed to include the homomeric variant previously called GABA C , to be designated GABA A0r w5x.; and GABA B , G-protein coupled receptors which regulate Ca2q and Kq channels. GABA receptors have been distinguished pharmacologically w5,12,14,15,22x, electrophysio-
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logically w13,29,56x, and more recently through molecular cloning w14,27,37,42,52,58x. A ubiquitous distribution of GABA A and GABA B receptor binding sites in the rat CNS has been described by receptor autoradiographic studies employing 3 H-GABA or 3 H-baclofen as ligands w9,10,16,21x. The distribution of the GABA A and GABA A0r receptor messenger RNA expression in the rat CNS and chick retina, respectively, has been described using in situ hybridization histochemistry w1,55,64x. Heterogeneity within the GABA B receptor family has been suggested on the basis of pharmacological studies w11,12,24,41,51,57 x. Recently, two isoforms of the GABA B1 receptor were cloned and designated GABA B1a and GABA B1b w37x, differing only in the length of their
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Table 1 The distribution of GABA B2 mRNA in the rat CNS. The strength of the hybridization signal observed in various regions of the rat brain was graded as weak Žq., moderate Žqq ., heavy Žqqq . or intense Žqqqq . Region Olfactory system Internal granule layer Glomerular layer External plexiform layer Mitral cell layer Anterior olfactory n. Olfactory tubercle Islands of Calleja Piriform cortex N. of the lateral olfactory tract Cortex Frontal Agranular insular Cingulate Retrosplenial Parietal Occipital Entorhinal Dorsal endopiriforn n. Basal ganglia and basal forebrain Accumbens nucleus Caudate–putamen Globus pallidus Ventral pallidum Horizontal diagonal band Claustrum Subthalamic n. Substantia nigra, reticular part Substantia nigra, compact part Septal region Medial septum Lateral septum Septohippocampal n. Amygdala Lateral n. Basolateral n. Medial amygdaloid n. Basomedial n. Central n. Anterior cortical n. Posteromedial cortical n. Bed n. stria terminalis Hippocampus Field CA1 Fields CA2rCA3 Taenia tecta Induseum griseum Dentate gyrus Polymorphic dentate gyrus Subiculum PrerParasubiculum Hypothalamus Suprachiasmatic n. Median preoptic area Paraventricular n. Arcuate n. Anterior hypothalamus, posterior Lateral hypothalamus Ventromedial n.
Density q q y y qqq q y qqq qq qqq qqq qqq qqq qqq qqq qqq qqq q q q qq qq qq qq q qq qq qq q qqq qqq qqq q qq qq qq qq qq qqqq qqq q qqqq qqq q qq q q qq qq q q qq
Region
Density
Dorsomedial n. Periventricular n. Supraoptic n. Supramammillary n. Premammillary n. Medial mammillary n. Thalamus and epithalamus Anterodorsal n. Laterodorsal n. Paraventricular n. Centromedial n. Paracentral n. Parafascicular n. Reuniens n. Rhomboid n. Ventrolateral n. Ventromedial n. Ventral posterolateral n. Reticular n. Lateral geniculate n. Medial geniculate n. Medial habenular n. Lateral habenular n. Midbrainr Pons r Medulla Superior colliculus Inferior colliculus Periaqueductal gray Dorsal raphe Deep mesencephalic n. Pontine n. Retrorubral field Ventral tegmental area Mesencephalic trigeminal n. Motor trigeminal n. Interpeduncular n. Raphe magnus Raphe pallidus Spinal trigeminal n. Pontine reticular n. Parvicellular reticular n. Locus coeruleus Parabrachial n. Vestibular n. Gigantocellular reticular n. Prepositus hypoglossal n. Ventral cochlear n. N. soltary tract A5 noradrenaline cells Facial n. Ž7. Cerebellum Granule cell layer Purkinje cells Deep cerebellar n. Spinal cord Dorsal horn Ventral horn Peripheral ganglia Trigeminal ganglion Dorsal root ganglion
qq q q qq q q qqq qqq qq qq qq qq qqq qqq qqq qqq qqq q qqq qq qqqq q qq q qq qq q qqq qq q qq q qq qq q q qqq q q q q qq q qq qq q q q qqq q q q qqq qqq
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amino terminal sequences. A similarly ubiquitous and overlapping distribution of message expression has been described in the rat brain for the GABA B1 splice variants w10x. We have recently reported w34x the isolation of an additional GABA B protein ŽGABA B2 ., which exhibits approximately 38% overall sequence homology to GABA B1 . In addition, recent reports demonstrated that the co-expression of GABA B1 and GABA B2 results in a single pharmacologically and functionally defined receptor with signal transduction properties similar to the native GABA B receptors w34,38,40,63x. These observations prompted us to explore the in vivo co-localization in neurons of the GABA B2 and GABA B1b genes. In the present study, the distribution of the GABA B2 messenger RNA expression in the rat CNS has been examined using radiolabeled oligonucleotide probes.
2. Materials and methods 2.1. Animals The Synaptic Pharmaceutical Animal Care and Use Committee approved all protocols. Male Sprague–Dawley rats Ž200–250 g, Charles Rivers, Rochester, NY. were euthanized using CO 2 , decapitated, and their brains immediately removed and rapidly frozen on crushed dry ice. Coronal sections of brain tissue were cut at 11 mm using a cryostat, thaw-mounted onto poly-L-lysine-coated slides, and stored at y208C until use. 2.2. Tissue preparation Prior to hybridization, the tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline ŽPBS. pH 7.4 ŽSpecialty Media, Lavallette, NJ. followed by two washes in PBS. Tissues were then treated in 5 mM dithiothreitol, rinsed in DEPC Ždiethyl pyrocarbonate.-treated PBS, acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride, rinsed twice in 2 = SSC Žstandard saline citrate: 1 = SSC is 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0., delipidated with chloroform, and then dehydrated through a series of graded alcohols. All reagents were purchased from Sigma ŽSt. Louis, MO.. 2.3. In situ hybridization histochemistry The oligonucleotide probes used to characterize the distribution of the GABA B2 mRNA corresponded to nucleotides 354–398 and 952–991 in the amino terminus of the rat GABA B2 cDNA. For co-localization studies the oligonucleotide probes corresponded to nucleotides 34–71 of the rat GABA B1b cDNA. The probes were synthesized
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using an Expedite Nucleic Acid Synthesis System ŽPerSeptive Biosystems, Framingham, MA.. The probes were air-dried using an Eppendorf Concentrator 5301 ŽBrinkmann Instruments, NY., reconstituted in Molecular Grade water, purified on a 12% polyacrylamide gel, and then reconstituted to a final concentration of 100 ngrml. The oligonucleotides were labeled at the 3X-end using terminal deoxynucleotidyl transferase ŽPharmacia, Piscataway, NJ. with w35 SxdATP Ž1200 Cirmmol, NEN, Boston, MA. to a specific activity of 10 9 dpmrmg. In situ hybridization was performed with modification of the method described previously w25x. 2.4. Hybridization specificity Hybridization specificity was established by performing in situ hybridization, as described above for tissue sections, with w35 SxdATP-labeled GABA B2 antisense and sense oligonucleotides on transiently-transfected HEK293 cells. The cells were transfected with eukaryotic expression vectors containing the cDNAs for rat GABA B2 , GABA B1b , and GABA B1a or mock transfected with only the vector. In addition, GABA B2 hybridization experiments were performed on the transfected cells and rat brain sections using oligonucleotides targeted to disparate regions of the GABA B2 mRNA. 2.5. Analysis The strength of the hybridization signal obtained in various region of the rat brain, as defined by Paxinos and Watson w54x, was graded as weak Žq., moderate Žqq ., heavy Žqqq . or intense Žqqqq .. These were qualitative evaluations for GABA B2 mRNA distribution based on the relative optical density on the autoradiographic film and on the relative number of silver grains observed over individual cells at the microscopic level ŽTable 1.. A total of four rat brains were analyzed for this study.
3. Results 3.1. Control experiments The specificity of the hybridization signal generated from the GABA B2 antisensersense oligonucleotide probes to the disparate regions of the GABA B2 gene was verified by in situ hybridization on transiently transfected HEK293 cells. The results indicate that the hybridization signal obtained using either antisense GABA B2 probe was specific to the HEK293 cells transfected with the GABA B2 cDNA. No hybridization signals were observed with GABA B2 oligonucleotide probes over cells transfected with the GABA B1a and GABA B1b cDNA or the mock-
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Fig. 1. Adjacent coronal rat brain sections hybridized with w35 Sx-labeled GABA B2 antisense ŽA. and sense ŽB. oligonucleotide probes. Arrows in A identify Purkinje cell layer. Note the absence of specific hybridization in B. Scale bar, 2 mm. Abbreviations: GiA, gigantocellular reticular nucleus, alpha; Lat, lateral Ždentate. cerebellar nucleus; MVe, medial vestibular nucleus; Sp5, spinal trigeminal nucleus; VCP, ventral cochlear nucleus, posterior.
transfected cells. Similarly, the GABA B1b probes hybridized specifically only to those cells transfected with the GABA B1b cDNA. Furthermore, no visible hybridization signals were detected with the sense probes in any of the transfected cells Ždata not shown.. As an additional criterion to exclude non-specific hybridization to unknown related RNA, adjacent rat brain sections were hybridized with oligonucleotide probes targeted to non-overlapping regions of the GABA B2 mRNA. This resulted in an identical distribution pattern and intensity of labeling in all regions of the rat CNS. Non-specific hybridization signal on CNS sections was determined using the sense probes and produced a weak non-specific image just evident above background ŽFig. 1.. These results indicate that the oligonucleotide probes used to characterize the distribution of GABA B2 in the rat CNS were specific for the target nucleic acid sequence. 3.2. OÕerÕiew The anatomical distribution of GABA B2 mRNA in the rat brain and spinal cord was determined by in situ hy-
bridization histochemistry using radiolabeled oligonucleotide probes ŽFig. 2A–H, Table 1.. Through observation of tissue sections by light microscopy, silver grains were determined to be distributed over neuronal profiles. The results indicate that the GABA B2 message is widely distributed throughout the rat CNS and is expressed in several sensory ganglia ŽFig. 7C, D.. However, the degree of expression of the GABA B2 transcripts throughout the brain was not uniform. Several regions exhibited higher levels of expression, such as the medial habenula ŽFig. 5A., CA2rCA3 fields of the hippocampus ŽFig. 2C–E and Fig. 4B., piriform cortex ŽFig. 3C. and thalamus ŽFig. 5B–F.. In the CA1 field of the hippocampus ŽFig. 4A. and cerebellar Purkinje cells ŽFig. 6F. moderate GABA B2 mRNA expression was observed. A weak GABA B2 mRNA hybridization signal was seen in the ventral forebrain, septum ŽFig. 2B., throughout most of the hypothalamus ŽFig. 2C, D., the mesencephalon ŽFig. 2E, F., several brainstem nuclei ŽFig. 2G and Fig. 6E., and spinal cord dorsal horn ŽFig. 2H and Fig. 7A, B.. The weakest hybridization signals in the rat brain were detected in the striatum and other nuclei of the basal ganglia.
Fig. 2. Autoradiographic images of in situ hybridization histochemistry showing the distribution of GABA B2 mRNA in coronal rat brain and spinal cord sections presented rostrocaudally ŽA–H.. Scale bar ŽG., 2 mm and applies for A–G. Scale bar ŽH., 2 mm. Abbreviations: AI, anterior insular cortex; AON, anterior olfactory nucleus; Arc, arcuate nucleus; BLA, basolateral amygdaloid nucleus; CA1rCA3, fields CA1rCA3 of hippocampus; Ce, central amygdaloid nucleus; Cg, cingulate cortex; CPu, caudate–putamen; DEn, dorsal endopiriform cortex; DG, dentate gyrus; dh, dorsal horn; DLG, dorsolateral geniculate; DpMe, deep mesencephalic nucleus; DR, dorsal raphe; Ent, entorhinal cortex; Fr, frontal cortex; Gi, gigantocellular reticular nucleus; GrO, granular cell layer, olfactory bulb; HDB, horizontal diagonal band; Int, interposed cerebellar nucleus; IP, interpeduncular nucleus; LD, laterodorsal thalamic nucleus; LSD, lateral septal nucleus, dorsal; LSI, lateral septal nucleus, intermediate; MeA, medial amygdaloid nucleus; MG, medial geniculate; MHb, medial habenular nucleus; MVe, medial vestibular nucleus; PAG, periaqueductal gray; PH, posterior hypothalamus; Pir, piriform cortex; PMCo, posteromedial cortical amygdaloid nucleus; Po, posterior thalamic nucleus; PoDG, polymorphic dentate gyrus; Purkinje cell layer Žarrows in G.; PVT, paraventricular thalamic nucleus; Re, reuniens nucleus; RMg, raphe magnus; RSG, retrosplenial cortex; S1, somatosensory cortex; SC, superior colliculus; SNC, substantia nigra, compact part; STh, subthalamic nucleus; vh, ventral horn; VMH, ventromedial hypothalamus; VP, ventroposterior thalamic nucleus.
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3.3. Olfactory system The main olfactory bulb contained a very light hybridization signal for GABA B2 mRNA in the granule and glomerular cell layers. Mitral cells were devoid of hybridization signal. A moderately heavy hybridization signal was observed over all cells in the anterior olfactory nucleus ŽFig. 2A.. Caudally, a heavy hybridization signal was detected over all the neuronal profiles of the piriform cortex ŽFig. 3C., and a moderate signal was observed in the nucleus of the lateral olfactory tract. A weak hybridization signal was seen on cells in the olfactory tubercle and no hybridization was detectable over granule cells of the islands of Calleja. 3.4. Telencephalon 3.4.1. Cerebral cortex In all divisions of the neocortex there was a diffuse and moderately strong GABA B2 mRNA hybridization signal. The signal appeared to be slightly more intense in the superficial layers of the cortex and in layer VI ŽFig. 2A–F.. This intensity pattern continued through the rostrocaudal extent of the cerebral cortex. By light microscopy, silver grains were localized over pyramidal cells ŽFig. 3A.. The density of silver grains was slightly higher over the cells in layer VI. The dorsal endopiriform nucleus ŽFig. 3B. displayed a moderately high expression of GABA B2 mRNA.
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3.4.4. Hippocampal region The pyramidal cell layer of the CA2 and CA3 fields of the hippocampus ŽFig. 2C and Fig. 4B. and the granule cells of the dentate gyrus ŽFig. 4C. were intensely labeled by the GABA B2 probes. The pyramidal cells of the CA1 field ŽFig. 4A. displayed a relatively weaker hybridization signal than both the CA2rCA3 fields, as did the neurons in the polymorphic layer of the dentate hilar region ŽFig. 4C, arrows.. The taenia tecta exhibited a strong GABA B2 mRNA hybridization and the induseum griseum showed light-to-moderate labeling.
3.4.5. Amygdala and bed nucleus of the stria terminalis The amygdaloid nuclei exhibited a variety of expression levels. Overall, the GABA B2 mRNA expression was moderate, with slightly higher expression levels over cells of the bed nucleus of the stria terminalis, posteromedial cortical, lateral, and basolateral nuclei of the amygdala. A moderate number of silver grains was observed over cell bodies in the medial anterior cortical, and the central nuclei ŽFig. 2C, D.. In addition, in each of these nuclei it appeared that practically all of the cells expressed the GABA B2 message.
3.5. Diencephalon
3.4.2. Basal ganglia and basal forebrain The weakest hybridization signals for GABA B2 message in the rat brain were detected throughout all the nuclei that comprise the basal ganglia ŽFig. 2B.. GABA B2 transcripts could be detected over background in the medium sized cells located within the patch matrix compartment of the caudate–putamen. Positive cells were also seen in the nucleus accumbens, globus pallidus, and the reticular part of the substantia nigra. There was a slightly stronger signal observed over cells in the substantia nigra, compact part ŽFig. 2E., and over cells in the ventral forebrain, including the nucleus of the diagonal band ŽFig. 2B.. The cells of the claustrum and the subthalamic nucleus ŽFig. 2D. displayed moderate hybridization signals.
3.5.1. Hypothalamus and preoptic area In general, most of the hypothalamic nuclei exhibited relatively weak GABA B2 mRNA hybridization ŽFig. 2C, D.. In this region of the brain, there was a slightly higher background that made interpretation of positive cells more difficult than in other regions. However, the expression of the GABA B2 mRNA was higher in neurons of the parvicellular and magnocellular divisions of the paraventricular nucleus and in the arcuate ŽFig. 2D., supraoptic, and supramammillary nuclei. The ventromedial ŽFig. 2C. and dorsomedial nuclei showed the highest hybridization signals in the hypothalamus. Throughout the preoptic area only a weak hybridization signal was detected.
3.4.3. Septal region A moderately weak GABA B2 hybridization signal was observed over cells of both the lateral ŽFig. 2B and Fig. 6A. and medial septal nuclei.
3.5.2. Thalamus and epithalamus For the most part, moderately high hybridization signals were observed throughout the thalamus ŽFig. 2C, D and Fig. 5B–F.. Relatively high GABA B2 mRNA signal was
Fig. 3. Localization of GABA B2 mRNA in telencephalic structures. High-magnification brightfield photomicrographs showing ŽA. virtually all of the pyramidal cells in layer V of the neocortex are labeled Žarrows.. ŽB. Silver grains localized over cells of the dorsal endopiriform nucleus Žarrows.. ŽC. Labeled cells are seen throughout all layers ŽI, II, and III. of the piriform cortex. Scale bar, 40 mm.
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detected over cell bodies in the anterior nuclear group namely: the anterodorsal and mediodorsal nuclei ŽFig. 5B.; the lateral nuclear group, including the laterodorsal nucleus ŽFig. 2C.; and the ventral nuclear group, specifically the ventral anterior, ventral lateral, and ventroposterior nuclei ŽFig. 2C, D and Fig. 5D, F.. A difference in expression levels was observed among the midline and intralaminar nuclei. GABA B2 mRNA expression was slightly higher in many of the cells in the reuniens ŽFig. 5E. and rhomboid nuclei than that observed over cells of the paraventricular ŽFig. 2C and Fig. 5C., centromedial, and paracentral nuclei. The dorsal lateral ŽFig. 2D. and medial geniculate nuclei ŽFig. 2E. displayed moderate GABA B2 message expression. Cell bodies in the reticular nucleus contained the lowest expression levels detected in the thalamus ŽFig. 5F, open arrows.. The most intense expression of GABA B2 mRNA in the rat brain was observed in the epithalamus over practically all of the densely packed dark staining small neurons in the medial habenula ŽFig. 2C and Fig. 5A.; lower expression was visible over the more loosely arranged cells in the lateral habenula. 3.6. Brainstemr pons r medulla A weak-to-moderate hybridization signal was observed over most of cells in the mesencephalic reticular formation, including the deep mesencephalic tegmentum ŽFig. 2F. and the large cells of the mesencephalic trigeminal nucleus. Slightly higher GABA B2 mRNA expression was detected in the superior colliculus, periaqueductal gray, retrorubral field, and interpeduncular nucleus ŽFig. 2E.. Caudally, in the pontine tegmentum, a light hybridization signal was seen over the medium sized cells of the locus coeruleus, cells of the A5 noradrenaline group, and the facial nucleus. A moderate-to-high hybridization signal was observed in the pontine nuclei ŽFig. 2F., while a light-to-moderate signal was present in the nucleus of the solitary tract, motor trigeminal nucleus, and dorsal raphe nuclei. A moderate number of silver grains were detected by light microscopy over medium-sized perikarya in the raphe magnus nucleus ŽFigs. 2G and 6E.. Expression levels were light-to-moderate within the medullary reticular formation, including the gigantocellular and spinal trigeminal nuclei ŽFig. 1A.. There appeared to be a higher degree of background hybridization in the brainstem similar to that seen in the hypothalamic area; however, this did not compromise the ability to determine positive cells in the region.
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3.7. Cerebellum The GABA B2 hybridization signal was not uniformly distributed over the cerebellar granule cell layer. The signal was scattered over many, but not all, of the small intensely stained granule cells. The large pale-staining Purkinje cells expressed higher levels of GABA B2 message than granule cells. In contrast to the granule cells, the hybridization signal was seen over all Purkinje cells ŽFig. 6F.. There was a weak hybridization signal evident over all of the medium-to-large sized neurons in all divisions of the deep cerebellar nuclei ŽFig. 2G..
3.8. Spinal cord and sensory ganglia In both the dorsal and ventral horns of the spinal cord the expression of GABA B2 mRNA was light-to-moderate ŽFig. 2H and Fig. 7A, B.. In the dorsal horn, the labeling appeared to be slightly denser in the cells of the superficial layer than the lighter labeling which was detected over most large neurons of the ventral horn. The hybridization signal was uniformly distributed over most of these cells. This pattern of expression continued throughout all segments of the spinal cord, including sympathetic and parasympathetic centers. In both the dorsal root and trigeminal ganglia, a moderately heavy hybridization signal was evenly distributed over small, medium, and large ganglion cells ŽFig. 7C, D..
3.9. Co-localization of the GABA B2 and GABA B1b subtypes The in vivo co-expression of GABA B2 and GABA B1b mRNAs in various regions of the rat brain was determined at the cellular level by in situ hybridization studies using w35 SxdATP-labeled-oligoprobes on adjacent rat brain sections. Co-localization was confirmed in brain regions with identifiable structural features, such as the hippocampus, piriform cortex, mesencephalic trigeminal nucleus, and cerebellum. In the hippocampus, all of the pyramidal cells in the CA1, CA2, and CA3 fields of the hippocampus exhibited expression of both GABA B2 and GABA B1b mRNAs ŽFig. 8A–D. as did all cells in the piriform cortex, motor trigeminal nucleus, and the cerebellar Purkinje cells
Fig. 4. High-magnification brightfield photomicrographs of GABA B2 mRNA expression in coronal sections of the hippocampal formation. ŽA. CA1 and ŽB. CA3 fields. Note the presence of silver grains over the pyramidal cell layer Žpcl. in each of the fields and the absence of signal in the stratum oriens Žso. and stratum lucidum Žsl.. ŽC. Silver grains are evident over cells in the granule cell layer Žgcl. of the dentate gyrus and pyramidal cells in the polymorphic dentate gyrus ŽPoDG. Žarrows.. Granule cells extend into the molecular layer Žml.. Scale bar ŽC., 40 mm.
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Fig. 5. GABA B2 mRNA hybridization signal obtained using w35 Sx-labeled GABA B2 oligonucleotide probes in select thalamic nuclei and epithalamus. High-magnification brightfield photomicrographs of emulsion-dipped tissues demonstrating GABA B2 mRNA expression in the ŽA. medial habenula, the asterisk marks the third ventricle, ŽB. anterodorsal thalamic nucleus, ŽC. paraventricular thalamic nucleus, ŽD. ventroposterior thalamic nucleus, ŽE. reuniens nucleus, and ŽF. reticular thalamic and ventromedial nuclei. In A–F arrows indicate positive cells, in F lower levels of GABA B2 mRNA expression are visible over reticular thalamic neurons Žopen arrows.. Scale bar ŽF., 40 mm.
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Fig. 6. The localization of the GABA B2 mRNA in select brain regions. High-magnification brightfield photomicrographs of emulsion-dipped tissues showing the distribution of GABA B2 mRNA in the ŽA. laterodorsal septum, the asterisk marks the third ventricle, ŽB. central amygdaloid, ŽC. basolateral amygdaloid, ŽD. medial amygdaloid nuclei, ŽE. raphe magnus, and ŽF. the cerebellum. In F both the Purkinje cells Žarrows. and scattered cells within the granule cell layer Žsmall arrowheads. are positive. Arrows in A–F identify cells expressing GABA B2 mRNA. Scale bar ŽE., 40 mm.
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Fig. 7. The distribution of GABA B2 mRNA in the spinal cord and sensory ganglia. High-magnification brightfield photomicrographs showing a weak GABA B2 mRNA expression in the spinal cord ŽA. superficial dorsal horn, lamina I, and ŽB. lamina VI. Arrows indicate cells expressing the GABA B2 mRNA. A moderate-to-high expression of GABA B2 mRNA is observed in all ganglion cells of the ŽC. trigeminal and ŽD. dorsal root ganglia. Scale bar ŽD., 40 mm.
ŽFig. 8E–H.. Individual neurons in the hippocampus and cerebellum expressing both mRNAs were identified on adjacent sections ŽFig. 8C,D,G,H.. While individual cells could not be discretely identified, microscopic examination of the neocortex, medial habenula, locus coeruleus, se-
lected thalamic, and brainstem nuclei suggested co-expression of the GABA B2 and GABA B1b mRNAs Ždata not shown.. In these regions, virtually all of the cells exhibited hybridization signal for both the GABA B2 and GABA B1b mRNAs.
Fig. 8. Co-localization of GABA B1 b and GABA B2 mRNA in selected regions of the rat brain. High-magnification photomicrographs of the autoradiographic images of adjacent coronal sections through the rat dorsal hippocampus and diencephalon ŽA,B. and cerebellum ŽE,F., illustrating overlapping hybridization signal for GABA B2 ŽA,E. and GABA B1b mRNA ŽB,F.. Arrows in E and F identify the Purkinje cell layer. ŽC,D. Brightfield photomicrographs from adjacent sections of the region outlined by the box in A showing silver grains over all of the pyramidal cells in the CA1 field of the hippocampus. Arrows indicate identical cells labeled for ŽC. GABA B2 and ŽD. GABA B1b mRNA. ŽG,H. Brightfield photomicrographs from adjacent sections of the region outlined by the box in E. Arrows indicate identical Purkinje cells expressing ŽG. GABA B2 and ŽH. GABA B1b mRNA. Scale bar ŽB., 40 mm and applies to A, B, E and F. Scale bar ŽD., 40 mm and applies to C,D,G, and H. The emulsion exposure times were shortened to 1 week so that the silver grains would not interfere with identification of individual cells.
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4. Discussion The anatomical distribution of mRNA encoding a recently reported protein, GABA B2 , has been revealed throughout the adult rat CNS by in situ hybridization histochemistry. The results indicate that the GABA B2 mRNA expression is restricted primarily to neuronal profiles. Furthermore, the GABA B2 messenger RNA is widely expressed throughout the rat CNS and primary sensory ganglia. Expression levels of the GABA B2 mRNA in the rat brain varied among the regions of the CNS. Several areas exhibited intense GABA B2 mRNA expression, including the medial habenula, piriform cortex, the CA2rCA3 fields and dentate gyrus of the hippocampus, while moderate GABA B2 expression was observed throughout the entire neocortex, the CA1 field of the hippocampus, the majority of thalamic nuclei, and the cerebellar Purkinje cells. Lower GABA B2 mRNA expression was seen throughout most of the hypothalamus, mesencephalon, and several brainstem nuclei. GABAergic neurons and terminals have been described to be widely distributed throughout the rat CNS w48x, and the localization of the GABA B2 message correlates well with the distribution of GABAergic neurons. Moreover, the pattern of distribution of the GABA B2 mRNA correlates reasonably well with the distribution described for GABA B receptor binding sites by receptor autoradiography w16,21,30x, and the distribution described for both GABA B1a and GABA B1b by in situ hybridization w10,37x. There were differences between the expression of the GABA B1 isoforms and GABA B2 , most notably in the hypothalamus, caudate–putamen, and locus coeruleus Žsee below.. In these regions, the GABA B2 message appeared much weaker than that observed for either of the GABA B1 mRNAs. Additionally, the cerebellar Purkinje cells express the GABA B2 and GABA B1b mRNAs, but not the GABA B1a mRNA. There have been numerous reviews of the actions of GABA in the mammalian brain w4,35,43,53,65x and on the physiological role for GABA B receptors w9,15,17,39, 43,45x. The extensive expression of both the GABA B2 and GABA B1 messages in the rat brain w34,38,40,63x implies that GABA mediates many of its effects through these proteins, thereby influencing a variety of neurotransmitter systems, including glutamate w6,8,50x, serotonin w6,62x, NPY w33x, acetylcholine w32x, histamine w60x and dopamine w26,28,49x. The expression of GABA B2 mRNA in primary sensory neurons, the spinal cord and the relay nuclei of several sensory pathways suggest the possible participation of GABA B2 in the modulation a variety of sensory modalities. In the olfactory and visual pathways, GABA B2 message appears to be located in a position to potentially modulate excitatory glutamatergic neurotransmission w46,59,61x. GABA B2 mRNA is expressed in the target regions of the
projection fibers from the main olfactory bulb, namely the anterior olfactory cortex, olfactory tubercle, taenia tecta, piriform, and entorhinal cortices. This is in addition to the target regions of the retina, such as the superior colliculus and the dorsal lateral geniculate nucleus. The extensive presence of GABA B2 message throughout both of these systems may allow for considerable regulation of neurotransmission by the GABA B2 . It has been well established that GABA B antagonists can block the antinociceptive effects of GABA and GABA B agonists w18x. Moreover, GABAergic neurons in the spinal cord dorsal horn have been reported to be presynaptic to Ad and C primary afferent terminals w2,7x. The expression of GABA B2 message in cells of the superficial dorsal horn supports a potential presynaptic autoreceptor localization for this protein. As such, GABA B2 may have a role in mediating GABA’s inhibition of neurotransmitter release in the spinal cord. Likewise, our studies supply evidence for GABA B2 to potentially modulate the influence of excitatory glutamatergic nociceptive primary afferents, namely, the terminals of glutamatergic dorsal root and trigeminal ganglion cells w20,36x. GABA B2 mRNA expression was evident in both of these sensory ganglia and in small and medium-sized ganglion cells. A further indication of a possible integral role for GABA B2 protein in processing nociceptive information comes from observation of the extensive and abundant expression of GABA B2 mRNA in the thalamus, neocortex, the raphe nuclei, and the periaqueductal gray. A potential involvement of GABA B2 in cognitive functioning as well as in absence epilepsy is suggested by its moderately abundant expression throughout the cerebral cortex, thalamus, and the hippocampal pyramidal and dentate gyrus granule cells. Several studies have shown that physiological as well as pathological neuronal activity in the hippocampus can be regulated by GABA B receptors w17,19,31,47x. Furthermore, pre- and post-synaptic GABA B receptors have been shown to be involved in the regulation of the induction of long term potentiation w23x. Additional evidence for the involvement of GABA B receptors in learning and memory comes from studies showing that the GABA B antagonist, CGP 36,742, can improve learning performance in aged rats and rhesus monkeys while baclofen, a GABA B agonist, impairs spatial learning w3,44,46x. The overlapping distribution of GABA B2 and GABA B1b mRNAs observed in coronal rat brain sections ŽFig. 8A,B,E,F. supports the recent reports that the GABA B1 and GABA B2 proteins assemble as a heterodimer in vivo w34,38,40,63x. In situ hybridization histochemistry employing w35 Sx-labeled GABA B2 and GABA B1b oligonucleotide probes on adjacent tissue sections in the hippocampus and cerebellum enabled us to identify individual neurons expressing both GABA B2 and GABA B1b mRNAs. In other regions of the brain Že.g., the neocortex, medial habenula, thalamus, locus coeruleus, and mesencephalic trigeminal
M.M. Durkin et al.r Molecular Brain Research 71 (1999) 185–200
nucleus., it was more difficult to identify individual neurons; however, co-expression could be implied since all of the cells in these regions appeared to express both the GABA B2 and GABA B1b mRNAs. In addition, extensive co-localization of the GABA B2 and GABA B1b proteins has been reported at extrasynaptic sites of cerebellar Purkinje cell dendritic spines w38x. Taken together, these observations support the view that the GABA B2 and GABA B1b proteins associate in vivo to produce a functional receptor and that the heterodimers may be localized to discrete neuronal sites such as dendritic spines. Other as yet unidentified GABA B receptor homologues may associate elsewhere to produce novel subtypes. The low level of expression of GABA B2 mRNA relative to either of the two GABA B1 isoforms seen in the hypothalamus, caudate–putamen, and locus coeruleus Žsee above. raises the possibility that other GABA B receptor homologues may associate with GABA B1 to produce novel subtypes in these regions. Alternatively, the mismatch in mRNA levels in these areas may reflect regional differences in the rate of mRNA turnover. Future studies using isoform-specific antibodies will reveal interesting detail of the regional heterogeneity of GABA B receptors. In conclusion, we have described the localization of mRNA in the adult rat brain for a new member of the GABA B family, GABA B2 . The distribution of the GABA B2 mRNA suggests its potential involvement in the modulation of a variety of neurotransmitters systems. These findings also imply that some of the physiological actions of GABA B pharmacological agents may be through interaction with GABA B2 . Acknowledgements The authors would like to thank Drs. Douglas Craig and Roman Artymyshyn for reviewing the manuscript, Mary Johnson for technical assistance, and Elisabeth Griggs for photographic assistance.
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Research report
An in situ hybridization study of the distribution of the GABA B2 protein mRNA in the rat CNS Margaret M. Durkin ) , Caryn A. Gunwaldsen, Beth Borowsky, Kenneth A. Jones, Theresa A. Branchek Department of Pharmacology, Synaptic Pharmaceutical Corporation, 215 College Road, Paramus, NJ 07652, USA Accepted 25 May 1999
Abstract g-Aminobutyric acid ŽGABA. is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA exerts its actions through two classes of receptors: GABA A , multimeric ligand-gated Cly ion channels Ža class which has been proposed to include the homomeric variant previously called GABA C , to be designated GABA A0r .; and GABA B , G-protein coupled receptors which regulate Ca2q and Kq channels. Currently, within the GABA B receptor family two proteins have been identified through molecular cloning techniques and designated GABA B1 and GABA B2 . Two N-terminal variants of GABA B1 were isolated and designated GABA B1a and GABA B1b . The distribution of neurons in the rat CNS expressing the mRNA for the GABA B1 isoforms have been previously described by in situ hybridization histochemistry. The recent isolation and identification of the GABA B2 protein by homology cloning has enabled the use of radiolabeled oligonucleotides to detect the distribution of the expression of GABA B2 mRNA in the rat CNS. The expression of GABA B2 mRNA was observed to be primarily related to neuronal profiles. The highest levels of GABA B2 mRNA expression were detected in the piriform cortex, hippocampus, and medial habenula. GABA B2 mRNA was abundant in all layers of the cerebral cortex, the thalamus and in cerebellar Purkinje cells. Moderate expression was observed in several hypothalamic and brainstem nuclei. In contrast to the distribution of GABA B1 mRNA, only a weak hybridization signal for GABA B2 was detected over cells of the basal ganglia, including the caudate–putamen, nucleus accumbens, olfactory tubercle and throughout most of the hypothalamus. Moderate-to-heavy GABA B2 mRNA expression was also seen over dorsal root and trigeminal ganglion cells. In general, the pattern of GABA B2 mRNA expression in the rat brain overlaps considerably with the distributions described for both GABA B1 mRNAs, and is concordant with the distribution described for GABA B receptor binding sites. However, differences between GABA B2 expression levels and GABA B binding sites were observed in the basal ganglia. q 1999 Elsevier Science B.V. All rights reserved. Keywords: GABA; GABA B2 protein; In situ hybridization; Rat CNS
1. Introduction g-Aminobutyric acid ŽGABA. is the main inhibitory neurotransmitter in the mammalian CNS. GABA exerts its actions through two classes of receptors: GABA A , multimeric ligand-gated Cly ion channels Ža class which has been proposed to include the homomeric variant previously called GABA C , to be designated GABA A0r w5x.; and GABA B , G-protein coupled receptors which regulate Ca2q and Kq channels. GABA receptors have been distinguished pharmacologically w5,12,14,15,22x, electrophysio-
) Corresponding author. [email protected]
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logically w13,29,56x, and more recently through molecular cloning w14,27,37,42,52,58x. A ubiquitous distribution of GABA A and GABA B receptor binding sites in the rat CNS has been described by receptor autoradiographic studies employing 3 H-GABA or 3 H-baclofen as ligands w9,10,16,21x. The distribution of the GABA A and GABA A0r receptor messenger RNA expression in the rat CNS and chick retina, respectively, has been described using in situ hybridization histochemistry w1,55,64x. Heterogeneity within the GABA B receptor family has been suggested on the basis of pharmacological studies w11,12,24,41,51,57 x. Recently, two isoforms of the GABA B1 receptor were cloned and designated GABA B1a and GABA B1b w37x, differing only in the length of their
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 1 8 2 - 5
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Table 1 The distribution of GABA B2 mRNA in the rat CNS. The strength of the hybridization signal observed in various regions of the rat brain was graded as weak Žq., moderate Žqq ., heavy Žqqq . or intense Žqqqq . Region Olfactory system Internal granule layer Glomerular layer External plexiform layer Mitral cell layer Anterior olfactory n. Olfactory tubercle Islands of Calleja Piriform cortex N. of the lateral olfactory tract Cortex Frontal Agranular insular Cingulate Retrosplenial Parietal Occipital Entorhinal Dorsal endopiriforn n. Basal ganglia and basal forebrain Accumbens nucleus Caudate–putamen Globus pallidus Ventral pallidum Horizontal diagonal band Claustrum Subthalamic n. Substantia nigra, reticular part Substantia nigra, compact part Septal region Medial septum Lateral septum Septohippocampal n. Amygdala Lateral n. Basolateral n. Medial amygdaloid n. Basomedial n. Central n. Anterior cortical n. Posteromedial cortical n. Bed n. stria terminalis Hippocampus Field CA1 Fields CA2rCA3 Taenia tecta Induseum griseum Dentate gyrus Polymorphic dentate gyrus Subiculum PrerParasubiculum Hypothalamus Suprachiasmatic n. Median preoptic area Paraventricular n. Arcuate n. Anterior hypothalamus, posterior Lateral hypothalamus Ventromedial n.
Density q q y y qqq q y qqq qq qqq qqq qqq qqq qqq qqq qqq qqq q q q qq qq qq qq q qq qq qq q qqq qqq qqq q qq qq qq qq qq qqqq qqq q qqqq qqq q qq q q qq qq q q qq
Region
Density
Dorsomedial n. Periventricular n. Supraoptic n. Supramammillary n. Premammillary n. Medial mammillary n. Thalamus and epithalamus Anterodorsal n. Laterodorsal n. Paraventricular n. Centromedial n. Paracentral n. Parafascicular n. Reuniens n. Rhomboid n. Ventrolateral n. Ventromedial n. Ventral posterolateral n. Reticular n. Lateral geniculate n. Medial geniculate n. Medial habenular n. Lateral habenular n. Midbrainr Pons r Medulla Superior colliculus Inferior colliculus Periaqueductal gray Dorsal raphe Deep mesencephalic n. Pontine n. Retrorubral field Ventral tegmental area Mesencephalic trigeminal n. Motor trigeminal n. Interpeduncular n. Raphe magnus Raphe pallidus Spinal trigeminal n. Pontine reticular n. Parvicellular reticular n. Locus coeruleus Parabrachial n. Vestibular n. Gigantocellular reticular n. Prepositus hypoglossal n. Ventral cochlear n. N. soltary tract A5 noradrenaline cells Facial n. Ž7. Cerebellum Granule cell layer Purkinje cells Deep cerebellar n. Spinal cord Dorsal horn Ventral horn Peripheral ganglia Trigeminal ganglion Dorsal root ganglion
qq q q qq q q qqq qqq qq qq qq qq qqq qqq qqq qqq qqq q qqq qq qqqq q qq q qq qq q qqq qq q qq q qq qq q q qqq q q q q qq q qq qq q q q qqq q q q qqq qqq
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amino terminal sequences. A similarly ubiquitous and overlapping distribution of message expression has been described in the rat brain for the GABA B1 splice variants w10x. We have recently reported w34x the isolation of an additional GABA B protein ŽGABA B2 ., which exhibits approximately 38% overall sequence homology to GABA B1 . In addition, recent reports demonstrated that the co-expression of GABA B1 and GABA B2 results in a single pharmacologically and functionally defined receptor with signal transduction properties similar to the native GABA B receptors w34,38,40,63x. These observations prompted us to explore the in vivo co-localization in neurons of the GABA B2 and GABA B1b genes. In the present study, the distribution of the GABA B2 messenger RNA expression in the rat CNS has been examined using radiolabeled oligonucleotide probes.
2. Materials and methods 2.1. Animals The Synaptic Pharmaceutical Animal Care and Use Committee approved all protocols. Male Sprague–Dawley rats Ž200–250 g, Charles Rivers, Rochester, NY. were euthanized using CO 2 , decapitated, and their brains immediately removed and rapidly frozen on crushed dry ice. Coronal sections of brain tissue were cut at 11 mm using a cryostat, thaw-mounted onto poly-L-lysine-coated slides, and stored at y208C until use. 2.2. Tissue preparation Prior to hybridization, the tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline ŽPBS. pH 7.4 ŽSpecialty Media, Lavallette, NJ. followed by two washes in PBS. Tissues were then treated in 5 mM dithiothreitol, rinsed in DEPC Ždiethyl pyrocarbonate.-treated PBS, acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride, rinsed twice in 2 = SSC Žstandard saline citrate: 1 = SSC is 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0., delipidated with chloroform, and then dehydrated through a series of graded alcohols. All reagents were purchased from Sigma ŽSt. Louis, MO.. 2.3. In situ hybridization histochemistry The oligonucleotide probes used to characterize the distribution of the GABA B2 mRNA corresponded to nucleotides 354–398 and 952–991 in the amino terminus of the rat GABA B2 cDNA. For co-localization studies the oligonucleotide probes corresponded to nucleotides 34–71 of the rat GABA B1b cDNA. The probes were synthesized
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using an Expedite Nucleic Acid Synthesis System ŽPerSeptive Biosystems, Framingham, MA.. The probes were air-dried using an Eppendorf Concentrator 5301 ŽBrinkmann Instruments, NY., reconstituted in Molecular Grade water, purified on a 12% polyacrylamide gel, and then reconstituted to a final concentration of 100 ngrml. The oligonucleotides were labeled at the 3X-end using terminal deoxynucleotidyl transferase ŽPharmacia, Piscataway, NJ. with w35 SxdATP Ž1200 Cirmmol, NEN, Boston, MA. to a specific activity of 10 9 dpmrmg. In situ hybridization was performed with modification of the method described previously w25x. 2.4. Hybridization specificity Hybridization specificity was established by performing in situ hybridization, as described above for tissue sections, with w35 SxdATP-labeled GABA B2 antisense and sense oligonucleotides on transiently-transfected HEK293 cells. The cells were transfected with eukaryotic expression vectors containing the cDNAs for rat GABA B2 , GABA B1b , and GABA B1a or mock transfected with only the vector. In addition, GABA B2 hybridization experiments were performed on the transfected cells and rat brain sections using oligonucleotides targeted to disparate regions of the GABA B2 mRNA. 2.5. Analysis The strength of the hybridization signal obtained in various region of the rat brain, as defined by Paxinos and Watson w54x, was graded as weak Žq., moderate Žqq ., heavy Žqqq . or intense Žqqqq .. These were qualitative evaluations for GABA B2 mRNA distribution based on the relative optical density on the autoradiographic film and on the relative number of silver grains observed over individual cells at the microscopic level ŽTable 1.. A total of four rat brains were analyzed for this study.
3. Results 3.1. Control experiments The specificity of the hybridization signal generated from the GABA B2 antisensersense oligonucleotide probes to the disparate regions of the GABA B2 gene was verified by in situ hybridization on transiently transfected HEK293 cells. The results indicate that the hybridization signal obtained using either antisense GABA B2 probe was specific to the HEK293 cells transfected with the GABA B2 cDNA. No hybridization signals were observed with GABA B2 oligonucleotide probes over cells transfected with the GABA B1a and GABA B1b cDNA or the mock-
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Fig. 1. Adjacent coronal rat brain sections hybridized with w35 Sx-labeled GABA B2 antisense ŽA. and sense ŽB. oligonucleotide probes. Arrows in A identify Purkinje cell layer. Note the absence of specific hybridization in B. Scale bar, 2 mm. Abbreviations: GiA, gigantocellular reticular nucleus, alpha; Lat, lateral Ždentate. cerebellar nucleus; MVe, medial vestibular nucleus; Sp5, spinal trigeminal nucleus; VCP, ventral cochlear nucleus, posterior.
transfected cells. Similarly, the GABA B1b probes hybridized specifically only to those cells transfected with the GABA B1b cDNA. Furthermore, no visible hybridization signals were detected with the sense probes in any of the transfected cells Ždata not shown.. As an additional criterion to exclude non-specific hybridization to unknown related RNA, adjacent rat brain sections were hybridized with oligonucleotide probes targeted to non-overlapping regions of the GABA B2 mRNA. This resulted in an identical distribution pattern and intensity of labeling in all regions of the rat CNS. Non-specific hybridization signal on CNS sections was determined using the sense probes and produced a weak non-specific image just evident above background ŽFig. 1.. These results indicate that the oligonucleotide probes used to characterize the distribution of GABA B2 in the rat CNS were specific for the target nucleic acid sequence. 3.2. OÕerÕiew The anatomical distribution of GABA B2 mRNA in the rat brain and spinal cord was determined by in situ hy-
bridization histochemistry using radiolabeled oligonucleotide probes ŽFig. 2A–H, Table 1.. Through observation of tissue sections by light microscopy, silver grains were determined to be distributed over neuronal profiles. The results indicate that the GABA B2 message is widely distributed throughout the rat CNS and is expressed in several sensory ganglia ŽFig. 7C, D.. However, the degree of expression of the GABA B2 transcripts throughout the brain was not uniform. Several regions exhibited higher levels of expression, such as the medial habenula ŽFig. 5A., CA2rCA3 fields of the hippocampus ŽFig. 2C–E and Fig. 4B., piriform cortex ŽFig. 3C. and thalamus ŽFig. 5B–F.. In the CA1 field of the hippocampus ŽFig. 4A. and cerebellar Purkinje cells ŽFig. 6F. moderate GABA B2 mRNA expression was observed. A weak GABA B2 mRNA hybridization signal was seen in the ventral forebrain, septum ŽFig. 2B., throughout most of the hypothalamus ŽFig. 2C, D., the mesencephalon ŽFig. 2E, F., several brainstem nuclei ŽFig. 2G and Fig. 6E., and spinal cord dorsal horn ŽFig. 2H and Fig. 7A, B.. The weakest hybridization signals in the rat brain were detected in the striatum and other nuclei of the basal ganglia.
Fig. 2. Autoradiographic images of in situ hybridization histochemistry showing the distribution of GABA B2 mRNA in coronal rat brain and spinal cord sections presented rostrocaudally ŽA–H.. Scale bar ŽG., 2 mm and applies for A–G. Scale bar ŽH., 2 mm. Abbreviations: AI, anterior insular cortex; AON, anterior olfactory nucleus; Arc, arcuate nucleus; BLA, basolateral amygdaloid nucleus; CA1rCA3, fields CA1rCA3 of hippocampus; Ce, central amygdaloid nucleus; Cg, cingulate cortex; CPu, caudate–putamen; DEn, dorsal endopiriform cortex; DG, dentate gyrus; dh, dorsal horn; DLG, dorsolateral geniculate; DpMe, deep mesencephalic nucleus; DR, dorsal raphe; Ent, entorhinal cortex; Fr, frontal cortex; Gi, gigantocellular reticular nucleus; GrO, granular cell layer, olfactory bulb; HDB, horizontal diagonal band; Int, interposed cerebellar nucleus; IP, interpeduncular nucleus; LD, laterodorsal thalamic nucleus; LSD, lateral septal nucleus, dorsal; LSI, lateral septal nucleus, intermediate; MeA, medial amygdaloid nucleus; MG, medial geniculate; MHb, medial habenular nucleus; MVe, medial vestibular nucleus; PAG, periaqueductal gray; PH, posterior hypothalamus; Pir, piriform cortex; PMCo, posteromedial cortical amygdaloid nucleus; Po, posterior thalamic nucleus; PoDG, polymorphic dentate gyrus; Purkinje cell layer Žarrows in G.; PVT, paraventricular thalamic nucleus; Re, reuniens nucleus; RMg, raphe magnus; RSG, retrosplenial cortex; S1, somatosensory cortex; SC, superior colliculus; SNC, substantia nigra, compact part; STh, subthalamic nucleus; vh, ventral horn; VMH, ventromedial hypothalamus; VP, ventroposterior thalamic nucleus.
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3.3. Olfactory system The main olfactory bulb contained a very light hybridization signal for GABA B2 mRNA in the granule and glomerular cell layers. Mitral cells were devoid of hybridization signal. A moderately heavy hybridization signal was observed over all cells in the anterior olfactory nucleus ŽFig. 2A.. Caudally, a heavy hybridization signal was detected over all the neuronal profiles of the piriform cortex ŽFig. 3C., and a moderate signal was observed in the nucleus of the lateral olfactory tract. A weak hybridization signal was seen on cells in the olfactory tubercle and no hybridization was detectable over granule cells of the islands of Calleja. 3.4. Telencephalon 3.4.1. Cerebral cortex In all divisions of the neocortex there was a diffuse and moderately strong GABA B2 mRNA hybridization signal. The signal appeared to be slightly more intense in the superficial layers of the cortex and in layer VI ŽFig. 2A–F.. This intensity pattern continued through the rostrocaudal extent of the cerebral cortex. By light microscopy, silver grains were localized over pyramidal cells ŽFig. 3A.. The density of silver grains was slightly higher over the cells in layer VI. The dorsal endopiriform nucleus ŽFig. 3B. displayed a moderately high expression of GABA B2 mRNA.
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3.4.4. Hippocampal region The pyramidal cell layer of the CA2 and CA3 fields of the hippocampus ŽFig. 2C and Fig. 4B. and the granule cells of the dentate gyrus ŽFig. 4C. were intensely labeled by the GABA B2 probes. The pyramidal cells of the CA1 field ŽFig. 4A. displayed a relatively weaker hybridization signal than both the CA2rCA3 fields, as did the neurons in the polymorphic layer of the dentate hilar region ŽFig. 4C, arrows.. The taenia tecta exhibited a strong GABA B2 mRNA hybridization and the induseum griseum showed light-to-moderate labeling.
3.4.5. Amygdala and bed nucleus of the stria terminalis The amygdaloid nuclei exhibited a variety of expression levels. Overall, the GABA B2 mRNA expression was moderate, with slightly higher expression levels over cells of the bed nucleus of the stria terminalis, posteromedial cortical, lateral, and basolateral nuclei of the amygdala. A moderate number of silver grains was observed over cell bodies in the medial anterior cortical, and the central nuclei ŽFig. 2C, D.. In addition, in each of these nuclei it appeared that practically all of the cells expressed the GABA B2 message.
3.5. Diencephalon
3.4.2. Basal ganglia and basal forebrain The weakest hybridization signals for GABA B2 message in the rat brain were detected throughout all the nuclei that comprise the basal ganglia ŽFig. 2B.. GABA B2 transcripts could be detected over background in the medium sized cells located within the patch matrix compartment of the caudate–putamen. Positive cells were also seen in the nucleus accumbens, globus pallidus, and the reticular part of the substantia nigra. There was a slightly stronger signal observed over cells in the substantia nigra, compact part ŽFig. 2E., and over cells in the ventral forebrain, including the nucleus of the diagonal band ŽFig. 2B.. The cells of the claustrum and the subthalamic nucleus ŽFig. 2D. displayed moderate hybridization signals.
3.5.1. Hypothalamus and preoptic area In general, most of the hypothalamic nuclei exhibited relatively weak GABA B2 mRNA hybridization ŽFig. 2C, D.. In this region of the brain, there was a slightly higher background that made interpretation of positive cells more difficult than in other regions. However, the expression of the GABA B2 mRNA was higher in neurons of the parvicellular and magnocellular divisions of the paraventricular nucleus and in the arcuate ŽFig. 2D., supraoptic, and supramammillary nuclei. The ventromedial ŽFig. 2C. and dorsomedial nuclei showed the highest hybridization signals in the hypothalamus. Throughout the preoptic area only a weak hybridization signal was detected.
3.4.3. Septal region A moderately weak GABA B2 hybridization signal was observed over cells of both the lateral ŽFig. 2B and Fig. 6A. and medial septal nuclei.
3.5.2. Thalamus and epithalamus For the most part, moderately high hybridization signals were observed throughout the thalamus ŽFig. 2C, D and Fig. 5B–F.. Relatively high GABA B2 mRNA signal was
Fig. 3. Localization of GABA B2 mRNA in telencephalic structures. High-magnification brightfield photomicrographs showing ŽA. virtually all of the pyramidal cells in layer V of the neocortex are labeled Žarrows.. ŽB. Silver grains localized over cells of the dorsal endopiriform nucleus Žarrows.. ŽC. Labeled cells are seen throughout all layers ŽI, II, and III. of the piriform cortex. Scale bar, 40 mm.
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detected over cell bodies in the anterior nuclear group namely: the anterodorsal and mediodorsal nuclei ŽFig. 5B.; the lateral nuclear group, including the laterodorsal nucleus ŽFig. 2C.; and the ventral nuclear group, specifically the ventral anterior, ventral lateral, and ventroposterior nuclei ŽFig. 2C, D and Fig. 5D, F.. A difference in expression levels was observed among the midline and intralaminar nuclei. GABA B2 mRNA expression was slightly higher in many of the cells in the reuniens ŽFig. 5E. and rhomboid nuclei than that observed over cells of the paraventricular ŽFig. 2C and Fig. 5C., centromedial, and paracentral nuclei. The dorsal lateral ŽFig. 2D. and medial geniculate nuclei ŽFig. 2E. displayed moderate GABA B2 message expression. Cell bodies in the reticular nucleus contained the lowest expression levels detected in the thalamus ŽFig. 5F, open arrows.. The most intense expression of GABA B2 mRNA in the rat brain was observed in the epithalamus over practically all of the densely packed dark staining small neurons in the medial habenula ŽFig. 2C and Fig. 5A.; lower expression was visible over the more loosely arranged cells in the lateral habenula. 3.6. Brainstemr pons r medulla A weak-to-moderate hybridization signal was observed over most of cells in the mesencephalic reticular formation, including the deep mesencephalic tegmentum ŽFig. 2F. and the large cells of the mesencephalic trigeminal nucleus. Slightly higher GABA B2 mRNA expression was detected in the superior colliculus, periaqueductal gray, retrorubral field, and interpeduncular nucleus ŽFig. 2E.. Caudally, in the pontine tegmentum, a light hybridization signal was seen over the medium sized cells of the locus coeruleus, cells of the A5 noradrenaline group, and the facial nucleus. A moderate-to-high hybridization signal was observed in the pontine nuclei ŽFig. 2F., while a light-to-moderate signal was present in the nucleus of the solitary tract, motor trigeminal nucleus, and dorsal raphe nuclei. A moderate number of silver grains were detected by light microscopy over medium-sized perikarya in the raphe magnus nucleus ŽFigs. 2G and 6E.. Expression levels were light-to-moderate within the medullary reticular formation, including the gigantocellular and spinal trigeminal nuclei ŽFig. 1A.. There appeared to be a higher degree of background hybridization in the brainstem similar to that seen in the hypothalamic area; however, this did not compromise the ability to determine positive cells in the region.
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3.7. Cerebellum The GABA B2 hybridization signal was not uniformly distributed over the cerebellar granule cell layer. The signal was scattered over many, but not all, of the small intensely stained granule cells. The large pale-staining Purkinje cells expressed higher levels of GABA B2 message than granule cells. In contrast to the granule cells, the hybridization signal was seen over all Purkinje cells ŽFig. 6F.. There was a weak hybridization signal evident over all of the medium-to-large sized neurons in all divisions of the deep cerebellar nuclei ŽFig. 2G..
3.8. Spinal cord and sensory ganglia In both the dorsal and ventral horns of the spinal cord the expression of GABA B2 mRNA was light-to-moderate ŽFig. 2H and Fig. 7A, B.. In the dorsal horn, the labeling appeared to be slightly denser in the cells of the superficial layer than the lighter labeling which was detected over most large neurons of the ventral horn. The hybridization signal was uniformly distributed over most of these cells. This pattern of expression continued throughout all segments of the spinal cord, including sympathetic and parasympathetic centers. In both the dorsal root and trigeminal ganglia, a moderately heavy hybridization signal was evenly distributed over small, medium, and large ganglion cells ŽFig. 7C, D..
3.9. Co-localization of the GABA B2 and GABA B1b subtypes The in vivo co-expression of GABA B2 and GABA B1b mRNAs in various regions of the rat brain was determined at the cellular level by in situ hybridization studies using w35 SxdATP-labeled-oligoprobes on adjacent rat brain sections. Co-localization was confirmed in brain regions with identifiable structural features, such as the hippocampus, piriform cortex, mesencephalic trigeminal nucleus, and cerebellum. In the hippocampus, all of the pyramidal cells in the CA1, CA2, and CA3 fields of the hippocampus exhibited expression of both GABA B2 and GABA B1b mRNAs ŽFig. 8A–D. as did all cells in the piriform cortex, motor trigeminal nucleus, and the cerebellar Purkinje cells
Fig. 4. High-magnification brightfield photomicrographs of GABA B2 mRNA expression in coronal sections of the hippocampal formation. ŽA. CA1 and ŽB. CA3 fields. Note the presence of silver grains over the pyramidal cell layer Žpcl. in each of the fields and the absence of signal in the stratum oriens Žso. and stratum lucidum Žsl.. ŽC. Silver grains are evident over cells in the granule cell layer Žgcl. of the dentate gyrus and pyramidal cells in the polymorphic dentate gyrus ŽPoDG. Žarrows.. Granule cells extend into the molecular layer Žml.. Scale bar ŽC., 40 mm.
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Fig. 5. GABA B2 mRNA hybridization signal obtained using w35 Sx-labeled GABA B2 oligonucleotide probes in select thalamic nuclei and epithalamus. High-magnification brightfield photomicrographs of emulsion-dipped tissues demonstrating GABA B2 mRNA expression in the ŽA. medial habenula, the asterisk marks the third ventricle, ŽB. anterodorsal thalamic nucleus, ŽC. paraventricular thalamic nucleus, ŽD. ventroposterior thalamic nucleus, ŽE. reuniens nucleus, and ŽF. reticular thalamic and ventromedial nuclei. In A–F arrows indicate positive cells, in F lower levels of GABA B2 mRNA expression are visible over reticular thalamic neurons Žopen arrows.. Scale bar ŽF., 40 mm.
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Fig. 6. The localization of the GABA B2 mRNA in select brain regions. High-magnification brightfield photomicrographs of emulsion-dipped tissues showing the distribution of GABA B2 mRNA in the ŽA. laterodorsal septum, the asterisk marks the third ventricle, ŽB. central amygdaloid, ŽC. basolateral amygdaloid, ŽD. medial amygdaloid nuclei, ŽE. raphe magnus, and ŽF. the cerebellum. In F both the Purkinje cells Žarrows. and scattered cells within the granule cell layer Žsmall arrowheads. are positive. Arrows in A–F identify cells expressing GABA B2 mRNA. Scale bar ŽE., 40 mm.
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Fig. 7. The distribution of GABA B2 mRNA in the spinal cord and sensory ganglia. High-magnification brightfield photomicrographs showing a weak GABA B2 mRNA expression in the spinal cord ŽA. superficial dorsal horn, lamina I, and ŽB. lamina VI. Arrows indicate cells expressing the GABA B2 mRNA. A moderate-to-high expression of GABA B2 mRNA is observed in all ganglion cells of the ŽC. trigeminal and ŽD. dorsal root ganglia. Scale bar ŽD., 40 mm.
ŽFig. 8E–H.. Individual neurons in the hippocampus and cerebellum expressing both mRNAs were identified on adjacent sections ŽFig. 8C,D,G,H.. While individual cells could not be discretely identified, microscopic examination of the neocortex, medial habenula, locus coeruleus, se-
lected thalamic, and brainstem nuclei suggested co-expression of the GABA B2 and GABA B1b mRNAs Ždata not shown.. In these regions, virtually all of the cells exhibited hybridization signal for both the GABA B2 and GABA B1b mRNAs.
Fig. 8. Co-localization of GABA B1 b and GABA B2 mRNA in selected regions of the rat brain. High-magnification photomicrographs of the autoradiographic images of adjacent coronal sections through the rat dorsal hippocampus and diencephalon ŽA,B. and cerebellum ŽE,F., illustrating overlapping hybridization signal for GABA B2 ŽA,E. and GABA B1b mRNA ŽB,F.. Arrows in E and F identify the Purkinje cell layer. ŽC,D. Brightfield photomicrographs from adjacent sections of the region outlined by the box in A showing silver grains over all of the pyramidal cells in the CA1 field of the hippocampus. Arrows indicate identical cells labeled for ŽC. GABA B2 and ŽD. GABA B1b mRNA. ŽG,H. Brightfield photomicrographs from adjacent sections of the region outlined by the box in E. Arrows indicate identical Purkinje cells expressing ŽG. GABA B2 and ŽH. GABA B1b mRNA. Scale bar ŽB., 40 mm and applies to A, B, E and F. Scale bar ŽD., 40 mm and applies to C,D,G, and H. The emulsion exposure times were shortened to 1 week so that the silver grains would not interfere with identification of individual cells.
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4. Discussion The anatomical distribution of mRNA encoding a recently reported protein, GABA B2 , has been revealed throughout the adult rat CNS by in situ hybridization histochemistry. The results indicate that the GABA B2 mRNA expression is restricted primarily to neuronal profiles. Furthermore, the GABA B2 messenger RNA is widely expressed throughout the rat CNS and primary sensory ganglia. Expression levels of the GABA B2 mRNA in the rat brain varied among the regions of the CNS. Several areas exhibited intense GABA B2 mRNA expression, including the medial habenula, piriform cortex, the CA2rCA3 fields and dentate gyrus of the hippocampus, while moderate GABA B2 expression was observed throughout the entire neocortex, the CA1 field of the hippocampus, the majority of thalamic nuclei, and the cerebellar Purkinje cells. Lower GABA B2 mRNA expression was seen throughout most of the hypothalamus, mesencephalon, and several brainstem nuclei. GABAergic neurons and terminals have been described to be widely distributed throughout the rat CNS w48x, and the localization of the GABA B2 message correlates well with the distribution of GABAergic neurons. Moreover, the pattern of distribution of the GABA B2 mRNA correlates reasonably well with the distribution described for GABA B receptor binding sites by receptor autoradiography w16,21,30x, and the distribution described for both GABA B1a and GABA B1b by in situ hybridization w10,37x. There were differences between the expression of the GABA B1 isoforms and GABA B2 , most notably in the hypothalamus, caudate–putamen, and locus coeruleus Žsee below.. In these regions, the GABA B2 message appeared much weaker than that observed for either of the GABA B1 mRNAs. Additionally, the cerebellar Purkinje cells express the GABA B2 and GABA B1b mRNAs, but not the GABA B1a mRNA. There have been numerous reviews of the actions of GABA in the mammalian brain w4,35,43,53,65x and on the physiological role for GABA B receptors w9,15,17,39, 43,45x. The extensive expression of both the GABA B2 and GABA B1 messages in the rat brain w34,38,40,63x implies that GABA mediates many of its effects through these proteins, thereby influencing a variety of neurotransmitter systems, including glutamate w6,8,50x, serotonin w6,62x, NPY w33x, acetylcholine w32x, histamine w60x and dopamine w26,28,49x. The expression of GABA B2 mRNA in primary sensory neurons, the spinal cord and the relay nuclei of several sensory pathways suggest the possible participation of GABA B2 in the modulation a variety of sensory modalities. In the olfactory and visual pathways, GABA B2 message appears to be located in a position to potentially modulate excitatory glutamatergic neurotransmission w46,59,61x. GABA B2 mRNA is expressed in the target regions of the
projection fibers from the main olfactory bulb, namely the anterior olfactory cortex, olfactory tubercle, taenia tecta, piriform, and entorhinal cortices. This is in addition to the target regions of the retina, such as the superior colliculus and the dorsal lateral geniculate nucleus. The extensive presence of GABA B2 message throughout both of these systems may allow for considerable regulation of neurotransmission by the GABA B2 . It has been well established that GABA B antagonists can block the antinociceptive effects of GABA and GABA B agonists w18x. Moreover, GABAergic neurons in the spinal cord dorsal horn have been reported to be presynaptic to Ad and C primary afferent terminals w2,7x. The expression of GABA B2 message in cells of the superficial dorsal horn supports a potential presynaptic autoreceptor localization for this protein. As such, GABA B2 may have a role in mediating GABA’s inhibition of neurotransmitter release in the spinal cord. Likewise, our studies supply evidence for GABA B2 to potentially modulate the influence of excitatory glutamatergic nociceptive primary afferents, namely, the terminals of glutamatergic dorsal root and trigeminal ganglion cells w20,36x. GABA B2 mRNA expression was evident in both of these sensory ganglia and in small and medium-sized ganglion cells. A further indication of a possible integral role for GABA B2 protein in processing nociceptive information comes from observation of the extensive and abundant expression of GABA B2 mRNA in the thalamus, neocortex, the raphe nuclei, and the periaqueductal gray. A potential involvement of GABA B2 in cognitive functioning as well as in absence epilepsy is suggested by its moderately abundant expression throughout the cerebral cortex, thalamus, and the hippocampal pyramidal and dentate gyrus granule cells. Several studies have shown that physiological as well as pathological neuronal activity in the hippocampus can be regulated by GABA B receptors w17,19,31,47x. Furthermore, pre- and post-synaptic GABA B receptors have been shown to be involved in the regulation of the induction of long term potentiation w23x. Additional evidence for the involvement of GABA B receptors in learning and memory comes from studies showing that the GABA B antagonist, CGP 36,742, can improve learning performance in aged rats and rhesus monkeys while baclofen, a GABA B agonist, impairs spatial learning w3,44,46x. The overlapping distribution of GABA B2 and GABA B1b mRNAs observed in coronal rat brain sections ŽFig. 8A,B,E,F. supports the recent reports that the GABA B1 and GABA B2 proteins assemble as a heterodimer in vivo w34,38,40,63x. In situ hybridization histochemistry employing w35 Sx-labeled GABA B2 and GABA B1b oligonucleotide probes on adjacent tissue sections in the hippocampus and cerebellum enabled us to identify individual neurons expressing both GABA B2 and GABA B1b mRNAs. In other regions of the brain Že.g., the neocortex, medial habenula, thalamus, locus coeruleus, and mesencephalic trigeminal
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nucleus., it was more difficult to identify individual neurons; however, co-expression could be implied since all of the cells in these regions appeared to express both the GABA B2 and GABA B1b mRNAs. In addition, extensive co-localization of the GABA B2 and GABA B1b proteins has been reported at extrasynaptic sites of cerebellar Purkinje cell dendritic spines w38x. Taken together, these observations support the view that the GABA B2 and GABA B1b proteins associate in vivo to produce a functional receptor and that the heterodimers may be localized to discrete neuronal sites such as dendritic spines. Other as yet unidentified GABA B receptor homologues may associate elsewhere to produce novel subtypes. The low level of expression of GABA B2 mRNA relative to either of the two GABA B1 isoforms seen in the hypothalamus, caudate–putamen, and locus coeruleus Žsee above. raises the possibility that other GABA B receptor homologues may associate with GABA B1 to produce novel subtypes in these regions. Alternatively, the mismatch in mRNA levels in these areas may reflect regional differences in the rate of mRNA turnover. Future studies using isoform-specific antibodies will reveal interesting detail of the regional heterogeneity of GABA B receptors. In conclusion, we have described the localization of mRNA in the adult rat brain for a new member of the GABA B family, GABA B2 . The distribution of the GABA B2 mRNA suggests its potential involvement in the modulation of a variety of neurotransmitters systems. These findings also imply that some of the physiological actions of GABA B pharmacological agents may be through interaction with GABA B2 . Acknowledgements The authors would like to thank Drs. Douglas Craig and Roman Artymyshyn for reviewing the manuscript, Mary Johnson for technical assistance, and Elisabeth Griggs for photographic assistance.
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