Expression of Cx36 in mammalian neurons

Brain Research Reviews 32 Ž2000. 72–85 www.elsevier.comrlocaterbres

Short review

Expression of Cx36 in mammalian neurons Daniele F. Condorelli a

a,)

, Natale Belluardo b, Angela Trovato-Salinaro a , Giuseppa Mudo`

c

Section of Biochemistry and Molecular Biology, Department of Chemical Sciences, UniÕersity of Catania, Viale A Doria 6 95125 Catania, Italy b Institute of Human Physiology, UniÕersity of Palermo, 90100 Palermo, Italy c Department of Physiological Sciences, UniÕersity of Catania, 95125 Catania, Italy

Abstract Cx36 is the first mammalian member of a novel subgroup of the connexin family, characterized by a long cytoplasmic loop, a peculiar gene structure and a preferential expression in cell types of neural origin. In the present review we summarize the evidence in favour of its predominant expression in neuronal cells in the mammalian central nervous system, such as results from experiments with specific neurotoxins and co-localization of Cx36 mRNA and a neuronal marker. We also report a detailed description of Cx36 mRNA distribution in the rat and human central nervous system by in situ hybridization and, for each brain region, we correlate the novel findings with previous morphological or functional demonstrations of neuronal gap junctions in the same area. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Molecular neuroscience; Gap junctions; Electrical synapses; Interneuronal communication

Contents 1. Introduction

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2. Cx36 protein and gene structure .

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3. Evidence for preferential expression of Cx36 mRNA in neural tissue . 4. In situ hybridization analysis in the rat central nervous system 4.1. Spinal cord and dorsal root ganglia . . . . . . . . . . . . 4.2. Inferior olivary complex and other brainstem nuclei . . . 4.3. Cerebellum . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Mesencephalon . . . . . . . . . . . . . . . . . . . . . . . 4.5. Hypothalamus . . . . . . . . . . . . . . . . . . . . . . . 4.6. Thalamus . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Habenular nuclei and pineal gland . . . . . . . . . . . . . 4.8. Basal ganglia . . . . . . . . . . . . . . . . . . . . . . . . 4.9. Septum, basal forebrain, amygdala and piriform cortex . . 4.10. Hippocampal formation . . . . . . . . . . . . . . . . . . 4.11. Cerebral cortex . . . . . . . . . . . . . . . . . . . . . . 4.12. Olfactory bulb . . . . . . . . . . . . . . . . . . . . . . . 4.13. Retina . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5. In situ hybridization analysis in the human central nervous system

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Corresponding author. Fax: q0039-095-580138; e-mail: [email protected]

0165-0173r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 1 7 3 Ž 9 9 . 0 0 0 6 8 - 5

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D.F. Condorelli et al.r Brain Research ReÕiews 32 (2000) 72–85 6. Evidence for neuronal expression of Cx36 . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Evidence for neuronal localization by neurotoxic experiments . . . . . . . . . . . . 6.2. Evidence for neuronal expression by morphological analysis of expressing cells . . 6.3. Evidence for neuronal expression by co-localization studies with neuronal markers .

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7. Postnatal developmental pattern of Cx36 expression in the rat brain 8. Conclusion . References

1. Introduction Gap junctions are specialized membrane regions composed of aggregates of transmembrane channels that directly connect the cytoplasm of adjacent cells. The passage of ions and small molecules Žmol.wt.- 1200 Da. through the gap junction-channels results in metabolic and electrical coupling of cells. These intercellular channels are present in almost all cell types of vertebrate organisms Žfor a review see Ref. w9x. and, in the nervous system, gap junction-mediated communication is the most common form of electrotonic coupling between neurons. In other words, interneuronal gap junctions provide the structural basis for the so-called ‘‘electrical synapses’’. The early demonstration of the existence of gap junctions and electrical synapses between neurons goes back to more than 30 years ago Žfor a review see Ref. w8x., but the fact that initial studies were performed in invertebrates and fishes generated the widely held opinion that gap junction–communication between neurons was characteristic of lower forms, but played only a minor role in the mammalian brain. Such idea was also strengthened by the technical difficulties for the demonstration of functional gap junctions in the mammalian central nervous system Žfor a discussion see Ref. w8x.. However, during the last 30 years, several studies have shown the existence of neuronal gap junctions in specific areas of the adult mammalian Žrat, guinea pigs, rabbit, cat, monkeys. brain, by functional assays Želectrophysiological recordings andror dye coupling. w1,2,7,38–40,45–48,74,91,95x and morphological analysis Želectron microscopy. w3,21,22,36,68,72,75,82,83 x. An important step forward in the field has been the cloning of the protein subunits that form the intercellular channels: the connexins w62x. It was soon clear that connexins were encoded by a large multigene family and, after 10 years, 13 different members of the connexin family were known in rodents Žfor a review see Ref. w9x.. The availability of specific cDNA probes and specific antibodies allowed an analysis of the pattern of expression of these connexins and of their cellular localization. Surprisingly, the analysis in the central nervous system showed that these connexins either were not expressed in the brain or were expressed predominantly in glial cells Ži.e., Cx43, Cx40, Cx30 and Cx45 in astrocytes and Cx32 and Cx45 in oligodendrocytes; for a review see Refs. w16,17x.. How-

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ever, expression of Cx32 and Cx43 was also revealed restricted in specific neuronal subpopulations in several regions of adult or developing rat brain w5,15,33,50– 52,56,77,93,94x. The analysis of the scientific literature regarding neuronal gap junctions and connexins in the CNS revealed a striking discrepancy between the high density of neuronal gap junctions in adult inferior olives and the relatively low levels of Cx32 expression in this specific brain nucleus. This discrepancy prompted us a search for novel connexin transcripts using RNA extracted from microdissected rat inferior olivary region. Our cloning method was based on the reverse transcription–polymerase chain reaction ŽRT-PCR. with degenerate primers, designed on the basis of two conserved regions in the connexin family. This strategy led us to the identification of a novel connexin, called Cx36, highly enriched in the inferior olivary nucleus and to the isolation of its mouse gene w12x.. Independently, Sohl ¨ et al. w80x cloned the rat Cx36 using RT-PCR with primers designed on the basis of the sequence of skate Cx35 w58x and analysed the mouse gene structure. In our first study on rat Cx36 we analysed the pattern of expression of rat Cx36 by in situ hybridization and showed, by neurodegeneration with specific toxins, its predominant expression in neuronal cells of the inferior olive and hippocampus. In the present review, we report a more detailed description of Cx36 distribution in the central nervous system and a confirmation of its predominant neuronal expression in several areas of CNS by co-localization of Cx36 mRNA and a neuronal marker.

2. Cx36 protein and gene structure Mouse and rat Cx36 is a 321 amino acid protein showing all the typical features of members of the connexin family w12,80x. It is characterized by a long cytoplasmic loop of 99 amino acids and by a relatively short carboxy-terminal domain. The intracellular loop of human and rodent Cx36 shows a glycine-rich tract of 18 amino acids, absent in the corresponding region of fish and skate proteins. Cx36 is the mammalian ortholog of the skate Cx35, a previously cloned connexin expressed in the retina of the

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primitive vertebrate Raja Erinacea w58x. Moreover, two other related connexins have been recently cloned from fish Žperch. retina: Cx35, which is the perch ortholog of skate Cx35 and mouse Cx36, and Cx34.7, which represents a novel gene w59x. At the level of gene structure Cx35r36 connexins are different from the a and b groups of connexins for the presence of a single intron located within the coding region, 71 bp after the translation initiation site w12,58,59,80x. Based on the gene structure, the brain andror retinal expression, and phylogenetic analysis it has been proposed that Cx36rCx35 connexins represent a distinct subgroup of the connexin family w59,80x.

3. Evidence for preferential expression of Cx36 mRNA in neural tissue An intense single band of approximately 2.9 kb can be detected by Northern blot using RNA extracted from rat inferior olive and retina. Results obtained by Northern blot w12x, RNAse protection assay ŽFig. 1. and RT-PCR ŽFig. 1. confirm an intense expression in the retina, inferior olive and olfactory bulb. A band of lower intensity is also observed in several brain regions and in endocrine glands, such as the pineal gland and the pituitary gland, while no hybridization band is detectable in non-neural tissues, such as spleen, liver, kidney, lung, lens, and heart ŽFig. 1.. By RT-PCR and ribonuclease protection assays the expression of Cx36 mRNA is detectable in primary neuronal cell cultures, but not in primary astroglial, oligodendroglial or microglial cultures Žour unpublished results..

Fig. 2. Representative autoradiograms of in situ hybridization for Cx36 mRNA in coronal section of adult rat brain. ŽA. antisense probe; ŽB. sense probe; CTX: cerebral cortex; Hi: hippocampus; Hb: habenula; TH: thalamus; Rt: reticular thalamic nucleus; Zi: zona incerta; Hy: hypothalamus; CPu: caudate–putamen; Pir: piriform cortex.

Fig. 1. ŽA. Cx36 mRNA levels in differerent adult rat tissues analysed by RNAse protection assay. Ž1. labeled riboprobe; Ž2. yeast RNA; Ž3. lung; Ž4. heart; Ž5. kidney; Ž6. liver; Ž7. lens; Ž8. pituitary gland; Ž9. spinal cord; Ž10. inferior olive; Ž11. brain stem; Ž12. cerebellum; Ž13. striatum; Ž14. hypothalamus; Ž15. olfactory bulb; Ž16. retina. ŽB. RT-PCR detection of Cx36 mRNA in different adult rat tissues. M: 100-bp ladder; Lu: lung; K: kidney; R: retina; Le: lens; H: heart; OB: olfactory bulb.

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4. In situ hybridization analysis in the rat central nervous system Using a specific antisense riboprobe for the intracellular domain we have analysed the distribution of Cx36 mRNA at different levels of the rat central nervous system. Control sense riboprobe resulted in a signal equivalent to background in all the regions examined ŽFig. 2.. In the following sections we will report, for each brain region, the distribution of Cx36 mRNA, the previous morphological or functional demonstrations of neuronal gap junctions in the same area, and a brief discussion on the possible physiological meaning.

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nexin from the embryonic life through adulthood. It has been also reported that nerve damage induces the reappearance of gap junction coupling among adult motoneurons w4x. A laborious combination of confocal light microscopy and ‘‘grid-mapped freeze-fracture’’ electron microscopy has revealed an unexpectedly high incidence of mixed synapses with the morphological characteristics of both gap junctions and chemical synapses in the spinal cord of adult rat w68x. The presence of a widespread expression of Cx36 in spinal cord is in agreement with these morphological observations and challenges the idea that synaptic activity in the spinal cord of adult mammals can be attributed exclusively to chemical neurotransmission.

4.1. Spinal cord and dorsal root ganglia 4.2. Inferior oliÕary complex and other brainstem nuclei Labeled cells are present in all the lamina of the gray matter of the spinal cord, with the highest grain density in the motoneurons of the anterior horn ŽFig. 3.. Scattered Cx36-expressing cells are also found in dorsal root ganglia ŽFig. 3.. Although a transient gap junctional coupling among spinal motoneurons can be observed only during early postnatal development w90x, Balice-Gordon et al. w4x have recently shown, by immunostaining and in situ hybridization, that most motoneurons express more than one con-

As expected, the most intense signal among the cerebral areas examined is observed in the nuclei of the inferior olivary complex. All the olivary neurons appear strongly labeled Žthe highest intensity of labeling per cell observed in this study., with no difference between the various subdivision of the olivary complex ŽFig. 4.. Indeed, the presence of a high density of neuronal gap junctions have been firmly demonstrated in this nucleus by physiological and morphological evidence w3,7,21,22,38–40,50,72,74,

Fig. 3. Dark and bright-field microautoradiographs from emulsion dipped slides hybridized with a riboprobe specific for Cx36 mRNA. ŽA. Schematic representation of atlas section through lumbar spinal cord. ŽB. Dark-field microautoradiographs of shadow area shown in A. ŽC. Dark-field microautoradiographs of dorsal root ganglia section. ŽD. Bright-field high magnification of labeled spinal neurons shown in B. Spc: spinal cord; DRG: dorsal root ganglia; Scale bar: B and C: 200 mm; D: 25 mm.

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4.4. Mesencephalon

Fig. 4. In situ hybridization for Cx36 mRNA in various hindbrain regions. ŽA. Dark-field microautoradiographs from inferior olivary complex; IOD: inferior olive dorsal nucleus; IODM: inferior olive dorsomedial cell group; IOM: inferior olive medial nucleus; IOPr: inferior olive principal nucleus; ŽB. Dark-field microautoradiographs from facial nucleus Ž7.; ŽC. Dark-field microautoradiographs from lateral cerebellar nucleus ŽLat.; ŽD. Bright-field micrograph from granular layer ŽGL. of cerebellar cortex showing scattered labeled cells with large pale nuclei Žarrows.; ŽE. Bright-field micrograph from molecular layer ŽMoL. of cerebellar cortex showing scattered labeled cells with large pale nuclei Žarrows.. Scale bar: A, B, C: 200 mm; D, E: 25 mm.

Cx36 mRNA is expressed in several nuclei such as the red, pararubral and oculomotor nuclei, the periaqueductal grey and the superior and inferior colliculi ŽFig. 5.. Scattered positive cells with a low intensity of labeling are present in the interpeduncular nucleus. Cx36-expressing cells can be also observed in the substantia nigra Žboth pars compacta and pars reticulata. and in the ventral tegmental area ŽFig. 5.. Double labeling by in situ hybridization for Cx36 and immunohistochemistry for thyrosine hydroxylase confirms that dopaminergic neurons of substantia nigra pars compacta express Cx36 mRNA Žour unpublished results.. Indeed, Grace and Bunney w29x have already reported the presence of dye- and electrotonic coupling between sets of rat zona compacta dopaminergic neurons, and suggested that electrical communication between these neurons could be involved in modulating burst firing and in synchronizing dopamine release. Moreover, electrophysiological recordings from substantia nigra dopaminergic neurons in freely moving rats revealed a form of interaction between dopaminergic cells Žpresumed electrical coupling. that is only rarely observed in anesthetized or paralyzed rats w27x. 4.5. Hypothalamus The hypothalamus can be considered one of the brain areas with a moderate expression of Cx36 mRNA ŽFig. 2..

82,83,91x. The functional implications of the microcircuitry of the inferior olive and of its neuronal gap junctions have been recently reviewed w23x. Several other brain-stem nuclei show a moderate expression of Cx36, including the lateral reticular nucleus, nucleus ambiguus, hypoglossal nucleus, the gracilis and cuneate, the vestibular and cochlear nuclei, the gigantocellular reticular nucleus, the nucleus facialis ŽFig. 4., the sensitive trigeminal nucleus and the pontine nuclei. 4.3. Cerebellum Cx36 expression is detected in subpopulations of scattered cells present in the inner third of the molecular layer and throughout the granular layer ŽFig. 4.. The nuclei of Cx36 expressing cells in the granule cell layer are larger and weakly stained compared to those of the large majority of Cx36-negative granule cells ŽFig. 4.. An attractive hypothesis, that can be easily tested, is that Cx36 is expressed in GABAergic interneurons, such as the Golgi cells in the granule cell layer or the basket cells in the molecular cell layer. Sotelo and Llinas ` w81x have previously reported the existence of electrotonic coupling in the vertebrate cerebellar cortex. Cx36 expressing cells are also detected in all deep cerebellar nuclei Žsee lateral cerebellar nucleus in Fig. 4..

Fig. 5. In situ hybridization for Cx36 mRNA in various mesencephalic nuclei. ŽA. Dark-field microautoradiographs showing labeled cells in substantia nigra pars compacta ŽSNC., supramammillary nucleus ŽSuM. and lateral hypothalamus ŽLH.; ŽB. Dark-field microautoradiographs showing oculomotor nucleus Ž3.; ŽC. Dark-field microautoradiographs showing red nucleus, magnocellular part ŽRMC. and parvicellular part ŽRPC.; ŽD. Bright-field micrograph showing labeled cells in SNC; ŽE. Bright-field micrograph showing labeled cells in oculomotor nucleus. Scale bars: A, C: 200 mm; B: 100 mm; D, E: 50 mm.

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Although the percentage of Cx36-positive neurons and the intensity of labeling per cells is not homogeneous, the vast majority of neuronal groups express Cx36 mRNA. Moderately to intensely labeled cells can be observed in nuclei of the preoptic region, of the anterior region Žparaventricular nucleus, anterior hypothalamic nucleus, lateral hypothalamic area, supraoptic nucleus., of the tuberal region Žsuch as arcuate nucleus, ventromedial and dorsomedial nucleus, perifornical nucleus. and of the mammillary region. The presence of dye- and electronic coupling in some hypothalamic nuclei, such as the supraoptic and the paraventricular nuclei, has been well documented w1,52,95x. Moreover, the intensity of dye-coupling and the number of dendrodendritic membrane contacts between neuroendocrine cells in supraoptic nucleus increase in response to dehydration, gestation and lactation, suggesting an important role for gap junctions in the secretion of oxytocin and vasopressin w52,64x. Milk ejections follow sharp rises in plasma oxytocin which are produced by synchronized bursts of highfrequency firing by the oxytocin neurons of the magnocellular nuclei of hypothalamus. An up-regulation of Cx32 mRNA levels in the rat supraoptic nucleus has been found during late pregnancy and during lactation w52x. The functional and structural relationship between Cx32 and Cx36 in these neuronal nuclei represents an interesting question that can be now addressed experimentally. It has also been suggested a role for gap junctions and Cx32 in the pulsatile release of gonadotropin-releasing hormone from the median eminence w33x. 4.6. Thalamus Several thalamic nuclei Žsuch as mediodorsal, ventrolateral, ventromedial, ventroposterior and posterior thalamic nuclei, lateral and medial geniculate nuclei. are completely devoid of hybridization grains, with the notable exception of the reticular thalamic nucleus that is strongly and homogeneously labeled ŽFigs. 2 and 6.. A careful analysis reveals a weak expression of Cx36 mRNA in the anterior Žanterodorsal, anteroventral, anteromedial. and lateral Žlaterodorsal and lateral posterior. thalamic nuclei, in the parafascicular nucleus, and in several intralaminar nuclei, such as centrolateral, paracentral, central medial nuclei, rhomboid and reuniens nuclei ŽFigs. 2 and 6.. A diffuse labeling is also observed in the zona incerta and in the subthalamic nucleus ŽFig. 7.. Reticular thalamic neurons are parvalbumin-containing GABAergic neurons that are involved in the genesis of thalamocortical oscillations w20,67x. All the thalamic nuclei receive GABAergic fibers from restricted zones of the reticular nucleus, and in turn, send excitatory projections to the same zone of the reticular nucleus. The reticular nucleus is especially active during sleep, when its GABAergic projections phasically hyperpolarize cells in other thalamic nuclei, thus inhibiting the transfer of sensory- or motor-related activity to the cortex and establish-

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Fig. 6. In situ hybridization for Cx36 mRNA in some diencephalic structures. ŽA. Dark-field microautoradiographs showing intensely labeled pineal gland ŽPi.; ŽB. Dark-field microautoradiographs showing habenular nuclei Žmedial: MHb; lateral: LHb.; ŽC. Dark-field microautoradiographs showing reticular thalamic nucleus ŽRt.; ŽD. Bright-field micrograph showing labeled cells in reticular thalamic nucleus; ŽE. Simultaneous detection of Cx36 transcripts and NeuN neuronal marker in reticular thalamic neurons: note the localization of hybridization grains in NeuN-positive cells. Scale bars: A, B: 400 mm; C: 200 mm; D, E: 25 mm.

ing a rhythmic firing mode. Mutually connected inhibitory neurons are thought to be responsible for rhythm generation in the reticular thalamic nucleus w18,61x. Dendrodendritic synapses have been found between dendrites of reticular thalamic neurons and the possible relationship andror association with dendrodendritic gap junctions deserves further analysis. It is also interesting the presence of Cx36 mRNA in intralaminar thalamocortical cells, such as neurons of the centrolateral nucleus, because of the possible involvement of these neurons in the distribution of the coherent 40 Hz oscillation that characterizes the magnetoencephalographic activity during wakefulness and REM sleep w42,84x. 4.7. Habenular nuclei and pineal gland An intense and widespread hybridization signal can be observed in the medial habenular nucleus, while only scattered Cx36-expressing cells are present in the lateral nucleus. An intense hybridization signal is also present in the pineal gland ŽFig. 6.. Saez ´ et al. w73x have reported the

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absence of Cx36 in the vast majority of striatal cells ŽFig. 7.. However, gap junctions have been shown among parvalbumin-positive striatal neurones w35x that constitute only 3–5% of striatal cells, and this observation is in agreement with the low number of scattered striatal cells that express Cx36 mRNA. Such relatively rare cells might form a continuous network that integrates excitatory input over a larger scale than that of a single neuron. Indeed, parvalbumin-positive cells are GABAergic interneurons that receive powerful excitation from the cerebral cortex and that could participate to the synchronization of striatal spiny neurons. 4.9. Septum, basal forebrain, amygdala and piriform cortex Fig. 7. In situ hybridization for Cx36 mRNA in the striatum and in the subthalamic nucleus. ŽA. Dark-field microautoradiographs showing scattered intensely labeled cells in the adult rat caudate–putamen; ŽB. Simultaneous detection of Cx36 transcripts and NeuN neuronal marker in striatal neurons, showing that a single labeled cell, representative of scattered labeled cells shown in A, is NeuN-positive Žarrow.. Note that the majority of NeuN positive cells are unlabeled. ŽC. Dark-field microautoradiographs showing labeled cells in the subthalamic nucleus ŽSTh; surrounded by dashed line.. ZIV: zona incerta ventral part; LH: lateral hypothalamus; cp: cerebral peduncle. Scale bar: A, C: 200 mm; B: 50 mm.

presence of dye- and electrical-coupling between cultured pinealocytes and suggested that metabolic and electrical synchronization mediated by gap junctions may favor melatonin secretion. The same research group have also shown that pinealocytes express Cx26, whereas Cx43 is confined to astrocytes and Cx32 is not expressed in the pineal gland. Although it is likely that Cx26 and Cx36 are co-expressed by pinealocytes, this hypothesis and the possible functional and structural interactions between Cx26 and Cx36 should be tested in a rigorous way. 4.8. Basal ganglia In basal ganglia structures, such as caudate putamen, globus pallidus and nucleus accumbens labeling was localized in a subpopulation of scattered cells with large pale nuclei. In particular, a low number of scattered striatal cells are intensely labeled ŽFig. 7.. Dye-coupling between neurons of nucleus accumbens and its modulation by dopamine has been reported w60x. In the striatum a membrane potential synchrony of simultaneously recorded striatal spiny neurons in vivo has been shown w85x. However, the same authors could not reveal direct synaptic or electrical interactions by simultaneous dual intracellular recordings from pairs of striatal spiny neurons in vivo, suggesting that other mechanisms are responsible for the synchronization of membranepotential state transitions. The lack of electrotonic coupling between striatal spiny neurons is in agreement with the

A moderate labeling for Cx36 mRNA is found in the medial septum, in the diagonal band, both vertical and horizontal limbs, and in the bed nucleus of the stria terminalis. In the amygdala, only the medial amygdaloid nucleus, the anterior and posteromedial cortical amygdaloid nucleus, the amygdalo-hippocampal transition area, and the medial and lateral part of the central nucleus express moderate amount of Cx36 mRNA. A diffuse labeling is observed in cells of the piriform cortex. The expression of Cx36 in cells of the piriform cortex is in agreement with the relatively high density of dendritic lamellar bodies, a neuronal organelle associated with dendrodendritic gap junctions, in the piriform cortex along the lateral olfactory tract w22x. 4.10. Hippocampal formation Cx36 expressing neurons are present in all the regions of the hippocampal formation, including the enthorinal cortex ŽFig. 8.. In the subicular area, a widespread moderate labeling is present in the vast majority of cells. It is also possible to distinguish a scattered subpopulation of intensely labeled cells. In the dentate gyrus, scattered labeled cells with large pale nuclei are observed within the stratum granulosum at the hilar border or just outside stratum granulosum either below or above the layer, and in the polymorph zone of the hilus ŽFig. 8.. However, principal cells in the stratum granulosum are unlabeled. Interestingly, all cells are labeled in the stratum pyramidale of CA3. On the contrary only scattered cells are labeled along the superior or inferior border of stratum pyramidale of CA1 and in the strata radiatum, oriens and lacunosummoleculare of CA3 and CA1 ŽFig. 8.. An interpretation of these results is that the principal pyramidal cells express Cx36 only in CA3 region, while GABAergic interneurons located in the various layers of CA1, CA3 and dentate gyrus represent the observed Cx36-positive scattered cells. Indeed, dendro-dendritic gap junctions are known to occur in rat and guinea-pig hippocampal hilar interneurons and it has been reported that a subpopulation of GABAergic

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shown that electrical coupling via gap junctions underlies a form of high-frequency oscillations Ž; 200 Hz. in the hippocampus in vitro w24x. 4.11. Cerebral cortex

Fig. 8. In situ hybridization for Cx36 mRNA in the hippocampal formation. ŽA. Dark-field microautoradiographs showing a coronal section of dorsal hippocampus; DG: dentate gyrus; PoDG: polymorph layer of dentate gyrus; Mol: molecular layer; CA1rCA3: pyramidal layers of hippocampus; Rad: stratum radiatum; Or, stratum oriens. ŽB. Bright-field micrograph showing labeled cells in dentate gyrus ŽDG.. Note labeled cells along the hilus border and in the hilus Žarrows.; ŽC. Bright-field micrograph showing labeled cells in the CA3 pyramidal layer; ŽD, E. Simultaneous detection of Cx36 transcripts and NeuN neuronal marker in hippocampal neurons of the dentate gyrus ŽD. and CA3 region ŽE.. Arrows indicate labeled NeuN positive cells. Scale bars: A: 200 mm; B, C, D, E: 50 mm.

hippocampal hilar interneurons are dye-coupled and can become synchronized by a mechanism probably involving electrotonic coupling w31,53x. Moreover, it is well-known that some specialized class of hippocampal interneurons ŽIS-1. form long dendrodendritic junctions with each other, in which typically two to three dendrites are intermingled for more than 100 um w28x. However, the presence of gap junctions in such dendritic structures, although very likely, has not been unequivocally demonstrated w28x. The presence of dye-coupling and electrotonic coupling between pyramidal neurons of the CA3 region has been first reported almost 19 years ago w45,46,48,49x and the intense and widespread Cx36 expression in the CA3 stratum pyramidale is in agreement with those results. Dyecoupling and electrotonic potentials have been shown also in CA1 neurons w66,89x and in dentate granule neurons w47x, but the possible involvement of Cx36 is less clear on the basis of its cellular localization. Other connexins w5,77x could play a role in these regions. It has been recently

Analysis of cerebral cortex reveals distinct variation in the density of labeled cells in the different neocortical layers. Labeled cells were absent in the most superficial Žlayer I. and were scattered in the remaining layers with the higher density in layer IV, V and VI ŽFig. 9.. The distribution of labeled cells and the degree of labeling per cell is similar in the cingulate and the retrosplenial cortices. It is generally believed that interneuronal coupling via gap junctions is extensive in the early postnatal cerebral cortex, where this form of neuronal communication precedes the formation of chemical transmission. It is well shown that neuronal coupling is stronger and widespread in the embryonic and early postnatal rat cortex w13,43,63,70,71x and declines rapidly during post-natal development. However, low values of neuronal dye-coupling have been also found in the adult rat cerebral cortex w13,56x and dendro-dendritic and dendro-somatic gap junctions between large stellate cells in layers IV and V have been observed in the primate sensori-motor cortex w65,78,79x. Indeed, the presence of several Cx36-expressing cells scattered through the various layers of cerebral cortex suggests that adult cortical neurons preserve their capability of expressing functional gap junctions more frequently than previously considered. The expression of Cx43 w77x and Cx32 w56x have also been detected in neuronal cells of adult neocortex. One possible function of interneuronal gap junctioncommunication in the adult neocortex and hippocampus might be the generation or modulation of synchronized oscillatory activity. Neurons can generate synchronized oscillations by a variety of mechanisms: some neurons may have an intrinsic propensity to oscillate and in the neocortex examples of intrinsic cellular oscillators at different rhythms include sparsely spiny layer 4 interneurons of guinea pig frontal cortex w41x, about 20% of long axon projection neurons in layers 5 and 6 in cat motor and association neocortex w57x, layer 5 pyramidal cells in rat sensorimotor cortex w76x, and layer 2r3 ‘‘chattering cells’’ in the cat striate cortex w30x. The existence of intrinsic oscillating cells does not in itself explain the synchronization of local population of neurones, but oscillating neurons can act as a pacemaker and entrain an entire neuronal network if cells are suitably coupled by chemical or electrical synapses or both. An alternative mechanism is that synchronized rhythms arise as a network property through a suitable interactions of neurons, that, as individuals, are non-rhythmic w10,34x. Both networks of inhibitory interneurones w11,34,86,87,92x or feedback loops between excitatory and inhibitory neurons w25,26x can generate the

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Fig. 9. In situ hybridization for Cx36 mRNA in the cerebral cortex. ŽA. Dark-field microautoradiographs showing a coronal section of primary somatosensory cortex; ŽB. Bright-field micrograph of section showed in A; ŽC. Bright-field high-magnification micrograph showing labeled cells from the VI layer of primary somatosensory cortex: arrows indicate labeled cells; note the presence of several unlabeled cells with large pale nucleus Žarrowhead.; ŽD. Simultaneous detection of Cx36 transcripts and NeuN neuronal marker in cortical neurons; arrows indicate Cx36-expressing NeuN-positive cells; note the presence of several NeuN-positive cells that do not express Cx36 mRNA Žarrowheads.. A, B: 200 mm; C, D: 50 mm.

synchronous neuronal oscillations in the 30–70 Hz range, known as gamma oscillations. When the features of the inhibitory network oscillation were examined using computer simulations, it was concluded that gap junctions were not required for generation of oscillations, but that they can enhance synchronization w92x. The analysis of electro-

physiological and neurochemical properties of Cx36-expressing cells in cerebral cortex and hippocampus could provide the necessary background for a better understanding of the possible role of neuronal gap junctions in generation or modulation of different types of cortical synchronous oscillatory activity.

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4.12. Olfactory bulb An intense and widespread labeling can be observed in the mitral cell layer. Cx36 expression is also detected in the glomerular layer and in scattered cells in the external plexiform layer Žperiglomerular cells and tufted cells. and in the granular cell layer. The presence of dendrodendritic gap junctions in the external plexiform layer of olfactory bulb has been shown several years ago w37x. Moreover, it has been reported that granule cells of the olfactory bulb are frequently arranged in rowlike aggregates of three to five cells. In such aggregates, neuronal somata are tightly packed and coupled by gap junctions, which may serve to synchronize the functional activity of these neurons w69x. Indeed, a characteristic feature of olfactory bulb function is its synchronous, rhythmical activity during sniffing and olfactory processing w44x. 4.13. Retina A strong labelling can be observed in the ganglion cell layer and in the inner border of the inner nuclear layer, where the amacrine cells are mainly localized. The presence of neuronal coupling in several cell types in mammalian retina is well established w32,88x. Moreover, Cx34.7, another member of the Cx35rCx36 subfamily, has been localised in fish retina w59x and the expression of Cx43 and Cx32 has been reported in mammalian retina Žsee discussion in Ref. w59x.. Interestingly, different properties Žsize permeability and regulation by second messengers. of two gap junctional pathways made by AII amacrine cells have been described w54x: identification of connexins expressed by amacrine cells should allow a correlation between connexin composition and functional properties.

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several myelencephalic nuclei, in specific cells of the cerebellar cortex, in a relatively large subpopulation of cells in the cerebral cortex, in the hilus of the dentate gyrus and in the strata radiatum and oriens of hippocampal subfields. Moreover, labeled cells can be revealed in all the lamina of the spinal cord gray matter.

6. Evidence for neuronal expression of Cx36 6.1. EÕidence for neuronal localization by neurotoxic experiments Two neuron-specific toxins were used: 3-acetylpyridine, which selectively destroys the inferior olive after i.p. injection w19x, and the ibotenic acid, which selectively destroys neuronal cell body, but spares glial cells, after intracerebral injection w14x. In agreement with a preferential neuronal localization, the stereotaxic intrahippocampal injection of ibotenic acid caused a complete disappearance of the hybridization signal for Cx36 in the hippocampal region w12x and the 3-acetylpyridine-treatment fully eliminated the strong signal normally present in the inferior olive w12x ŽFig. 10.. 6.2. EÕidence for neuronal expression by morphological analysis of expressing cells The staining of the brain sections with Cresyl violet allowed us to distinguish cells with large pale nuclei from

5. In situ hybridization analysis in the human central nervous system As a prerequisite for studies devoted to the investigation of the possible role of Cx36 in human neurological diseases, we have recently sequenced the human Cx36 gene and analysed its pattern of expression in the human brain by radioactive in situ hybridization w6x. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved from skate to man and that the gene structure is that typical of the Cx35r36 subgroup observed in the other species. The distribution of Cx36 in several regions of the human central nervous system is similar to that previously observed in rat brain. The most intense signal, among the cerebral areas examined by in situ hybridization, is present in the inferior olivary complex, both in principal and accessory nuclei. A moderate labeling is also found in

Fig. 10. In situ hybridization for Cx36 mRNA showing an intense labeled inferior olive complex ŽIO; arrowheads. in normal animals ŽA. and the complete disappearance of hybridization signal in the IO region Žarrowheads. 2 weeks after 3-acetylpyridine treatment ŽB..

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cells with small dark nuclei. Overall, the former cells can be potentially representative of neuronal cells and the latter of glial cells. By bright field microscopic observation the Cx36 hybridization grain were localized on cells with large pale nuclei in all the brain regions examined, in agreement with a neuronal localization of Cx36. No hybridization signal was detected in cells with small dark nuclei, in ependymal and meningeal cells, in choroid plexus and in white matter tracts. However, the limitation of the discrimination of neuronal and non-neuronal cells based on their nuclear size and staining must be emphasized. Another indication in favour of the neuronal expression of Cx36 derives from non-radioactive-digoxigenin in situ hybridization analysis: in these experiments, positive cells show a neuronal morphology w12x.

6.3. EÕidence for neuronal expression by co-localization studies with neuronal markers In order to confirm the neuronal localization of Cx36 we performed double labeling experiments by in situ hybridization for Cx36 mRNA and immunohistochemistry for a neuronal marker. We used a monoclonal antibody that specifically recognizes the DNA-binding, neuronspecific protein NeuN w55x. This antibody reacts with most neuronal cell types throughout the nervous system, staining primarily the nucleus of the neurons and only weakly the cytoplasm. As shown in Figs. 6–9 the hybridization grains were present only in NeuN-positive cells in several brain regions examined, such as reticular thalamic nucleus, striatum, hippocampus and cerebral cortex.

Fig. 11. Developmental expression of Cx36 mRNA. Bright- and dark-field microautoradiographs of cerebral cortex sections ŽA, B. at postnatal days 8 ŽP8, A. and 20 ŽP20, B. and of olfactory bulb ŽC, D. at postnatal day 4 ŽP4, C. and 8 ŽP8, D.. Gl: glomerular layer; EPl: external plexiform layer; Mi: mitral cell layer; iGr: internal granular layer. Scale bar: 200 mm.

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7. Postnatal developmental pattern of Cx36 expression in the rat brain The well established existence of a network of coupled neurons in the developing rat cortex w13,43,63,70,96x prompted us to analyse the cortical expression of Cx36 in early stage of postnatal brain development. At postnatal day 4 and 8, a widespread labeling was observed in superficial cortical plate layer ŽII–IV., while scattered labeled cells are present in layers V–VI ŽFig. 11.. At later stage Žpostnatal day 14. the diffuse labeling persists in layers II–III, while scattered cells with a higher grain density are detectable in layers IV–VI. At postnatal day 20, the adult pattern is already established and only scattered intensely labeled cells can be observed in layers II–VI with a higher density in layer IV and VI ŽFig. 11.. Therefore, a pattern of diffuse labeling seems to convert in a scattered pattern during the differentiation of the various layers of cerebral cortex, starting from the deeper layers and progressing toward the surface. The widespread pattern of Cx36 expression in the cortical plate is compatible with a participation of Cx36 in the extensive dye-coupling observed between neurons in the first two postnatal weeks w63x. Indeed, it has been shown by Northern blot analysis that Cx36 mRNA in the mouse brain is very high in the perinatal period reaching a peak of expression at seven days of postnatal life and then progressively declining to low adult levels w80x. Other connexins, such as Cx26 w15,56x, could also participate to the extensive coupling of cortical neurons during early postnatal development. Distinct changes in the pattern of Cx36 expression during early postnatal development can be observed in olfactory bulb. At postnatal day 4, a band of hybridization grains is clearly detectable over the entire mitral layer, but no Cx36 mRNA expression can be observed in the glomerular layer ŽFig. 11.. At postnatal day 8, the adult pattern is already established with labeling distributed over the mitral cell layer, the glomerular layer and scattered cells in the granular layer. This pattern is in agreement with the fact that the olfactory bulb is a site of postnatal neurogenesis in the rat and that the major portion of these postnatally formed cells are interneurons, such as granule cells of the granule cell layer or periglomerular cells of the glomerular layer. The projection neurons of olfactory bulb, the mitral cells, are generated prenatally, in accordance with the observation that Cx36 expression in the mitral cell layer is already evident at the first day of postnatal life.

8. Conclusion The identification of Cx36 and of other members of the same subfamily, and the demonstration of its preferential neuronal expression marks the beginning of a novel phase of research on the role of gap junctions in the nervous system. The distribution of Cx36 mRNA in the nervous

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system is not only in agreement with a wealth of previous functional and morphological studies on neuronal gap junctions, but stimulates further functional analysis in several other regions of the mammalian brain. Moreover, it has been possible for the first time to evaluate the expression pattern of a neuronal gap junction component in the human brain. Novel molecular tools are now available for a deeper understanding of interneuronal gap junction-mediated communication.

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