GAP JUNCTIONAL COMMUNICATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM

Cell Biology International 1998, Vol. 22, No. 11/12, 751–763 Article No. cb980391, available online at http://www.idealibrary.com on

GAP JUNCTIONAL COMMUNICATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM CHRISTIAN C. G. NAUS* and MAHMUD BANI-YAGHOUB Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario N6A 5C1, Canada Accepted 25 November 1998

The development of the central nervous system is a complex process involving multiple interactions between various cell types undergoing mitosis, migration, differentiation, axonal outgrowth, synaptogenesis and programmed cell death. For example, neocortical development is characterized by a series of transient events that ultimately leads to the formation of a discrete pattern of laminar and columnar organization. While neuron–glial cell–cell interactions have been shown to be involved in neuronal migration, recent observations that neurons are extensively coupled by gap junctions in the developing neocortex have implicated this phenomenon in the process of neocortical differentiation. The present review will examine the putative role of gap junctional intercellular communication in development of the central nervous system, with specific reference to recent studies in the development of the cerebral cortex.  1998 Academic Press

K: gap junctions; connexin; development; intercellular communication

INTRODUCTION The gap junction is the site of the intercellular membrane channels which provide for direct cytoplasmic continuity between adjacent cells (Bruzzone et al., 1996). The structural unit of the gap junction is the connexon, a proteinaceous cylinder with a hydrophilic channel. Connexons spanning the plasma membranes of closely apposed cells align end-to-end, forming intercellular channels which provide for the exchange of small molecules (less than 1200 Daltons) including second messengers and ions. Since the initial report of gap junctions by Furshpan and Potter (1959) describing a novel form of synaptic transmission in the giant motor synapse of the crayfish which involved the rapid transfer of electrical activity from the pre- to post-synaptic fibre, the complexity of the family of gap junction proteins and their constituent channels has increased dramatically (for recent reviews see Goodenough et al., 1996; Nicholson and Bruzzone, 1997). Following the isolation of gap junction proteins and the cloning of their mRNAs, it has been established that these *To whom correspondence should be addressed. 1065–6995/98/110751+13 $30.00/0

proteins, the connexins (Cx), are encoded by a multigene family consisting of at least 14 members in mammals. Alignment comparisons of connexins show that the transmembrane regions are highly homologous whereas the amino terminus, cytoplasmic loop and, particularly the carboxy terminus, are divergent. Gap junctions can be found in almost all mammalian tissues, attesting to a likely homeostatic role for these channels in cell function (Loewenstein, 1987). Several connexins exhibit a characteristic tissue and cellular distribution in the adult animal, implying functional differentiation among the different types of channels. To further complicate matters, many tissues and their constituent cells have been shown to express multiple connexins.

GAP JUNCTIONS IN THE CENTRAL NERVOUS SYSTEM Gap junctions were previously considered prevalent in invertebrate and lower vertebrate nervous systems, presumably functioning as a ‘primitive’ form of interneuronal communication via electro 1998 Academic Press

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Table 1. Expression of connexin genes in the nervous system Connexin

Cell type In vitro

Cx26

In vivo Neuroepithelium Neuron

Dermietzel et al., 1989; Matesic et al., 1993; Belliveau et al., 1997; Nadarajah et al., 1997; Nagy et al., 1997

Cx30

Astrocyte

Dahl et al., 1996

Cx31

Rhombomeres

Dahl et al., 1997

Cx32

Neuron

Reference

Oligodendrocyte Oligodendrocyte Neuron Neuron Schwann cell

Cx36

Micevych and Abelson, 1991; Belliveau and Naus, 1995; Nagy et al., 1988; Shiosaka et al., 1989; Yamamoto et al., 1990; Nadarajah et al., 1996; Li et al., 1997

Neuron

Condorelli et al., 1998; Sohl et al., 1998

Cx37

Neuroblast

Neuroblast

Willecke et al., 1991; Dermietzel et al., 1995; Haefliger et al., 1992

Cx40

Astrocyte Neuron

Astrocyte Neuron

Zheng et al., 1995

Cx43

Astrocyte Neuron

Astrocyte Neuron

Giaume et al., 1991a; Dermietzel et al., 1991; Micevych and Abelson, 1991; Naus et al., 1991a; Belliveau and Naus, 1995; Fushiki and Kinoshita, 1995; Nadarajah et al., 1997; Simburger et al., 1997

Cx45

Oligodendrocyte Oligodendrocyte Astrocyte

tonic synapses (Bennett, 1997). In addition to intercellular coupling between neurons, gap junctions exist between glial cells, including astrocytes, oligodendrocytes and ependymal cells (Dermietzel and Spray, 1993; Bruzzone and Ressot, 1997). These latter non-neuronal junctions presumably play a role in ion transfer and metabolic cooperation. While the in vivo electrophysiological and morphological demonstration of gap junctions in the mammalian CNS suggested a relatively restricted distribution, the presence of gap junctions has been more readily demonstrated in in vitro slice preparations of the mammalian CNS (reviewed in Jefferys, 1995). With the advent of biochemical and molecular advances in the gap junction field, several investigators have used immunohistochemistry or in situ hybridization to demonstrate that gap junctions are more widely expressed than previously thought throughout various regions of the CNS. Thus mRNA and/or protein for Cx26, Cx30, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43 and Cx45 have been demonstrated in brain, and in some cases resolved at the cellular level (see Table 1). Many of these connexins are differentially expressed during neural development (see below). Given the heterogeneous composition of brain tissue, some of this

Kunzelmann et al., 1997

expression is probably accounted for by endothelial cells which are known to express Cx37 (Reed et al., 1993) and Cx40 (Bruzzone et al., 1993), as well as Cx43 (Pepper et al., 1992), and meningeal cells which express Cx43 and Cx26 (Spray et al., 1991). However, the expression of many of these connexins has not been examined at the cellular level. GAP JUNCTIONS AND NEURAL DEVELOPMENT During development, there is extensive regulation of the spatial and temporal expression of gap junctions, this regulation being implicated in the control of differentiation and growth (Caveney, 1985). In general, there is high intercellular coupling during development, with a loss or attenuation of coupling during terminal differentiation. Resticted gap junctional coupling has been shown to delineate communication compartments in development (Kalimi and Lo, 1989). During development, reduced gap junctional permeability has been shown at interrhombomeric boundaries of the avian CNS (Martinez et al., 1992). The

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transient expression of Cx31 in mouse hindbrain rhombomeres provides support for such a role for gap junctional intercellular communication (GJIC) in neural development (Dahl et al., 1998). Dramatic evidence for the necessity of gap junctions during embryonic development has been provided by utilizing antibodies to gap junction proteins. Injection of such antibodies into blastomeres of amphibian or rodent embryos resulted in severe developmental anomalies, particularly in the CNS (Lee et al., 1987; Warner, 1992). Similar disturbances have also been reported following injection of RNA encoded by dominant negative connexin cDNA constructs (Paul et al., 1995). Although GJIC and cellular expression of some connexins has been partially carried out in the developing nervous system (for reviews see Fulton, 1995; Giaume and Venance, 1995; Kandler and Katz, 1995; Kandler, 1997), this characterization is far from complete. Furthermore, the functional significance of connexin expression in astrocytes, oligodendrocytes and neurons is only beginning to be resolved. A variety of other reports mention the presence of several connexins in the brain (Table 1). However, only in some cases have the temporal and spatial (i.e. cellular) expression patterns been characterized. Analysis by immunocytochemistry indicated that Cx26, Cx43 and Cx45 are present in the ventricular zone of the developing brain, with Cx32 appearing later at postnatal times (Dermietzel et al., 1989; Dermietzel, 1996). We have also characterized the expression of Cx32 and Cx43 mRNA in postnatal developing brain (Belliveau et al., 1991; Belliveau and Naus, 1995). While Cx43 is expressed prenatally, Cx32 is predominantly expressed at postnatal timepoints, when myelination occurs. These results are in agreement with those reported by others (Matsumoto et al., 1991; Micevych and Abelson, 1991), and are consistent with reported immunohistochemical analyses (Dermietzel et al., 1989; Yamamoto et al., 1992; Nadarajah et al., 1997). Recently, Condorelli et al. (1998) cloned the first mammalian neuronal connexin, Cx36. The unique structure of this gene, as well as the recent report of homologous connexin genes expressed in fish retina (i.e. Cx35, Cx34.7) (O’Brien et al., 1996, 1998) suggests that this may be the first of a family of mammalian neuronal connexins. Of particular interest is the suggestion that Cx36 is developmentally regulated (Sohl et al., 1998), with high expression in the brain at the time when gap junctional coupling has been demonstrated to be very extensive between neurons.

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CELLULAR FUNCTIONS OF GAP JUNCTIONS IN THE NERVOUS SYSTEM To specifically assess the putative role(s) of GJIC in CNS development, the functions of this form of intercellular communication must be examined in specific cell types. The developmental roles played by these cell types may be determined, to some extent, by GJIC. Astrocytes The main cell type in the brain coupled by gap junctions is the astrocyte. Gap junctions in astrocytes are primarily composed of Cx43, forming channels with voltage-dependent properties (Dermietzel et al., 1991; Giaume et al., 1991a). More recently, evidence suggests other connexins are expressed in astrocytes (Nagy et al., 1997), including Cx40 (Zheng et al., 1995), Cx30 (Dahl et al., 1996) and Cx45 (Kunzelmann et al., 1997). Gap junctions contribute to the formation of a functional astrocytic syncytium (Dermietzel et al., 1991; Giaume et al., 1991a). This has been implicated in the spatial buffering capacity of astrocytes, particularly dealing with extracellular K + arising from neuronal activity (Walz and Hertz, 1983; Jefferys, 1995). The level of GJIC in astrocytes has been shown to be regulated by a number of factors, including neurotransmitters and neuromodulators (Giaume et al., 1991b, 1992; Giaume, 1994; Vukelic et al., 1991), extracellular ion concentrations (Enkvist and McCarthy, 1994) and various pharmacological agents (Anders, 1988; Mantz et al., 1993; MacVicar and Jahnsen, 1985). GJIC between astrocytes has been shown to increase with maturation (reviewed in Giaume and Venance, 1995). Serum added to cerebellar astrocyte precursors, previously cultured in defined medium without serum and deficient in GJIC, led to reduced proliferation and increased dyecoupling (Fischer and Kettenmann, 1985). The induction of GJIC was much more rapid and extensive in co-cultures with neurons. In slices of neonatal hippocampus, astrocytic dye-coupling was readily detected at the earliest time examined, postnatal day (PD) 4 (Konietzko and Muller, 1994). However, in vivo, no dye-coupling was detected in astrocytes in the rat visual cortex before PD 11, compared to 80% of astrocytes in adult (Binmoller and Muller, 1992). This may reflect regional differences in astrocytic GJIC (Lee et al., 1994) and Cx43 expression (Batter et al., 1992). Alternatively, differences could also be explained based on the molecular tracer used to demonstrate

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GJIC. Neurobiotin has been shown to more readily demonstrate coupling. Nevertheless, this functional data correlates in some cases with the presence of Cx43 mRNA and protein in the brain (Dermietzel et al., 1989; Belliveau et al., 1991; Yamamoto et al., 1992) as well as specifically in astrocytes (Belliveau and Naus, 1995; Yamamoto et al., 1992). Concerning the heterogeneity of astrocytes, it is also interesting that, in contrast to type 1 astrocytes which are highly coupled, we could not detect GJIC in type 2 astrocytes which do not express Cx26, 32 or 43 (Belliveau and Naus, 1994). Similar results have also been reported for type 2 astrocytes cultured from optic nerve (Sontheimer et al., 1990). Finally, it is also apparent that astrocytes mature in the absence of Cx43 expression, as revealed by their morphology and expression of GFAP (Naus et al., 1997). Oligodendrocytes Early morphological evidence indicated that gap junctions could be seen between oligodendrocytes (Mugnaini, 1986; Dermietzel et al., 1978). Several reports have indicated that oligodendrocyte gap junctions are composed of Cx32 in vivo (Dermietzel et al., 1989; Micevych and Abelson, 1991; Belliveau and Naus, 1995; Spray and Dermietzel, 1995; Li et al., 1997) and in vitro (Belliveau and Naus, 1994; Giaume and Venance, 1995). We have shown that expression of Cx32 coincides with maturation of oligodendrocytes temporally and spatially (Belliveau et al., 1991; Belliveau and Naus, 1995). The function of GJIC in oligodendrocytes is likely to be primarily metabolic to allow ions and nutrients to pass from somata to all the layers of the myelin (Paul, 1995; Suter and Snipes, 1995). The importance of this channel has recently been realized by the reported mutations of Cx32 associated with X-linked Charcot-Marie-Tooth disease (CMTX), a peripheral demyelinating disorder (Bergoffen et al., 1993). Some of these mutations have been shown to lead to loss of functional gap junction channels, while others still encode functional channels (Bruzzone et al., 1994; Omori et al., 1996). The recent production of Cx32 knockout mice has shed some light on the role of gap junctions in peripheral myelination (Anzini, 1997). However, CNS demyelination has not been reported in CMTX or in these transgenic mice. The recent observation of the expression of Cx45 in oligodendrocytes may explain this lack of a CNS demyelinating phenotype (Kunzelmann et al., 1997).

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Neurons Electrotonic coupling between mammalian neurons has been shown in many areas, including neocortex, hippocampus, inferior olive, locus coeruleus, hypothalamus, striatum and retina, and has been implicated in neuronal synchronization (Dermietzel and Spray, 1993; Cook and Becker, 1995). During development, there appears to be a high degree of intercellular coupling between neurons (Kandler and Katz, 1995). Detailed analysis in the rat reveal that neuroblasts are coupled with approximately 30–60 others in columns within the ventricular zone of the developing cerebral cortex (Connors et al., 1983; Lo Turco and Kriegstein, 1991; Yuste et al., 1992; Peinado et al., 1993a). This extensive coupling occurs throughout all neocortical layers and decreases as neuroblasts undergo their final mitosis and migrate away from the ventricular zone. Similar changes in GJIC have been reported in postnatal human brain tissue, obtained from surgical resections for epilepsy, where a significant decrease in neuronal dyecoupling occurs between the first year (38% of neurons coupled) to the third year (13%) to the eighth year (4%) (Cepeda et al., 1993). To clarify the role of this GJIC in the development of the CNS, some progress has been made in the identification of cell-specific connexin expression. Cx43 has been reported in neurons of the cortical plate, with some Cx32 appearing as neurons matured (Fushiki and Kinoshita, 1995). While several other connexins are expressed in the brain (see Table 1), the connexin underlying this high level of neuronal coupling during development in vivo remains to be definitively clarified. Recently, expression of Cx26 has been reported in neurons, with this expression decreasing as neuronal coupling decreases (Nadarajah et al., 1997). Of particular interest is the recent report of a decrease in Cx36 expression in the brain with increasing maturing (Sohl et al., 1998). In the adult cerebral cortex, Cx32 and Cx43 have been localized to neurons (Nadarajah et al., 1996), and Cx36 appears to be highly expressed in specific neuronal populations, including the inferior olivary nucleus and other brainstem nuclei, the olfactory bulb and hippocampus, with moderate expression in other areas including the cerebral cortex (Condorelli et al., 1998). Additional insight into the specificity of connexin expression in neurons has been gained from in vitro studies. A similar decrease in gap junctional coupling has been reported during differentiation of hippocampal progenitor cells in vitro, where the

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onset of voltage-dependent responses coincides with decreased electrical coupling (Rozental et al., 1995). We have extensively characterized two neuronal cell culture models with regard to differentiation and gap junctions. The P19 mouse embryonal carcinoma cell line differentiates into neurons and astrocytes in response to retinoic acid treatment (Jones-Villeneuve et al., 1982). Similarly, the NT2 human teratocarcinoma cell line, which has been considered a stem cell line and reported to possess many features of neuroepithelial precursor cells, differentiates in response to retinoic acid, with the additional advantage of providing pure neuronal cultures (Pleasure et al., 1992). In both models, gap junctional communication decreases with neuronal differentiation such that mature neurons are not coupled (Bani-Yaghoub et al., 1997; Belliveau et al., 1997). Our preliminary studies on primary cultures of mouse neural progenitor cells reveal that these cells are highly coupled and express Cx43 prior to neuronal differentiation (Fig. 1). It has been suggested that this early electrotonic coupling via gap junctions may function in determining positional fate of neurons in the cortex, providing a mechanism for laminar organization (Lo Turco and Kriegstein, 1991). In addition, it has recently been suggested that gap junctional coupling in the embryonic neocortical proliferative epithelium plays a role in cell cycle regulation, possibly synchronizing cells in the same phase (Bittman et al., 1997; Goto et al., 1997). GJIC may establish cortical domains in the developing neocortex that underlie the adult pattern of functional architecture (Yuste et al., 1992; Peinado et al., 1993a,b). Disruption of the processes controlling the generation and migration of neurons has been implicated in the etiology of a number of neurodevelopmental defects (Caviness and Williams, 1984; Rakic, 1984), many of which are associated with mental deficits and seizure disorders (reviewed in Aicardi, 1994). Recently, Bittman et al. (1997) have shown that cortical precursors in the ventricular zone are coupled by gap junctions to radial glia prior to migration, consistent with a role for GJIC between neuroblasts and glia in this process. Our preliminary findings demonstrating a disturbance in neocortical neuronal migration in Cx43 knockout mice support a role for GJIC in this phenomenon (see below). The establishment of an early system of electrotonic synapses may be the precursor of the more differentiated chemical synapses which follow (Kandler and Katz, 1995). Alternatively, it has been suggested that this transient neuronal GJIC

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provides a means of biochemical coordination which is required for neuronal differentiation (Kandler and Katz, 1998). For example, in the retina, the spatiotemporal pattern of activity mediated by GJIC between retinal ganglion and amacrine cells is proposed to play a role in determining retinal circuitry (Wong et al., 1995). In addition, recent findings have provided evidence for neuronal GJIC in the formation of commissures, as well as in plasticity of CNS projections in the leech (Wolszon et al., 1994, 1995). A more definitive role for GJIC in the mammalian CNS may arise from careful analysis of transgenic mice with targeted mutations of connexin genes expressed in the nervous system. The exchange of developmental signals through gap junctions has also been suggested to play a role in neuronal differentiation (Warner, 1992). Support for this hypothesis comes from our recent findings with two in vitro models of neuronal differentiation. When NT2 human embryonal carcinoma cells differentiate into neurons in response to retinoic acid (RA), the expression of Cx43, and the level of GJIC, progressively disappear (Bani-Yaghoub et al., 1997). However, when GJIC is pharmacologically blocked during RA treatment, neuronal differentiation is blocked (Bani-Yaghoub et al., 1998). Similar results were obtained with the P19 mouse embryonal carcinoma cell line (Bani-Yaghoub et al., 1999). It therefore appears that the temporal pattern of connexin expression and GJIC during neuronal differentiation is critical. The putative transjunctional signal(s) remains to be determined. However, precedent for such signals can be found in invertebrate models of differentiation (Fraser et al., 1987). Novel approaches to isolate and characterize transjunctional molecules could provide insight into this process (Goldberg et al., 1998). Neuronal migration disorders are the main cause of mental retardation and seizures in children (Aicardi, 1994). Interestingly, it is possible that the relatively high level of GJIC seen in developing neocortical neurons may underlie the higher susceptibility to seizures in infants (Moshe et al., 1983).There is some evidence implicating a role for GJIC in the development of synchronization of neuronal discharges characteristic of seizure disorders (Dudek et al., 1986; Dermietzel and Spray, 1993). We have documented increased expression of connexins in human epileptic neocortex (Naus et al., 1991b). This is supported by the observation that convulsants have been shown to increase transmission at electrical synapses in invertebrates (Rayport and Kandel, 1981). More recently, it has

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Fig. 2. Histology of Cx43 +/+ (A) and Cx43
/
(B) cortex and hippocampus at postnatal day 6 suggests thinner cortex, denser neuropil and poor lamination in the latter.

been shown that, in zero Ca2+ perfusate, which blocks all nerve-evoked synaptic transmission, epileptiform activity occurs associated with increased interneuronal dye-coupling and increased frequency of fast prepotentials (FPPs) as measured by whole cell voltage recordings (Perez-Velazquez et al., 1994). This epileptiform activity was decreased by factors which decrease gap junctional conductance (e.g. intracellular acidosis, octanol) and increased by intracellular alkalosis. INSIGHTS FROM CONNEXIN MUTATIONS The changes in the expression of gap junction genes during postnatal brain development that we and others have observed coincides with various major differentiative events. These include cessation of cellular migration, increases in axonal growth and synaptogenesis, myelination, neurotransmitter differentiation, neuronal death and retraction of exuberant projections (reviewed in Jacobson, 1991). For the most part, it remains to be determined if gap junctions play any direct role in such developmental events. Several approaches may be

used to interfere with GJIC, including the use of pharmacological blockers and various molecular approaches to alter gene expression. One of the most promising approaches to interfering with gene expression in vivo utilizes targeted gene knockout through homologous recombination. This approach has been used to eliminate expression of selective connexin genes in transgenic mice. Thus Cx43 null mutant mice were recently produced which are proving very useful in defining some of the roles of GJIC in the CNS (Perez Velazquez et al., 1996; Naus et al., 1997). Initial reports indicated that the homozygous Cx43 null mutation in C57BL/6 mice was lethal at birth (Reaume et al., 1995). However, we have been studying this null mutation in a CD1 background and have found some of these mice are able to survive 1 week after birth (Fig. 2). No abnormalities are readily apparent in the brain (Fig. 2A,B). However, cardiac function is compromised (Fig. 2C,D), and all homozygotes die perinatally. We have begun to examine additional aspects of brain development in Cx43 null mutant mice. To assess neuronal migration, pregnant mice were injected with bromodeoxyuridine to label dividing

Fig. 1. Preliminary analysis of neural progenitor cells obtained for fetal mice. (A) EGF- generated sphere of neural cells at 10 days in vitro. (B) Nestin immunoreactivity in some of the cells which migrated away from spheres, 14 days in vitro. (C, D) Cx43 immunoreactivity is apparent in EGF-generated cells at 14 days in vitro. (E) Phase contrast image of a field of cells which migrated away from the sphere. (F) LY injection of a single cell ( in E) shows extensive dye-coupling. Bar=30 (A), 50 (B), 75 (C), 60 (D), 100 (E) and 15 (F)
m.

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Fig. 3. Brdu was injected at GD 17 and processed at postnatal day 3. Sections were treated with an antibody to Brdu and visualized with a biotinylated secondary antibody and avidin-biotin-alkaline phosphatase. Many cells which were dividing at GD17 appear labeled in the ventricular zone (arrows). In wild type cortex (B), labeled cells appear throughout the cortex, however fewer labeled cells are distributed the Cx43 null mutant cortex (A). Magnification 100, reproduced at 85%.

neuroblasts in the fetus (Fushiki et al., 1997). In homozygous Cx43 null mutant cortex, we have noted an apparent delay in neuronal migration in the developing neocortex (Fig. 3). These data suggest a role for Cx43 in neuronal migration. Current status on other connexin knockouts has recently been summarized (Nicholson and Bruzzone, 1997). Studies have also been carried out with Cx26 and Cx32. Cx26 knockout mice die at 10–11 days in utero (Willecke, 1995). More interestingly, Cx32 knockout mice display late-onset progressive peripheral neuropathy (Anzini et al., 1997). No neural abnormalities have been reported for knockouts of other connexins known to be expressed in the CNS, including Cx37 (Simon et al., 1997) and Cx40 (Goodenough et al., 1997). Preliminary analysis of Cx43/Cx32 double null mutation reveal no apparent CNS defects in late gestation (Houghton et al., 1998). The recent findings of connexin mutations in human neurological disorders indicates significant differences from those found in transgenic mice. While the reported mutation of Cx32 in X-linked Charcot-Marie-Tooth disease, a human peripheral demyelinating disorder (Bergoffen et al., 1993), is consistent with the findings in Cx32 knockout mice, Cx26 null mutations are not lethal in humans. In fact, Cx26 mutations have been found in association with hereditary deafness (Kelsell et al., 1997; Zelante et al., 1997). Interestingly, in the rat

cochlea, the epithelial and connective tissue gap junction systems lining the cochlear duct and stria vascularis, where Cx26 is localized, have been implicated in the recirculation of extracellular K + released by hair cells (Kikuchi et al., 1995). It has been consistently noted that neuronal maturation is accompanied by loss of GJIC. One might thus expect that abnormal continued expression of connexin might interfere with development. One report has described some CNS effects in transgenic mice overexpressing Cx43 with the CMV promoter (Ewart et al., 1997). Some of these mice exhibited cranial neural tube defects, as well as heart malformations. In addition, forced constitutive expression of Cx43 in immortalized rat hippocampal neuronal cells (Rozental and Spray, 1996) or in PC12 cells (Rozental et al., 1995) interferes with expression of a neuronal phenotype. GAP JUNCTIONS AND NEURON–GLIAL INTERACTIONS Astrocytes have traditionally been viewed to have a role in the metabolic and trophic support of neurons (Jensen and Chiu, 1993). Their position at the interface of blood vessels and synapses is consistent with these roles. Intimate interactions have been proposed for the role of radial glia in directing migration of neurons in the cortex (Warner, 1992).

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Disruption of this process can lead to migrational disturbances which are seen in many neurodevelopmental disorders. It is clear that astrocytes and neurons interact at many levels (Chiu and Kriegler, 1994; Kimelberg, 1995), including GJIC. Neurons can regulate GJIC through neurotransmitters: stimulation of adrenergic (Giaume et al., 1991b) and purinergic (Enkvist and McCarthy, 1992) receptors decreases astrocytic GJIC, as do endothelins (Giaume et al., 1992) and eicosanoids (Giaume et al., 1991b; Venance et al., 1995). Nitric oxide, which can be released by astrocytes, decreases neuronal GJIC (Ando et al., 1994; Mills and Massey, 1995). When astrocytes are co-cultured with neurons, they exhibit higher GJIC (Fischer and Kettenmann, 1985). In addition, astrocytic intercellular Ca2+ signaling, as well as asynchronous intracellular Ca2+ oscillations, are more extensive in cultures with neurons that in purified astrocyte cultures (Charles, 1994; Dani and Smith, 1995). In fact, direct calcium signaling from neurons to astrocytes has been proposed to occur via GJIC (Nedergaard, 1994; Charles, 1994). The developmental significance of this Ca2+ signaling remains to be determined.

SUMMARY The presence of gap junctions in the CNS is well documented, but many of their function remains to be resolved. This is particularly evident with regard to development of the CNS. It is clear that neural progenitor cells express connexin43 and are coupled by gap junctions. Interference with GJIC in models of neuronal and glial differentiation compromises this cellular phenotype. In neocortical development, there is a well established pattern of neuronal gap junctional coupling which has been implicated in various aspects of neuronal migration, cortical lamination and functional organization. The manipulation of gap junction expression at the cellular level will aid in clarifying some aspects of the role(s) of these junctions in neural development.

ACKNOWLEDGEMENT The research presented in this article was made possible by a grant from the Ontario Mental Health Foundation (Effects of connexin mutations on neural development).

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