Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS

This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels.     

Freeze fracture analysis of the axolemma of cultured dorsal root ganglion neurons in the absence of Schwann cells

The distribution of intramembranous particles within the axolemma of cultured dorsal root ganglion neurons was determined by freeze-fracture microscopy. Utilizing culture conditions which eliminate Schwann cells, the particle distribution of the P-face, 735 +/- 119 microns2, and E-face, 100 +/- 39 microns2 resembled that of pre- and non-myelinated axons in vivo and no node-like E-face particle patching was seen. These results indicate that cultured neurite development is similar to that seen in vivo and that axons maintained in a glial-free environment do not develop nodal, E-face membrane specializations.     

Gap junctions couple astrocytes but not neurons in dissociated cultures of rat suprachiasmatic nucleus

Individual neurons dissociated from rat suprachiasmatic nucleus can express independently phased circadian firing rhythms in culture. The phases of these rhythms are unperturbed by reversible blockade of neuronal firing lasting 2.5 days, indicating that multiple circadian clocks continue to operate in the absence of conventional synaptic transmission. The possibility remains, however, that these circadian rhythms might depend on some other form of intercellular communication. In the present study, a potential role for gap junctional coupling in SCN cultures was evaluated by introduction of the tracer molecule Neurobiotin into both neurons (n = 98) and astrocytes (n = 10), as well as by immunolabeling for specific connexins, the molecular components of gap junctions. Astrocytes were extensively coupled to each other by connexin43-positive gap junctions, but no evidence was found for coupling of neurons to each other or to astrocytes. These data support the hypothesis that neurons expressing independently phased circadian rhythms in SCN cultures (‘clock cells’) are autonomous, single cell circadian oscillators, but do not exclude a role for glia in synchronizing neuronal clock cells in vivo.     

An identified pleural ganglion interneuron inhibits patterned motor activity in the buccal ganglia of the snail, Helisoma

Neuron, Pl1, an interneuron that inhibits patterned motor output underlying feeding in the snail, Helisoma, is identified. The soma of neuron Pl1 is in the pleural ganglion and its axon projects through the pedal and cerebral ganglia to the buccal ganglia. A train of action potentials in neuron Pl1 suppresses rhythmic activity in the buccal pattern generator even in the presence of strong pharmacological stimulation with serotonin.     

Lack of biocytin transfer at gap junctions in the chicken vestibular nuclei

In vivo experiments were designed to test for functional gap junctions at ‘mixed' synapses that were morphologically characterized between the large-diameter, primary vestibular fibers and second-order vestibular neurons in the chicken, Gallus gallus. In previous intracellular recordings and dye injections into these neurons from brain slice preparations of chick embryos (E15/16) and also newborn hatchlings (H1-2), no evidence was obtained for functional gap junctions. Therefore, biocytin, a low molecular weight tracer that permeates gap junction channels, was extracellularly applied to either the ampullary nerves or to the vestibular ganglion of 3–6 day old hatchlings and adult chickens (9 months). This procedure resulted in the uptake of the dye and heavy staining of both the thick and thin fibers composing the vestibular nerve and in loading of vestibular efferent neurons. However, no dye transfer was observed between the large-diameter, primary vestibular fibers and second-order vestibular neurons. This observation, which was performed using a relatively non-invasive approach on intact animals, suggests that the gap junctions at these mixed synapses are probably not functional under the conditions of these experiments.     

Neuronal gap junctions in the polymorph layer of the rat dentate gyrus

In thin sections of the rat dentate gyrus, neuronal gap junctions were observed in the polymorph layer. Gap junctions were seen on dendritic stems, on smooth and/or varicose dendrites, on spine-like appendages, and in one case on a soma. Somata of gap junction-bearing neurons showed indented nuclei with intranuclear inclusions, and received many asymmetrical and a few symmetrical synapses.     

Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression-injured spinal cord was compared by using two different sequence-specific anti-Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1- and 3-day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post-SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post-SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP-positive profiles were present at sites where Cx43 labeling was absent. By 7 days post-SCI, Cx43 again co-localized with GFAP-positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin-induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co-localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression. J. Comp. Neurol. 382:199-214, 1997. © 1997 Wiley-Liss, Inc.     

Freeze-fracture ultrastructure of the perinodal astrocyte and associated glial junctions

Freeze-fracture examination of nodes of Ranvier from adult rat optic nerve demonstrates the presence of astrocytic processes at the majority of nodes of Ranvier. Astrocytic processes often run along the entire length of the nodal gap, although they do not necessarily encircle the entire nodal circumference. The E- and P-fracture faces and the cross-fractured cytoplasm of these astrocytes (termed 'perinodal astrocytes') were examined. The cytoplasm of perinodal astrocytes contains 10-nm filaments. The P-faces of perinodal astrocytic membranes are characterized by orthogonal arrays of intramembranous particles ('assemblies'), with a center-to-center periodicity of approximately equal to 6 nm. Complementary orthogonally arranged pits are observed on the E-faces of the astrocytic membranes. The density of these arrays in perinodal astrocytic membranes is similar to that in parenchymal astrocytic membranes, but is substantially lower than that at pericapillary astrocytic membranes. In addition, gap junctions are present between astrocytes, and between astrocytes and paranodal oligodendroglial layers. These findings indicate that astrocytic processes comprise an important structural component of central nodes of Ranvier, and provide a morphological basis for a possible astrocytic role in nodal function.     

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.     

Co-culture of neurons and glia in a novel microfluidic platform

We have prepared the poly-d-lysine (PDL) bound surfaces for neuron cell culture by covalent binding between the poly-d-Lysine and substrates and investigated neuronal cell adhesion properties and cell growth morphology. The number of neuronal cell and the number of neurite per neuronal cell on PDL bound surfaces was much more than those on PDL coated surfaces and also the neuronal cells on PDL bounded surfaces survived a longer time. On the pattern of covalently bound PDL, neuronal cells and their neurites are confined within the grid line leading to patterned neuronal networks with the long-term survival.     

Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.     

Neuronal gap junctions and morphologically mixed synapses in the spinal cord of a teleost, Sternarchus albifrons (gymnotoidei)

Spinal cord motoneurons in the gymnotid, Sternarchus albifrons, were studied electron microscopically with special reference to the freeze-fracture method. Two types of motoneurons were identified. Electromotor neurons are monopolar and are located in a midline column dorsal to the ventral gray. These cells have a small fraction of their surface covered by synapses from descending axons, often at nodes. The synapses have multiple gap junctions, but few presynaptic vesicles or other correlates of chemical transmission. The gap junctions have an ordinary appearance in freeze-fracture replicas and exhibit a highly ordered substructure. The not infrequent appositions between the cell bodies of electromotor neurons exhibit no junctional specializations. Ordinary motoneurons are multipolar and densely covered with axosomatic and axodendritic synapses. In thin sections these synapses can be divided into two groups according to whether the vesicles are spherical or flattened. Gap junctions occur only at the first type, thus forming ‘morphologically mixed’ synapses. In freeze-etch replicas of motoneurons, the gap junctions are often found near clusters of postsynaptic E face particles elsewhere associated with excitatory chemical transmission. In addition, vesicle attachment sites occur in the presynaptic membranes of some synapses with gap junctions. The morphological observations are consistent with dual chemical and electrical transmission at these particular synapses, i.e. electrical excitation across gap junctions and chemical excitation at active zones with spherical vesicles and post-synaptic E face particles.     

Age-related changes in junctional and non-junctional conductances in two electrically coupled peptidergic neurons of the mollusc Lymnaea stagnalis

Age-related changes in electrotonic coupling ratio of two identified neurons in Lymnaea stagnalis were studied together with the underlying changes in the steady-state conductance properties of the network. Two phases were distinguished in the development of coupling ratio across lifespan. During the first phase (age of 3-13 months), coupling ratio decreased from decreased from 60% to 30%. The second phase (age 13-20 months) was characterized by an increase in coupling ratio. Values of up to 60% were reached again in the oldest animals. Voltage clamp measurements showed that the biphasic trend of the age-related changes in coupling ratio is paralleled by changes in conductance properties of the junction between VD1 and RPD2. During the first phase junctional conductance decreased, whereas during the second phase junctional conductance increased. In addition to the decrease in junctional conductance, a growth-related increase in non-junctional conductance of VD1 and RPD2 contributed to the decrease in coupling ratio observed during the first phase. Thus our results indicate that in Lymnaea junctional connections between neurons may undergo considerable and discontinuous changes after sexual maturation. In addition to these changes in steady-state electrical properties, indications were obtained that age-related changes of kinetically slower conductance(s) may occur in the non-junctional membrane of VD1 and RPD2.     

Chemical phenotypes of muscle and cutaneous afferent neurons in the rat trigeminal ganglion

Retrograde labeling was combined with cytochemistry to investigate phenotypic differences in primary afferent neurons relaying sensory information from deep and superficial craniofacial tissues. Calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM) immunoreactivity and isolectin IB4, and cholera toxin B (ChTB) binding were examined for trigeminal masticatory muscle and cutaneous afferent neurons. Somata labeled from muscle were larger than cutaneous afferent neurons. Muscle afferent neurons exhibited positive staining as follows: 22% CGRP, 5% SP, 0% SOM; 18% ChTB, 5% IB4. The somata of CGRP- and SP-positive muscle afferent neurons were smaller than that of the overall muscle afferent population. Size differences were not detected between IB4- or ChTB-binding muscle afferent neurons and the total muscle afferent population. The following distribution was found for cutaneous afferent neurons: 26% CGRP, 7% SP, 1% SOM, 26% ChTB, 44% IB4. Cutaneous afferent neurons positive for SP were smaller, while ChTB-binding cutaneous afferents were larger than the overall cutaneous afferent population. No size differences were found between cutaneous CGRP-, SOM-, or IB4-positive neurons and the total cutaneous afferent population. Target-specific differences exist for SOM and IB4. The percentage of cutaneous afferent neurons positive for SOM and IB4 exceeds that for SOM- or IB4-positive muscle afferents. The number of retrogradely labeled neurons never differed between sexes. The percentage of retrogradely labeled muscle afferent neurons that were CGRP-positive was greater in males than females. These data indicate the presence of phenotypic, target, and sex differences in trigeminal ganglion primary afferent neurons.     

Connexin36 is required for gap junctional coupling of most ganglion cell subtypes in the mouse retina

Converging evidence indicates that electrical synaptic transmission via gap junctions plays a crucial role in signal processing in the retina. In particular, amacrine and ganglion cells express numerous gap junctions, resulting in extensive electrical networks in the proximal retina. Both connexin36 (Cx36) and connexin45 (Cx45) subunits are widely distributed in the inner plexiform layer (IPL) and therefore are likely contribute to gap junctions formed by a number of ganglion cell subtypes. In the present study, we used the gap junction‐permeant tracer Neurobiotin to compare the coupling pattern of different ganglion cell subtypes in wild‐type (WT) and Cx36 knockout (KO) mouse retinas. We found that homologous ganglion‐to‐ganglion cell coupling was lost for two subtypes after deletion of Cx36, whereas two other ganglion cell subtypes retained homologous coupling in the KO mouse. In contrast, deletion of Cx36 resulted in a partial or complete loss of ganglion‐to‐amacrine cell heterologous coupling in 9 of 10 ganglion cell populations studied. Overall, our results indicate that Cx36 is the predominant subunit of gap junctions in the proximal mouse retina, expressed by most ganglion cell subtypes, and thereby likely plays a major role in the concerted activity generated by electrical synapses. J. Comp. Neurol. 518:911–927, 2010. © 2009 Wiley‐Liss, Inc.     

阿霉素中毒大鼠背根神经节神经元及细胞核孔的改变

目的 探讨阿霉素中毒所致神经元坏死过程中胞核,胞质及核孔的变化,是否存在凋亡。方法 ＳＤ大鼠静脉注射阿霉素８ｍｇ／ｋｇ,使其腰背根神经节细胞发生变性。利用光镜,电镜、聚焦激光扫描显微镜,ＴＵＮＥＬ染色等方法观察不同时期神经元和核孔的变化,寻找凋亡存在的证据。结果 阿霉素应用１４ｄ后,核膜切线位核孔的结构变得模糊不清,数量减少;垂直位核膜隔明显稀疏,甚至消失,神经核中度变性及胞质明显变性。结论 核孔     

Ionic mechanism of a hyperpolarizing glutamate effect on two identified neurons in the buccal ganglion of Aplysia

The ionic mechanism of a membrane effect of L-glutamate on two identified neurons in the buccal ganglion of Aplysia kurodai was investigated with conventional microelectrode techniques and glutamate iontophoresis. Bath-applied and iontophoresed glutamate hyperpolarized the membrane and increased the membrane conductance. The hyperpolarizing glutamate response decreased in amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of the hyperpolarizing glutamate response was close to the ECl (-60 mV). The reversal potential changed by 22.4 mV when the external chloride concentration was altered by a factor of 5. The relationship between the iontophoretically applied current and the membrane conductance changes was suggestive of two glutamate molecules reacting with a single receptor site. The hyperpolarizing glutamate response was essentially unaffected by 2-amino-4-phosphonobutyric acid (2-APB), L-proline, and quinuclidinyl benzilate (QNB). It was concluded that the hyperpolarizing glutamate response was generated by an activation of Cl- conductance.     

Localization of glutamic-acid-decarboxylase-immunoreactive axon terminals in the inferior olive of the rat, with special emphasis on anatomical relations between GABAergic synapses and dendrodendritic gap junctions

Immunocytochemical and electron microscopic methods were used to examine the GABAergic innervation of the inferior olivary nucleus in adult rats. This neuronal system was visualized with an antibody against glutamic acid decarboxylase (GAD, EC 4.1.1.15), the GABA-synthesizing enzyme. A GAD-positive reaction product was encountered only in short segments of preterminal axons and in axon terminals. Their relative number per unit area of neuropil was very similar in all olivary subnuclei. Despite this homogeneity in density, obvious intraregional differences existed. Some regions were strongly immunoreactive (the "c" subgroup, the beta nucleus, and the mediolateral outgrowth of the medial accessory olive), whereas others were weakly labeled (the dorsomedial cell column and the central zones of the medial accessory and principal olives). The strongly immunoreactive areas contained the largest and most intensively labeled axon terminals. Areas of weak labeling were filled with small, weakly immunoreactive nerve terminals. Thus, variations in size and in intensity of labeling create a specific pattern of GABA innervation, revealed by an almost continuous gradient between the above-mentioned extremes. The GAD-positive axon terminals established conventional synapses with dendrites (94% of the samples) or with cell bodies (6%). The vast majority of these synapses were type II (84%) and only a small proportion formed type I synaptic contacts (16%), regardless of the nature of the postsynaptic element. Immunoreactive terminals were also involved in the complex synaptic arrangements--the glomeruli, which characterize the olivary neuropil. Within these formations, olivary neurons were electrotonically coupled through dendrodendritic gap junctions. There was a constant association between GAD-positive axon terminals and small dendritic appendages linked by gap junctions. This association was revealed not only by the systematic presence of immunolabeled terminals directly apposed to the dendritic appendages but, more importantly, by the frequent presence of type II synapses straddling both elements. These synapses were in close proximity to the low-resistance pathways represented by the gap junctions. The strategic location of these GABA synapses is discussed in relation to recent findings indicating the possibility of a synaptic modulation of the electrical coupling: the release of GABA, by increasing nonjunctional membrane conductance, could shunt the coupling between olivary neurons. The functional decoupling of selected gap junctions would be responsible for the spatial organization of the olivary electrotonic coupling.     

Role of activity in the selection of new electrical synapses between adult Helisoma neurons

Our aim was to determine whether neural activity in the form of sodium-dependent action potentials plays a role in the formation, maintenance and specificity of electrical synapses between regenerating neurons. We axotomized buccal neurons of the mollusc, Helisoma trivolvis, and placed ganglia into organ culture in the absence or presence of tetrodotoxin (TTX), a specific sodium channel blocker. Electrical coupling was measured using intracellular microelectrodes positioned within the soma of identified neurons. Neurite outgrowth was assessed by epifluorescence microscopy after filling neurons by iontophoresis with Lucifer yellow. Previous studies found that two days after axotomy transient electrical synapses form between heterologous neurons (e.g. buccal neurons 4 and 5). Five days after axotomy these transient connections disappeared and a new electrical synapse was stabilized between the paired buccal neurons 5. To determine whether blocking neural activity with TTX affected the specificity and formation of new electrical synapses, we examined electrical coupling between the heterologous neurons 4 and 5 two days after axotomy, and the paired buccal neurons 5 five days after axotomy. Our electrophysiological recordings indicated that different neurons in the buccal ganglion varied in their sensitivity to TTX (i.e. sensitivity of buccal neurons 19 > 5 > 4), but spontaneous activity was abolished in all 3 neurons by 2 × 10⁻⁵ M TTX. Furthermore, the inhibitory effects of TTX occurred within seconds of superfusion and persisted for at least 6 days. Inhibition of activity by TTX could be reversed after superfusion with normal saline. Neurite outgrowth from axotomized neurons was not appreciably altered in the presence of TTX. Furthermore, no differences in the incidence of electrical coupling or the coupling resistance were detected between neurons 4 and 5 two days after axotomy and organ culture in the presence of TTX. However, electrical coupling between the symmetrically paired neurons 5 was elevated in the presence of TTX after 5 days. We conclude from these results that neural activity in the form of sodium-dependent action potentials does not play an important role in the formation or breaking of transient electrical synapses during neuronal regeneration in the mollusc Helisoma trivolvis.     

Differential connexin expression,gap junction intercellular coupling,and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.