Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Effect of high fat diet on maternal behavior, brain-derived neurotrophic factor and neural stem cell proliferation in mice expressing human placental lactogen during pregnancy.

Maternal obesity is a serious health concern because it increases risks of neurological disorders, including anxiety and peripartum depression. In mice, a high fat diet (HFD) in pregnancy can negatively affect placental structure and function as well as maternal behavior reflected by impaired nest building and pup-retrieval. In humans, maternal obesity in pregnancy is associated with reduced placental lactogen (PL) gene expression, which has been linked to a higher risk of depression. PL acting predominantly through the prolactin receptor maintains energy homeostasis and is a marker of placenta villous trophoblast differentiation during pregnancy. Impaired neurogenesis and low serum levels of brain-derived neurotrophic factor (BDNF) have also been implicated in depression. Augmented neurogenesis in brain during pregnancy was reported in the subventricular zone (SVZ) of mice at gestation day 7 and linked to increased prolactin receptor signaling. Here, we used transgenic CD-1 mice that express human (h) PL during pregnancy to investigate whether the negative effects of diet on maternal behavior are mitigated in these (CD-1[hGH/PL]) mice. Specifically, we examined the effect of a HFD on nest building prepartum and pup retrieval postpartum, as well as on brain BDNF levels and neurogenesis. In contrast to wild-type CD-1[WT]mice, CD-1[hGH/PL] mice displayed significantly less anxiety-like behavior, and showed no impairment in prepartum nest building or postpartum pup-retrieval when fed a HFD. Furthermore, the HFD decreased prepartum and increased postpartum BDNF levels in CD-1[WT] but not CD-1[hGH/PL] mice. Finally, neurogenesis in the SVZ as well as phosphorylated mitogen-activated protein kinase, indicative of lactogenic signaling, appeared unaffected by pregnancy and diet at gestation day 7 in CD-1[hGH/PL] mice. These observations indicate that CD-1[hGH/PL] mice are resistant to the negative effects of HFD reported for CD-1[WT] mice, including effects on maternal behaviors and BDNF levels, and potentially, neurogenesis. This difference probably reflects a direct or indirect effect of the products of the hGH/PL transgene.

Gap junction connexin43 is a key element in mediating phagocytosis activity in human trabecular meshwork cells.

Human trabecular meshwork (TM) cells play pivotal roles in maintaining homeostasis of intraocular pressure via regulation of aqueous humor outflow. These cells are capable of phagocytosis, which is considered to be essential for their regulatory function. In addition, there is a strong expression of the gap junction protein connexin43 (Cx43) in the TM. Here, we investigated functional relationships between phagocytosis activity of TM cells and their expression of Cx43. Phagocytosis was measured by showing the ability of TM cells to engulf inert fluorescent particles consisting of pHrodo. We found that internalized pHrodo was partially co-localized with Cx43 and that the phagocytic activity was dramatically reduced after knockdown of Cx43 using lentiviral Cx43 shRNA. These results suggest that Cx43 is involved in the regulation of phagocytosis by TM cells.

ZO-1 associates with α3 integrin and connexin43 in trabecular meshwork and Schlemm's canal cells.

Cellular structures that perform essential homeostatic functions include tight junctions, gap junctions, desmosomes and adherens junctions. The aqueous humor, produced by the ciliary body, passes into the anterior chamber of the eye and is filtered by the trabecular meshwork (TM), a tiny tissue found in the angle of the eye. This tissue, along with Schlemm's canal (SC) inner wall cells, is thought to control intraocular pressure (IOP) homeostasis for normal, optimal vision. The actin cytoskeleton of the tissue plays a regulatory role in maintaining IOP. One of the key risk factors for primary open angle glaucoma is persistent elevation of IOP, which compromises the optic nerve. The ZO-1 (Zonula Occludens-1), extracellular matrix protein integrins, and gap junction protein connexin43 (Cx43) are widely expressed in many different cell populations. Here, we investigated the localization and interactions of ZO-1, α3 integrin, ß1 integrin, and Cx43 in cultured porcine TM and SC cells using RT-PCR, western immunoblotting and immunofluorescence labeling with confocal microscopy, along with co-immunoprecipitation. ZO-1 partially co-localized with α3 integrin, but not with ß1 integrin, and co-immunoprecipitated with Cx43, as well as with α3 integrin. The association of ZO-1 with α3 integrin and Cx43 suggests that these proteins may form a multiple protein complex in porcine TM and SC cells. Since integrins interact with the actin cytoskeleton via scaffolding proteins, these results implicate junctional and scaffolding protein ZO-1 as a potential control point in regulation of IOP to normal levels for glaucoma therapy.

Connexin36 localization along axon initial segments in the mammalian CNS.

Electrical synapses formed by gap junctions occur at a variety of neuronal subcellular sites in the mammalian central nervous system (CNS), including at somatic, dendritic and axon terminal compartments. Numerous electrophysiological studies using mice and rats, as well as computer modelling approaches, have predicted the additional occurrence of electrical synapses between axons near their emergence from neuronal somata. Here, we used immunofluorescence methods to search for localization of the neuronal gap junction-forming protein connexin36 (Cx36) along axon initial segments (AISs) labelled for the AIS marker ankyrinG. Immunofluorescent Cx36-puncta were found to be associated with AISs in several CNS regions of mice, including the spinal cord, inferior olive and cerebral cortex. Localization of Cx36-puncta at AISs was confirmed by confocal single scan and 3D imaging, immunofluorescence intensity profiling and high resolution structured illumination microscopy (SIM). AISs measuring up to 30 µm in length displayed typically a single Cx36-punctum and the incidence of these long AISs displaying Cx36-puncta ranged from 3% to 7% in the inferior olive and in various layers of the cerebral cortex. In the inferior olive, the gap junction associated protein zonula occludens-1 (ZO-1) was found to be co-localized with Cx36-puncta on AISs, indicating that these puncta have some of the molecular constituents of gap junctions. Our results add to the neuronal subcellular locations at which Cx36 is deployed, and raise possibilities for its involvement in novel functions in the AIS compartment.

Could electrical coupling contribute to the formation of cell assemblies?

Cell assemblies and central pattern generators (CPGs) are related types of neuronal networks: both consist of interacting groups of neurons whose collective activities lead to defined functional outputs. In the case of a cell assembly, the functional output may be interpreted as a representation of something in the world, external or internal; for a CPG, the output 'drives' an observable (i.e. motor) behavior. Electrical coupling, via gap junctions, is critical for the development of CPGs, as well as for their actual operation in the adult animal. Electrical coupling is also known to be important in the development of hippocampal and neocortical principal cell networks. We here argue that electrical coupling - in addition to chemical synapses - may therefore contribute to the formation of at least some cell assemblies in adult animals.

On the occurrence and enigmatic functions of mixed (chemical plus electrical) synapses in the mammalian CNS.

Electrical synapses with diverse configurations and functions occur at a variety of interneuronal appositions, thereby significantly expanding the physiological complexity of neuronal circuitry over that provided solely by chemical synapses. Gap junctions between apposed dendritic and somatic plasma membranes form "purely electrical" synapses that allow for electrical communication between coupled neurons. In addition, gap junctions at axon terminals synapsing on dendrites and somata allow for "mixed" (dual chemical+electrical) synaptic transmission. "Dual transmission" was first documented in the autonomic nervous system of birds, followed by its detection in the central nervous systems of fish, amphibia, and reptiles. Subsequently, mixed synapses have been detected in several locations in the mammalian CNS, where their properties and functional roles remain undetermined. Here, we review available evidence for the presence, complex structural composition, and emerging functional properties of mixed synapses in the mammalian CNS.

Astrocytes drive cortical vasodilatory signaling by activating endothelial NMDA receptors.

Astrocytes express neurotransmitter receptors that serve as sensors of synaptic activity and initiate signals leading to activity-dependent local vasodilation and increases in blood flow. We previously showed that arteriolar vasodilation produced by activation of cortical astrocytes is dependent on endothelial nitric oxide synthase (eNOS) and endogenous agonists of N-methyl-D-aspartate (NMDA) receptors. Here, we tested the hypothesis that these effects are mediated by NMDA receptors expressed by brain endothelial cells. Primary endothelial cultures expressed NMDA receptor subunits and produced nitric oxide in response to co-agonists, glutamate and D-serine. In cerebral cortex in situ, immunoelectron microscopy revealed that endothelial cells express the GluN1 NMDA receptor subunit at basolateral membrane surfaces in an orientation suitable for receiving intercellular messengers from brain cells. In cortical slices, activation of astrocytes by two-photon flash photolysis of a caged Ca2+ compound or application of a metabotropic glutamate receptor agonist caused endothelial NO generation and local vasodilation. These effects were mitigated by NMDA receptor antagonists and conditional gene silencing of endothelial GluN1, indicating at least partial dependence on endothelial NMDA receptors. Our observations identify a novel astrocyte-endothelial vasodilatory signaling axis that could contribute to endothelium-dependent vasodilation in brain functional hyperemia.

E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36.

Electrical synapses in the mammalian central nervous system (CNS) are increasingly recognized as highly complex structures for mediation of neuronal communication, both with respect to their capacity for dynamic short- and long-term modification in efficacy of synaptic transmission and their multimolecular regulatory and structural components. These two characteristics are inextricably linked, such that understanding of mechanisms that contribute to electrical synaptic plasticity requires knowledge of the molecular composition of electrical synapses and the functions of proteins associated with these synapses. Here, we provide evidence that the key component of gap junctions that form the majority of electrical synapses in the mammalian CNS, namely connexin36 (Cx36), directly interacts with the related E3 ubiquitin ligase proteins Ligand of NUMB protein X1 (LNX1) and Ligand of NUMB protein X2 (LNX2). This is based on immunofluorescence colocalization of LNX1 and LNX2 with Cx36-containing gap junctions in adult mouse brain versus lack of such coassociation in LNX null mice, coimmunoprecipitation of LNX proteins with Cx36, and pull-down of Cx36 with the second PDZ domain of LNX1 and LNX2. Furthermore, cotransfection of cultured cells with Cx36 and E3 ubiquitin ligase-competent LNX1 and LNX2 isoforms led to loss of Cx36-containing gap junctions between cells, whereas these junctions persisted following transfection with isoforms of these proteins that lack ligase activity. Our results suggest that a LNX protein mediates ubiquitination of Cx36 at neuronal gap junctions, with consequent Cx36 internalization, and may thereby contribute to intracellular mechanisms that govern the recently identified modifiability of synaptic transmission at electrical synapses.

Electrical synapses in mammalian CNS: Past eras, present focus and future directions.

Gap junctions provide the basis for electrical synapses between neurons. Early studies in well-defined circuits in lower vertebrates laid the foundation for understanding various properties conferred by electrical synaptic transmission. Knowledge surrounding electrical synapses in mammalian systems unfolded first with evidence indicating the presence of gap junctions between neurons in various brain regions, but with little appreciation of their functional roles. Beginning at about the turn of this century, new approaches were applied to scrutinize electrical synapses, revealing the prevalence of neuronal gap junctions, the connexin protein composition of many of those junctions, and the myriad diverse neural systems in which they occur in the mammalian CNS. Subsequent progress indicated that electrical synapses constitute key elements in synaptic circuitry, govern the collective activity of ensembles of electrically coupled neurons, and in part orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie fundamental integrative processes. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Electrical Synapses: New Rules for Assembling an Old Structure Asymmetrically.

Electrical synapses are emerging as complex structures, consisting of gap junction-forming connexin proteins and also multiple scaffolding and regulatory protein components, which were assumed to be symmetrically organized across these synapses; however, new findings reveal their synaptic asymmetry.

KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction.

Saltatory conduction in mammalian myelinated axons was thought to be well understood before recent discoveries revealed unexpected subcellular distributions and molecular identities of the K(+)-conductance pathways that provide for rapid axonal repolarization. In this study, we visualize, identify, localize, quantify, and ultrastructurally characterize axonal KV1.1/KV1.2 channels in sciatic nerves of rodents. With the use of light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling electron microscopy, KV1.1/KV1.2 channels are localized to three anatomically and compositionally distinct domains in the internodal axolemmas of large myelinated axons, where they form densely packed "rosettes" of 9-nm intramembrane particles. These axolemmal KV1.1/KV1.2 rosettes are precisely aligned with and ultrastructurally coupled to connexin29 (Cx29) channels, also in matching rosettes, in the surrounding juxtaparanodal myelin collars and along the inner mesaxon. As >98% of transmembrane proteins large enough to represent ion channels in these specialized domains, ∼500,000 KV1.1/KV1.2 channels define the paired juxtaparanodal regions as exclusive membrane domains for the voltage-gated K(+)conductance that underlies rapid axonal repolarization in mammals. The 1:1 molecular linkage of KV1 channels to Cx29 channels in the apposed juxtaparanodal collars, plus their linkage to an additional 250,000-400,000 Cx29 channels along each inner mesaxon in every large-diameter myelinated axon examined, supports previously proposed K(+)conductance directly from juxtaparanodal axoplasm into juxtaparanodal myeloplasm in mammalian axons. With neither Cx29 protein nor myelin rosettes detectable in frog myelinated axons, these data showing axon-to-myelin linkage by abundant KV1/Cx29 channels in rodent axons support renewed consideration of an electrically active role for myelin in increasing both saltatory conduction velocity and maximum propagation frequency in mammalian myelinated axons.

Elevated auditory brainstem response thresholds in mice with Connexin36 gene ablation.

CONCLUSION: Expression of connexin36 (Cx36) and electrical synapses formed by Cx36-containing gap junctions contribute to normal auditory brainstem response thresholds in mice. OBJECTIVES: Electrical synaptic transmission mediated by gap junctions has not been intensively studied in the auditory system. This study used transgenic mice with knockout of the gene coding for the major protein that forms neuronal gap junctions in mammalian brain (Cx36) to evaluate the role of Cx36 in murine hearing. METHODS: Auditory brainstem response (ABR) thresholds and distortion product otoacoustic emissions (DPOAEs) were measured in 26 wild-type and 26 Cx36 knockout mice. ABR thresholds were used to assess auditory brainstem function at four frequencies. DPOAEs were delivered for seven frequency pairs to assess cochlear function. RESULTS: The magnitudes of the 2f1-f2 distortion products were not different between Cx36 knockout and wild-type mice, suggesting similar cochlear function in the two groups. ABR thresholds were significantly elevated in the Cx36 knockout compared with the wild-type groups, suggesting impaired function in the auditory brainstem. The results suggest that electrical synapses formed by Cx36-containing gap junctions contribute to auditory sound processing and function at the level of the brainstem, not the cochlea. These findings may be important for understanding human auditory pathology.

Molecular determinants of magnesium-dependent synaptic plasticity at electrical synapses formed by connexin36.

Neuronal gap junction (GJ) channels composed of connexin36 (Cx36) play an important role in neuronal synchronization and network dynamics. Here we show that Cx36-containing electrical synapses between inhibitory neurons of the thalamic reticular nucleus are bidirectionally modulated by changes in intracellular free magnesium concentration ([Mg(2+)]i). Chimeragenesis demonstrates that the first extracellular loop of Cx36 contains a Mg(2+)-sensitive domain, and site-directed mutagenesis shows that the pore-lining residue D47 is critical in determining high Mg(2+)-sensitivity. Single-channel analysis of Mg(2+)-sensitive chimeras and mutants reveals that [Mg(2+)]i controls the strength of electrical coupling mostly via gating mechanisms. In addition, asymmetric transjunctional [Mg(2+)]i induces strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. We suggest that Mg(2+)-dependent synaptic plasticity of Cx36-containing electrical synapses could underlie neuronal circuit reconfiguration via changes in brain energy metabolism that affects neuronal levels of intracellular ATP and [Mg(2+)]i.

Molecular and functional asymmetry at a vertebrate electrical synapse.

Electrical synapses are abundant in the vertebrate brain, but their functional and molecular complexities are still poorly understood. We report here that electrical synapses between auditory afferents and goldfish Mauthner cells are constructed by apposition of hemichannels formed by two homologs of mammalian connexin 36 (Cx36) and that, while Cx35 is restricted to presynaptic hemiplaques, Cx34.7 is restricted to postsynaptic hemiplaques, forming heterotypic junctions. This molecular asymmetry is associated with rectification of electrical transmission that may act to promote cooperativity between auditory afferents. Our data suggest that, in similarity to pre- and postsynaptic sites at chemical synapses, one side in electrical synapses should not necessarily be considered the mirror image of the other. While asymmetry based on the presence of two Cx36 homologs is restricted to teleost fish, it might also be based on differences in posttranslational modifications of individual connexins or in the complement of gap junction-associated proteins.

Grafting of fetal brainstem 5-HT neurons into the sublesional spinal cord of paraplegic rats restores coordinated hindlimb locomotion.

In rodent models of spinal cord injury, there is increasing evidence that activation of the locomotor central pattern generator (CPG) below the site of injury with 5-hydroxytryptamine (5-HT) agonists improves locomotor recovery and restores coordination. A promising means of replacing 5-HT control of locomotion is to graft brainstem 5-HT neurons into the spinal cord below the level of the spinal cord injury. However, it is not known whether this approach improves limb coordination because recovery of coordinated stepping has not been documented in detail in previous studies employing this transplantation strategy. Here, adult rats with complete spinal cord transections at the T9/10 level were grafted with E14 fetal neurons from the medulla at the T10/11 vertebra level one month after injury. The B1, B2 and B3 fetal anlagen of brainstem 5-HT neurons, a grouping that included the presumed precursors of recently described 5-HT locomotor command neurons, were used in these grafts. EMG and video recordings of treadmill locomotion evoked by tail stimulation showed full recovery of inter- and intralimb coordination in the grafted rats. We showed, using systemically applied antagonists, that 5-HT2 and 5-HT7 receptors mediate the improved locomotion after grafting, but through actions on different populations of spinal locomotor neurons. Specifically, 5-HT2 receptors control CPG activation as well as motoneuron output, while 5-HT7 receptors contribute primarily to activity of the locomotor CPG. These results are consistent with the roles for these receptors during locomotion in intact rodents and in rodent brainstem-spinal cord in vitro preparations.

Evidence for connexin36 localization at hippocampal mossy fiber terminals suggesting mixed chemical/electrical transmission by granule cells.

Electrical synaptic transmission via gap junctions has become an accepted feature of neuronal communication in the mammalian brain, and occurs often between dendrites of interneurons in major brain structures, including the hippocampus. Electrical and dye-coupling has also been reported to occur between pyramidal cells in the hippocampus, but ultrastructurally-identified gap junctions between these cells have so far eluded detection. Gap junctions can be formed by nerve terminals, where they contribute the electrical component of mixed chemical/electrical synaptic transmission, but mixed synapses have only rarely been described in mammalian CNS. Here, we used immunofluorescence localization of the major gap junction forming protein connexin36 to examine its possible association with hippocampal pyramidal cells. In addition to labeling associated with gap junctions between dendrites of parvalbumin-positive interneurons, a high density of fine, punctate immunolabeling for Cx36, non-overlapping with parvalbumin, was found in subregions of the stratum lucidum in the ventral hippocampus of rat brain. A high percentage of Cx36-positive puncta in the stratum lucidum was localized to mossy fiber terminals, as indicated by co-localization of Cx36-puncta with the mossy terminal marker vesicular glutamate transporter-1, as well as with other proteins that are highly concentrated in, and diagnostic markers of, these terminals. These results suggest that mossy fiber terminals abundantly form mixed chemical/electrical synapses with pyramidal cells, where they may serve as intermediaries for the reported electrical and dye-coupling between ensembles of these principal cells. This article is part of a Special Issue entitled Electrical Synapses.

Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

Electrical transmission between mammalian neurons is supported by a small fraction of gap junction channels.

Electrical synapses formed by gap junctions between neurons create networks of electrically coupled neurons in the mammalian brain, where these networks have been found to play important functional roles. In most cases, interneuronal gap junctions occur at remote dendro-dendritic contacts, making difficult accurate characterization of their physiological properties and correlation of these properties with their anatomical and morphological features of the gap junctions. In the mesencephalic trigeminal (MesV) nucleus where neurons are readily accessible for paired electrophysiological recordings in brain stem slices, our recent data indicate that electrical transmission between MesV neurons is mediated by connexin36 (Cx36)-containing gap junctions located at somato-somatic contacts. We here review evidence indicating that electrical transmission between these neurons is supported by a very small fraction of the gap junction channels present at cell-cell contacts. Acquisition of this evidence was enabled by the unprecedented experimental access of electrical synapses between MesV neurons, which allowed estimation of the average number of open channels mediating electrical coupling in relation to the average number of gap junction channels present at these contacts. Our results indicate that only a small proportion of channels (~0.1 %) appear to be conductive. On the basis of similarities with other preparations, we postulate that this phenomenon might constitute a general property of vertebrate electrical synapses, reflecting essential aspects of gap junction function and maintenance.

Requirement of neuronal connexin36 in pathways mediating presynaptic inhibition of primary afferents in functionally mature mouse spinal cord.

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.