Cx36 Knockout Mice Research Articles

Retinal ganglion cells encode differently in the myopic mouse retina?

The etiology of myopia remains unclear. This study investigated whether retinal ganglion cells (RGCs) in the myopic retina encode visual information differently from the normal retina and to determine the role of Connexin (Cx) 36 in this process. Generalized linear models (GLMs), which can capture stimulus-dependent changes in real neurons with spike timing precision and reliability, were used to predict RGCs responses to focused and defocused images in the retinas of wild-type (normal) and Lens-Induced Myopia (LIM) mice. As the predominant subunit of gap junctions in the mouse retina and a plausible modulator in myopia development, Cx36 knockout (KO) mice were used as a control for an intact retinal circuit. The kinetics of excitatory postsynaptic currents (EPSCs) of a single αRGC could reflect projection of both focused and defocused images in the retinas of normal and LIM, but not in the Cx36 knockout mice. Poisson GLMs revealed that RGC encoding of visual stimuli in the LIM retina was similar to that of the normal retina. In the LIM retinas, the linear-Gaussian GLM model with offset was a better fit for predicting the spike count under a focused image than the defocused image. Akaike information criterion (AIC) indicated that nonparametric GLM (np-GLM) model predicted focused/defocused images better in both LIM and normal retinas. However, the spike counts in 33% of αRGCs in LIM retinas were better fitted by exponential GLM (exp-GLM) under defocus, compared to only 13% αRGCs in normal retinas. The differences in encoding performance between LIM and normal retinas indicated the possible amendment and plasticity of the retinal circuit in myopic retinas. The absence of a similar response between Cx36 KO mice and normal/LIM mice might suggest that Cx36, which is associated with myopia development, plays a role in encoding focused and defocused images.

Interrelationships Between Spinal Sympathetic Preganglionic Neurons, Autonomic Systems and Electrical Synapses Formed by Connexin36-containing Gap Junctions

Spinal sympathetic preganglionic neurons (SPNs) are among the many neuronal populations in the mammalian central nervous system (CNS) where there is evidence for electrical coupling between cell pairs linked by gap junctions composed of connexin36 (Cx36). Understanding the organization of this coupling in relation to autonomic functions of spinal sympathetic systems requires knowledge of how these junctions are deployed among SPNs. Here, we document the distribution of immunofluorescence detection of Cx36 among SPNs identified by immunolabelling of their various markers, including choline acetyltransferase, nitric oxide and peripherin in adult and developing mouse and rat. In adult animals, labelling of Cx36 was exclusively punctate and dense concentrations of Cx36-puncta were distributed along the entire length of the spinal thoracic intermediolateral cell column (IML). These puncta were also seen in association with SPN dendritic processes in the lateral funiculus, the intercalated and central autonomic areas and those within and extending medially from the IML. All labelling for Cx36 was absent in spinal cords of Cx36 knockout mice. High densities of Cx36-puncta were already evident among clusters of SPNs in the IML of mouse and rat at postnatal days 10–12. In Cx36BAC::eGFP mice, eGFP reporter was absent in SPNs, thus representing false negative detection, but was localized to some glutamatergic and GABAergic synaptic terminals. Some eGFP+ terminals were found contacting SPN dendrites. These results indicate widespread Cx36 expression in SPNs, further supporting evidence of electrical coupling between these cells, and suggest that SPNs are innervated by neurons that themselves may be electrically coupled.

Modulation of Gap Junction Coupling Within the Islet of Langerhans During the Development of Type 1 Diabetes.

In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca2+ homeostasis. Connexin36 (Cx36) gap junctions electrically couple β-cells to coordinate glucose-stimulated Ca2+ and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca2+ dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6–12 weeks age. Glucose-stimulated insulin secretion, Ca2+ dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and KATP or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca2+ dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by KATP activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca2+. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca2+ homeostasis protects against islet decline in T1D.

Molecular and functional architecture of the mouse photoreceptor network.

Mouse photoreceptors are electrically coupled via gap junctions, but the relative importance of rod/rod, cone/cone, or rod/cone coupling is unknown. Furthermore, while connexin36 (Cx36) is expressed by cones, the identity of the rod connexin has been controversial. We report that FACS-sorted rods and cones both express Cx36 but no other connexins. We created rod- and cone-specific Cx36 knockout mice to dissect the photoreceptor network. In the wild type, Cx36 plaques at rod/cone contacts accounted for more than 95% of photoreceptor labeling and paired recordings showed the transjunctional conductance between rods and cones was ~300 pS. When Cx36 was eliminated on one side of the gap junction, in either conditional knockout, Cx36 labeling and rod/cone coupling were almost abolished. We could not detect direct rod/rod coupling, and cone/cone coupling was minor. Rod/cone coupling is so prevalent that indirect rod/cone/rod coupling via the network may account for previous reports of rod coupling.

Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart–brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.—Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36–containing gap junctions.

Connexin 36 expression is required for electrical coupling between mouse rods and cones.

Rod-cone gap junctions mediate the so-called "secondary rod pathway", one of three routes that convey rod photoreceptor signals across the retina. Connexin 36 (Cx36) is expressed at these gap junctions, but an unidentified connexin protein also seems to be expressed. Cx36 knockout mice have been used extensively in the quest to dissect the roles in vision of all three pathways, with the assumption, never directly tested, that rod-cone electrical coupling is abolished by deletion of this connexin isoform. We previously showed that when wild type mouse cones couple to rods, their apparent dynamic range is extended toward lower light intensities, with the appearance of large responses to dim flashes (up to several mV) originating in rods. Here we recorded from the cones of Cx36del[LacZ]/del[LacZ] mice and found that dim flashes of the same intensity evoked at most small sub-millivolt responses. Moreover, these residual responses originated in the cones themselves, since: (i) their spectral preference matched that of the recorded cone and not of rods, (ii) their time-to-peak was shorter than in coupled wild type cones, (iii) a pharmacological block of gap junctions did not reduce their amplitude. Taken together, our data show that rod signals are indeed absent in the cones of Cx36 knockout mice. This study is the first direct demonstration that Cx36 is crucial for the assembly of functional rod-cone gap junctional channels, implying that its genetic deletion is a reliable experimental approach to eliminate rod-cone coupling.

Electrical synapses and the development of inhibitory circuits in the thalamus.

The thalamus is a structure critical for information processing and transfer to the cortex. Thalamic reticular neurons are inhibitory cells interconnected by electrical synapses, most of which require the gap junction protein connexin36 (Cx36). We investigated whether electrical synapses play a role in the maturation of thalamic networks by studying neurons in mice with and without Cx36. When Cx36 was deleted, inhibitory synapses were more numerous, although both divergent inhibitory connectivity and dendritic complexity were reduced. Surprisingly, we observed non-Cx36-dependent electrical synapses with unusual biophysical properties interconnecting some reticular neurons in mice lacking Cx36. The results of the present study suggest an important role for Cx36-dependent electrical synapses in the development of thalamic circuits. Neurons within the mature thalamic reticular nucleus (TRN) powerfully inhibit ventrobasal (VB) thalamic relay neurons via GABAergic synapses. TRN neurons are also coupled to one another by electrical synapses that depend strongly on the gap junction protein connexin36 (Cx36). Electrical synapses in the TRN precede the postnatal development of TRN-to-VB inhibition. We investigated how the deletion of Cx36 affects the maturation of TRN and VB neurons, electrical coupling and GABAergic synapses by studying wild-type (WT) and Cx36 knockout (KO) mice. The incidence and strength of electrical coupling in TRN was sharply reduced, but not abolished, in KO mice. Surprisingly, electrical synapses between Cx36-KO neurons had faster voltage-dependent decay kinetics and conductance asymmetry (rectification) than did electrical synapses between WT neurons. The properties of TRN-mediated inhibition in VB also depended on the Cx36 genotype. Deletion of Cx36 increased the frequency and shifted the amplitude distributions of miniature IPSCs, whereas the paired-pulse ratio of evoked IPSCs was unaffected, suggesting that the absence of Cx36 led to an increase in GABAergic synaptic contacts. VB neurons from Cx36-KO mice also tended to have simpler dendritic trees and fewer divergent inputs from the TRN compared to WT cells. The findings obtained in the present study suggest that proper development of thalamic inhibitory circuitry, neuronal morphology, TRN cell function and electrical coupling requires Cx36. In the absence of Cx36, some TRN neurons express asymmetric electrical coupling mediated by other unidentified connexin subtypes.

Role of neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemic neuronal death.

Role of neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemic neuronal death.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

Aberrant activity in retinal degeneration impairs central visual processing and relies on Cx36-containing gap junctions

In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD.

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.

Investigation into the role of gap junction modulation of intracortical connectivity in mouse neocortical brain slices

General anesthetics are hypothesized to cause unconsciousness by interrupting communication pathways within the cerebral cortex. A correlate of this has been demonstrated in mouse neocortical slices, where anesthetics disrupt the spread of population field potential activity—resulting in a “decoupling” of activity recorded across spatial locations within the slice. In this study we investigated whether this decoupling can be explained by gap junction blockade, with a particular focus on the connexin36 (Cx36) subtype. Baseline, coupled seizure-like event (SLE) activity was recorded from two extracellular electrodes in slices perfused with no-magnesium artificial cerebrospinal fluid (aCSF). The connexin36 gap junction blocker mefloquine (25µM) failed to decouple SLE activity in wild-type mice (median(range) decoupling rate of 0.70(0.03–3.00)%, not significantly different from controls). Slices from Cx36 knock-out mice exhibited coupled SLE activity under baseline conditions and readily decoupled when exposed to the general anesthetic etomidate. The general gap junction blocker carbenoxolone (CBX, 100µM) strongly decoupled SLE activity compared to controls in wild-type mice (2.7(0.1–42.5) % compared to 0.03(0.0–0.5)%, p=0.0001). Taken together, the results show that Cx36 gap junction blockade does not cause decoupling of intracortical population activity, but the involvement of other gap junction subtypes cannot be ruled out.

Connexin 36 mediates blood cell flow in mouse pancreatic islets.

The insulin-secreting β-cells are contained within islets of Langerhans, which are highly vascularized. Blood cell flow rates through islets are glucose-dependent, even though there are no changes in blood cell flow within in the surrounding exocrine pancreas. This suggests a specific mechanism of glucose-regulated blood flow in the islet. Pancreatic islets respond to elevated glucose with synchronous pulses of electrical activity and insulin secretion across all β-cells in the islet. Connexin 36 (Cx36) gap junctions between islet β-cells mediate this synchronization, which is lost in Cx36 knockout mice (Cx36(-/-)). This leads to glucose intolerance in these mice, despite normal plasma insulin levels and insulin sensitivity. Thus, we sought to investigate whether the glucose-dependent changes in intraislet blood cell flow are also dependent on coordinated pulsatile electrical activity. We visualized and quantified blood cell flow using high-speed in vivo fluorescence imaging of labeled red blood cells and plasma. With the use of a live animal glucose clamp, blood cell flow was measured during either hypoglycemia (∼50 mg/dl) or hyperglycemia (∼300 mg/dl). In contrast to the large glucose-dependent islet blood velocity changes observed in wild-type mice, only minimal differences are observed in both Cx36(+/-) and Cx36(-/-) mice. This observation supports a novel model where intraislet blood cell flow is regulated by the coordinated electrical activity in the islet β-cells. Because Cx36 expression and function is reduced in type 2 diabetes, the resulting defect in intraislet blood cell flow regulation may also play a significant role in diabetic pathology.

Morphologically mixed chemical–electrical synapses formed by primary afferents in rodent vestibular nuclei as revealed by immunofluorescence detection of connexin36 and vesicular glutamate transporter-1

Axon terminals forming mixed chemical/electrical synapses in the lateral vestibular nucleus of rat were described over 40years ago. Because gap junctions formed by connexins are the morphological correlate of electrical synapses, and with demonstrations of widespread expression of the gap junction protein connexin36 (Cx36) in neurons, we investigated the distribution and cellular localization of electrical synapses in the adult and developing rodent vestibular nuclear complex, using immunofluorescence detection of Cx36 as a marker for these synapses. In addition, we examined Cx36 localization in relation to that of the nerve terminal marker vesicular glutamate transporter-1 (vglut-1). An abundance of immunolabeling for Cx36 in the form of Cx36-puncta was found in each of the four major vestibular nuclei of adult rat and mouse. Immunolabeling was associated with somata and initial dendrites of medium and large neurons, and was absent in vestibular nuclei of Cx36 knockout mice. Cx36-puncta were seen either dispersed or aggregated into clusters on the surface of neurons, and were never found to occur intracellularly. Nearly all Cx36-puncta were localized to large nerve terminals immunolabeled for vglut-1. These terminals and their associated Cx36-puncta were substantially depleted after labyrinthectomy. Developmentally, labeling for Cx36 was already present in the vestibular nuclei at postnatal day 5, where it was only partially co-localized with vglut-1, and did not become fully associated with vglut-1-positive terminals until postnatal day 20–25. The results show that vglut-1-positive primary afferent nerve terminals form mixed synapses throughout the vestibular nuclear complex, that the gap junction component of these synapses contains Cx36, that multiple Cx36-containing gap junctions are associated with individual vglut-1 terminals and that the development of these mixed synapses is protracted over several postnatal weeks.

Connexin-36 Knock-Out Mice have Increased Threshold for Kindled Seizures: Role of GABA Inhibition

Blockade of gap junctions (GJs) has been shown to reduce seizures in different epilepsy models. Gap junction mediated, electrically-coupled neuronal networks have been implicated in neuronal synchronization, which is the hallmark of seizure activity. To further understand the role of GJs in seizures, in particular hippocampal seizures, we evaluated electrophysiological responses in the dentate gyrus subfield of the hippocampus, including GABA-mediated recurrent inhibition, seizuregenic stimulation, and seizure activity in response to perforant path kindling in Cx36 knockout (KO) mice compared to wild-type (WT) controls. In anesthetized mice, Cx36 KO mice were characterized by enhanced GABA-mediated recurrent inhibition. Stimulation of the perforant path at 10 Hz for 10 sec (i.e., 100 pulses) markedly enhanced population spike (PS) amplitudes and reduced paired-pulse GABA-mediated inhibition during the stimulation, induced an after discharge for approximately 10 sec at 7-10 sec into the stimulation, and suppressed PS amplitudes postictally for 5-10 min in both KO and WT mice. Repeated epochs of 10 Hz for 10 sec stimulation of the perforant path at 20 min intervals resulted in progressive and persistent disinhibition of paired-pulse responses in WT, but not KO mice. In freely-behaving seizure studies, stimulation of the perforant path (10 Hz for 10 sec) once/day resulted in progressive Stage I-IV seizures in both WT and KO mice. Once Stage IV was achieved, another Stage IV seizure could be elicited each day with the same behavioral response (i.e., Stage V). The threshold for kindled seizures was significantly greater in KO mice compared to WT controls for most stages of seizures. The Cx36 antagonist mefloquine (MFQ) and the typical anticonvulsant pentobarbital reduced Stage IV seizures in both WT and KO mice. Knock-out mice were more sensitive to the anti-epileptic effects of the typical anti-convulsant pentobarbital (20 mg/ kg). Taken together, these findings support the emerging view that reduction in GJ-mediated GABA electrical coupling reduces seizures, perhaps through enhancement of GABAergic feedback inhibition, which results from the uncoupling of the GABA recurrent interneurons from the resistive load that is inherent in their electrical connectivity.

Connexin36 (Cx36) expression and protein detection in the mouse carotid body and myenteric plexus

Although connexin36 (Cx36) has been studied in several tissues, it is notable that no data are available on Cx36 expression in the carotid body and the intestine. The present study was undertaken to evaluate using immunohistochemistry, PCR and Western blotting procedures, whether Cx36 was expressed in the mouse carotid body and in the intestine at ileum and colon level. In the carotid body, Cx36 was detected as diffuse punctate immunostaining and as protein by Western blotting and mRNA by RT-PCR. Cx36 punctate immunostaining was also evident in the intestine with localization restricted to the myenteric plexus of both the ileum and the colon, and this detection was also confirmed by Western blotting and RT-PCR. All the data obtained were validated using Cx36 knockout mice. Taken together the present data on localization of Cx36 gap-junctions in two tissues of neural crest-derived neuroendocrine organs may provide an anatomical basis for future functional investigations.

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.

Behavioral alterations and changes in Ca/calmodulin kinase II levels in the striatum of connexin36 deficient mice

Gap junctions (GJ) are intercellular channels which directly connect the cytoplasm of adjacent cells. GJ allow direct cell-to-cell communication via the diffusion of ions, metabolites and second messengers such as IP 3. The connexin36 (Cx36) protein has been detected in GJ between interneurons of the hippocampus, cerebral cortex, striatum, amygdala, the inferior olive, cerebellum and other brain structures, such as the olfactory bulb. Cx36 knockout (Cx36 KO) mice display changes in synchronous network oscillations in the hippocampus, neocortex and inferior olive and exhibit impaired spatial alternation and one-trial object recognition in a Y-maze. Here, we further characterized the behavioral changes induced by Cx36 deficiency in the mouse by using different behavioral measures and experimental procedures. Additionally, we examined the effects of Cx36 deficiency on acetylcholine esterase (AChE) activity and calcium calmodulin kinase II alpha (CaMKII) protein levels in the striatum. The homozygous Cx36 KO mice displayed increased locomotion and running speed in the open-field, reduced object exploration and impaired one-trial object-place recognition. Furthermore, they exhibited more anxiety-like behavior as compared to the heterozygous controls in the light–dark box. Homozygous Cx36 KO mice exhibited reduced CaMKII levels in the striatum as compared to the heterozygous mice. AChE activity in the striatum was not significantly different between groups. The present results suggest that Cx36 deficiency in the mouse leads to reduced CaMKII levels in the striatum and behavioral changes in open-field activity, anxiety-related behavior in the light–dark box and one-trial object-place recognition.

Deletion of neuronal gap junction protein connexin 36 impairs hippocampal LTP

In the mammalian CNS, deletion of neuronal gap junction protein, connexin 36 (Cx36), causes deficiencies in learning and memory. Here we tested whether Cx36 deletion affects the hippocampal long-term potentiation (LTP), which is considered as a cellular model of learning and memory mechanisms. We report that in acute slices of the hippocampal CA1 area, LTP is reduced in Cx36 knockout mice as compared to wild-type mice. Western blot analysis of NMDA receptor subunits indicates a higher NR2A/NR2B ratio in Cx36 knockout mice, indicating that there is shift in the threshold for LTP induction in knockout animals. Data suggest a possibility that learning and memory deficiencies in Cx36 knockout mice are due to deficiencies in LTP mechanisms.

The role of connexin‐36 gap junctions in alcohol intoxication and consumption

Ventral tegmental area (VTA) GABA neurons appear to be critical substrates underlying the acute and chronic effects of ethanol on dopamine (DA) neurotransmission in the mesocorticolimbic system implicated in alcohol reward. The aim of this study was to examine the role of midbrain connexin-36 (Cx36) gap junctions (GJs) in ethanol intoxication and consumption. Using behavioral, molecular, and electrophysiological methods, we compared the effects of ethanol in mature Cx36 knockout (KO) mice and age-matched wild-type (WT) controls. Compared to WT mice, Cx36 KO mice exhibited significantly more ethanol-induced motor impairment in the open field test, but less disruption in motor coordination in the rotarod paradigm. Cx36 KO mice, and WT mice treated with the Cx36 antagonist mefloquine (MFQ), consumed significantly less ethanol than their WT controls in the drink-in-the-dark procedure. The firing rate of VTA GABA neurons in WT mice was inhibited by ethanol with an IC₅₀ of 0.25 g/kg, while VTA GABA neurons in KO mice were significantly less sensitive to ethanol. Dopamine neuron GABA-mediated sIPSC frequency was reduced by ethanol (30 mM) in WT mice, but not affected in KO mice. Cx36 KO mice evinced a significant up-regulation in DAT and D2 receptors in the VTA, as assessed by quantitative RT-PCR. These findings demonstrate the behavioral relevance of Cx36 GJ-mediated electrical coupling between GABA neurons in mature animals, and suggest that loss of coupling between VTA GABA neurons results in disinhibition of DA neurons, a hyper-DAergic state and lowered hedonic valence for ethanol consumption.