Altered olivocerebellar activity patterns in the connexin36 knockout mouse

The inferior olive (IO) has among the highest densities of neuronal gap junctions in the nervous system. These gap junctions are proposed to be the underlying mechanism for generating synchronous Purkinje cell complex spike (CS) activity. Gap junctions between neurons are formed mostly by connexin 36 proteins. Thus, the connexin 36 knockout (Cx36KO) mouse provides an opportunity to test whether gap junction coupling between IO neurons is the basis of CS synchrony. Multiple electrode recordings of crus 2 CSs were obtained from wildtype (Wt) and Cx36KO mice. Wts showed statistically significant levels of CS synchrony, with the same spatial distribution as has been reported for other species: high CS synchrony levels occurred mostly among Purkinje cells within the same parasagittally-oriented cortical strip. In contrast, in Cx36KOs, synchrony was at chance levels and had no preferential spatial orientation, supporting the gap junction hypothesis. CS firing rates for Cx36KOs were significantly lower than for Wts, suggesting that electrical coupling is an important determinant of IO excitability. Rhythmic CS activity was present in both Wt and Cx36KOs, suggesting that individual IO cells can act as intrinsic oscillators. In addition, the climbing fiber reflex was absent in the Cx36KOs, validating its use as a tool for assessing electrical coupling of IO neurons. Zebrin II staining and anterograde tracing showed that cerebellar cortical organization and the topography of the olivocerebellar projection are normal in the Cx36KO. Thus, the differences in CS activity between Wts and Cx36KOs likely reflect the loss of electrical coupling of IO cells.     

Cellular expression of connexins in the rat brain: neuronal localization, effects of kainate-induced seizures and expression in apoptotic neuronal cells

The identification of connexins (Cxs) expressed in neuronal cells represents a crucial step for understanding the direct communication between neurons and between neuron and glia. In the present work, using a double-labelling method combining in situ hybridization for Cx mRNAs with immunohistochemical detection for neuronal markers, we provide evidence that, among cerebral connexins (Cx26, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45 and Cx47), only Cx45 and Cx36 mRNAs are localized in neuronal cells in both developing and adult rat brain. In order to establish whether connexin expression is influenced in vivo by abnormal neuronal activity, we examined the short-term effects of kainate-induced seizures. The results revealed an unexpected expression of Cx26 and Cx45 mRNA in neuronal cells undergoing apoptotic cell death in the CA3-CA4, in the hilus of the hippocampus and in other brain regions involved in seizure-induced lesion. However, the expression of Cx26 and Cx45 mRNAs was not associated with detectable expression of corresponding proteins as evaluated by immunohistochemistry with specific antibodies. Moreover, in the same brain regions Cx32 and Cx43 were up-regulated in non-neruronal cells whereas the neuronal Cx36 was down-regulated. Taken together the present results provide novel information regarding the specific subpopulation of neurons expressing Cx45 and raise the question of the meaning of connexin mRNA expression in the neuronal apoptotic process.     

Expression of connexin30.2 in interneurons of the central nervous system in the mouse

Electrical synapses, particularly gap junctions composed of connexin (Cx) 36, have been suggested to synchronize neuronal network oscillations. Recently, we generated Cx30.2-deficient mice which express beta-galactosidase under control of Cx30.2 gene regulatory elements. In the central nervous system beta-galactosidase activity representing Cx30.2 expression was restricted to NeuN-positive cells, thus identifying Cx30.2 as new neuronal connexin. In the hippocampus, co-immunofluorescence analyses revealed beta-galactosidase/Cx30.2 expression in GABAergic inhibitory interneurons such as parvalbumin- and somatostatin-positive basket, axo-axonic, bistratified or oriens lacunosum-moleculare cells. approximately 94% of the Cx30.2 expressing parvalbumin-positive interneurons also expressed Cx36. Performing field potential recordings from hippocampal slices we found no differences in basal excitation and excitation-inhibition balance between Cx30.2+/+ and Cx30.2LacZ/LacZ)mice. Furthermore, frequency and power of gap junction dependent gamma and ripples oscillations were similar in these animals. This suggests that the lack of Cx30.2 in interneurons can be largely compensated by other connexins, most likely Cx36.     

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

Role of astroglial connexin30 in hippocampal gap junction coupling

The impact of connexin30 (Cx30) on interastrocytic gap junction coupling in the normal hippocampus is matter of debate; reporter gene analyses indicated a weak expression of Cx30 in the mouse hippocampus. In contrast, mice lacking connexin43 (Cx43) in astrocytes exhibited only 50% reduction in coupling. Complete uncoupling of hippocampal astrocytes in mice lacking both Cx30 and Cx43 suggested that Cx30 participates in interastrocytic gap junction coupling in the hippocampus. With comparative reporter gene assays, immunodetection, and cre/loxP-based reporter approaches we demonstrate that Cx30 is more abundant than previously thought. The specific role of Cx30 in interastrocytic coupling has never been investigated. Employing tracer coupling analyses in acute slices of Cx30 deficient mice here we show that Cx30 makes a substantial contribution to interastrocytic gap junctional communication in the mouse hippocampus.     

Re‐evaluation of connexins associated with motoneurons in rodent spinal cord,sexually dimorphic motor nuclei and trigeminal motor nucleus

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) are a common feature in mammalian brain circuitry, but less is known about their deployment in spinal cord. It has been reported based on connexin mRNA and/or protein detection that developing and/or mature motoneurons express a variety of connexins, including Cx26, Cx32, Cx36 and Cx43 in trigeminal motoneurons, Cx36, Cx37, Cx40, Cx43 and Cx45 in spinal motoneurons, and Cx32 in sexually dimorphic motoneurons. We re‐examined the localization of these connexins during postnatal development and in adult rat and mouse using immunofluorescence labeling for each connexin. We found Cx26 in association only with leptomeninges in the trigeminal motor nucleus (Mo5), Cx32 only with oligodendrocytes and myelinated fibers among motoneurons in this nucleus and in the spinal cord, and Cx37, Cx40 and Cx45 only with blood vessels in the ventral horn of spinal cord, including those among motoneurons. By freeze‐fracture replica immunolabeling, > 100 astrocyte gap junctions but no neuronal gap junctions were found based on immunogold labeling for Cx43, whereas 16 neuronal gap junctions at postnatal day (P)4, P7 and P18 were detected based on Cx36 labeling. Punctate labeling for Cx36 was localized to the somatic and dendritic surfaces of peripherin‐positive motoneurons in the Mo5, motoneurons throughout the spinal cord, and sexually dimorphic motoneurons at lower lumbar levels. In studies of electrical synapses and electrical transmission between developing and between adult motoneurons, our results serve to focus attention on mediation of this transmission by gap junctions composed of Cx36.     

Expression of connexin36 in the adult and developing rat brain

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.     

Unique distributions of the gap junction proteins connexin29, connexin32, and connexin47 in oligodendrocytes

Oligodendrocytes of adult rodents express three different connexins: connexin29 (Cx29), Cx32, and Cx47. In this study, we show that Cx29 is localized to the inner membrane of small myelin sheaths, whereas Cx32 is localized on the outer membrane of large myelin sheaths; Cx29 does not colocalize with Cx32 in gap junction plaques. All oligodendrocytes appear to express Cx47, which is largely restricted to their perikarya. Cx32 and Cx47 are colocalized in many gap junction plaques on oligodendrocyte somata, particularly in gray matter. Cx45 is detected in the cerebral vasculature, but not in oligodendrocytes or myelin sheaths. This diversity of connexins in oligodendrocytes (in different populations of cells and in different subcellular compartments) likely reflects functional differences between these connexins and perhaps the oligodendrocytes themselves.     

听源性惊厥增强大鼠星形胶质细胞Cx43基因表达

星形胶质细胞之间通过缝隙连接形成偶联，其缝隙连接蛋白为Cx43。该缝隙连接对神经元兴奋时释放到细胞外的K＋起空间缓冲作用。本研究以遗传性癫痫易感大鼠P77PMC为模型，采用原位杂交的方法，观察了一次听源性惊厥后脑内星形胶质细胞Cx43基因表达的变化，结果显示，P77PMC大鼠一次听源性惊厥后在大脑皮质、海马Cx43mRNA表达呈时间依赖性增加，从惊厥后2h开始增加，4～8h达高峰，24h仍高于对照。结果表明：惊厥时，皮质和海马星形胶质细胞Cx43基因表达增强，可能有利于星形胶质细胞对神经元兴奋时释放到细胞外的K＋产生空间缓冲作用，以便维持神经辕围的的K＋平衡。     

Changes in object recognition and anxiety-like behaviour in mice expressing a cx47 mutation that causes pelizaeus-merzbacher-like disease

Pelizaeus-Merzbacher-like disease is characterized by impaired psychomotor development, ataxia, progressive spasticity and mental retardation. It is induced by mutations in the gap junction gene GJC2 that encodes for the gap junction protein connexin 47. Mice bearing a human Cx47M283T missense mutation have been generated as a transgenic mouse model of Pelizaeus-Merzbacher-like disease. Homozygous expression of the mutant connexin 47 gene in oligodendrocytes resulted in a complex and variable neuropathologic phenotype, which was associated with impairments in motor coordination in juvenile, but not adult mice. In the present study, we have investigated anxiety-like behaviour and spatial working memory in juvenile (P23) and adult (3-month-old) Cx47M282T mutant mice. Adult Cx47M282T mice were also evaluated in terms of neuromotor functions and in the novel object recognition test. Juvenile Cx47M282T mutant mice exhibited an increase in anxiety-like behaviour in the open field test, but no changes in spatial working memory performance. No significant changes in anxiety-like behaviour, spatial working memory or neuromotor functions were observed in the adult Cx47M282T mutant mice. However, novel object recognition was significantly impaired in adult Cx47M282T mice. Our results suggest that the expression of the human Cx47M282T mutation in the mouse causes changes in anxiety-like behaviour in juvenile and novel object recognition impairments in adult mice. It appears that the distortion of panglial gap junction coupling in white and grey matter tissue in the Cx47M282T mice is associated with a complex age-dependent behavioural phenotype including changes in psychomotor, emotional and memory functions.     

Expression of connexin36 mRNA in adult rodent brain

A new member of the connexin gene family, named Connexin36 (Cx36) has, recently, been identified in rodents and shown to be preferentially, if not exclusively, expressed in neurones of the adult CNS. In this study we present a detailed in situ hybridization analysis of the expression pattern of mouse Connexin 36 (mCx36) mRNA in the adult mouse brain, with particular regards to the correlation of mCx36 expression to specific neuronal cell classes and systems. We found that mCx36 was strongly and widely expressed in the brain, including areas where the presence of gap junctions was never detected before. Quantitative analysis of the hybridization signal indicated varying levels of expression in different areas. In particular mCx36 was highly expressed in the neurones at different levels of the motor pathway, the olfactory pathway, the hippocampus, and areas related to the generation of respiratory rhythm. On the contrary, mCx36 was more heterogeneously expressed in nuclei of the sensory pathways. These findings show that mCx36 is the first connexin specifically expressed in neuronal cells in the adult rodent brain. The profiles of expression clearly indicate that mCx36 might play specific roles within different neuronal systems.     

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat

Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36‐containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase‐positive and 5‐hydroxytryptamine‐positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens‐1 (ZO‐1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36‐containing gap junctions that also incorporate the scaffolding protein ZO‐1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36‐containing gap junctions, which may now be tested by the use of Cx36 null mice.