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.

AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions.

Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36-EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced - as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin.

Pannexins in ischemia-induced neurodegeneration.

Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) form large-pore nonselective channels in the plasma membrane of cells and were suggested to play a role in the pathophysiology of cerebral ischemia. To directly test a potential contribution of pannexins in ischemia-related mechanisms, we performed experiments in Px1(-/-), Px2(-/-), and Px1(-/-)Px2(-/-) knockout mice. IL-1ß release, channel function in astrocytes, and cortical spreading depolarization were not altered in Px1(-/-)Px2(-/-) mice, indicating that, in contrast to previous concepts, these processes occur normally in the absence of pannexin channels. However, ischemia-induced dye release from cortical neurons was lower, indicating that channel function in Px1(-/-)Px2(-/-) neurons was impaired. Furthermore, Px1(-/-)Px2(-/-) mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. In conclusion, our data demonstrate that Px1 and Px2 underlie channel function in neurons and contribute to ischemic brain damage.

Connexin36 mediates spike synchrony in olfactory bulb glomeruli.

Neuronal synchrony is important to network behavior in many brain regions. In the olfactory bulb, principal neurons (mitral cells) project apical dendrites to a common glomerulus where they receive a common input. Synchronized activity within a glomerulus depends on chemical transmission but mitral cells are also electrically coupled. We examined the role of connexin-mediated gap junctions in mitral cell coordinated activity. Electrical coupling as well as correlated spiking between mitral cells projecting to the same glomerulus was entirely absent in connexin36 (Cx36) knockout mice. Ultrastructural analysis of glomeruli confirmed that mitral-mitral cell gap junctions on distal apical dendrites contain Cx36. Coupled AMPA responses between mitral cell pairs were absent in the knockout, demonstrating that electrical coupling, not transmitter spillover, is responsible for synchronization. Our results indicate that Cx36-mediated gap junctions between mitral cells orchestrate rapid coordinated signaling via a novel form of electrochemical transmission.

Synaptic imbalance, stereotypies, and impaired social interactions in mice with altered neuroligin 2 expression.

The level of excitation in the brain is kept under control through inhibitory signals mainly exerted by GABA neurons. However, the molecular machinery that regulates the balance between excitation and inhibition (E/I) remains unclear. Candidate molecules implicated in this process are neuroligin (NL) adhesion molecules, which are differentially enriched at either excitatory or inhibitory contacts. In this study, we use transgenic mouse models expressing NL1 or NL2 to examine whether enhanced expression of specific NLs results in synaptic imbalance and altered neuronal excitability and animal behavior. Our analysis reveals several abnormalities selectively manifested in transgenic mice with enhanced expression of NL2 but not NL1. A small change in NL2 expression results in enlarged synaptic contact size and vesicle reserve pool in frontal cortex synapses and an overall reduction in the E/I ratio. The frequency of miniature inhibitory synaptic currents was also found to be increased in the frontal cortex of transgenic NL2 mice. These animals also manifested stereotyped jumping behavior, anxiety, impaired social interactions, and enhanced incidence of spike-wave discharges, as depicted by EEG analysis in freely moving animals. These findings may provide the neural basis for E/I imbalance and altered behavior associated with neurodevelopmental disorders.

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-ß (CaMKII-ß) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-ß Antibody With Connexin36.

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-ß at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-ß. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-ß-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-ß. However, a monoclonal antibody (CB-ß-1) recognized CaMKII-ß but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-ß expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-ß also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-ß influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-ß-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-ß is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-ß with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-ß may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.

A novel network of multipolar bursting interneurons generates theta frequency oscillations in neocortex.

GABAergic interneurons can phase the output of principal cells, giving rise to oscillatory activity in different frequency bands. Here we describe a new subtype of GABAergic interneuron, the multipolar bursting (MB) cell in the mouse neocortex. MB cells are parvalbumin positive but differ from fast-spiking multipolar (FS) cells in their morphological, neurochemical, and physiological properties. MB cells are reciprocally connected with layer 2/3 pyramidal cells and are coupled with each other by chemical and electrical synapses. MB cells innervate FS cells but not vice versa. MB to MB cell as well as MB to pyramidal cell synapses exhibit paired-pulse facilitation. Carbachol selectively induced synchronized theta frequency oscillations in MB cells. Synchrony required both gap junction coupling and GABAergic chemical transmission, but not excitatory glutamatergic input. Hence, MB cells form a distinct inhibitory network, which upon cholinergic drive can generate rhythmic and synchronous theta frequency activity, providing temporal coordination of pyramidal cell output.

Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head.

Thrombospondin 3 (TSP3) is structurally similar to cartilage oligomeric matrix protein (COMP/TSP5), but its function is unknown. To determine the functional significance of TSP3, we generated mice with a targeted disruption of Thbs3. TSP3-null mice are viable and fertile and show normal prenatal skeletal patterning, based on Alcian blue/Alizarin red S staining. However, subtle and transient abnormalities were detected in the developing postnatal skeleton. Young adult TSP3-null mice are heavier than controls, and analyses of the geometric and biomechanical properties of long bones show increases in the moments of inertia, endocortical and periostal radii, and failure load. The bones of 9-week-old TSP3-null male mice also have a significantly greater cortical area. Most of these differences were no longer detected in 15-week-old mice. Micro-computed tomography scans showed that the trabecular bone proximal to the femoral head growth plate developed at an earlier time in TSP3-null mice than in wild-type mice. Thus, vascular invasion and ossification start in the femoral heads of TSP3-null mice at 9 weeks, whereas the wild-type femoral head is still composed of hypertrophic chondroctyes in a calcified matrix at 15 weeks. These results provide evidence for a role for TSP3 in the regulation of skeletal maturation in mice.

Cannabinoid modulation of neuronal function after oxygen/glucose deprivation in area CA1 of the rat hippocampus.

Endocannabinoids released during cerebral ischemia have been implicated as neuroprotective agents. We assessed the role of cannabinoid receptors in modulating the response of neurons to oxygen/glucose deprivation (OGD), a model for in vitro ischemia, in rat hippocampal slices using extracellular recording techniques. Under control conditions, 15 min OGD resulted in only 50% recovery of CA1 field excitatory postsynaptic potentials (fEPSPs) 60 min post-insult. This post-OGD depression of function was primarily NMDA receptor-dependent as the NMDA receptor antagonist MK-801 (50 microM) promoted recovery of synaptic transmission to 76% of the baseline. Treatment with the CB1 receptor antagonist AM251 (1 microM), which prevented the depression of excitatory synaptic transmission caused by WIN55,212-2 (1 microM), also markedly enhanced recovery of function (71% of control). The enhanced recovery after OGD in the presence of AM251 was independent of both GABA(A) receptors and NMDA receptors since co-application of AM251 with either bicuculline (10 microM) or MK-801 (50 microM) did not alter recovery, or further improved recovery, respectively. These results suggest endocannabinoids released during OGD may modulate synaptic transmission and post-OGD neuronal outcome via activation of an AM251-sensitive cannabinoid receptor.

Differential Distribution of Retinal Ca2+/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-ß and -δ as Specific Elements of Electrical Synapses Made of Connexin36 (Cx36).

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-ß was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-ß colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-ß and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-ß and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway.

Mice with a deletion in the first intron of the Col1a1 gene develop age-dependent aortic dissection and rupture.

The functional significance of the first intron of the Col1a1 gene in regulation of type I collagen synthesis remains uncertain. A previous study in mice established that a mutated Col1a1 allele that lacked a large fraction of the first intron, but retained the sequences required for normal splicing, was subject to an age- and tissue-dependent decrease in expression. In this study, we report that mice homozygous for this deletion are predisposed to dissection and rupture of the aorta during their adult life. Aortic dissection was not detected in autopsies of heterozygous animals or their littermate controls. Electron micrographs revealed fewer collagen fibrils and less compacted, irregular elastic lamellae in the aortic walls of homozygous mutant animals. Northern analysis of aortic RNA from 2.5- and 12-month-old homozygous mutant mice revealed that Col1a1 mRNA levels were decreased by 29% and 42%, respectively, relative to those of control littermates. In 12-month-old heterozygotes, the decrease was 32%. Allele-specific amplification of heterozygous cDNAs demonstrated that this reduction was limited to transcripts from the mutant allele. The collagen content of the aortas of homozygous mutant mice was also significantly lower in comparison to that of age-matched, control animals. These data establish that the integrity of the aortic wall depends on an adequate content of type I collagen, and that continued synthesis of collagen in the aorta as a function of age is critically dependent on sequences in the first intron of the Col1a1 gene.

Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina.

Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36-both expressed in AII amacrine cells-are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.

Selective impairment of hippocampal gamma oscillations in connexin-36 knock-out mouse in vivo.

The physiological roles of neuronal gap junctions in the intact brain are not known. The recent generation of the connexin-36 knock-out (Cx36 KO) mouse has offered a unique opportunity to examine this problem. Recent in vitro recordings in Cx36 KO mice suggested that Cx36 gap junction contributes to various oscillatory patterns in the theta (approximately 5-10 Hz) and gamma (approximately 30-80 Hz) frequency ranges and affects certain aspects of high-frequency (>100 Hz) patterns. However, the relevance of these pharmacologically induced patterns to the intact brain is not known. We recorded field potentials and unit activity in the CA1 stratum pyramidale of the hippocampus in the behaving wild-type (WT) and Cx36 KO mice. Fast-field "ripple" oscillations (140-200 Hz) were present in both WT and KO mice and did not differ significantly in power, intraepisode frequency, or probability of occurrence. Thus, fast-field oscillations either may not require electrical synapses or may be mediated by a hitherto unknown class of gap junctions. Theta oscillations, recorded during either wheel running or rapid eye movement sleep, were not different either. However, the power in the gamma frequency band and the magnitude of theta-phase modulation of gamma power were significantly decreased in KO mice compared with WT controls during wheel running. This suggests that Cx36 interneuronal gap junctions selectively contribute to gamma oscillations.

Electrical synapses: a dynamic signaling system that shapes the activity of neuronal networks.

Gap junctions consist of intercellular channels dedicated to providing a direct pathway for ionic and biochemical communication between contacting cells. After an initial burst of publications describing electrical coupling in the brain, gap junctions progressively became less fashionable among neurobiologists, as the consensus was that this form of synaptic transmission would play a minimal role in shaping neuronal activity in higher vertebrates. Several new findings over the last decade (e.g. the implication of connexins in genetic diseases of the nervous system, in processing sensory information and in synchronizing the activity of neuronal networks) have brought gap junctions back into the spotlight. The appearance of gap junctional coupling in the nervous system is developmentally regulated, restricted to distinct cell types and persists after the establishment of chemical synapses, thus suggesting that this form of cell-cell signaling may be functionally interrelated with, rather than alternative to chemical transmission. This review focuses on gap junctions between neurons and summarizes the available data, derived from molecular, biological, electrophysiological, and genetic approaches, that are contributing to a new appreciation of their role in brain function.

Connexin36 mediates gap junctional coupling of alpha-ganglion cells in mouse retina.

Alpha-ganglion cells are present in all vertebrate retinae and are subdivided into ON and OFF types according to their level of dendritic ramification within the inner plexiform layer. They have large dendritic fields and usually a good responsiveness to moving stimuli. They were the first ganglion cells in which tracer coupling was observed, suggesting the presence of gap junctions composed of unknown connexins. Here we show that ON-alpha-ganglion cells in the mouse retina are coupled to amacrine cells, whereas OFF-alpha-ganglion cells are coupled to other OFF-alpha-ganglion cells and to amacrine cells. These tracer coupling patterns were completely absent in mice deficient in connexin36 (Cx36). The expression of Cx36 protein in alpha-ganglion cells but not in coupled amacrine cells was confirmed in mice in which the Cx36 coding DNA was replaced by the lacZ reporter gene. The dendritic localization and the distribution pattern of Cx36 patches, analyzed in mice in which the enhanced green fluorescent protein (EGFP) was linked to the C-terminal region of the Cx36 protein, revealed a rather small number of fluorescent plaques and different patterns for ON- and OFF-alpha-ganglion cells. Furthermore, tracer coupling between OFF-alpha-ganglion cells could be inhibited by quinine, a gap junctional blocker with a slight preference for gap junctions formed by Cx36. These data strongly suggest that Cx36 gap junction channels are functional not only in interneurons but also in output neurons of the retina and are responsible for distinct coupling patterns of ganglion cells.

Pannexin1 and Pannexin2 expression in the developing and mature rat brain.

Recent studies have identified a new family of gap junction-forming proteins in vertebrates, called pannexins. Although their function in vivo is still not known, studies in Xenopus oocytes have indicated that pannexin1 (Px1) and pannexin2 (Px2) can form functional gap junction channels and can contribute to functional hemichannels. In this study, we have utilized a combination of radioactive and non-radioactive in situ hybridization experiments to characterize the expression pattern of the two pannexin genes during development and maturation of the rat brain. Expression analysis revealed a widespread and similar mRNA distribution for both genes, but indicated that Px1 and Px2 are inversely regulated during the development of the rat brain. Px1 is expressed at a high level in the embryonic and young postnatal brain and declines considerably in the adult, whereas Px2 mRNA is low in the prenatal brain but increases substantially during subsequent postnatal development. Immunohistochemical studies using different antibodies confirm the neuronal origin of pannexin-expressing cells and ascertain the presence of both pannexins in the majority of pyramidal cells and in GABAergic interneurons. The abundant presence of both pannexins in most neurons suggests that they may play a role in intercellular communication in many neuronal circuits. Furthermore, the temporal difference in the expression of the two genes indicates that the relative contribution of the two pannexins in immature and mature neuronal circuits may vary.

Could tuning of the inhibitory tone involve graded changes in neuronal chloride transport?

Hyperpolarizing synaptic inhibition through GABAA and glycine receptors depends on the presence of the neuronal cation-chloride-cotransporter protein, KCC2. Several transcriptional and post-transcriptional mechanisms have been shown to regulate KCC2 and thereby influence the polarity and efficacy of inhibitory synaptic transmission. It is unclear however whether regulation of KCC2 enables the transporter to attain different levels of activity thus allowing a neuron to modulate the strength of inhibitory synaptic transmission to its changing requirements. We therefore investigated whether phosphorylation can allow KCC2 to achieve distinct levels of [Cl(-)]i in neurons. We generated a variety of KCC2 alanine dephosphorylation mimics and used NH4(+)-induced pHi shifts in cultured hippocampal neurons to quantify the rate of KCC2 transport activity exhibited by these mutants. To explore the relationship between KCC2 transport and GABAA receptor-mediated current amplitudes we performed gramicidine perforated-patch recordings. The correlation between EGABA and NH4(+)-induced pHi shifts enabled an estimate of the range of chloride extrusion possible by kinase/phosphatase regulation of KCC2. Our results demonstrate that KCC2 transport can vary considerably in magnitude depending on the combination of alanine mutations present on the protein. Transport can be enhanced to sufficiently high levels that hyperpolarizing GABAA responses may be obtained even in neurons with an extremely negative resting membrane potential and at high extracellular K(+) concentrations. Our findings highlight the significant potential for regulating the inhibitory tone by KCC2-mediated chloride extrusion and suggest that cellular signaling pathways may act combinatorially to alter KCC2 phosphorylation/dephosphorylation and thereby tune the strength of synaptic inhibition.

Gap junction signalling is a stress-regulated component of adrenal neuroendocrine stimulus-secretion coupling in vivo.

Elucidating the mechanisms whereby neuroendocrine tissues coordinate their input and output signals to ensure appropriate hormone secretion is currently a topical issue. In particular, whether a direct communication mediated by gap junctions between neurosecretory cells contributes to hormone release in vivo still remains unknown. Here we address this issue using a microsurgical approach allowing combined monitoring of adrenal catecholamine secretion and splanchnic nerve stimulation in anaesthetised mice. Pharmacological blockade of adrenal gap junctions by the uncoupling agent carbenoxolone reduces nerve stimulation-evoked catecholamine release in control mice and to a larger extent in stressed mice. In parallel, the gap junction-coupled cell network is extended in stressed mice. Altogether, this argues for a significant contribution of adrenomedullary gap junctions to catecholamine secretion in vivo. As such, gap junctional signalling appears to be a substantial component for neuroendocrine function in the adrenal medulla, as it may represent an additional lever regulating hormone release.

Effects of electrically coupled inhibitory networks on local neuronal responses to intracortical microstimulation.

Using in vivo multielectrode electrophysiology in mice, we investigated the underpinnings of a local, long-lasting firing rate suppression evoked by intracortical microstimulation. Synaptic inhibition contributes to this suppression as it was reduced by pharmacological blockade of gamma-aminobutyric acid type B (GABAB) receptors. Blockade of GABAB receptors also abolished the known sublinear addition of inhibitory response duration after repetitive electrical stimulation. Furthermore, evoked inhibition was weaker and longer in connexin 36 knockout (KO) mice that feature decoupled cortical inhibitory networks. In supragranular layers of KO mice even an unusually long excitatory response (< or = 50 ms) appeared that was never observed in wild-type (WT) mice. Furthermore, the spread and duration of very fast oscillations (> 200 Hz) evoked by microstimulation at a short latency were strongly enhanced in KO mice. In the spatial domain, lack of connexin 36 unmasked a strong anisotropy of inhibitory spread. Although its reach along layers was almost the same as that in WT mice, the spread across cortical depth was severely hampered. In summary, the present data suggest that connexin 36-coupled networks significantly shape the electrically evoked cortical inhibitory response. Electrical coupling renders evoked cortical inhibition more precise and strong and ensures a uniform spread along the two cardinal axes of neocortical geometry.

A reporter allele for investigating connexin 26 gene expression in the mouse brain.

A variety of connexins are expressed in the diverse cell types of the central nervous system and are thought to regulate some of the functional properties exhibited by immature and mature cells. A proper understanding of the role of specific connexins in these processes requires an unambiguous characterization of their spatial and temporal pattern of expression. In order to define the cellular distribution of connexin 26 (Cx26) in the mouse we have generated a reporter allele (Cx26lacZ) by genetically manipulating the locus so that the beta-galactosidase (lacZ) gene is expressed from the endogenous Cx26 promoter. This modification decreased expression from the allele and resulted in embryonic lethality for the Cx26lacZ/lacZ genotype in accordance with previous studies on Cx26 knock-out animals indicating that Cx26-containing gap junctions are necessary for embryonic development. Despite the lower than expected transcript levels, the amount of lacZ protein produced in heterozygous mice was sufficient to label tissues known to contain Cx26, such as liver, kidney, skin, cochlea, small intestine, placenta and thyroid gland. In the embryonic and mature central nervous system, however, lacZ was restricted to meningeal cells and could not be detected in either neurons or glia. The absence of Cx26 mRNA in these cells could also be confirmed by reverse transcription-polymerase chain reaction and in situ hybridization. Our experiments indicate that the Cx26lacZ mouse line can be used as a reporter of Cx26 gene expression and suggest that Cx26, contrary to previous reports, is restricted to the meninges in both embryonic and adult brain.