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

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca/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 phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown.

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).

Differential Distribution of Retinal Ca/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-β 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.

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.

Neuronal chloride transport tuning.

Background: Epilepsy is characterised by disturbed neuronal activity in the brain rendering it more susceptible to seizures. An understanding of the molecular mechanisms by which the balance between excitability and inhibition in neuronal networks is controlled will help to devise better treatment options. Hyperpolarising synaptic inhibition through GABAA (γ aminobutyric acid type A) and glycine receptors depends on the presence of the neuronal cation-chloride-cotransporter protein, KCC2.

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.

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.

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.

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-1β 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.

In vivo evidence for the involvement of the carboxy terminal domain in assembling connexin 36 at the electrical synapse.

Connexin 36 (Cx36)-containing electrical synapses contribute to the timing and amplitude of neural responses in many brain regions. A Cx36-EGFP transgenic was previously generated to facilitate their identification and study. In this study we demonstrate that electrical coupling is normal in transgenic mice expressing Cx36 from the genomic locus and suggest that fluorescent puncta present in brain tissue represent distributed electrical synapses.

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.

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.

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.

Intracellular acidification in neurons induced by ammonium depends on KCC2 function.

The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons.

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.

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.

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.

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.

Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina.

Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced bybeta-galactosidase (beta-gal). In the outer nuclear layer,beta-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin.

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.

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.

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.

Pannexins, a family of gap junction proteins expressed in brain.

Database search has led to the identification of a family of proteins, the pannexins, which share some structural features with the gap junction forming proteins of invertebrates and vertebrates. The function of these proteins has remained unclear so far. To test the possibility that pannexins underlie electrical communication in the brain, we have investigated their tissue distribution and functional properties.

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.

Sharp wave-like activity in the hippocampus in vitro in mice lacking the gap junction protein connexin 36.

Bath application of kainate (100-300 nM) induced a persistent gamma-frequency (30-80 Hz) oscillation that could be recorded in stratum radiatum of the CA3 region in vitro. We have previously described that in knockout mice lacking the gap junction protein connexin 36 (Cx36KO), gamma-frequency oscillations are reduced but still present. We now demonstrate that in the Cx36KO mice, but not in wild-type (WT), large population field excitatory postsynaptic potentials, or sharp wave-burst discharges, also occurred during the on-going gamma-frequency oscillation.