Sheriar G. Hormuzdi

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. Here, we show that two of these genes, pannexin 1 (Px1) and Px2, are abundantly expressed in the CNS. In many neuronal cell populations, including hippocampus, olfactory bulb, cortex and cerebellum, there is coexpression of both pannexins, whereas in other brain regions, e.g., white matter, only Px1-positive cells were found. On expression in Xenopus oocytes, Px1, but not Px2 forms functional hemichannels. Coinjection of both pannexin RNAs results in hemichannels with functional properties that are different from those formed by Px1 only. In paired oocytes, Px1, alone and in combination with Px2, induces the formation of intercellular channels. The functional characteristics of homomeric Px1 versus heteromeric Px1/Px2 channels and the different expression patterns of Px1 and Px2 in the brain indicate that pannexins form cell type-specific gap junctions with distinct properties that may subserve different functions.

Impaired Electrical Signaling Disrupts Gamma Frequency Oscillations in Connexin 36-Deficient Mice

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.

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.

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. 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.

Contrasting roles of axonal (pyramidal cell) and dendritic (interneuron) electrical coupling in the generation of neuronal network oscillations

Electrical coupling between pyramidal cell axons, and between interneuron dendrites, have both been described in the hippocampus. What are the functional roles of the two types of coupling? Interneuron gap junctions enhance synchrony of γ oscillations (25–70 Hz) in isolated interneuron networks and also in networks containing both interneurons and principal cells, as shown in mice with a knockout of the neuronal (primarily interneuronal) connexin36. We have recently shown that pharmacological gap junction blockade abolishes kainate-induced γ oscillations in connexin36 knockout mice; without such gap junction blockade, γ oscillations do occur in the knockout mice, albeit at reduced power compared with wild-type mice. As interneuronal dendritic electrical coupling is almost absent in the knockout mice, these pharmacological data indicate a role of axonal electrical coupling in generating the γ oscillations. We construct a network model of an experimental γ oscillation, known to be regulated by both types of electrical coupling. In our model, axonal electrical coupling is required for the γ oscillation to occur at all; interneuron dendritic gap junctions exert a modulatory effect.

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 byβ-galactosidase (β-gal). In the outer nuclear layer,β-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36–EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36–EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2–GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.

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. J. Comp. Neurol. 485:191–201, 2005.

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 β‐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.