Neurexin1⍺ differentially regulates synaptic efficacy within striatal circuits.

Mutations in genes essential for synaptic function, such as the presynaptic adhesion molecule Neurexin1α (Nrxn1α), are strongly implicated in neuropsychiatric pathophysiology. As the input nucleus of the basal ganglia, the striatum integrates diverse excitatory projections governing cognitive and motor control, and its impairment may represent a recurrent pathway to disease. Here, we test the functional relevance of Nrxn1α in striatal circuits by employing optogenetic-mediated afferent recruitment of dorsal prefrontal cortical (dPFC) and parafascicular thalamic connections onto dorsomedial striatal (DMS) spiny projection neurons (SPNs). For dPFC-DMS circuits, we find decreased synaptic strength specifically onto indirect pathway SPNs in both Nrxn1α+/- and Nrxn1α-/- mice, driven by reductions in neurotransmitter release. In contrast, thalamic excitatory inputs to DMS exhibit relatively normal excitatory synaptic strength despite changes in synaptic N-methyl-D-aspartate receptor (NMDAR) content. These findings suggest that dysregulation of Nrxn1α modulates striatal function in an input- and target-specific manner.

Rewiring Endogenous Bioelectric Circuits in the Xenopus laevis Embryo Model.

Embryogenesis, as well as regeneration, is increasingly recognized to be orchestrated by an interplay of transcriptional and bioelectric networks. Spatiotemporal patterns of resting potentials direct the size, shape, and locations of numerous organ primordia during patterning. These bioelectrical properties are established by the function of ion channels and pumps that set voltage potentials of individual cells, and gap junctions (electrical synapses) that enable physiological states to propagate across tissue networks. Functional experiments to probe the roles of bioelectrical states can be carried out by targeting endogenous ion channels during development. Here, we describe protocols, optimized for the highly tractable Xenopus laevis embryo, for molecular genetic targeting of ion channels and connexins based on CRISPR, and monitoring of resting potential states using voltage-sensing fluorescent dye. Similar strategies can be adapted to other model species.

INX-18 and INX-19 play distinct roles in electrical synapses that modulate aversive behavior in Caenorhabditis elegans.

In order to respond to changing environments and fluctuations in internal states, animals adjust their behavior through diverse neuromodulatory mechanisms. In this study we show that electrical synapses between the ASH primary quinine-detecting sensory neurons and the neighboring ASK neurons are required for modulating the aversive response to the bitter tastant quinine in C. elegans. Mutant worms that lack the electrical synapse proteins INX-18 and INX-19 become hypersensitive to dilute quinine. Cell-specific rescue experiments indicate that inx-18 operates in ASK while inx-19 is required in both ASK and ASH for proper quinine sensitivity. Imaging analyses find that INX-19 in ASK and ASH localizes to the same regions in the nerve ring, suggesting that both sides of ASK-ASH electrical synapses contain INX-19. While inx-18 and inx-19 mutant animals have a similar behavioral phenotype, several lines of evidence suggest the proteins encoded by these genes play different roles in modulating the aversive quinine response. First, INX-18 and INX-19 localize to different regions of the nerve ring, indicating that they are not present in the same synapses. Second, removing inx-18 disrupts the distribution of INX-19, while removing inx-19 does not alter INX-18 localization. Finally, by using a fluorescent cGMP reporter, we find that INX-18 and INX-19 have distinct roles in establishing cGMP levels in ASK and ASH. Together, these results demonstrate that electrical synapses containing INX-18 and INX-19 facilitate modulation of ASH nociceptive signaling. Our findings support the idea that a network of electrical synapses mediates cGMP exchange between neurons, enabling modulation of sensory responses and behavior.

Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses.

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B-microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

Acute control of the sleep switch in Drosophila reveals a role for gap junctions in regulating behavioral responsiveness.

Sleep is a dynamic process in most animals, involving distinct stages that probably perform multiple functions for the brain. Before sleep functions can be initiated, it is likely that behavioral responsiveness to the outside world needs to be reduced, even while the animal is still awake. Recent work in Drosophila has uncovered a sleep switch in the dorsal fan-shaped body (dFB) of the fly's central brain, but it is not known whether these sleep-promoting neurons also govern the acute need to ignore salient stimuli in the environment during sleep transitions. We found that optogenetic activation of the sleep switch suppressed behavioral responsiveness to mechanical stimuli, even in awake flies, indicating a broader role for these neurons in regulating arousal. The dFB-mediated suppression mechanism and its associated neural correlates requires innexin6 expression, suggesting that the acute need to reduce sensory perception when flies fall asleep is mediated in part by electrical synapses.

Gap junctional coupling between retinal amacrine and ganglion cells underlies coherent activity integral to global object perception.

Coherent spike activity occurs between widely separated retinal ganglion cells (RGCs) in response to a large, contiguous object, but not to disjointed objects. Since the large spatial separation between the RGCs precludes common excitatory inputs from bipolar cells, the mechanism underlying this long-range coherence remains unclear. Here, we show that electrical coupling between RGCs and polyaxonal amacrine cells in mouse retina forms the synaptic mechanism responsible for long-range coherent activity in the retina. Pharmacological blockade of gap junctions or genetic ablation of connexin 36 (Cx36) subunits eliminates the long-range correlated spiking between RGCs. Moreover, we find that blockade of gap junctions or ablation of Cx36 significantly reduces the ability of mice to discriminate large, global objects from small, disjointed stimuli. Our results indicate that synchronous activity of RGCs, derived from electrical coupling with amacrine cells, encodes information critical to global object perception.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans.

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9-containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9-based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits.

DE NOVO MUTATIONS IN AUTISM IMPLICATE THE SYNAPTIC ELIMINATION NETWORK.

Autism has been shown to have a major genetic risk component; the architecture of documented autism in families has been over and again shown to be passed down for generations. While inherited risk plays an important role in the autistic nature of children, de novo (germline) mutations have also been implicated in autism risk. Here we find that autism de novo variants verified and published in the literature are Bonferroni-significantly enriched in a gene set implicated in synaptic elimination. Additionally, several of the genes in this synaptic elimination set that were enriched in protein-protein interactions (CACNA1C, SHANK2, SYNGAP1, NLGN3, NRXN1, and PTEN) have been previously confirmed as genes that confer risk for the disorder. The results demonstrate that autism-associated de novos are linked to proper synaptic pruning and density, hinting at the etiology of autism and suggesting pathophysiology for downstream correction and treatment.

Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila melanogaster. The purpose of this study was to identify which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakB(N+16) isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. However, the identity of the innexin that forms the presynaptic hemichannels in the JONs remains unknown. We used electrophysiology, immunocytochemistry and dye injection, along with presynaptically-driven RNA interference, to investigate this question. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakB(L) or ShakB(N) isoforms alone did not reduce the synaptic strength, implying that it is ShakB(N+16) that is required in the presynaptic neurons. Overexpression of ShakB(N+16) in JONs caused the formation of ectopic dye coupling, whereas ShakB(N) prevented it altogether, supporting this conclusion and also suggesting that gap junction proteins may have an instructive role in synaptic target choice.

Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Neuronal target identification requires AHA-1-mediated fine-tuning of Wnt signaling in C. elegans.

Electrical synaptic transmission through gap junctions is a vital mode of intercellular communication in the nervous system. The mechanism by which reciprocal target cells find each other during the formation of gap junctions, however, is poorly understood. Here we show that gap junctions are formed between BDU interneurons and PLM mechanoreceptors in C. elegans and the connectivity of BDU with PLM is influenced by Wnt signaling. We further identified two PAS-bHLH family transcription factors, AHA-1 and AHR-1, which function cell-autonomously within BDU and PLM to facilitate the target identification process. aha-1 and ahr-1 act genetically upstream of cam-1. CAM-1, a membrane-bound receptor tyrosine kinase, is present on both BDU and PLM cells and likely serves as a Wnt antagonist. By binding to a cis-regulatory element in the cam-1 promoter, AHA-1 enhances cam-1 transcription. Our study reveals a Wnt-dependent fine-tuning mechanism that is crucial for mutual target cell identification during the formation of gap junction connections.

Anhedonia requires MC4R-mediated synaptic adaptations in nucleus accumbens.

Chronic stress is a strong diathesis for depression in humans and is used to generate animal models of depression. It commonly leads to several major symptoms of depression, including dysregulated feeding behaviour, anhedonia and behavioural despair. Although hypotheses defining the neural pathophysiology of depression have been proposed, the critical synaptic adaptations in key brain circuits that mediate stress-induced depressive symptoms remain poorly understood. Here we show that chronic stress in mice decreases the strength of excitatory synapses on D1 dopamine receptor-expressing nucleus accumbens medium spiny neurons owing to activation of the melanocortin 4 receptor. Stress-elicited increases in behavioural measurements of anhedonia, but not increases in measurements of behavioural despair, are prevented by blocking these melanocortin 4 receptor-mediated synaptic changes in vivo. These results establish that stress-elicited anhedonia requires a neuropeptide-triggered, cell-type-specific synaptic adaptation in the nucleus accumbens and that distinct circuit adaptations mediate other major symptoms of stress-elicited depression.

New perspectives on the biology of fragile X syndrome.

Fragile X syndrome (FXS) is a trinucleotide repeat disorder caused by a CGG repeat expansion in FMR1, and loss of its protein product FMRP. Recent studies have provided increased support for the role of FMRP in translational repression via ribosomal stalling and the microRNA pathway. In neurons, particular focus has been placed on identifying the signaling pathways such as PI3K and mTOR downstream of group 1 metabotropic glutamate receptors (mGluR1/5) that regulate FMRP. New evidence also suggests that loss of FMRP causes presynaptic dysfunction and abnormal adult neurogenesis. In addition, studies on FXS stem cells especially induced pluripotent stem (iPS) cells and new sequencing efforts hold out promise for deeper understanding of the silencing process and mutation spectrum of FMR1.

Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses.

Collybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA(A) receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2(SH3+) and CB2(SH3-), and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2(SH3+) and CB2(SH3-) into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2(SH3-), but not CB2(SH3+), can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.

Synaptogenesis of electrical and GABAergic synapses of fast-spiking inhibitory neurons in the neocortex.

Parvalbumin-expressing fast-spiking (FS) cells are interconnected via GABAergic and electrical synapses and represent a major class of inhibitory interneurons in the neocortex. Synaptic connections among FS cells are critical for regulating network oscillations in the mature neocortex. However, it is unclear whether synaptic connections among FS interneurons also play a central role in the generation of patterned neuronal activity in the immature brain, which is thought to underlie the formation of neocortical circuits. Here, we investigated the developmental time course of synaptogenesis of FS cell in mouse visual cortex. In layer 5/6 (L5/6), we recorded from two or three FS and/or pyramidal (PYR) neurons to study the development of electrical and chemical synaptic interactions from postnatal day 3 (P3) to P18. We detected no evidence for functional connectivity for FS-FS or FS-PYR pairs at P3-P4. However, by P5-P6, we found that 20% of FS pairs were electrically coupled, and 24% of pairs were connected via GABAergic synapses; by P15-P18, 42% of FS pairs had established functional electrical synapses, and 47% of FS pairs were connected via GABAergic synapses. FS cell GABAergic inhibition of pyramidal cells showed a similar developmental time line, but no electrical coupling was detected for FS-PYR pairs. We found that synaptogenesis of electrical and GABAergic connections of FS cells takes place in the same period. Together, our results suggest that chemical and electrical connections among FS cells can contribute to patterned neocortical activity only by the end of the first postnatal week.

A novel mutation of gap junction protein ß 1 gene in X-linked Charcot-Marie-Tooth disease.

INTRODUCTION: In this study we report a novel mutation in the gap junction protein beta 1 (GJB1) gene of a Chinese X-linked Charcot-Marie-Tooth disease (CMTX1) family, which has specific electrophysiological characteristics. METHODS: Twenty members in the family were studied by clinical neurological examination and GJB1 gene mutation analysis, and 3 patients were studied electrophysiologically. The proband and his mother also underwent sural nerve biopsy. RESULTS: All patients have the CMT phenotype, except for 2 asymptomatic carriers. Electrophysiological examinations showed non-uniform slowing of motor conduction velocities and partial motor conduction blocks and temporal dispersion. Sural nerve biopsy confirmed a predominantly demyelinating neuropathy, and an Asn2Lys mutation in the amino-terminal domain was found in 9 members of this family, but not in 25 normal controls in the family. CONCLUSIONS: This family represents a novel mutation in the GJB1 form of CMTX1. The mutation in the amino-terminus has an impact on the electrophysiological characteristics of the disease.

A novel pathway regulates memory and plasticity via SIRT1 and miR-134.

The NAD-dependent deacetylase Sir2 was initially identified as a mediator of replicative lifespan in budding yeast and was subsequently shown to modulate longevity in worms and flies. Its mammalian homologue, SIRT1, seems to have evolved complex systemic roles in cardiac function, DNA repair and genomic stability. Recent studies suggest a functional relevance of SIRT1 in normal brain physiology and neurological disorders. However, it is unknown if SIRT1 has a role in higher-order brain functions. We report that SIRT1 modulates synaptic plasticity and memory formation via a microRNA-mediated mechanism. Activation of SIRT1 enhances, whereas its loss-of-function impairs, synaptic plasticity. Surprisingly, these effects were mediated via post-transcriptional regulation of cAMP response binding protein (CREB) expression by a brain-specific microRNA, miR-134. SIRT1 normally functions to limit expression of miR-134 via a repressor complex containing the transcription factor YY1, and unchecked miR-134 expression following SIRT1 deficiency results in the downregulated expression of CREB and brain-derived neurotrophic factor (BDNF), thereby impairing synaptic plasticity. These findings demonstrate a new role for SIRT1 in cognition and a previously unknown microRNA-based mechanism by which SIRT1 regulates these processes. Furthermore, these results describe a separate branch of SIRT1 signalling, in which SIRT1 has a direct role in regulating normal brain function in a manner that is disparate from its cell survival functions, demonstrating its value as a potential therapeutic target for the treatment of central nervous system disorders.

Angelman syndrome: current understanding and research prospects.

Angelman syndrome is a neurogenetic disorder characterized by developmental delay, severe intellectual disability, absent speech, exuberant behavior with happy demeanor, motor impairment, and epilepsy, due to deficient UBE3A gene expression that may be caused by various abnormalities of chromosome 15. Recent findings in animal models demonstrated altered dendritic spine formation as well as both synaptic [including gamma-aminobutyric acid (GABA)(A) and N-methyl-D-aspartate (NMDA) transmission] and nonsynaptic (including gap junction) influences in various brain regions, including hippocampus and cerebellar cortex. Reversal of selected abnormalities in rescue genetically engineered animal models is encouraging, although it should not be misinterpreted as promising "cure" for affected patients. Much research is still required to fully understand the functional links between lack of UBE3A expression and clinical manifestations of Angelman syndrome. Studies of regulation of UBE3A expression, including imprinting-related methylation, may point to possibilities of therapeutic upregulation. Understanding relevant roles of the gene product might lead to targeted intervention. Further documentation of brain network dynamics, with particular emphasis on hippocampus, thalamocortical, and cerebellar networks is needed, including in a developmental perspective. There is also a need for further clinical research for improving management of problems such as epilepsy, behavior, communication, learning, motor impairment, and sleep disturbances.

Interactions between innexins UNC-7 and UNC-9 mediate electrical synapse specificity in the Caenorhabditis elegans locomotory nervous system.

BACKGROUND: Approximately 10% of Caenorhabditis elegans nervous system synapses are electrical, that is, gap junctions composed of innexins. The locomotory nervous system consists of several pairs of interneurons and three major classes of motor neurons, all with stereotypical patterns of connectivity that include gap junctions. Mutations in the two innexin genes unc-7 and unc-9 result in identical uncoordinated movement phenotypes, and their respective gene products were investigated for their contribution to electrical synapse connectivity. RESULTS: unc-7 encodes three innexin isoforms. Two of these, UNC-7S and UNC-7SR, are functionally equivalent and play an essential role in coordinated locomotion. UNC-7S and UNC-7SR are widely expressed and co-localize extensively with green fluorescent protein-tagged innexin UNC-9 in the ventral and dorsal nerve cords. A subset of UNC-7S/SR expression visualizes gap junctions formed between the AVB forward command interneurons and their B class motor neuron partners. Experiments indicate that expression of UNC-7S/SR in AVB and expression of UNC-9 in B motor neurons is necessary for these gap junctions to form. In Xenopus oocyte pairs, both UNC-7S and UNC-9 form homomeric gap junctions, and together they form heterotypic channels. Xenopus oocyte studies and co-localization studies in C. elegans suggest that UNC-7S and UNC-9 do not heteromerize in the same hemichannel, leading to the model that hemichannels in AVB:B motor neuron gap junctions are homomeric and heterotypic. CONCLUSION: UNC-7S and UNC-9 are widely expressed and contribute to a large number of the gap junctions identified in the locomotory nervous system. Proper AVB:B gap junction formation requires UNC-7S expression in AVB interneurons and UNC-9 expression in B motor neurons. More broadly, this illustrates that innexin identity is critical for electrical synapse specificity, but differential (compartmentalized) innexin expression cannot account for all of the specificity seen in C. elegans, and other factors must influence the determination of synaptic partners.

The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptors.

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of (35)S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.