Soticlestat, a novel cholesterol 24-hydroxylase inhibitor, reduces seizures and premature death in Dravet syndrome mice.

OBJECTIVE: Dravet syndrome is a severe developmental and epileptic encephalopathy (DEE) most often caused by de novo pathogenic variants in SCN1A. Individuals with Dravet syndrome rarely achieve seizure control and have significantly elevated risk for sudden unexplained death in epilepsy (SUDEP). Heterozygous deletion of Scn1a in mice (Scn1a+/- ) recapitulates several core phenotypes, including temperature-dependent and spontaneous seizures, SUDEP, and behavioral abnormalities. Furthermore, Scn1a+/- mice exhibit a similar clinical response to standard anticonvulsants. Cholesterol 24-hydroxlase (CH24H) is a brain-specific enzyme responsible for cholesterol catabolism. Recent research has indicated the therapeutic potential of CH24H inhibition for diseases associated with neural excitation, including seizures. METHODS: In this study, the novel compound soticlestat, a CH24H inhibitor, was administered to Scn1a+/- mice to investigate its ability to improve Dravet-like phenotypes in this preclinical model. RESULTS: Soticlestat treatment reduced seizure burden, protected against hyperthermia-induced seizures, and completely prevented SUDEP in Scn1a+/- mice. Video-electroencephalography (EEG) analysis confirmed the ability of soticlestat to reduce occurrence of electroclinical seizures. SIGNIFICANCE: This study demonstrates that soticlestat-mediated inhibition of CH24H provides therapeutic benefit for the treatment of Dravet syndrome in mice and has the potential for treatment of DEEs.

CRISPR/dCas9-based Scn1a gene activation in inhibitory neurons ameliorates epileptic and behavioral phenotypes of Dravet syndrome model mice.

Dravet syndrome is a severe infantile-onset epileptic encephalopathy which begins with febrile seizures and is caused by heterozygous loss-of-function mutations of the voltage-gated sodium channel gene SCN1A. We designed a CRISPR-based gene therapy for Scn1a-haplodeficient mice using multiple guide RNAs (gRNAs) in the promoter regions together with the nuclease-deficient Cas9 fused to transcription activators (dCas9-VPR) to trigger the transcription of SCN1A or Scn1a in vitro. We tested the effect of this strategy in vivo using an adeno-associated virus (AAV) mediated system targeting inhibitory neurons and investigating febrile seizures and behavioral parameters. In both the human and mouse genes multiple guide RNAs (gRNAs) in the upstream, rather than downstream, promoter region showed high and synergistic activities to increase the transcription of SCN1A or Scn1a in cultured cells. Intravenous injections of AAV particles containing the optimal combination of 4 gRNAs into transgenic mice with Scn1a-haplodeficiency and inhibitory neuron-specific expression of dCas9-VPR at four weeks of age increased Nav1.1 expression in parvalbumin-positive GABAergic neurons, ameliorated their febrile seizures and improved their behavioral impairments. Although the usage of transgenic mice and rather modest improvements in seizures and abnormal behaviors hamper direct clinical application, our results indicate that the upregulation of Scn1a expression in the inhibitory neurons can significantly improve the phenotypes, even when applied after the juvenile stages. Our findings also suggest that the decrease in Nav1.1 is directly involved in the symptoms seen in adults with Dravet syndrome and open a way to improve this condition.

Potentiation of excitatory synaptic transmission ameliorates aggression in mice with Stxbp1 haploinsufficiency.

Genetic studies point to a major role of de novo mutations in neurodevelopmental disorders of intellectual disability, autism spectrum disorders, and epileptic encephalopathy. The STXBP1 gene encodes the syntaxin-binding protein 1 (Munc18-1) that critically controls synaptic vesicle exocytosis and synaptic transmission. This gene harbors a high frequency of de novo mutations, which may play roles in these neurodevelopmental disorders. However, the system and behavioral-level pathophysiological changes caused by these genetic defects remain poorly understood. Constitutional (Stxbp1+/-), dorsal-telencephalic excitatory (Stxbp1fl/+/Emx), or global inhibitory neuron-specific (Stxbp1fl/+/Vgat) mice were subjected to a behavioral test battery examining locomotor activity, anxiety, fear learning, and social interactions including aggression. Furthermore, measurements of local field potentials in multiple regions of the brain were performed. Stxbp1+/- male mice exhibited enhanced aggressiveness and impaired fear learning associated with elevated gamma activity in several regions of the brain including the prefrontal cortex. Stxbp1fl/+/Emx mice showed fear-learning deficits, but neither Stxbp1fl/+/Emx nor Stxbp1fl/+/Vgat mice showed increased aggressiveness. Pharmacological potentiation of the excitatory transmission at active synapses via the systemic administration of ampakine CX516, which enhances the excitatory postsynaptic function, ameliorated the aggressive phenotype of Stxbp1+/- mice. These findings suggest that synaptic impairments of the dorsal telencephalic and subcortical excitatory neurons cause learning deficits and enhanced aggression in Stxbp1+/- mice, respectively. Additionally, normalizing the excitatory synaptic transmission is a potential therapeutic option for managing aggressiveness in patients with STXBP1 mutations.

Impairments in social novelty recognition and spatial memory in mice with conditional deletion of Scn1a in parvalbumin-expressing cells.

Loss of function mutations in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1, have been described in the majority of Dravet syndrome patients presenting with epileptic seizures, hyperactivity, autistic traits, and cognitive decline. We previously reported predominant Nav1.1 expression in parvalbumin-expressing (PV+) inhibitory neurons in juvenile mouse brain and observed epileptic seizures in mice with selective deletion of Scn1a in PV+ cells mediated by PV-Cre transgene expression (Scn1afl/+/PV-Cre-TG). Here we investigate the behavior of Scn1afl/+/PV-Cre-TG mice using a comprehensive battery of behavioral tests. We observed that Scn1afl/+/PV-Cre-TG mice display hyperactive behavior, impaired social novelty recognition, and altered spatial memory. We also generated Scn1afl/+/SST-Cre-KI mice with a selective Scn1a deletion in somatostatin-expressing (SST+) inhibitory neurons using an SST-IRES-Cre knock-in driver line. We observed that Scn1afl/+/SST-Cre-KI mice display no spontaneous convulsive seizures and that Scn1afl/+/SST-Cre-KI mice have a lowered threshold temperature for hyperthermia-induced seizures, although their threshold values are much higher than those of Scn1afl/+/PV-Cre-TG mice. We finally show that Scn1afl/+/SST-Cre-KI mice exhibited no noticeable behavioral abnormalities. These observations suggest that impaired Nav1.1 function in PV+ interneurons is critically involved in the pathogenesis of hyperactivity, autistic traits, and cognitive decline, as well as epileptic seizures, in Dravet syndrome.

Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Heterotrimeric guanosine triphosphate-binding protein-coupled modulatory actions of motilin on K+ channels and postsynaptic γ-aminobutyric acid receptors in mouse medial vestibular nuclear neurons.

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice-patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti-motilin receptor antibody. Bath application of motilin (0.1 µm) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose-dependent manner (K(d) , 0.03 µm). The motilin action on spontaneous action potentials was blocked by apamin (100 nm), a blocker of small-conductance Ca(2+) -activated K(+) channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 µg/µL) or guanosine triphosphate-γ-S (1 mm) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild-type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ-aminobutyric acid transporter-Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small-conductance Ca(2+) -activated K(+) channels and postsynaptic γ-aminobutyric acid receptors through heterotrimeric guanosine triphosphate-binding protein-coupled receptor in a group of glutamatergic MVNs.

Lipid signaling in cytosolic phospholipase A2alpha-cyclooxygenase-2 cascade mediates cerebellar long-term depression and motor learning.

In this study, we show the crucial roles of lipid signaling in long-term depression (LTD), that is, synaptic plasticity prevailing in cerebellar Purkinje cells. In mouse brain slices, we found that cPLA(2)alpha knockout blocked LTD induction, which was rescued by replenishing arachidonic acid (AA) or prostaglandin (PG) D(2) or E(2). Moreover, cyclooxygenase (COX)-2 inhibitors block LTD, which is rescued by supplementing PGD(2)/E(2). The blockade or rescue occurs when these reagents are applied within a time window of 5-15 min following the onset of LTD-inducing stimulation. Furthermore, PGD(2)/E(2) facilitates the chemical induction of LTD by a PKC activator but is unable to rescue the LTD blocked by a PKC inhibitor. We conclude that PGD(2)/E(2) mediates LTD jointly with PKC, and suggest possible pathways for their interaction. Finally, we demonstrate in awake mice that cPLA(2)alpha deficiency or COX-2 inhibition attenuates short-term adaptation of optokinetic eye movements, supporting the view that LTD underlies motor learning.

Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons.

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

CUX2 deficiency causes facilitation of excitatory synaptic transmission onto hippocampus and increased seizure susceptibility to kainate.

CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

A recurrent PJA1 variant in trigonocephaly and neurodevelopmental disorders.

OBJECTIVE: Neurodevelopmental disorders (NDDs) often associate with epilepsy or craniofacial malformations. Recent large-scale DNA analyses identified hundreds of candidate genes for NDDs, but a large portion of the cases still remain unexplained. We aimed to identify novel candidate genes for NDDs. METHODS: We performed exome sequencing of 95 patients with NDDs including 51 with trigonocephaly and subsequent targeted sequencing of additional 463 NDD patients, functional analyses of variant in vitro, and evaluations of autism spectrum disorder (ASD)-like phenotypes and seizure-related phenotypes in vivo. RESULTS: We identified de novo truncation variants in nine novel genes; CYP1A1, C14orf119, FLI1, CYB5R4, SEL1L2, RAB11FIP2, ZMYND8, ZNF143, and MSX2. MSX2 variants have been described in patients with cranial malformations, and our present patient with the MSX2 de novo truncation variant showed cranial meningocele and partial epilepsy. MSX2 protein is known to be ubiquitinated by an E3 ubiquitin ligase PJA1, and interestingly we found a PJA1 hemizygous p.Arg376Cys variant recurrently in seven Japanese NDD patients; five with trigonocephaly and one with partial epilepsy, and the variant was absent in 886 Japanese control individuals. Pja1 knock-in mice carrying p.Arg365Cys, which is equivalent to p.Arg376Cys in human, showed a significant decrease in PJA1 protein amount, suggesting a loss-of-function effect of the variant. Pja1 knockout mice displayed moderate deficits in isolation-induced ultrasonic vocalizations and increased seizure susceptibility to pentylenetetrazole. INTERPRETATION: These findings propose novel candidate genes including PJA1 and MSX2 for NDDs associated with craniofacial abnormalities and/or epilepsy.

Scn2a haploinsufficient mice display a spectrum of phenotypes affecting anxiety, sociability, memory flexibility and ampakine CX516 rescues their hyperactivity.

Background: Mutations of the SCN2A gene encoding a voltage-gated sodium channel alpha-II subunit Nav1.2 are associated with neurological disorders such as epilepsy, autism spectrum disorders, intellectual disability, and schizophrenia. However, causal relationships and pathogenic mechanisms underlying these neurological defects, especially social and psychiatric features, remain to be elucidated. Methods: We investigated the behavior of mice with a conventional or conditional deletion of Scn2a in a comprehensive test battery including open field, elevated plus maze, light-dark box, three chambers, social dominance tube, resident-intruder, ultrasonic vocalization, and fear conditioning tests. We further monitored the effects of the positive allosteric modulator of AMPA receptors CX516 on these model mice. Results: Conventional heterozygous Scn2a knockout mice (Scn2aKO/+) displayed novelty-induced exploratory hyperactivity and increased rearing. The increased vertical activity was reproduced by heterozygous inactivation of Scn2a in dorsal-telencephalic excitatory neurons but not in inhibitory neurons. Moreover, these phenotypes were rescued by treating Scn2aKO/+ mice with CX516. Additionally, Scn2aKO/+ mice displayed mild social behavior impairment, enhanced fear conditioning, and deficient fear extinction. Neuronal activity was intensified in the medial prefrontal cortex of Scn2aKO/+ mice, with an increase in the gamma band. Conclusions: Scn2aKO/+ mice exhibit a spectrum of phenotypes commonly observed in models of schizophrenia and autism spectrum disorder. Treatment with the CX516 ampakine, which ameliorates hyperactivity in these mice, could be a potential therapeutic strategy to rescue some of the disease phenotypes.

Impaired cortico-striatal excitatory transmission triggers epilepsy.

STXBP1 and SCN2A gene mutations are observed in patients with epilepsies, although the circuit basis remains elusive. Here, we show that mice with haplodeficiency for these genes exhibit absence seizures with spike-and-wave discharges (SWDs) initiated by reduced cortical excitatory transmission into the striatum. Mice deficient for Stxbp1 or Scn2a in cortico-striatal but not cortico-thalamic neurons reproduce SWDs. In Stxbp1 haplodeficient mice, there is a reduction in excitatory transmission from the neocortex to striatal fast-spiking interneurons (FSIs). FSI activity transiently decreases at SWD onset, and pharmacological potentiation of AMPA receptors in the striatum but not in the thalamus suppresses SWDs. Furthermore, in wild-type mice, pharmacological inhibition of cortico-striatal FSI excitatory transmission triggers absence and convulsive seizures in a dose-dependent manner. These findings suggest that impaired cortico-striatal excitatory transmission is a plausible mechanism that triggers epilepsy in Stxbp1 and Scn2a haplodeficient mice.

Loss of GPRC5B impairs synapse formation of Purkinje cells with cerebellar nuclear neurons and disrupts cerebellar synaptic plasticity and motor learning.

GPRC5B is a membrane glycoprotein robustly expressed in mouse cerebellar Purkinje cells (PCs). Its function is unknown. In Gprc5b-/- mice that lack GPRC5B, PCs develop distal axonal swellings in deep cerebellar nuclei (DCN). Numerous misshapen mitochondria, which generated excessive amounts of reactive oxygen species (ROS), accumulated in these distal axonal swellings. In primary cell cultures of Gprc5b-/- PCs, pharmacological reduction of ROS prevented the appearance of such swellings. To examine the physiological role of GPRC5B in PCs, we analyzed cerebellar synaptic transmission and cerebellum-dependent motor learning in Gprc5b-/- mice. Patch-clamp recordings in cerebellum slices in vitro revealed that the induction of long-term depression (LTD) at parallel fiber-PC synapses was normal in adult Gprc5b-/- mice, whereas the induction of long-term potentiation (LTP) at mossy fiber-DCN neuron synapses was attenuated in juvenile Gprc5b-/- mice. In Gprc5b-/- mice, long-term motor learning was impaired in both the rotarod test and the horizontal optokinetic response eye movement (HOKR) test. These observations suggest that GPRC5B plays not only an important role in the development of distal axons of PCs and formation of synapses with DCN neurons, but also in the synaptic plasticity that underlies long-term motor learning.

Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice.

Mutations in the SCN2A gene encoding a voltage-gated sodium channel Nav1.2 are associated with epilepsies, intellectual disability, and autism. SCN2A gain-of-function mutations cause early-onset severe epilepsies, while loss-of-function mutations cause autism with milder and/or later-onset epilepsies. Here we show that both heterozygous Scn2a-knockout and knock-in mice harboring a patient-derived nonsense mutation exhibit ethosuximide-sensitive absence-like seizures associated with spike-and-wave discharges at adult stages. Unexpectedly, identical seizures are reproduced and even more prominent in mice with heterozygous Scn2a deletion specifically in dorsal-telencephalic (e.g., neocortical and hippocampal) excitatory neurons, but are undetected in mice with selective Scn2a deletion in inhibitory neurons. In adult cerebral cortex of wild-type mice, most Nav1.2 is expressed in excitatory neurons with a steady increase and redistribution from proximal (i.e., axon initial segments) to distal axons. These results indicate a pivotal role of Nav1.2 haplodeficiency in excitatory neurons in epilepsies of patients with SCN2A loss-of-function mutations.

Involvement of basal protein kinase C and extracellular signal-regulated kinase 1/2 activities in constitutive internalization of AMPA receptors in cerebellar Purkinje cells.

AMPA receptor (AMPAR) internalization provides a mechanism for long-term depression (LTD) in both hippocampal pyramidal neurons and cerebellar Purkinje cells (PCs). Cerebellar LTD at the parallel fiber (PF)-PC synapse is the underlying basis of motor learning and requires AMPAR activation, a large Ca2+ influx, and protein kinase C (PKC) activation. However, whether these requirements affect the constitutive AMPAR internalization in PF-PC synapses remains unclarified. Tetanus toxin (TeTx) infusion into PCs decreased PF-EPSC amplitude to 60% within 20-30 min (TeTx rundown), without change in paired-pulse facilitation ratio or receptor kinetics. Immunocytochemically measured glutamate receptor 2 (GluR2) internalization ratio decreased at the steady state of TeTx rundown. TeTx rundown did not require AMPAR activity nor an increase in intracellular Ca2+ concentration. TeTx rundown was suppressed partially by the inhibition of either conventional PKC or mitogen-activated protein kinase kinase (MEK) and completely by the inhibition of both kinases. The background PKC activity was shown to be sufficient, because a PKC activator did not facilitate TeTx rundown. The inhibition of protein phosphatase 1/2A (PP1/2A) enhanced TeTx rundown slightly, and both inhibition of PP1/2A and activation of PKC maximized it, but one-half of AMPARs at PF-PC synapses remained in the TeTx-resistant pool. The inhibition of actin depolymerization suppressed TeTx rundown and decreased the GluR2 internalization ratio. In contrast, the inhibition of actin polymerization enhanced TeTx rundown and increased the GluR2 internalization ratio. We suggest that the regulation of actin polymerization is involved in the surface expression of AMPARs and the surface expressing AMPARs are constitutively internalized through both basal PKC and MEK-ERK1/2 (extracellular signal-regulated kinase 1/2) activities at PF-PC synapses.

Endogenous zinc can be a modulator of glycinergic signaling pathway in the rat retina.

Zinc is a modulator of glutamatergic inputs in the hippocampus. In the retina, however, we previously reported that endogenous zinc is present in the non-glutamatergic neural processes and earlier electrophysiological studies suggest that zinc is a modulator of inhibitory signaling pathways, which are mediated by glycine and GABA. AII amacrine cells, a subpopulation of glycinergic amacrine cells, are identified by selective immunoreactivity for parvalbumin in the rat retina. In the present study, therefore, we focused on whether zinc is present in AII amacrine cells using silver amplification combined with immunohistochemistry in the rat retina. We also examined whether zinc modulate glycine response in the rat retina by the patch clamp technique. Association of silver precipitates with the parvalbumin-immunoreactive neural processes was observed at the ultrastructural level. We also found that zinc existed in the neural processes which were not parvalbumin-immunoreactive. Glycine-induced responses were augmented when the concentration of Zn(2+) was below 10 microM, but inhibited at Zn(2+) concentrations of 50 microM or more. Our results suggest the notion that zinc in neural processes of retinal neurons modulates the inhibitory signaling pathway, particularly that mediated by glycine receptors in AII amacrine cells.

Lack of Connexin43-mediated bergmann glial gap junctional coupling does not affect cerebellar long-term depression, motor coordination, or eyeblink conditioning.

Bergmann glial cells are specialized astrocytes in the cerebellum. In the mature cerebellar molecular layer, Bergmann glial processes are closely associated with Purkinje cells, enclosing Purkinje cell dendritic synapses with a glial sheath. There is intensive gap junctional coupling between Bergmann glial processes, but their significance in cerebellar functions is not known. Connexin43 (Cx43), a major component of astrocytic gap junction channels, is abundantly expressed in Bergmann glial cells. To examine the role of Cx43-mediated gap junctions between Bergmann glial cells in cerebellar functions, we generated Cx43 conditional knockout mice with the S100b-Cre transgenic line (Cx43(fl/fl):S100b-Cre), which exhibited a significant loss of Cx43 in the Bergmann glial cells and astrocytes in the cerebellum with a postnatal onset. The Cx43(fl/fl):S100b-Cre mice had normal cerebellar architecture. Although gap junctional coupling between the Bergmann glial cells measured by spreading of microinjected Lucifer yellow was virtually abolished in Cx43(fl/fl):S100b-Cre mice, electrophysiologic analysis revealed that cerebellar long-term depression could be induced and maintained normally in their cerebellar slices. In addition, at the behavioral level, Cx43(fl/fl):S100b-Cre mice had normal motor coordination in the rotarod task and normal conditioned eyelid response. Our findings suggest that Cx43-mediated gap junctional coupling between Bergmann glial cells is not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning.

Connexin43 and bergmann glial gap junctions in cerebellar function.

Connexin43 (Cx43), a major component of astrocytic gap junctions, is abundantly expressed in Bergmann glial cells (BGCs) in the cerebellum, but the function of Cx43 in BGCs is largely unknown. BGCs are specialized astrocytes closely associated with Purkinje cells. Here, we review our recent studies of the role of Cx43 in gap junctional coupling between BGCs and in cerebellar function. We generated Cx43 conditional knockout mice with an S100b-Cre transgenic line (Cx43(fl/fl):S100b-Cre), in which there was a significant postnatal loss of Cx43 in BGCs and cerebellar astrocytes. Gap junctional coupling between BGCs measured by dye coupling was virtually abolished in Cx43(fl/fl):S100b-Cre mice. Electrophysiologic and behavioral analyses suggested that Cx43-mediated gap junctions and Cx43 hemichannels in BGCs are not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning. These findings raise questions regarding the regional differences in the impact of the loss of Cx43 in the brain.