An intersectional genetic approach for simultaneous cell type-specific labelling and gene knockout in the mouse.

Precise genome manipulation in specific cell types and subtypes in vivo is crucial for neurobiological research because of the cellular heterogeneity of the brain. Site-specific recombinase systems in the mouse, such as Cre-loxP, improve cell type-specific genome manipulation; however, undesirable expression of cell type-specific Cre can occur. This could be due to transient expression during early development, natural expression in more than one cell type, kinetics of recombinases, sensitivity of the Cre reporter, and disruption in cis-regulatory elements by transgene insertion. Moreover, cell subtypes cannot be distinguished in cell type-specific Cre mice. To address these issues, we applied an intersectional genetic approach in mouse using triple recombination systems (Cre-loxP, Flp-FRT and Dre-rox). As a proof of principle, we labelled heterogeneous cell subtypes and deleted target genes within given cell subtypes by labelling neuropeptide Y (NPY)-, calretinin (calbindin 2) (CR)- and cholecystokinin (CCK)-expressing GABAergic neurons in the brain followed by deletion of RNA-binding Fox-1 homolog 3 (Rbfox3) in our engineered mice. Together, our study applies an intersectional genetic approach in vivo to generate engineered mice serving dual purposes of simultaneous cell subtype-specific labelling and gene knockout.

Loss of mGlu5 receptors in somatostatin-expressing neurons alters negative emotional states.

Subtype 5 metabotropic glutamate receptors (mGlu5) are known to play an important role in regulating cognitive, social and valence systems. However, it remains largely unknown at which circuits and neuronal types mGlu5 act to influence these behavioral domains. Altered tissue- or cell-specific expression or function of mGlu5 has been proposed to contribute to the exacerbation of neuropsychiatric disorders. Here, we examined how these receptors regulate the activity of somatostatin-expressing (SST+) neurons, as well as their influence on behavior and brain rhythmic activity. Loss of mGlu5 in SST+ neurons elicited excitatory synaptic dysfunction in a region and sex-specific manner together with a range of emotional imbalances including diminished social novelty preference, reduced anxiety-like behavior and decreased freezing during retrieval of fear memories. In addition, the absence of mGlu5 in SST+ neurons during fear processing impaired theta frequency oscillatory activity in the medial prefrontal cortex and ventral hippocampus. These findings reveal a critical role of mGlu5 in controlling SST+ neurons excitability necessary for regulating negative emotional states.

Neuronal splicing regulator RBFOX3 mediates seizures via regulating Vamp1 expression preferentially in NPY-expressing GABAergic neurons.

Epilepsy is a common neurological disorder, which has been linked to mutations or deletions of RNA binding protein, fox-1 homolog (Caenorhabditis elegans) 3 (RBFOX3)/NeuN, a neuronal splicing regulator. However, the mechanism of seizure mediation by RBFOX3 remains unknown. Here, we show that mice with deletion of Rbfox3 in gamma-aminobutyric acid (GABA) ergic neurons exhibit spontaneous seizures and high premature mortality due to increased presynaptic release, postsynaptic potential, neuronal excitability, and synaptic transmission in hippocampal dentate gyrus granule cells (DGGCs). Attenuating early excitatory gamma-aminobutyric acid (GABA) action by administering bumetanide, an inhibitor of early GABA depolarization, rescued premature mortality. Rbfox3 deletion reduced hippocampal expression of vesicle-associated membrane protein 1 (VAMP1), a GABAergic neuron-specific presynaptic protein. Postnatal restoration of VAMP1 rescued premature mortality and neuronal excitability in DGGCs. Furthermore, Rbfox3 deletion in GABAergic neurons showed fewer neuropeptide Y (NPY)-expressing GABAergic neurons. In addition, deletion of Rbfox3 in NPY-expressing GABAergic neurons lowered intrinsic excitability and increased seizure susceptibility. Our results establish RBFOX3 as a critical regulator and possible treatment path for epilepsy.

Hypothalamic Glutamate/GABA Cotransmission Modulates Hippocampal Circuits and Supports Long-Term Potentiation.

Subcortical input engages in cortico-hippocampal information processing. Neurons of the hypothalamic supramammillary nucleus (SuM) innervate the dentate gyrus (DG) by coreleasing two contrasting fast neurotransmitters, glutamate and GABA, and thereby support spatial navigation and contextual memory. However, the synaptic mechanisms by which SuM neurons regulate the DG activity and synaptic plasticity are not well understood. The DG comprises excitatory granule cells (GCs) as well as inhibitory interneurons (INs). Combining optogenetic, electrophysiological, and pharmacological approaches, we demonstrate that the SuM input differentially regulates the activities of different DG neurons in mice of either sex via distinct synaptic mechanisms. Although SuM activation results in synaptic excitation and inhibition in all postsynaptic cells, the ratio of these two components is variable and cell type-dependent. Specifically, dendrite-targeting INs receive predominantly synaptic excitation, whereas soma-targeting INs and GCs receive primarily synaptic inhibition. Although SuM excitation alone is insufficient to excite GCs, it enhances the GC spiking precision and reduces the latencies in response to excitatory drives. Furthermore, SuM excitation enhances the GC spiking in response to the cortical input, thereby promoting induction of long-term potentiation at cortical-GC synapses. Collectively, these findings provide physiological significance of the cotransmission of glutamate/GABA by SuM neurons in the DG network.SIGNIFICANCE STATEMENT The cortical-hippocampal pathways transfer mnemonic information during memory acquisition and retrieval, whereas subcortical input engages in modulation of communication between the cortex and hippocampus. The supramammillary nucleus (SuM) neurons of the hypothalamus innervate the dentate gyrus (DG) by coreleasing glutamate and GABA onto granule cells (GCs) and interneurons and support memories. However, how the SuM input regulates the activity of various DG cell types and thereby contributes to synaptic plasticity remains unexplored. Combining optogenetic and electrophysiological approaches, we demonstrate that the SuM input differentially regulates DG cell dynamics and consequently enhances GC excitability as well as synaptic plasticity at cortical input-GC synapses. Our findings highlight a significant role of glutamate/GABA cotransmission in regulating the input-output dynamics of DG circuits.

Connectivity and synaptic features of hilar mossy cells and their effects on granule cell activity along the hippocampal longitudinal axis.

The hippocampus is an elongated brain structure which runs along a ventral-to-dorsal axis in rodents, corresponding to the anterior-to-posterior axis in humans. A glutamatergic cell type in the dentate gyrus (DG), the mossy cells (MCs), establishes extensive excitatory collateral connections with the DG principal cells, the granule cells (GCs), and inhibitory interneurons in both hippocampal hemispheres along the longitudinal axis. Although coupling of two physically separated GC populations via long-axis projecting MCs is instrumental for information processing, the connectivity and synaptic features of MCs along the longitudinal axis are poorly defined. Here, using channelrhodopsin-2 assisted circuit mapping, we showed that MC excitation results in a low synaptic excitation-inhibition (E/I) balance in the intralamellar (local) GCs, but a high synaptic E/I balance in the translamellar (distant) ones. In agreement with the differential E/I balance along the ventrodorsal axis, activation of MCs either enhances or suppresses the local GC response to the cortical input, but primarily promotes the distant GC activation. Moreover, activation of MCs enhances the spike timing precision of the local GCs, but not that of the distant ones. Collectively, these findings suggest that MCs differentially regulate the local and distant GC activity through distinct synaptic mechanisms. KEY POINTS: Hippocampal mossy cell (MC) pathways differentially regulate granule cell (GC) activity along the longitudinal axis. MCs mediate a low excitation-inhibition balance in intralamellar (local) GCs, but a high excitation-inhibition balance in translamellar (distant) GCs. MCs enhance the spiking precision of local GCs, but not distant GCs. MCs either promote or suppress local GC activity, but primarily promote distant GC activation.

Endoplasmic reticulum stress induces Alzheimer disease-like phenotypes in the neuron derived from the induced pluripotent stem cell with D678H mutation on amyloid precursor protein.

Alzheimer disease (AD), a progressive neurodegenerative disorder, is mainly caused by the interaction of genetic and environmental factors. The impact of environmental factors on the genetic mutation in the amyloid precursor protein (APP) is not well characterized. We hypothesized that endoplasmic reticulum (ER) stress would promote disease for the patient carrying the APP D678H mutation. Therefore, we analyzed the impact of a familial AD mutation on amyloid precursor protein (APP D678H) under ER stress. Induced pluripotent stem cells (iPSCs) from APP D678H mutant carrier was differentiated into neurons, which were then analyzed for AD-like changes. Immunocytochemistry and whole-cell patch-clamp recording revealed that the derived neurons on day 28 after differentiation showed neuronal markers and electrophysiological properties similar to those of mature neurons. However, the APP D678H mutant neurons did not have significant alterations in the levels of amyloid-ß (Aß) and phosphorylated tau (pTau) compared to its isogenic wild-type neurons. Only under ER stress, the neurons with the APP D678H mutation had more Aß and pTau via immune detection assays. The higher level of Aß in the APP D678H mutant neurons was probably due to the increased level of ß-site APP cleaving enzyme (BACE1) and decreased level of Aß-degrading enzymes under ER stress. Increased Aß and pTau under ER stress reduced the N-methyl-D-aspartate receptor (NMDAR) in Western blot analysis and altered electrophysiological properties in the mutant neurons. Our study provides evidence that the interaction between genetic mutation and ER stress would induce AD-like changes. Cover Image for this issue: https://doi.org/10.1111/jnc.15420.

CGRP-dependent sensitization of PKC-δ positive neurons in central amygdala mediates chronic migraine.

BACKGROUND: To investigate specific brain regions and neural circuits that are responsible for migraine chronification. METHODS: We established a mouse model of chronic migraine with intermittent injections of clinically-relevant dose of nitroglycerin (0.1 mg/kg for 9 days) and validated the model with cephalic and extracephalic mechanical sensitivity, calcitonin gene-related peptide (CGRP) expression in trigeminal ganglion, and responsiveness to sumatriptan or central CGRP blockade. We explored the neurons that were sensitized along with migraine chronification and investigated their roles on migraine phenotypes with chemogenetics. RESULTS: After repetitive nitroglycerin injections, mice displayed sustained supraorbital and hind paw mechanical hyperalgesia, which lasted beyond discontinuation of nitroglycerin infusion and could be transiently reversed by sumatriptan. The CGRP expression in trigeminal ganglion was also upregulated. We found the pERK positive cells were significantly increased in the central nucleus of the amygdala (CeA), and these sensitized cells in the CeA were predominantly protein kinase C-delta (PKC-δ) positive neurons co-expressing CGRP receptors. Remarkably, blockade of the parabrachial nucleus (PBN)-CeA CGRP neurotransmission by CGRP8-37 microinjection to the CeA attenuated the sustained cephalic and extracephalic mechanical hyperalgesia. Furthermore, chemogenetic silencing of the sensitized CeA PKC-δ positive neurons reversed the mechanical hyperalgesia and CGRP expression in the trigeminal ganglion. In contrast, repetitive chemogenetic activation of the CeA PKC-δ positive neurons recapitulated chronic migraine-like phenotypes in naïve mice. CONCLUSIONS: Our data suggest that CeA PKC-δ positive neurons innervated by PBN CGRP positive neurons might contribute to the chronification of migraine, which may serve as future therapeutic targets for chronic migraine.

Enhanced Na+ -K+ -2Cl- cotransporter 1 underlies motor dysfunction in huntington's disease.

BACKGROUND: Altered γ-aminobutyric acid signaling is believed to disrupt the excitation/inhibition balance in the striatum, which may account for the motor symptoms of Huntington's disease. Na-K-2Cl cotransporter-1 is a key molecule that controls γ-aminobutyric acid-ergic signaling. However, the role of Na-K-2Cl cotransporter-1 and efficacy of γ-aminobutyric acid-ergic transmission remain unknown in Huntington's disease. METHODS: We determined the levels of Na-K-2Cl cotransporter-1 in brain tissue from Huntington's disease mice and patients by real-time quantitative polymerase chain reaction, western blot, and immunocytochemistry. Gramicidin-perforated patch-clamp recordings were used to measure the Eγ-aminobutyric acid in striatal brain slices. To inhibit Na-K-2Cl cotransporter-1 activity, R6/2 mice were treated with an intraperitoneal injection of bumetanide or adeno-associated virus-mediated delivery of Na-K-2Cl cotransporter-1 short-hairpin RNA into the striatum. Motor behavior assays were employed. RESULTS: Expression of Na-K-2Cl cotransporter-1 was elevated in the striatum of R6/2 and Hdh150Q/7Q mouse models. An increase in Na-K-2Cl cotransporter-1 transcripts was also found in the caudate nucleus of Huntington's disease patients. Accordingly, a depolarizing shift of Eγ-aminobutyric acid was detected in the striatum of R6/2 mice. Expression of the mutant huntingtin in astrocytes and neuroinflammation were necessary for enhanced expression of Na-K-2Cl cotransporter-1 in HD mice. Notably, pharmacological or genetic inhibition of Na-K-2Cl cotransporter-1 rescued the motor deficits of R6/2 mice. CONCLUSIONS: Our findings demonstrate that aberrant γ-aminobutyric acid-ergic signaling and enhanced Na-K-2Cl cotransporter-1 contribute to the pathogenesis of Huntington's disease and identify a new therapeutic target for the potential rescue of motor dysfunction in patients with Huntington's disease. © 2019 International Parkinson and Movement Disorder Society.

K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones.

Membrane excitability in the axonal growth cones of embryonic neurons influences axon growth. Voltage-gated K+ (Kv) channels are key factors in controlling membrane excitability, but whether they regulate axon growth remains unclear. Here, we report that Kv3.4 is expressed in the axonal growth cones of embryonic spinal commissural neurons, motoneurons, dorsal root ganglion neurons, retinal ganglion cells, and callosal projection neurons during axon growth. Our in vitro (cultured dorsal spinal neurons of chick embryos) and in vivo (developing chick spinal commissural axons and rat callosal axons) findings demonstrate that knockdown of Kv3.4 by a specific shRNA impedes axon initiation, elongation, pathfinding, and fasciculation. In cultured dorsal spinal neurons, blockade of Kv3.4 by blood depressing substance II suppresses axon growth via an increase in the amplitude and frequency of Ca2+ influx through T-type and L-type Ca2+ channels. Electrophysiological results show that Kv3.4, the major Kv channel in the axonal growth cones of embryonic dorsal spinal neurons, is activated at more hyperpolarized potentials and inactivated more slowly than it is in postnatal and adult neurons. The opening of Kv3.4 channels effectively reduces growth cone membrane excitability, thereby limiting excessive Ca2+ influx at subthreshold potentials or during Ca2+-dependent action potentials. Furthermore, excessive Ca2+ influx induced by an optogenetic approach also inhibits axon growth. Our findings suggest that Kv3.4 reduces growth cone membrane excitability and maintains [Ca2+]i at an optimal concentration for normal axon growth.SIGNIFICANCE STATEMENT Accumulating evidence supports the idea that impairments in axon growth contribute to many clinical disorders, such as autism spectrum disorders, corpus callosum agenesis, Joubert syndrome, Kallmann syndrome, and horizontal gaze palsy with progressive scoliosis. Membrane excitability in the growth cone, which is mainly controlled by voltage-gated Ca2+ (Cav) and K+ (Kv) channels, modulates axon growth. The role of Cav channels during axon growth is well understood, but it is unclear whether Kv channels control axon outgrowth by regulating Ca2+ influx. This report shows that Kv3.4, which is transiently expressed in the axonal growth cones of many types of embryonic neurons, acts to reduce excessive Ca2+ influx through Cav channels and thus permits normal axon outgrowth.

K+ Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control.

The K+ channel pore-forming subunit Kv4.3 is expressed in a subset of nonpeptidergic nociceptors within the dorsal root ganglion (DRG), and knockdown of Kv4.3 selectively induces mechanical hypersensitivity, a major symptom of neuropathic pain. K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are coexpressed in Kv4.3+ DRG neurons, but whether they participate in Kv4.3-mediated pain control is unknown. Here, we show the existence of a Kv4.3/KChIP1/KChIP2/DPP10 complex (abbreviated as the Kv4 complex) in the endoplasmic reticulum and cell surface of DRG neurons. After intrathecal injection of a gene-specific antisense oligodeoxynucleotide to knock down the expression of each component in the Kv4 complex, mechanical hypersensitivity develops in the hindlimbs of rats in parallel with a reduction in all components in the lumbar DRGs. Electrophysiological data further indicate that the excitability of nonpeptidergic nociceptors is enhanced. The expression of all Kv4 complex components in DRG neurons is downregulated following spinal nerve ligation (SNL). To rescue Kv4 complex downregulation, cDNA constructs encoding Kv4.3, KChIP1, and DPP10 were transfected into the injured DRGs (defined as DRGs with injured spinal nerves) of living SNL rats. SNL-evoked mechanical hypersensitivity was attenuated, accompanied by a partial recovery of Kv4.3, KChIP1, and DPP10 surface levels in the injured DRGs. By showing an interdependent regulation among components in the Kv4 complex, this study demonstrates that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 participate in Kv4.3-mediated mechanical pain control. Thus, these modulatory subunits could be potential drug targets for neuropathic pain.SIGNIFICANCE STATEMENT Neuropathic pain, a type of moderate to severe chronic pain resulting from nerve injury or disorder, affects 6.9%-10% of the global population. However, less than half of patients report satisfactory pain relief from current treatments. K+ channels, which act to reduce nociceptor activity, have been suggested to be novel drug targets for neuropathic pain. This study is the first to show that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are potential drug targets for neuropathic pain because they form a channel complex with the K+ channel pore-forming subunit Kv4.3 in a subset of nociceptors to selectively inhibit mechanical hypersensitivity, a major symptom of neuropathic pain.

Defective GABAergic neurotransmission in the nucleus tractus solitarius in Mecp2-null mice, a model of Rett syndrome.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (Mecp2) gene. GABAergic dysfunction has been implicated contributing to the respiratory dysfunction, one major clinical feature of RTT. The nucleus tractus solitarius (NTS) is the first central site integrating respiratory sensory information that can change the nature of the reflex output. We hypothesized that deficiency in Mecp2 gene reduces GABAergic neurotransmission in the NTS. Using whole-cell patch-clamp recordings in NTS slices, we measured spontaneous inhibitory postsynaptic currents (sIPSCs), miniature IPSCs (mIPSCs), NTS-evoked IPSCs (eIPSCs), and GABAA receptor (GABAA-R) agonist-induced responses. Compared to those from wild-type mice, NTS neurons from Mecp2-null mice had significantly (p<0.05) reduced sIPSC amplitude, sIPSC frequency, and mIPSC amplitude but not mIPSC frequency. Mecp2-null mice also had decreased eIPSC amplitude with no change in paired-pulse ratio. The data suggest reduced synaptic receptor-mediated phasic GABA transmission in Mecp2-null mice. In contrast, muscimol (GABAA-R agonist, 0.3-100µM) and THIP (selective extrasynaptic GABAA-R agonist, 5µM) induced significantly greater current response in Mecp2-null mice, suggesting increased extrasynaptic receptors. Using qPCR, we found a 2.5 fold increase in the delta subunit of the GABAA-Rs in the NTS in Mecp2-null mice, consistent with increased extrasynaptic receptors. As the NTS was recently found required for respiratory pathology in RTT, our results provide a mechanism for NTS dysfunction which involves shifting the balance of synaptic/extrasynaptic receptors in favor of extrasynaptic site, providing a target for boosting GABAergic inhibition in RTT.

Wiring Specificity and Synaptic Diversity in the Mouse Lateral Central Amygdala.

The central amygdala (CeA) nucleus, a subcortical structure composed of mostly GABA-releasing (GABAergic) neurons, controls fear expression via projections to downstream targets in the hypothalamus and brainstem. The CeA consists of the lateral (CeL) and medial (CeM) subdivisions. The CeL strongly gates information transfer to the CeM, the main output station of the amygdala, but little is known about the functional organization of local circuits in this region. Using cluster analysis, we identified two major electrophysiologically distinct CeL neuron classes in mouse amygdala slices, the early-spiking (ES) and late-spiking (LS) neurons. These two classes displayed distinct autaptic transmission. Compared with LS neurons, ES neurons had strong and depressing autapses, which enhanced spike-timing precision. With multiple patch-clamp recordings, we found that CeL neurons made chemical, but not electrical, synapses. Analysis of individual connections revealed cannabinoid type 1 receptor-mediated suppression of the ES, but not of the LS cell output synapse. More interestingly, the efficacy of the ES→LS or LS→ES synapse was ~2-fold greater than that of the LS→LS or ES→ES synapse. When tested at 20 Hz, synapses between different neurons, but not within the same class, were markedly depressing and were more powerful to sculpt activity of postsynaptic neurons. Moreover, neurons of different classes also form synapses with higher degree of connectivity. We demonstrate that ES and LS neurons represent two functionally distinct cell classes in the CeL and interactions between presynaptic and postsynaptic neurons dictate synaptic properties between neurons. SIGNIFICANCE STATEMENT: The central lateral amygdala (CeL) is a key node in fear circuits, but the functional organization of local circuits in this region is largely unknown. The CeL consists of mostly GABAergic inhibitory neurons with different functional and molecular features. Here, we report that the presynaptic cell class determines functional properties of autapses and cannabinoid-mediated modulation of synaptic transmission between neurons, whereas presynaptic versus postsynaptic cell classes dictate the connectivity, efficacy, and dynamics of GABAergic synapses between any two neurons. The wiring specificity and synaptic diversity have a great impact on neuronal output in amygdala inhibitory networks. Such synaptic organizing principles advance our understanding of the significance of physiologically defined neuronal phenotypes in amygdala inhibitory networks.

Differential Recruitment of Dentate Gyrus Interneuron Types by Commissural Versus Perforant Pathways.

Gamma-aminobutyric acidergic (GABAergic) interneurons (INs) in the dentate gyrus (DG) provide inhibitory control to granule cell (GC) activity and thus gate incoming signals to the hippocampus. However, how various IN subtypes inhibit GCs in response to different excitatory input pathways remains mostly unknown. By using electrophysiology and optogenetics, we investigated neurotransmission of the hilar commissural pathway (COM) and the medial perforant path (MPP) to the DG in acutely prepared mouse slices. We found that the short-term dynamics of excitatory COM-GC and MPP-GC synapses was similar, but that the dynamics of COM- and MPP-mediated inhibition measured in GCs was remarkably different, during theta-frequency stimulation. This resulted in the increased inhibition-excitation (I/E) ratios in single GCs for COM stimulation, but decreased I/E ratios for MPP stimulation. Further analysis of pathway-specific responses in identified INs revealed that basket cell-like INs, total molecular layer- and molecular layer-like cells, received greater excitation and were more reliably recruited by the COM than by the MPP inputs. In contrast, hilar perforant path-associated and hilar commissural-associational pathway-related-like cells were minimally activated by both inputs. These results demonstrate that distinct IN subtypes are preferentially recruited by different inputs to the DG, and reveal their relative contributions in COM-mediated feedforward inhibition.

Rapid dynamic changes of dendritic inhibition in the dentate gyrus by presynaptic activity patterns.

The dentate gyrus (DG) serves as a primary gate to control information transfer from the cortex to the hippocampus. Activation of incoming cortical inputs results in rapid synaptic excitation followed by slow GABA-mediated (GABAergic) synaptic inhibition onto DG granule cells (GCs). GABAergic inhibitory interneurons (INs) in the DG comprise fast-spiking (FS) and non-fast-spiking (non-FS) cells. Anatomical analyses of DG INs reveal that FS cells are soma-targeting INs, whereas non-FS cells are dendrite-targeting INs. These two IN classes are differentially recruited by excitatory inputs and in turn provide exquisite spatiotemporal control over GC activity. Yet, little is known how FS and non-FS cells transform their presynaptic dynamics into varying postsynaptic response amplitudes. Using paired recordings in rat hippocampal slices, we show that inhibition in the DG is dominated by somatic GABAergic inputs during periods of sparse presynaptic activity, whereas dendritic GABAergic inputs are rapidly shifted to powerful and sustained inhibition during periods of intense presynaptic activity. The variant dynamics of dendritic inhibition is dependent on presynaptic IN subtypes and their activity patterns and is attributed to Ca(2+)-dependent increases in the probability of release and the size of the readily releasable pool. Furthermore, the degree of dynamic GABA release can be reduced by blocking voltage-gated K(+) channels, which increases the efficacy of dendrite-targeting IN output synapses during sparse firing. Such rapid dynamic modulation of dendritic inhibition may act as a frequency-dependent filter to prevent overexcitation of GC dendrites and thus set the excitatory-inhibitory synaptic balance in the DG circuits.

Genetic mapping of ASIC4 and contrasting phenotype to ASIC1a in modulating innate fear and anxiety.

Although ASIC4 is a member of the acid-sensing ion channel (ASIC) family, we have limited knowledge of its expression and physiological function in vivo. To trace the expression of this ion channel, we generated the ASIC4-knockout/CreERT(2)-knockin (Asic4(Cre) (ERT) (2)) mouse line. After tamoxifen induction in the Asic4(Cre) (ERT)(2)::CAG-STOP(floxed)-Td-tomato double transgenic mice, we mapped the expression of ASIC4 at the cellular level in the central nervous system (CNS). ASIC4 was expressed in many brain regions, including the olfactory bulb, cerebral cortex, striatum, hippocampus, amygdala, thalamus, hypothalamus, brain stem, cerebellum, spinal cord and pituitary gland. Colocalisation studies further revealed that ASIC4 was expressed mainly in three types of cells in the CNS: (i) calretinin (CR)-positive and/or vasoactive intestine peptide (VIP)-positive interneurons; (ii) neural/glial antigen 2 (NG2)-positive glia, also known as oligodendrocyte precursor cells; and (iii) cerebellar granule cells. To probe the possible role of ASIC4, we hypothesised that ASIC4 could modulate the membrane expression of ASIC1a and thus ASIC1a signaling in vivo. We conducted behavioral phenotyping of Asic4(Cre) (ERT)(2) mice by screening many of the known behavioral phenotypes found in Asic1a knockouts and found ASIC4 not involved in shock-evoked fear learning and memory, seizure termination or psychostimulant-induced locomotion/rewarding effects. In contrast, ASIC4 might play an important role in modulating the innate fear response to predator odor and anxious state because ASIC4-mutant mice showed increased freezing response to 2,4,5-trimethylthiazoline and elevated anxiety-like behavior in both the open-field and elevated-plus maze. ASIC4 may modulate fear and anxiety by counteracting ASIC1a activity in the brain.

Acid-sensing ion channel-1a is not required for normal hippocampal LTP and spatial memory.

Acid-sensing ion channel-1a (ASIC1a) is localized in brain regions with high synaptic density and is thought to contribute to synaptic plasticity, learning, and memory. A prominent hypothesis is that activation of postsynaptic ASICs promotes depolarization, thereby augmenting N-methyl-d-aspartate receptor function and contributing to the induction of long-term potentiation (LTP). However, evidence for activation of postsynaptic ASICs during neurotransmission has not been established. Here, we re-examined the role of ASIC1a in LTP in the hippocampus using pharmacological and genetic approaches. Our results showed that a tarantula peptide psalmotoxin, which profoundly blocked ASIC currents in the hippocampal neurons, had no effect on LTP. Similarly, normal LTP was robustly generated in ASIC1a-null mice. A further behavioral analysis showed that mice lacking ASIC1a had normal performance in hippocampus-dependent spatial memory. In summary, our results indicate that ASIC1a is not required for hippocampal LTP and spatial memory. We therefore propose that the role of ASIC1a in LTP and spatial learning should be reassessed.

GABA is depolarizing in hippocampal dentate granule cells of the adolescent and adult rats.

GABAergic signaling in hippocampal pyramidal neurons undergoes a switch from depolarizing to hyperpolarizing during early neuronal development. Whether such a transformation of GABAergic action occurs in dentate granule cells (DGCs), located at the first stage of the hippocampal trisynaptic circuit, is unclear. Here, we use noninvasive extracellular recording to monitor the effect of synaptically released GABA on the DGC population. We find that GABAergic responses in adolescent and adult rat DGCs are still depolarizing from rest. Using a morphologically realistic DGC model, we show that GABAergic action, depending on its precise timing and location, can have either an excitatory or inhibitory role in signal processing in the dentate gyrus.

Ba2+- and bupivacaine-sensitive background K+ conductances mediate rapid EPSP attenuation in oligodendrocyte precursor cells.

Glutamatergic transmission onto oligodendrocyte precursor cells (OPCs) may regulate OPC proliferation, migration and differentiation. Dendritic integration of excitatory postsynaptic potentials (EPSPs) is critical for neuronal functions, and mechanisms regulating dendritic propagation and summation of EPSPs are well understood. However, little is known about EPSP attenuation and integration in OPCs. We developed realistic OPC models for synaptic integration, based on passive membrane responses of OPCs obtained by simultaneous dual whole-cell patch-pipette recordings. Compared with neurons, OPCs have a very low value of membrane resistivity, which is largely mediated by Ba(2+)- and bupivacaine-sensitive background K(+) conductances. The very low membrane resistivity not only leads to rapid EPSP attenuation along OPC processes but also sharpens EPSPs and narrows the temporal window for EPSP summation. Thus, background K(+) conductances regulate synaptic responses and integration in OPCs, thereby affecting activity-dependent neuronal control of OPC development and function.

Mutations in KCND3 cause spinocerebellar ataxia type 22.

OBJECTIVE: To identify the causative gene in spinocerebellar ataxia (SCA) 22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21-q23. METHODS: We previously characterized a large Chinese family with progressive ataxia designated SCA22, which overlaps with the locus of SCA19. The disease locus in a French family and an Ashkenazi Jewish American family was also mapped to this region. Members from all 3 families were enrolled. Whole exome sequencing was performed to identify candidate mutations, which were narrowed by linkage analysis and confirmed by Sanger sequencing and cosegregation analyses. Mutational analyses were also performed in 105 Chinese and 55 Japanese families with cerebellar ataxia. Mutant gene products were examined in a heterologous expression system to address the changes in protein localization and electrophysiological functions. RESULTS: We identified heterozygous mutations in the voltage-gated potassium channel Kv4.3-encoding gene KCND3: an in-frame 3-nucleotide deletion c.679_681delTTC p.F227del in both the Chinese and French pedigrees, and a missense mutation c.1034G>T p.G345V in the Ashkenazi Jewish family. Direct sequencing of KCND3 further identified 3 mutations, c.1034G>T p.G345V, c.1013T>C p.V338E, and c.1130C>T p.T377M, in 3 Japanese kindreds. Immunofluorescence analyses revealed that the mutant p.F227del Kv4.3 subunits were retained in the cytoplasm, consistent with the lack of A-type K(+) channel conductance in whole cell patch-clamp recordings. INTERPRETATION: Our data identify the cause of SCA19/22 in patients of diverse ethnic origins as mutations in KCND3. These findings further emphasize the important role of ion channels as key regulators of neuronal excitability in the pathogenesis of cerebellar degeneration.

WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release.

Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) in response to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs (CVOs) of the brain focus on mechanosensitive membrane proteins. The present study demonstrated that intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuron-specific conditional KO (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1 cKO also blunted mannitol-induced AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.