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Elucidating the neural basis of fear allows for more effective treatments for maladaptive fear often observed in psychiatric disorders. Although the basal forebrain (BF) has an essential role in fear learning, its function in fear expression and the underlying neuronal and circuit substrates are much less understood. Here we report that BF glutamatergic neurons are robustly activated by social stimulus following social fear conditioning in male mice. And cell-type-specific inhibition of those excitatory neurons largely reduces social fear expression. At the circuit level, BF glutamatergic neurons make functional contacts with the lateral habenula (LHb) neurons and these connections are potentiated in conditioned mice. Moreover, optogenetic inhibition of BF-LHb glutamatergic pathway significantly reduces social fear responses. These data unravel an important function of the BF in fear expression via its glutamatergic projection onto the LHb, and suggest that selective targeting BF-LHb excitatory circuitry could alleviate maladaptive fear in relevant disorders., (© 2024. The Author(s).)

Acid-sensing ion channels (ASICs), the main H + receptors in the central nervous system, sense extracellular pH fluctuations and mediate cation influx. ASIC1a, the major subunit responsible for acid-activated current, is widely expressed in brain neurons, where it plays pivotal roles in diverse functions including synaptic transmission and plasticity. However, the underlying molecular mechanisms for these functions remain mysterious. Using extracellular epitope tagging and a novel antibody recognizing the hASIC1a ectodomain, we examined the membrane targeting and dynamic trafficking of hASIC1a in cultured cortical neurons. Surface hASIC1a was distributed throughout somata and dendrites, clustered in spine heads, and co-localized with postsynaptic markers. By extracellular pHluorin tagging and fluorescence recovery after photobleaching, we detected movement of hASIC1a in synaptic spine heads. Single-particle tracking along with use of the anti-hASIC1a ectodomain antibody revealed long-distance migration and local movement of surface hASIC1a puncta on dendrites. Importantly, enhancing synaptic activity with brain-derived neurotrophic factor accelerated the trafficking and lateral mobility of hASIC1a. With this newly-developed toolbox, our data demonstrate the synaptic location and high dynamics of functionally-relevant hASIC1a on the surface of excitatory synapses, supporting its involvement in synaptic functions.

Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na + and Ca 2+ sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na + and Ca 2+ signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na + and Ca 2+ signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na + and Ca 2+ waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na + and Ca 2+ signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na + signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor., (© 2020 International Society for Neurochemistry.)

Mutations in the macroautophagy/autophagy gene WDR45 cause β-propeller protein-associated neurodegeneration (BPAN); however the molecular and cellular mechanism of the disease process is largely unknown. Here we generated constitutive wdr45 knockout (KO) mice that displayed cognitive impairments, abnormal synaptic transmission and lesions in several brain regions. Immunohistochemistry analysis showed loss of neurons in prefrontal cortex and basal ganglion in aged mice, and increased apoptosis in prefrontal cortex, recapitulating a hallmark of neurodegeneration. Quantitative proteomic analysis showed accumulation of endoplasmic reticulum (ER) proteins in KO mouse. At the cellular level, accumulation of ER proteins due to WDR45 deficiency resulted in increased ER stress and impaired ER quality control. The unfolded protein response (UPR) was elevated through ERN1/IRE1 or EIF2AK3/PERK pathway, and eventually led to neuronal apoptosis. Suppression of ER stress or activation of autophagy through MTOR inhibition alleviated cell death. Thus, the loss of WDR45 cripples macroautophagy machinery in neurons and leads to impairment in organelle autophagy, which provides a mechanistic understanding of cause of BPAN and a potential therapeutic strategy to treat this genetic disorder. Abbreviations: 7-ADD: 7-aminoactinomycin D; ASD: autistic spectrum disorder; ATF6: activating transcription factor 6; ATG: autophagy-related; BafA1: bafilomycin A 1 ; BCAP31: B cell receptor associated protein 31; BPAN: β-propeller protein-associated neurodegeneration; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CDIPT: CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase); DDIT3/CHOP: DNA-damage inducible transcript 3; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GFP: green fluorescent protein; HIP: hippocampus; HSPA5/GRP78: heat shock protein family A (HSP70) member 5; KO: knockout; LAMP1: lysosomal-associated membrane 1; mEPSCs: miniature excitatory postsynaptic currents; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MIB: mid-brain; MTOR: mechanistic target of rapamycin kinase; PCR: polymerase chain reaction; PFA: paraformaldehyde; PFC: prefrontal cortex; PRM: parallel reaction monitoring; RBFOX3/NEUN: RNA binding protein, fox-1 homolog [C. elegans] 3; RTN3: reticulon 3; SEC22B: SEC22 homolog B, vesicle trafficking protein; SEC61B: SEC61 translocon beta subunit; SEM: standard error of the mean; SNR: substantia nigra; SQSTM1/p62: sequestosome 1; TH: tyrosine hydroxylase; Tm: tunicamycin; TMT: tandem mass tag; TUDCA: tauroursodeoxycholic acid; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling; UPR: unfolded protein response; WDR45: WD repeat domain 45; WT: wild type; XBP1: X-box binding protein 1.

We reported previously that acid-sensing ion channel 1a (ASIC1a) mediates acidic neuronal necroptosis via recruiting receptor-interacting protein kinase 1 (RIPK1) to its C terminus (CT), independent of its ion-conducting function. Here we show that the N-terminus (NT) of ASIC1a interacts with its CT to form an auto-inhibition that prevents RIPK1 recruitment/activation under resting conditions. The interaction involves glutamate residues at distal NT and is disrupted by acidosis. Expression of mutant ASIC1a bearing truncation or glutamate-to-alanine substitutions at distal NT causes constitutive cell death. The NT-CT interaction is further disrupted by N-ethylmaleimide-sensitive fusion ATPase (NSF), which associates with ASIC1a-NT under acidosis, facilitating RIPK1 interaction with ASIC1a-CT. Importantly, a membrane-penetrating synthetic peptide representing the distal 20 ASIC1a NT residues, NT 1-20 , reduced neuronal damage in both in vitro model of acidotoxicity and in vivo mouse model of ischemic stroke, demonstrating the therapeutic potential of targeting the auto-inhibition of ASIC1a for neuroprotection against acidotoxicity.

The quiescence of radial neural stem cells (rNSCs) in adult brain is regulated by environmental stimuli. However, little is known about how the neurogenic niche couples the external signal to regulate activation and transition of quiescent rNSCs. Here, we reveal that long-term excitation of hippocampal dentate granule cells (GCs) upon voluntary running leads to activation of adult rNSCs in the subgranular zone and thereby generation of newborn neurons. Unexpectedly, the role of these excited GC neurons in NSCs depends on direct GC-rNSC interaction in the local niche, which is through down-regulated ephrin-B3, a GC membrane-bound ligand, and attenuated transcellular EphB2 kinase-dependent signaling in the adjacent rNSCs. Furthermore, constitutively active EphB2 kinase sustains the quiescence of rNSCs during running. These findings thus elucidate the physiological significance of GC excitability on adult rNSCs under external environments and indicate a key-lock switch regulation via cell-cell contact for functional transition of rNSCs.

The autonomic nervous system controls various internal organs and executes crucial functions through sophisticated neural connectivity and circuits. Its dysfunction causes an imbalance of homeostasis and numerous human disorders. In the past decades, great efforts have been made to study the structure and functions of this system, but so far, our understanding of the classification of autonomic neuronal subpopulations remains limited and a precise map of their connectivity has not been achieved. One of the major challenges that hinder rapid progress in these areas is the complexity and heterogeneity of autonomic neurons. To facilitate the identification of neuronal subgroups in the autonomic nervous system, here we review the well-established and cutting-edge technologies that are frequently used in peripheral neuronal tracing and profiling, and discuss their operating mechanisms, advantages, and targeted applications.

Extinction of conditioned fear necessitates the dynamic involvement of hippocampus, medial prefrontal cortex (mPFC), and basolateral amygdala (BLA), but key molecular players that regulate these circuits to achieve fear extinction remain largely unknown. Here, we report that acid-sensing ion channel 1a (ASIC1a) is a crucial molecular regulator of fear extinction, and that this function requires ASIC1a in ventral hippocampus (vHPC), but not dorsal hippocampus, mPFC, or BLA. While genetic disruption or pharmacological inhibition of ASIC1a in vHPC attenuated the extinction of conditioned fear, overexpression of the channel in this area promoted fear extinction. Channelrhodopsin-2-assisted circuit mapping revealed that fear extinction involved an ASIC1a-dependent modification of the long-range hippocampal-prefrontal correlates in a projection-specific manner. Gene expression profiling analysis and validating experiments identified several neuronal activity-regulated and memory-related genes, including Fos , Npas4 , and Bdnf , as the potential mediators of ASIC1a regulation of fear extinction. Mechanistically, genetic overexpression of brain-derived neurotrophic factor (BDNF) in vHPC or supplement of BDNF protein in mPFC both rescued the deficiency in fear extinction and the deficits on extinction-driven adaptations of hippocampal-prefrontal correlates caused by the Asic1a gene inactivation in vHPC. Together, these results establish ASIC1a as a critical constituent in fear extinction circuits and thus a promising target for managing adaptive behaviors.

Acid-sensing ion channel 1a (ASIC1a) is abundant in multiple brain regions, including the striatum, which serves as the input nucleus of the basal ganglia and is critically involved in procedural learning and motor memory. We investigated the functional role of ASIC1a in striatal neurons. We found that ASIC1a was critical for striatum-dependent motor coordination and procedural learning by regulating the synaptic plasticity of striatal medium spiny neurons. Global deletion of Asic1a in mice led to increased dendritic spine density but impaired spine morphology and postsynaptic architecture, which were accompanied by the decreased function of N -methyl-d-aspartate (NMDA) receptors at excitatory synapses. These structural and functional changes caused by the loss of ASIC1a were largely mediated by reduced activation (phosphorylation) of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated protein kinases (ERKs). Consequently, Asic1a null mice exhibited poor performance on multiple motor tasks, which was rescued by striatal-specific expression of either ASIC1a or CaMKII. Together, our data reveal a previously unknown mechanism mediated by ASIC1a that promotes the excitatory synaptic function underlying striatum-related procedural learning and memory., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (I Kv ) and Ca 2+ -activated (I K(Ca) ) K +  currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating I Kv , mediated by the Shaker (Sh) channel to a large extent, while in the same neurons I K(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained I Kv and prominent I K(Ca) , encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the Sh M null mutant removed inactivating, transient I Kv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient I Kv . In comparison, the slo 1 mutation abolished the vast majority of I K(Ca) , particularly at the pupal stage. Strikingly, the deficiency of I K(Ca) in slo pupae was compensated by the transient component of I Kv mediated by Sh channels. Thus, I K(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient I Kv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K +  currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

Acidotoxicity is common among neurological disorders, such as ischemic stroke. Traditionally, Ca(2+) influx via homomeric acid-sensing ion channel 1a (ASIC1a) was considered to be the leading cause of ischemic acidotoxicity. Here we show that extracellular protons trigger a novel form of neuronal necroptosis via ASIC1a, but independent of its ion-conducting function. We identified serine/threonine kinase receptor interaction protein 1 (RIP1) as a critical component of this form of neuronal necroptosis. Acid stimulation recruits RIP1 to the ASIC1a C-terminus, causing RIP1 phosphorylation and subsequent neuronal death. In a mouse model of focal ischemia, middle cerebral artery occlusion causes ASIC1a-RIP1 association and RIP1 phosphorylation in affected brain areas. Deletion of the Asic1a gene significantly prevents RIP1 phosphorylation and brain damage, suggesting ASIC1a-mediated RIP1 activation has an important role in ischemic neuronal injury. Our findings indicate that extracellular protons function as a novel endogenous ligand that triggers neuronal necroptosis during ischemia via ASIC1a independent of its channel function.

Methyleugenol (ME) is a natural constituent isolated from many plant essential oils having multiple biological effects including anticonvulsant and anesthetic activities, although the underlying mechanisms remain unclear. Here, we identify ME as a novel agonist of ionotropic γ-aminobutyric acid (GABA) receptors. At lower concentrations (∼30 μM), ME significantly sensitized GABA-induced, but not glutamate- or glycine-induced, currents in cultured hippocampal neurons, indicative of a preferentially modulatory role of this compound for A type GABA receptors (GABAARs). In addition, ME at higher concentrations (≥100 μM) induced a concentration-dependent, Cl(-)-permeable current in hippocampal neurons, which was inhibited by a GABAAR channel blocker, picrotoxin, and a competitive GABAAR antagonist, bicuculline, but not a specific glycine receptor inhibitor, strychnine. Moreover, ME activated a similar current mediated by recombinant α1-β2-γ2 or α5-β2-γ2 GABAARs in human embryonic kidney (HEK) cells. Consequently, ME produced a strong inhibition of synaptically driven neuronal excitation in hippocampal neurons. Together, these results suggest that ME represents a novel agonist of GABAARs, shedding additional light on future development of new therapeutics targeting GABAARs. The present study also adds GABAAR activation to the list of molecular targets of ME that probably account for its biological activities.

Rhizoma Curcumae is a common Chinese dietary spice used in South Asia and China for thousands of years. As the main extract, Rhizoma Curcumae oil has attracted a great interest due to its newly raised therapeutic activities including its pharmacological effects upon central nervous system such as neuroprotection, cognitive enhancement, and anti-seizure efficacy; however the molecular mechanisms and the target identification remain to be established. Here we characterize an inhibitory effect of curcumol, a major bioactive component of Rhizoma Curcumae oil, on the excitability of hippocampal neurons in culture, the basal locomotor activity of freely moving animals, and the chemically induced seizure activity in vivo. Electrophysiological recording showed that acute application of curcumol significantly facilitated the γ-aminobutyric acid (GABA)-activated current in cultured mouse hippocampal neurons and in human embryonic kidney cells expressing α1- or α5-containing A type GABA (GABAA) receptors in a concentration-dependent manner. Measurement of tonic and miniature inhibitory postsynaptic GABAergic currents in hippocampal slices indicated that curcumol enhanced both forms of inhibition. In both pentylenetetrazole and kainate seizure models, curcumol suppressed epileptic activity in mice by prolonging the latency to clonic and tonic seizures and reducing the mortality as well as the susceptibility to seizure, presumably by facilitating the activation of GABAA receptors. Taken together, our results identified curcumol as a novel anti-seizure agent which inhibited neuronal excitability through enhancing GABAergic inhibition., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Astrocytic glutamate transporter 1 (GLT-1) is responsible for the majority of extracellular glutamate clearance and is essential for preventing excitotoxicity in the brain. Up-regulation of GLT-1 shows benefit effect on ischemia-induced neuronal damage. In present study, we examined the effect of histamine, a neurotransmitter or neuromodulator, on GLT-1 expression and function. In acute hippocampal slices, histamine selectively increased GLT-1 expression independent of neuronal activities. Similar up-regulation of GLT-1 was also observed after histamine treatment in pure cultured astrocytes, which was abolished by H1 receptor antagonist or PKC inhibitor. Cell surface biotinylation and whole-cell patch recordings of glutamate transporter current confirmed the up-regulation of functional GLT-1 following histamine exposure. Histamine treatment decreased the extracellular glutamate content and alleviated neuronal cell death induced by exogenous glutamate challenge. Moreover, we found a significant neuroprotective effect of histamine in brain slices after oxygen-glucose deprivation (OGD). In addition, histidine, the precursor of histamine, also showed neuroprotection against ischemic injury, which was accompanied by reversion of declined expression of GLT-1 in adult rats subjected to middle cerebral artery occlusion (MCAO). These neuroprotective effects of histamine/histidine were blocked by GLT-1 specific inhibitor dihydrokainate or H1 receptor antagonist. In summary, our results suggest that histamine up-regulates GLT-1 expression and function via astrocytic H1 receptors, thus resulting in neuroprotection against excitotoxicity and ischemic injury., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The pentameric A type γ-aminobutyric acid receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the nervous system and have long been considered as important pharmaceutical targets for the treatment of multiple neurological or psychological disorders. Here, we show that 2-guanidine-4-methylquinazoline (GMQ), a recently identified acid-sensing ion channel (ASIC) modulator, strongly and preferentially inhibits GABAAR among the major neurotransmitter-gated ion channels in cultured rat hippocampal neurons. GMQ inhibited GABA (1 μM)-induced currents in a competitive manner, with an IC50 (0.39±0.05 μM) comparable to that of bicuculline. Schild analysis revealed a slope of 1.04±0.06 for GMQ on α1β2 GABAARs expressed in HEK293T cells. Single-channel analysis showed that GMQ decreased open probability of GABAARs without affecting conductance. Moreover, GMQ inhibited GABAergic neurotransmission in hippocampal neurons, while having no significant effect on the basal field excitatory postsynaptic potentials (fEPSPs) and the intrinsic excitability of neurons. Using site-directed mutagenesis, we further demonstrated that mutations at Glu155 of β2 subunit and Phe64 of α1 subunit, both located inside the GABA binding pocket, profoundly decreased the sensitivity of the receptor to both GABA and GMQ. Interestingly, these mutations did not significantly affect the inhibition by amiloride, a diuretic structurally similar to GMQ and a known GABAAR inhibitor. We conclude that GMQ represents a novel chemical structure that acts, possibly, by competing with GABA binding to GABAARs. It is anticipated that GMQ and its analogs will facilitate the development of new chemical probes for GABAARs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The proper function of the nervous system depends on precise production and connection of distinct neurons and glia. Cell fate determination of neurons and glia is tightly controlled by complex gene expression regulation in the developing and adult nervous system. Emerging evidence has demonstrated the importance of noncoding microRNAs (miRNAs) in neural development and function. This review highlights current discoveries of miRNA functions in specifying neuronal and glial cell fate. We summarize the roles of miRNAs in expansion and differentiation of neural stem cells, specification of neuronal subtypes and glial cells, reprogramming of functional neurons from embryonic stem cells and fibroblasts, and left-right asymmetric organization of neuronal subtypes. Investigating the network of interactions between miRNAs and target genes will reveal new gene regulation machinery involved in tuning the cell fate decisions of neurons and glia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) μ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues (465)LCRRG(469) at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.

Acidosis is a common feature of many neuronal diseases and often accompanied with adverse consequences such as pain and neuronal injury. Before the discovery of acid-sensing ion channels (ASICs), protons were usually considered as a modulator of other ion channels, such as voltage-gated calcium channels, N-methyl-D-aspartate, and γ-amino butyric acid(A) receptor channels. Accordingly, the functional effects of acidosis were considered as consequences of modulations of these channels. Since the first cloning of ASICs in 1997, the conventional view on acidosis-mediated pain and cell injury has been dramatically changed. To date, ASICs, which are directly activated by extracellular protons, are shown to mediate most of the acidosis-associated physiological and pathological functions. For example, ASIC1a channels are reported to mediate acidosis-induced ischemic neuronal death. In this article, we will review the possible mechanisms that underlie ASIC1a channel-mediated neuronal death and discuss ASIC1a channel modulators involved in this process.

Ischemic brain injury is a major problem associated with stroke. It has been increasingly recognized that acid-sensing ion channels (ASICs) contribute significantly to ischemic neuronal damage, but the underlying mechanism has remained elusive. Here, we show that extracellular spermine, one of the endogenous polyamines, exacerbates ischemic neuronal injury through sensitization of ASIC1a channels to extracellular acidosis. Pharmacological blockade of ASIC1a or deletion of the ASIC1 gene greatly reduces the enhancing effect of spermine in ischemic neuronal damage both in cultures of dissociated neurons and in a mouse model of focal ischemia. Mechanistically, spermine profoundly reduces desensitization of ASIC1a by slowing down desensitization in the open state, shifting steady-state desensitization to more acidic pH, and accelerating recovery between repeated periods of acid stimulation. Spermine-mediated potentiation of ASIC1a activity is occluded by PcTX1 (psalmotoxin 1), a specific ASIC1a inhibitor binding to its extracellular domain. Functionally, the enhanced channel activity is accompanied by increased acid-induced neuronal membrane depolarization and cytoplasmic Ca(2+) overload, which may partially explain the exacerbated neuronal damage caused by spermine. More importantly, blocking endogenous spermine synthesis significantly attenuates ischemic brain injury mediated by ASIC1a but not that by NMDA receptors. Thus, extracellular spermine contributes significantly to ischemic neuronal injury through enhancing ASIC1a activity. Our data suggest new neuroprotective strategies for stroke patients via inhibition of polyamine synthesis and subsequent spermine-ASIC interaction.

Glycine is a primary inhibitory neurotransmitter in the spinal cord and brainstem. It acts at glycine receptor (GlyR)-chloride channels, as well as a co-agonist of NMDA receptors (NMDARs). In the hippocampus, the study of GlyRs has largely been under-appreciated due to the apparent absence of glycinergic synaptic transmission. Emerging evidence has shown the presence of extrasynaptic GlyRs in the hippocampus, which exert a tonic inhibitory role, and can be highly regulated under many pathophysiological conditions. On the other hand, besides d-serine, glycine has also been shown to modulate NMDAR function in the hippocampus. The simultaneous activation of excitatory NMDARs and inhibitory GlyRs may provide a homeostatic regulation of hippocampal network function. Furthermore, glycine can regulate hippocampal neuronal activity through GlyR-mediated cross-inhibition of GABAergic inhibition, or through the glycine binding site-dependent internalization of NMDARs. Therefore, hippocampal glycine and its receptors may operate in concert to finely regulate hippocampus-dependent high brain function such as learning and memory. Finally, dysfunction of hippocampal glycine signaling is associated with neuropsychiatric disorders. We speculate that further studies of hippocampal glycine-mediated regulation may help develop novel glycine-based approaches for therapeutic developments., ((c) 2010 Elsevier Ltd. All rights reserved.)

The development of chronic pain involves increased sensitivity of peripheral nociceptors and elevated neuronal activity in many regions of the central nervous system. Much of these changes are caused by the amplification of nociceptive signals resulting from the modulation and altered expression of specific ion channels and receptors in the central and peripheral nervous system. Understanding the processes by which these ion channels and receptors are regulated and how these mechanisms malfunction may lead to new treatments for chronic pain. Here we review the contribution of the Ca2+-permeable acid-sensing ion channel (ASIC(Ca)) in the development and persistence of chronic pain, and the potential underlying mechanisms. Accumulating evidence suggests that ASIC(Ca) represents an attractive new target for developing effective therapies for chronic pain.

Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR alpha2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

Fluoxetine is a selective serotonin reuptake inhibitor widely used for treating depression. However, fluoxetine treatment may lead to seizures at higher doses, which underlying mechanism remains largely unknown. In this study, we examined the effects of fluoxetine on glycine receptor (GlyR) activity. Using the whole-cell patch-clamp recording method, we found that fluoxetine and its metabolite norfluoxetine inhibited glycine-induced currents in cultured rat hippocampal neurons. This inhibition was dose-dependent, and voltage-independent. Fluoxetine shifted the glycine concentration-response curve to the right without altering the maximal current. Both Lineweaver-Burk and Schild plots suggest competitive inhibition. The amount of fluoxetine inhibition significantly increased when homomeric GlyRs were selectively inhibited with picrotoxin. Moreover, fluoxetine inhibited the current mediated by heteromeric alpha2beta- but not homomeric alpha2-GlyRs transiently expressed in HEK293T cells. These results suggest that fluoxetine is a competitive and subtype-selective GlyR inhibitor, which may explain its capacity to induce seizures.

The GABA system is highly involved in the pathophysiology of mood disorders such as depression. Altered GABAergic function is evident in depressed patients and animal models of depression. Currently, the most widely used antidepressants are selective 5-HT reuptake inhibitors, such as fluoxetine. However, the effects of fluoxetine on GABAergic synaptic neurotransmission remain poorly investigated. Whole-cell patch-clamp recordings from cultured rat hippocampal neurons were therefore conducted to investigate the effects of fluoxetine on GABAergic neurotransmission. The spontaneous inhibitory postsynaptic current (sIPSC) was completely blocked by 10 microM bicuculline and reversibly potentiated by 30 microM fluoxetine. The fluoxetine potentiation on either amplitude or frequency of sIPSCs was dose-dependent, with the EC(50) values of 10.96 and 14.26 microM, respectively. This potentiation was also TTX-insensitive, suggesting independence of presynaptic action potentials. The ritanserin (5 microM), a selective 5-HT(2) receptor antagonist, did not alter the fluoxetine potentiation on miniature inhibitory postsynaptic currents. Taken together, our data suggest that fluoxetine can potentiate GABAergic neurotransmission without depending on presynaptic firing of action potentials and its elevating of 5-HT receptor activities. This potentiation by fluoxetine may normalize the hippocampal GABA deficit during depression and in part exert its antidepressant activity.

Salicylate is the major metabolite and active component of aspirin (acetylsalicylic acid), which is widely used in clinical medicine for treating inflammation, pain syndromes and cardiovascular disorders. The well-known mechanism underlying salicylate's action mainly involves the inhibition of cyclooxygenase and subsequent decrease in prostaglandin production. Recent evidence suggests that salicylate also affects neuronal function through interaction with specific membrane channels/receptors. However, the effect of salicylate on synaptic and neural network function remains largely unknown. In this study, we investigated the effect of sodium salicylate on the synaptic transmission and neuronal excitation in the hippocampal CA1 area of rats, a key structure for many complex brain functions. With electrophysiological recordings in hippocampal slices, we found that sodium salicylate significantly enhanced neuronal excitation through reducing inhibitory GABAergic transmission without affecting the basal excitatory synaptic transmission. Salicylate significantly inhibited the amplitudes of both evoked and miniature inhibitory postsynaptic currents, and directly reduced gamma-aminobutyric acid type A (GABA(A)) receptor-mediated responses in cultured rat hippocampal neurons. Together, our results suggest that the widely used aspirin might impair hippocampal synaptic and neural network functions through its actions on GABAergic neurotransmission. Given the capability of aspirin to penetrate the blood-brain barrier, the present data imply that aspirin intake may cause network hyperactivity and be potentially harmful in susceptible subpopulations.

Changes of extracellular pH values can have profound effects on neuronal function. For example, the low pH (also called acidosis) generated in brain ischemia causes acute neuronal injury. For years the receptors that detect pH variations surrounding neurons and their physiological/pathological importance remain uncertain. The recent finding that acidosis activates a distinct family of membrane ion channels, the acid-sensing ion channels (ASICs) in both peripheral and central neurons has dramatically changed the view of acidosis-associated signaling and provided a new strategy for therapeutic inventions. Although proton is the only known agonist for the activation of ASICs, a variety of extracellular and intracellular signaling molecules can modulate the activities of ASICs and have profound influence on the functions of these channels in both physiological and pathological processes. The goal of this article is therefore to provide a comprehensive review of the modulators of ASICs that adapt ASIC activity to changes of extracellular and intracellular environments.

Amiloride, a potassium sparing diuretic, is well known to interact with many ion transport systems and modulate the activity of several membrane receptors. However, relatively little information is available as to how amiloride affects membrane receptors of neurons in the brain areas. In the present study, we investigated the effects of amiloride on glycine-induced currents (I(Gly)) in cultured neurons of rat inferior colliculus with whole-cell patch-clamp recordings. Amiloride itself did not activate any current across the neuronal membrane but it reversibly inhibited the amplitude of the I(Gly) in a reversible and concentration-dependent manner, with an IC(50) of 487.4+/-25.3microM (n=5). Amiloride shifted the concentration-response relationship to the right without changing Hill coefficient and without changing the maximum response of the I(Gly). The pre-perfusion of amiloride produced an inhibitory effect on the I(Gly). In addition, amiloride was shown with a voltage ramp protocol to significantly reduce the conductance induced by glycine but not to change the reversal potential of the I(Gly). These results demonstrate that amiloride competitively inhibits the I(Gly) in rat inferior colliculus neurons by decreasing the affinity of glycine to its receptor. Our finding suggests that attention should be paid to the possible side effects of amiloride used as a drug on brain functions in the case of a defective blood-brain barrier and in the case of direct application of this drug into the cerebrospinal fluid for treatment of brain tumors.

Sodium salicylate (SS) is a medicine for anti-inflammation and for chronic pain relief with a side effect of tinnitus. To understand the cellular mechanisms of tinnitus induced by SS in the central auditory system, we examined effects of SS on evoked and miniature inhibitory postsynaptic currents (eIPSCs and mIPSCs) recorded from layer II/III pyramidal neurons of rat auditory cortex in a brain slice preparation with whole-cell patch-clamp techniques. Both eIPSCs and mIPSCs recorded from the auditory cortex could be completely blocked by bicuculline, a selective GABA(A) receptor antagonist. SS did not change the input resistance of neurons but was found to reversibly depress eIPSCs in a concentration-dependent manner. SS reduced eIPSCs to 82.3% of the control level at 0.5 mM (n=7) and to 60.9% at 1.4 mM (n=12). In addition, SS at 1.4 mM significantly reduced the amplitude of mIPSCs from 24.12+/-1.44 pA to 19.92+/-1.31 pA and reduced the frequency of mIPSCs from 1.34+/-0.23 Hz to 0.89+/-0.13 Hz (n=6). Our results demonstrate that SS attenuates inhibitory postsynaptic currents in the auditory cortex, suggesting that the alteration of inhibitory neural circuits may be one of the cellular mechanisms for tinnitus induced by SS in the central auditory region.

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca(2+) permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASIC1a-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca(2+) elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K+ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd2+ enhanced the peak inactivating current (I(A)) without affecting the delayed component (I(K)). The same results were obtained in Ca(2+)-free external solution, and from slo1 mutation, which eliminates transient Ca(2+)-activated K+ current. Micromolar Cd2+ and Zn2+, and millimolar Ca2+ and Mg2+ all shifted the steady-state inactivation curve of I(A) without affecting the voltage-dependence of I(A) activation, whereas millimolar Cd2+ markedly affected both the activation and steady-state inactivation curves for I(A). Divalent cations affected I(A) with different potency; the sequence was: Zn2+ > Cd2+ > Ca2+ > Mg2+. The modulation of I(A) by Cd2+ was partially inhibited in Sh(M), a null Shaker (one of I(A)-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on I(A) activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K+ channels through specific binding to extracellular sites of the channels.

Increasing evidence suggests that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal apoptosis in brain ischemia. To further study the roles of JNK activation in hippocampal CA1 neurons in a rat model of transient global ischemia, we assessed the effect of JNK inhibition by SP600125 on the degree of brain injury. Our results demonstrated that SP600125 significantly increased the number of surviving cells in hippocampal CA1 subfield and decreased the activation of p-JNK1/2 and p-JNK3 at 30 min and 3 days after brain ischemia. Moreover, SP600125 significantly diminished the increased levels of phosphorylated-c-Jun (Ser63/73) and phosphorylated-Bcl-2 (Ser87) at 3 h after brain ischemia. These results indicate that SP600125, a new inhibitor of JNK, protected transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region at least via suppressing the activation of nuclear substrate (c-Jun) and inactivating non-nuclear substrate (Bcl-2) induced by ischemic insult. Thus, inhibiting JNK activity by SP600125 may represent a new and effective strategy to treat ischemic stoke.

Taurine (Tau) is one of the most abundant free amino acids in the mammalian central nervous system. Whether the neurotransmission of the central auditory system is regulated or modulated by Tau is not clear. In the present study, we investigated the electrophysiological and pharmacological properties of Tau-activated currents in acutely dissociated neurons of the rat inferior colliculus (IC) using whole cell patch clamp recordings. At a holding potential of -60 mV and under a condition of chloride equilibrium potential near 0 mV, Tau activated an inward current and its half-maximal activation concentration was equal to 0.37 mM. The measured reversal potential of Tau-activated currents was close to theoretical chloride equilibrium potential. The currents evoked by Tau at both low (1 mM) and high (10 mM) concentrations were almost completely inhibited by strychnine, a glycine receptor antagonist. The Tau-activated current, however, was not affected by bicuculline, a GABA(A) receptor antagonist. Tau at increased concentrations progressively reduced the current response to subsequent glycine application. At saturated concentrations, Tau-activated current and glycine-activated current were mutually cross-desensitized by each other. These findings indicate that Tau activates glycine receptors in neurons of the rat IC and thus may have a functional role in regulating or modulating the neurotransmission of the central auditory system in mammals.

The characterization of acid-sensing ion channel (ASIC)-like currents has been reported in hippocampal neurons in primary culture. However, it is suggested that the profile of expression of ASICs changes in culture. In this study, we investigated the properties of proton-activated current and its modulation by extracellular Ca(2+) and Zn(2+) in neurons acutely dissociated from the rat hippocampal CA1 using conventional whole-cell patch-clamp recording. A rapidly decaying inward current and membrane depolarization was induced by exogenous application of acidic solution. The current was sensitive to the extracellular proton with a response threshold of pH 7.0-6.8 and the pH(50) of 6.1, the reversal potential close to the Na(+) equilibrium potential. It had a characteristic of acid-sensing ion channels (ASICs) as demonstrated by its sensitivity to amiloride (IC(50)=19.6+/-2.1 microM). Either low [Ca(2+)](o) or high [Zn(2+)](o) increased the amplitude of the current. All these characteristics are consistent with a current mediated through a mixture of homomeric ASIC1a and heteromeric ASIC1a+2a channels and closely replicate many of the characteristics that have been previously reported for hippocampal neurons cultured for a week or more, indicating that culture artifacts do not necessarily flaw the properties of ASICs. Interestingly, we found that high [Zn(2+)](o) (>10(-4) M) slowed the decay time constant of the ASIC-like current significantly in both acutely dissociated and cultured hippocampal neurons. In addition, the facilitating effects of low [Ca(2+)](o) and high [Zn(2+)](o) on the ASIC-like current were not additive. Since tissue acidosis, extracellular Zn(2+) elevation and/or Ca(2+) reduction occur concurrently under some physiological and/or pathological conditions, the present observations suggest that hippocampal ASICs may offer a novel pharmacological target for therapeutic invention.

Many ionotropic receptors are modulated by extracellular H+. So far, few studies have directly addressed the role of such modulation at synapses. In the present study, we investigated the effects of changes in extracellular pH on glycinergic miniature inhibitory postsynaptic currents (mIPSCs) as well as glycine-evoked currents (IGly) in mechanically dissociated spinal neurons with native synaptic boutons preserved. H+ modulated both the mIPSCs and IGly biphasically, although it activated an amiloride-sensitive inward current by itself. Decreasing extracellular pH reversibly inhibited the amplitude of the mIPSCs and IGly, while increasing external pH reversibly potentiated these parameters. Blockade of acid-sensing ion channels (ASICs) with amiloride, the selective antagonist of ASICs, or decreasing intracellular pH did not alter the modulatory effect of H+ on either mIPSCs or IGly. H+ shifted the EC50 of the glycine concentration-response curve from 49.3 +/- 5.7 microM at external pH 7.4 to 131.5 +/- 8.1 microM at pH 5.5, without altering the Cl- selectivity of the glycine receptor (GlyR), the Hill coefficient and the maximal IGly, suggesting a competitive inhibition of IGly by H+. Both Zn2+ and H+ inhibited IGly. However, H+ induced no further inhibition of IGly in the presence of a saturating concentration of Zn2+. In addition, H+ significantly affected the kinetics of glycinergic mIPSCs and IGly. It is proposed that H+ and/or Zn2+ compete with glycine binding and inhibit the amplitude of glycinergic mIPSCs and IGly. Moreover, binding of H+ induces a global conformational change in GlyR, which closes the GlyR Cl- channel and results in the acceleration of the seeming desensitization of IGly as well as speeding up the decay time constant of glycinergic mIPSCs. However, the deprotonation rate is faster than the unbinding rate of glycine from the GlyR, leading to reactivation of the undesensitized GlyR after washout of agonist and the appearance of a rebound IGly. H+ also modulated the glycine cotransmitter, GABA-activated current (IGABA). Taken together, the results support a "conformational coupling" model for H+ modulation of the GlyR and suggest that H+ may act as a novel modulator for inhibitory neurotransmission in the mammalian spinal cord.

By electrophysiological methods, cultured Drosophila embryonic and larval central neurons have been widely used to study ion channels, neurotransmitter release and intracellular message regulation. Voltage-activated K(+) channels play a crucial role in repolarizing the membrane following action potentials, stabilizing membrane potentials and shaping firing patterns of cells. In this study, a mechanical vibration-isolation system was used to produce a sufficient number of acutely dissociated larval central neurons, of which the majority were type II neurons (2~5 microm in diameter). Using patch clamp technique, the whole-cell K(+) currents in type II neurons were characterized by containing a transient 4-AP-sensitive current (I(A)) and a more slowly inactivating, TEA-sensitive component (I(K)). According to their kinetic properties, five types of whole-cell K(+) currents were identified. Type A current exhibited primarily fast transient K(+) currents that activated and inactivated rapidly. The majority of the neurons, however, slowly inactivated K(+) currents with variable inactivation time course (type B current). Type C current, being present in a small number of the cells, was mainly composed of noninactivating components. Some of the neurons expressed both transient and slow inactivating components, but the slowly inactivating components could reach more than 50% of the peak current (type D current). Type E current showed distinct voltage-dependent activation properties, characterized by its bell-shaped activation curve. Type E current was inhibited by application of Ca(2+)-free solution or 0.1 mmol/L Cd(2+). Moreover, this novel current ran down much more rapidly than other types. These results indicate that different K(+) channels, which have different kinetic and pharmacological properties, underlie the whole-cell K(+) currents in type II neurons of Drosophila larval central nervous system.

The effect of Radix paeoniae rubra (RPR) on voltage-gated sodium channel (VGSC) currents (I(Na)) was examined in freshly isolated rat hippocampal CA1 neurons using whole-cell patch-clamp technique under voltage-clamp conditions. RPR suppressed I(Na) without affecting the current activation, inactivation and deactivation. The amplitude of I(Na) decreased by approximately 18.4% within a few seconds of 0.8 mg/ml RPR exposure. RPR (0.8 mg/ml) shifted the steady-state inactivation curves of I(Na) to negative potentials, with hyperpolarizing direction shift of V(1/2) of 10.0 mV. The time course of I(Na) recovery from inactivation was prolonged significantly by 0.8 mg/ml RPR. RPR (0.8 mg/ml) also enhanced the activity-dependent attenuation of I(Na) and decreased the fraction of activated channels. These results suggested that RPR suppressed hippocampal CA1 I(Na) by shifting the inactivation curve in hyperpolarizing direction, slowing the recovery time course from inactivation, enhancing the activity-dependent attenuation and decreasing the number of activatable channels. RPR suppression on I(Na) might predict the protective effect during brain ischemia in hippocampal CA1 neurons.

Using whole-cell patch-clamp recording of acutely isolated rat hippocampal CA1 neurons, we studied the state-dependent cross-inhibition between anionic GABA(A) and glycine (Gly) ionotropic receptors. Co-application of relatively high concentrations of GABA and Gly produced a total current (IGABA + Gly) much smaller than the linear summation of the two individual responses. The mutual inhibition between IGABA and IGly was voltage-independent. However, no current inhibition was observed with co-application of low concentrations of these two agonists. The hippocampal neurons lacking Gly response (IGly) showed no current inhibition with GABA current (IGABA), whereas the neurons losing GABA response (IGABA) (after complete rundown of IGABA) showed no current inhibition with IGly. The current inhibition was also absent in the presence of Gly or GABA(A) receptor antagonists, strychnine or bicuculline, respectively. The results indicate that cross-inhibition between GABA(A) and Gly receptors is state-dependent and a direct receptor-receptor interaction might enable the cross-inhibition to occur.

Mitochondrial dysfunction has been regarded as one of the major contributors of ischemic neuronal death after stroke. Recently, intercellular mitochondrial transfer between different cell types has been widely studied and suggested as a potential therapeutic approach. However, whether mitochondria are involved in the neuron-glia cross-talk following ischemic stroke and the underlying mechanisms have not been explored yet. In this study, we demonstrated that under physiological condition, neurons release few mitochondria into the extracellular space, and the mitochondrial release increased when subjected to the challenges of acidosis, hydrogen peroxide (H2O2), N -methyl-D-aspartate (NMDA), or glutamate. Acidosis reduced the mitochondrial basal respiration and lowered the membrane potential in primary-cultured mouse cortical neurons. These defective mitochondria were prone to be expelled to the extracellular space by the injured neurons, and were engulfed by adjacent astrocytes, leading to increased astrocytic expressions of mitochondrial Rho GTPase 1 (Miro 1) and mitochondrial transcription factor A (TFAM) at mRNA level. In mice subjected to transient focal cerebral ischemia, the number of defective mitochondria in the cerebrospinal fluid increased. Our results suggested that the neuron-derived mitochondria may serve as a "help-me" signaling and mediate the neuron-astrocyte cross-talk following ischemic stroke. Promoting the intercellular mitochondrial transfer by accelerating the neuronal releasing or astrocytic engulfing might be a potential and attractive therapeutic strategy for the treatment of ischemic stroke in the future. [ABSTRACT FROM AUTHOR]

Abstract: The potassium-chloride cotransporter 2 (KCC2)-dependent intracellular chloride level determines whether neurons respond to GABA and/or glycine by depolarization or hyperpolarization. However, still unknown is the role of KCC2-dependent chloride homeostasis in regulating the spontaneous activity of neuronal circuits via GABAA receptor (GABAAR) and the glycine receptor (GlyR). In this study, patch-clamp recordings were performed to measure the change of spontaneous neuronal activity in cultured hippocampal neurons. Our results showed that inhibition of KCC2 with furosemide, as well as blockade of GABAAR with bicuculline, significantly enhanced circuit activity. Perfusion with bicuculline further enhanced the effects of furosemide on spontaneous circuit activity, while furosemide did not alter the effects of bicuculline. Surprisingly, blockade of GlyR not only induced obvious tonic currents, but also significantly decreased spontaneous synaptic activity. Moreover, inhibition of KCC2 did not change the depressive effect of strychnine on neuronal circuits. Our findings suggest that KCC2-dependent chloride homeostasis is mainly involved in GABAAR-mediated synaptic inhibition whereas GlyR-mediated tonic action plays a totally different role in regulating hippocampal circuit activity. [Copyright &y& Elsevier]

Abstract: Whole-cell patch-clamp was used to assess the modulatory effect of thiopental (Thio) on glycine (Gly) receptor in mechanically dissociated rat spinal dorsal horn neurons. It was found that Thio inhibited the amplitude, accelerated the desensitization and prolonged the deactivation of Gly-induced currents (I Gly) in a concentration-dependent manner. In addition, a rebound current occurred after washout of the co-application of Gly and Thio in most neurons tested. Moreover, the inhibitory effect of Thio was not the result of cross-inhibition between Gly and GABAA receptors. Furthermore, taurine-induced currents, a low-affinity agonist for Gly receptors, were also markedly inhibited by Thio in a similar way to I Gly. These results indicate that Thio suppresses Gly receptor function and suggest that Thio anesthetic actions might not be mediated by Gly receptors. We speculate that the weak muscle relaxation and the limited analgesic effects observed during Thio anesthesia may attribute to its inhibitory effects on Gly receptors. [Copyright &y& Elsevier]

The effect of amiloride on glycinergic transmission in mechanically dissociated rat spinal dorsal horn neurons was examined with the use of whole-cell patch-clamp recording. Amiloride reversibly reduced both the frequency and amplitude of spontaneous glycinergic miniature IPSCs (mIPSCs) and its inhibitory effect on glycinergic mIPSCs persisted in either Ca2+-free or Na+-free external solutions while it disappeared in K+-free external solution. Analysis of the relationship between mIPSCs amplitude and frequency at various holding potentials shows that amiloride inhibition of glycinergic mIPSC frequency could result indirectly from its inhibition of amplitude. [Copyright &y& Elsevier]

The use of body-focused repetitive behaviors (BFRBs) is conceptualized as a means of coping with stress. However, the neurological mechanism by which repetitive behaviors affect anxiety regulation is unclear. Here, we identify that the excitatory somatostatin-positive neurons in the medial paralemniscal nucleus (MPLSST neurons) in mice promote self-grooming and encode reward. MPLSST neurons display prominent grooming-related neuronal activity. Loss of function of MPLSST neurons impairs both self-grooming and post-stress anxiety alleviation. Activation of MPLSST neurons is rewarding and sufficient to drive reinforcement by activating dopamine (DA) neurons in the ventral tegmental area (VTA) and eliciting dopamine release. The neuropeptide SST facilitates the rewarding impact of MPLSST neurons. MPLSST neuron-mediated self-grooming is triggered by the input from the central amygdala (CeA). Our study reveals a dual role of CeA-MPLSST-VTADA circuit in self-grooming and post-stress anxiety regulation and conceptualizes MPLSST neurons as an interface linking the stress and reward systems in mice. [Display omitted] • MPLSST neurons promote self-grooming and show grooming-related neuronal activity • MPLSST neurons are glutamatergic and encode reward value • The neuropeptide SST facilitates the rewarding impact of MPLSST neurons • Inhibition of MPLSST neurons impairs post-stress anxiety regulation Sun et al. identify a CeA-MPLSST-VTADA circuit controlling self-grooming and post-stress anxiety alleviation by activation of the reward system in mice. This study provides insights into how repetitive behavior-related neurons connect to and influence emotion regulation-related neurons, with significant implications for the neuropsychological mechanisms of body-focused repetitive behaviors (BFRBs). [ABSTRACT FROM AUTHOR]

Summary: Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H+)-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular “palm” domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis. [ABSTRACT FROM AUTHOR]

Abstract: Taurine is an important endogenous amino acid for neural development and for many physiological functions, but little is known about its functional role in the central auditory system. We investigated in young rats (P10–P14) the effects of taurine on the neuronal responses and synaptic transmissions in the central nucleus of the inferior colliculus (ICC) with a brain slice preparation and with whole-cell patch-clamp recordings. Perfusion of taurine at 1mM reliably evoked a current across the membrane and decreased the input resistance in neurons of the ICC. Taurine also depressed the spontaneous and current-evoked firing of ICC neurons. All these effects were reversible after washout and could be blocked by 3μM strychnine, an antagonist of glycine receptors, but not by 10μM bicuculline, an antagonist of GABA(A) receptors. When the inhibitory receptors were not pharmacologically blocked, taurine reversibly reduced the postsynaptic currents/potentials evoked by electrically stimulating the commissure of the inferior colliculus or the ipsilateral lateral lemniscus. The results demonstrate that taurine reduces the neuronal excitability and depresses the synaptic transmission in the ICC by activating glycine-gated chloride channels. Our findings suggest that taurine acts as a ligand of glycine receptors in the ICC and can be involved in the information processing of the central auditory system similarly like the neurotransmitter glycine. [Copyright &y& Elsevier]

The characterization of acid-sensing ion channel (ASIC)-like currents has been reported in hippocampal neurons in primary culture. However, it is suggested that the profile of expression of ASICs changes in culture. In this study, we investigated the properties of proton-activated current and its modulation by extracellular Ca2+ and Zn2+ in neurons acutely dissociated from the rat hippocampal CA1 using conventional whole-cell patch-clamp recording. A rapidly decaying inward current and membrane depolarization was induced by exogenous application of acidic solution. The current was sensitive to the extracellular proton with a response threshold of pH 7.0–6.8 and the pH50 of 6.1, the reversal potential close to the Na+ equilibrium potential. It had a characteristic of acid-sensing ion channels (ASICs) as demonstrated by its sensitivity to amiloride (IC50=19.6±2.1 μM). Either low [Ca2+]o or high [Zn2+]o increased the amplitude of the current. All these characteristics are consistent with a current mediated through a mixture of homomeric ASIC1a and heteromeric ASIC1a+2a channels and closely replicate many of the characteristics that have been previously reported for hippocampal neurons cultured for a week or more, indicating that culture artifacts do not necessarily flaw the properties of ASICs. Interestingly, we found that high [Zn2+]o (>10-4 M) slowed the decay time constant of the ASIC-like current significantly in both acutely dissociated and cultured hippocampal neurons. In addition, the facilitating effects of low [Ca2+]o and high [Zn2+]o on the ASIC-like current were not additive. Since tissue acidosis, extracellular Zn2+ elevation and/or Ca2+ reduction occur concurrently under some physiological and/or pathological conditions, the present observations suggest that hippocampal ASICs may offer a novel pharmacological target for therapeutic invention. [Copyright &y& Elsevier]

Electrophysiological properties of etomidate (ET)-induced current (IET) at different concentrations and effects of ET at clinically relevant concentrations (1–10 μM) on postsynaptic GABAA receptor function were investigated using whole-cell patch-clamp technique in mechanically dissociated rat spinal dorsal horn neurons. The results showed that ET actions were concentration-dependent: low concentrations (10 μM) of ET potentiated GABA-activated current (IGABA), slowed activation, desensitization and deactivation of GABAA receptors; moderate concentrations (10–1000 μM) of ET directly activated and desensitized GABAA receptors; high concentrations (>1000 μM) of ET produced an inhibitory effect on IET. In addition, ET prolonged the duration of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the mechanically dissociated rat dorsal horn neurons. These results suggest that general anesthetics-induced changes at spinal level could significantly contribute to analgesia and general anesthesia. [Copyright &y& Elsevier]

Itch is an aversive sensation that evokes a desire to scratch. Paradoxically, scratching the itch also produces a hedonic experience. The specific brain circuits processing these different aspects of itch, however, remain elusive. Here, we report that GABAergic (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) are activated with different temporal patterns during acute and chronic itch. DA neuron activation lags behind GABA neurons and is dependent on scratching of the itchy site. Optogenetic manipulations of VTA GABA neurons rapidly modulated scratching behaviors through encoding itch-associated aversion. In contrast, optogenetic manipulations of VTA DA neurons revealed their roles in sustaining recurrent scratching episodes through signaling scratching-induced reward. A similar dichotomy exists for the role of VTA in chronic itch. These findings advance understanding of circuit mechanisms of the unstoppable itch-scratch cycles and shed important insights into chronic itch therapy. • Itch elicits distinct activation patterns of GABA and DA neurons in VTA • GABA neurons signal aversion toward itch • DA neurons mediate reward to scratch-induced relief of itch • VTA neurons regulate scratching behaviors in chronic itch Su et al. demonstrate that VTA GABA neurons signal aversion to itch and DA neurons mediate scratching-associated reward. Differential activation and function of VTA neurons may underlie the progression of vicious itch-scratch cycles. [ABSTRACT FROM AUTHOR]