Search

Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca2+] increase. The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gβγ subunits, but not by 8-bromoadenosine 3′,5′-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gβγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.

Neurotransmitters can be released either synchronously or asynchronously with respect to action potential timing. Synapsins (Syns) are a family of synaptic vesicle (SV) phosphoproteins that assist gamma-aminobutyric acid (GABA) release and allow a physiological excitation/inhibition balance. Consistently, deletion of either or both Syn1 and Syn2 genes is epileptogenic. In this work, we have characterized the effect of SynI knockout (KO) in the regulation of GABA release dynamics. Using patch-clamp recordings in hippocampal slices, we demonstrate that the lack of SynI impairs synchronous GABA release via a reduction of the readily releasable SVs and, in parallel, increases asynchronous GABA release. The effects of SynI deletion on synchronous GABA release were occluded by ω-AgatoxinIVA, indicating the involvement of P/Q-type Ca2+channel-expressing neurons. Using in situ hybridization, we show that SynI is more expressed in parvalbumin (PV) interneurons, characterized by synchronous release, than in cholecystokinin or SOM interneurons, characterized by a more asynchronous release. Optogenetic activation of PV and SOM interneurons revealed a specific reduction of synchronous release in PV/SynIKO interneurons associated with an increased asynchronous release in SOM/SynIKO interneurons. The results demonstrate that SynI is differentially expressed in interneuron subpopulations, where it boosts synchronous and limits asynchronous GABA release.

The escape response and rhythmic swimming in zebrafish are distinct behaviors mediated by two functionally distinct motoneuron (Mn) types. The primary (1°Mn) type depresses, has a large quantal content (Qc), and a high release probability (Pr). Conversely, the secondary (2°Mn) type facilitates and has low and variable Qc and Pr. This functional duality matches well the distinct associated behaviors, with the 1°Mn providing the strong, singular C-bend initiating escape and the 2°Mn confers weaker, rhythmic contractions. Contributing to these functional distinctions is our identification of P/Q type calcium channels mediating transmitter release in 1°Mns and N type channels in 2°Mns. Remarkably, despite these functional and behavioral distinctions, all ~15 individual synapses on each muscle cell are shared by a 1°Mn bouton and at least one 2°Mn bouton. This novel blueprint of synaptic sharing provides an efficient way of controlling two different behaviors at the level of a single postsynaptic cell.

Indole-3-carbinol (I3C), found in cruciferous vegetables, has been proposed to exhibit neuroprotective effects. This study aimed to investigate the effect of the I3C derivative [1(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (CIM), which has superior pharmacokinetic properties to I3C, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). We observed that CIM dose-dependently inhibited glutamate release evoked by the potassium channel blocker 4-aminopyridine (4-AP). CIM-mediated inhibition of glutamate release was attributed to reduced exocytosis, as it correlated with the removal of extracellular calcium and blocking of the vesicular glutamate transporter but not the glutamate transporter. In addition, CIM decreased 4-AP-evoked intrasynaptosomal Ca

The glutamatergic neurotransmitter system has received substantial attention in research on the pathophysiology and treatment of neurological disorders. The study investigated the effect of the polyphenolic compound chlorogenic acid (CGA) on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). CGA inhibited 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes. This inhibition was prevented in the absence of extracellular Ca2+ and was associated with the inhibition of 4-AP-induced elevation of Ca2+ but was not attributed to changes in synaptosomal membrane potential. In line with evidence observed through molecular docking, CGA did not inhibit glutamate release in the presence of P/Q-type Ca2+ channel inhibitors, therefore, CGA-induced inhibition of glutamate release may be mediated by P/Q-type Ca2+ channels. CGA-induced inhibition of glutamate release was also diminished by the calmodulin and Ca2+/calmodilin-dependent kinase II (CaMKII) inhibitors, and CGA reduced the phosphorylation of CaMKII and its substrate, synapsin I. Furthermore, pretreatment with intraperitoneal CGA injection attenuated the glutamate increment and neuronal damage in the rat cortex that were induced by kainic acid administration. These results indicate that CGA inhibits glutamate release from cortical synaptosomes by suppressing P/Q-type Ca2+ channels and CaMKII/synapsin I pathways, thereby preventing excitotoxic damage to cortical neurons.

The medial prefrontal cortex (mPFC) plays a key role in higher functions such as memory and attention. In order to demonstrate sensory responses in the mPFC, we used electrophysiological recordings of urethane-anesthetized rats to record somatosensory-evoked potentials (SEPs) or auditory-evoked potentials (AEPs) elicited by whisker deflections and click stimulation, respectively. Contralateral whisker stimulation or auditory stimuli were also applied to study sensory interference in the mPFC. Interference with other sensory stimuli or recent stimulation history reduced whisker responses in the infralimbic and prelimbic cortices of the ventral mPFC. This effect could be mediated by activation of parvalbumin (PV) interneurons since the effect was blocked by the P/Q calcium channel antagonist ω-agatoxin. In contrast, sensory interference or the recent stimulation history was not detected by the dorsal mPFC or the primary somatosensory cortex. Results obtained from retrograde tracer injections in the dorsal and ventral regions of the mPFC indicated that somatosensory and auditory sensory inputs may arrive at the dorsal mPFC through secondary sensory cortical areas, and through the insular and temporal cortical areas. The ventral mPFC may receive sensory information through the strong anatomical connections between the dorsal and ventral mPFC areas. In conclusion, results suggest mPFC plays an important role in sensory processing, which may have important implications in attentional and memory processes.

Presynaptic active zones (AZs) contain many molecules essential for neurotransmitter release and are assembled in a highly organized manner. A network of adaptor proteins known as cytomatrix at the AZ (CAZ) is important for shaping the structural characteristics of AZ. Rab3-interacting molecule (RIM)-binding protein (RBP) family are binding partners of the CAZ protein RIM and also bind the voltage-gated calcium channels (VGCCs) in mice and flies. Here, we investigated the physiological roles of RIMB-1, the homolog of RBPs in the nematodeCaenorhabditis elegans. RIMB-1 is expressed broadly in neurons and predominantly localized at presynaptic sites. Loss-of-function animals ofrimb-1displayed slight defects in motility and response to pharmacological inhibition of synaptic transmission, suggesting a modest involvement ofrimb-1in synapse function. We analyzed genetic interactions ofrimb-1by testing candidate genes and by an unbiased forward genetic screen forrimb-1enhancer. Both analyses identified the RIM homolog UNC-10 that acts together with RIMB-1 to regulate presynaptic localization of the P/Q-type VGCC UNC-2/Cav2. We also find that the precise localization of RIMB-1 to presynaptic sites requires presynaptic UNC-2/Cav2. RIMB-1 has multiple FN3 and SH3 domains. Our transgenic rescue analysis with RIMB-1 deletion constructs revealed a functional requirement of a C-terminal SH3 in regulating UNC-2/Cav2 localization. Together, these findings suggest a redundant role of RIMB-1/RBP and UNC-10/RIM to regulate the abundance of UNC-2/Cav2 at the presynaptic AZ inC. elegans, depending on the bidirectional interplay between CAZ adaptor and channel proteins.SIGNIFICANCE STATEMENTPresynaptic active zones (AZs) are highly organized structures for synaptic transmission with characteristic networks of adaptor proteins called cytomatrix at the AZ (CAZ). In this study, we characterized a CAZ protein RIMB-1, named for RIM-binding protein (RBP), in the nematodeCaenorhabditis elegans. Through systematic analyses of genetic interactions and an unbiased genetic enhancer screen ofrimb-1, we revealed a redundant role of two CAZ proteins RIMB-1/RBP and UNC-10/RIM in regulating presynaptic localization of UNC-2/Cav2, a voltage-gated calcium channel (VGCC) critical for proper neurotransmitter release. Additionally, the precise localization of RIMB-1/RBP requires presynaptic UNC-2/Cav2. These findings provide new mechanistic insight about how the interplay among multiple CAZ adaptor proteins and VGCC contributes to the organization of presynaptic AZ.

Small-conductance Ca

It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by CaExocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic CaCells stimulated by brief depolatizations in absence of extracellular CaWe demonstrated that Ca

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+(Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+coscillations have not been elucidated. As β-cell Ca2+coscillations are regulated in part by Ca2+-activated K+(Kslow) currents, this work investigated the role of Kslowin α-cell Ca2+handling and GCG secretion. α-Cells displayed Kslowcurrents that were dependent on Ca2+influx through L- and P/Q-type voltage-dependent Ca2+channels (VDCCs) as well as Ca2+released from endoplasmic reticulum stores. α-Cell Kslowwas decreased by small-conductance Ca2+-activated K+(SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+(BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+(IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslowwith apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+influx. Total α-cell Ca2+cwas similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+cfollowing prolonged Kslowinhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslowactivation provides a hyperpolarizing influence on α-cell Vmthat sustains Ca2+entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+cis elevated during secretagogue stimulation, Kslowactivation helps to preserve GCG secretion.

Epigenetic mechanisms (i.e., histone post-translational modification and DNA methylation) play a role in regulation of gene expression. The pedunculopontine nucleus (PPN), part of the reticular activating system, manifests intrinsic gamma oscillations generated by voltage-dependent, high threshold N- and P/Q-type Ca2+ channels. We studied whether PPN intrinsic gamma oscillations are affected by inhibition of histone deacetylation. We showed that, a) acute in vitro exposure to the histone deacetylation Class I and II inhibitor trichostatin A (TSA, 1 μM) eliminated oscillations in the gamma range, but not lower frequencies, b) pre-incubation with TSA (1 μM, 90–120 min) also decreased gamma oscillations, c) Ca2+ currents (ICa) were reduced by TSA, especially on cells with P/Q-type channels, d) a HDAC Class I inhibitor MS275 (500 nM), and a Class IIb inhibitor Tubastatin A (150–500 nM), failed to affect gamma oscillations, e) MC1568, a HDAC Class IIa inhibitor (1 μM), blocked gamma oscillations, and f) the effects of both TSA and MC1568 were blunted by blockade of CaMKII with KN-93 (1 μM). These results suggest a cell type specific effect on gamma oscillations when histone deacetylation is blocked, suggesting that gamma oscillations through P/Q-type channels modulated by CaMKII may be linked to processes related to gene transcription. Fil: Urbano Suarez, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. University of Arkansas for Medical Sciences; Estados Unidos Fil: Bisagno, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentina Fil: Mahaffey, Susan. University of Arkansas for Medical Sciences; Estados Unidos Fil: Lee, Sang-hun. University of Arkansas for Medical Sciences; Estados Unidos Fil: Garcia Rill, Edgar. University of Arkansas for Medical Sciences; Estados Unidos

Enteric inhibitory motoneurons use nitric oxide and a purine neurotransmitter to relax gastrointestinal smooth muscle. Enteric P/Q-type Ca2+ channels contribute to excitatory neuromuscular transmission; their contribution to inhibitory transmission is less clear. We used the colon from tottering mice ( tg/tg, loss of function mutation in the α1A pore-forming subunit of P/Q-type Ca2+ channels) to test the hypothesis that P/Q-type Ca2+ channels contribute to inhibitory neuromuscular transmission and colonic propulsive motility. Fecal pellet output in vivo and the colonic migrating motor complex (ex vivo) were measured. Neurogenic circular muscle relaxations and inhibitory junction potentials (IJPs) were also measured ex vivo. Colonic propulsive motility in vivo and ex vivo was impaired in tg/tg mice. IJPs were either unchanged or somewhat larger in tissues from tg/tg compared with wild-type (WT) mice. Nifedipine (L-type Ca2+ channel antagonist) inhibited IJPs by 35 and 14% in tissues from tg/tg and WT mice, respectively. The contribution of N- and R-type channels to neuromuscular transmission was larger in tissues from tg/tg compared with WT mice. The resting membrane potential of circular muscle cells was similar in tissues from tg/tg and WT mice. Neurogenic relaxations of circular muscle from tg/tg and WT mice were similar. These results demonstrate that a functional deficit in P/Q-type channels does not alter propulsive colonic motility. Myenteric neuron L-type Ca2+ channel function increases to compensate for loss of functional P/Q-type Ca2+ channels. This compensation maintains inhibitory neuromuscular transmission and normal colonic motility.

ω-Agatoxin IV A, a peptidyl toxin from Agelenopsis aperta venom, selectively binds to voltage-gated P/Q-type calcium channels. ω-Agatoxin IV A has been used as a selective tool in pharmacological and electrophysiological studies. Visualization of P/Q-type calcium channels has previously been accomplished using biotin-conjugated ω-Agatoxin IV A in freshly prepared mouse cerebellar and hippocampal slices (Nakanishi et al, J. Neurosci. Res., 41: , 532, 1995). Here biotinylated ω-agatoxin IV A was applied to transcardially fixed brain slices prepared with various fixatives. ω-Agatoxin IV A did not bind to fixed tissues from P/Q-type calcium channel knockout mice, confirming that binding to normal, fixed tissues was not an artifact. Using transmission electron microscopy, locations of biotinylated ω-agatoxin IV A binding sites visualized with gold-conjugated streptavidin showed a similar pattern to those visualized with antibody. The ability of biotinylated ω-agatoxin IV A to bind to fixed tissue provides a new cytochemical technique to study molecular architecture of synapses.

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon‐like peptide‐1 (GLP‐1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP‐1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only

Patient: Female, 55 Final Diagnosis: Cerebellar ataxia due to voltage-gated calcium channel and glutamic acid decarboxylase autoantibodies Symptoms: Ataxia Medication: — Clinical Procedure: — Specialty: Neurology Objective: Rare disease Background: Autoimmune cerebellar ataxia can be paraneoplastic in nature or can occasionally present without evidence of an ongoing malignancy. The detection of specific autoantibodies has been statistically linked to different etiologies. Case Report: A 55-year-old African-American woman with hypertension and a past history of morbid obesity and uncontrolled diabetes status post gastric bypass four years prior to the visit (with significantly improved body mass index and hemoglobin A1c controlled at the time of the clinical encounter) presented to the office complaining of gradual onset of unsteadiness and recurrent falls for the past three years, as well as difficulties coordinating routine daily activities. The neurologic exam showed moderate dysarthria and ataxic gait with bilateral dysmetria and positive Romberg test. Routine laboratory test results were only remarkable for a mild elevation of erythrocyte sedimentation rate, and most laboratory and imaging tests for common causes of ataxia failed to demonstrate an etiology. Upon further workup, evidence of anti-voltage-gated calcium channel and anti-glutamic acid decarboxylase antibody was demonstrated. She was then treated with intravenous immunoglobulins with remarkable clinical improvement. Conclusions: We present a case of antibody-mediated ataxia not associated with malignancy. While ataxia is rarely related to autoantibodies, in such cases it is critical to understand the etiology of this disabling condition in order to treat it correctly. Clinicians should be aware of the possible association with specific autoantibodies and the necessity to rule out an occult malignancy in such cases.

β-Subunits of voltage-gated calcium channels (VGCCs) regulate assembly and membrane localization of the pore-forming α1-subunit and strongly influence channel function. β4-Subunits normally coassociate with α1A-subunits which comprise P/Q-type (Cav2.1) VGCCs. These control acetylcholine (ACh) release at adult mammalian neuromuscular junctions (NMJs). The naturally occurring lethargic (lh) mutation of the β4-subunit in mice causes loss of the α1-binding site, possibly affecting P/Q-type channel expression or function, and thereby ACh release. End-plate potentials and miniature end-plate potentials were recorded at hemidiaphragm NMJs of 5–7-week and 3–5-month-old lh and wild-type (wt) mice. Sensitivity to antagonists of P/Q- [ω-agatoxin IVA (ω-Aga-IVA)], L- (nimodipine), N- (ω-conotoxin GVIA), and R-type [C192H274N52O60S7 (SNX-482)] VGCCs was compared in juvenile and adult lh and wt mice. Quantal content (m) of adult, but not juvenile, lh mice was reduced compared to wt. ω-Aga-IVA (~60%) and SNX-482 (~ 45%) significantly reduced m in adult lh mice. Only Aga-IVA affected wt adults. In juvenile lh mice, ω-Aga-IVA and SNX-482 decreased m by >75% and ~20%, respectively. Neither ω-conotoxin GVIA nor nimodipine affected ACh release in any group. Immunolabeling revealed α1E and α1A, β1, and β3 staining at adult lh, but not wt NMJs. Therefore, in lh mice, when the β-subunit that normally coassociates with α1A to form P/Q channels is missing, P/Q-type channels partner with other β-subunits. However, overall participation of P/Q-type channels is reduced and compensated for by R-type channels. R-type VGCC participation is age-dependent, but is less effective than P/Q-type at sustaining NMJ function.

Although ghrelin receptors have been demonstrated to be widely expressed in the central nervous system and peripheral tissues of mammals, it is still unknown whether ghrelin functions in cerebellar Purkinje neurons. In this study, we identified a novel functional role for ghrelin in modulating P-type Ca 2 + channel (P-type channel) currents ( I Ba ) as well as action-potential firing in rat Purkinje neurons. Our results show that ghrelin at 0.1 μM reversibly decreased I Ba by ~ 32.3%. This effect was growth hormone secretagogue receptor 1a (GHS-R1a)-dependent and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Intracellular application of GDP-β-S and pretreatment with pertussis toxin abolished the inhibitory effects of ghrelin. Dialysis of cells with the peptide QEHA (but not the scrambled peptide SKEE), and a selective antibody raised against the G-protein α o subunit both blocked the ghrelin-induced response. Ghrelin markedly increased protein kinase A (PKA) activity, and intracellular application of PKI 5-24 as well as pretreatment of the cells with the PKA inhibitor KT-5720 abolished ghrelin-induced I Ba decrease, while inhibition of PKC had no such effects. At the cellular level, ghrelin induced a significant increase in action-potential firing, and blockade of GHS-R1a by BIM-28163 abolished the ghrelin-induced hyperexcitability. In summary, these results suggest that ghrelin markedly decreases I Ba via the activation of GHS-R1a, which is coupled sequentially to the activities of G o -protein βγ subunits and the downstream PKA pathway. This could contribute to its physiological functions, including the spontaneous firing of action potentials in cerebellar Purkinje neurons.

The corticothalamic network is responsible for producing many forms of neural oscillations, such as sleep spindles and the slow-wave (

The central amygdala (CeA) GABAergic system is hypothesized to drive the development of alcohol dependence, due to its pivotal roles in the reinforcing actions of alcohol and the expression of negative emotion, anxiety and stress. Recent work has also identified an important role for the CeA corticotropin-releasing factor (CRF) system in the interaction between anxiety/stress and alcohol dependence. We have previously shown that acute alcohol and CRF each increase action potential-independent GABA release in the CeA via their actions at presynaptic CRF type 1 receptors (CRF1s); however, the shared mechanism employed by these two compounds requires further investigation. Here we report that acute alcohol interacts with the CRF/CRF1 system, such that CRF and alcohol act via presynaptic CRF1s and P/Q-type voltage-gated calcium channels to promote vesicular GABA release and that both compounds occlude the effects of each other at these synapses. Chronic alcohol exposure does not alter P/Q-type voltage-gated calcium channel membrane abundance or this CRF1/P/Q-type voltage-gated calcium channel mechanism of acute alcohol-induced GABA release, indicating that alcohol engages this molecular mechanism at CeA GABAergic synapses throughout the transition to dependence. Thus, P/Q-type voltage-gated calcium channels, like CRF1s, are key regulators of the effects of alcohol on GABAergic signaling in the CeA.

The authors studied two patients with Lambert-Eaton myasthenic syndrome (LEMS) in whom the repeated examination did not find specific of LEMS P/Q type voltage-gates calcium channel autoantibodies. The results of clinical testing and electrophysiological examination showed the typical character of movement disorders with the absence of tendon reflexes and signs of disautonomia as well as a decrease in M-response amplitude and phenomena of decrement with low frequency- and increment with high frequency stimulation. Both patients revealed no signs of paraneoplastic process. Autoimmune character of the damage was confirmed by the effectiveness of treatment with glucocorticoid hormones.Обследовали 2 пациентов, у которых повторно проведенные исследования не выявили характерного для миастенического синдрома Ламберта-Итона наличия антител к потенциалзависимым кальциевым каналам класса P/Q. Однако данные клинического тестирования и электрофизиологического обследования выявляли типичный характер двигательных расстройств с отсутствием сухожильных рефлексов и явлениями дизавтономии, а также уменьшением амплитуды М-ответа и феноменов декремента при низкочастотной и инкремента при высокочастотной стимуляции. У пациентов не было выявлено признаков карциноматозного процесса. Аутоиммунный характер поражения подтверждался эффективностью терапии глюкокортикоидными препаратами.

Aims Both N-type and P/Q-type voltage-gated Ca2 + channels (VGCCs) are involved in the induction of long-term potentiation (LTP), the long-lasting increase of synaptic strength, in the central nervous system. To provide further information on the roles of N-type and P/Q-type VGCCs in the induction of LTP at excitatory synapses of trigeminal primary afferents in the spinal trigeminal subnucleus oralis (Vo), we investigated whether they contribute to the induction of LTP by activation of group I metabotropic glutamate receptors (mGluRs). Main methods (S)-3,5-Dihydroxyphenylglycine (DHPG; 10 μM for 5 min), the group I mGluR agonist, was used to induce LTP of excitatory postsynaptic currents that were evoked in the Vo neurons by stimulating the trigeminal track. Key findings Weak blockade of the N-type or P/Q-type VGCCs by ω-conotoxin GVIA or ω-agatoxin IVA, respectively, which inhibited only 20 − 40% of Ca2 + currents recorded in isolated trigeminal ganglion neurons but had no effect on the basal excitatory synaptic transmission, completely blocked the induction of LTP. In contrast, stronger blockade of the channels, which inhibited > 50% of Ca2 + currents and about 30% of basal synaptic transmission, resulted in the development of long-term depression (LTD), the long-lasting decrease of synaptic strength. Interestingly, the postsynaptic mechanism of DHPG-induced LTP, which was determined by paired-pulse ratio, disappeared when LTP was blocked, or LTD occurred, while a presynaptic mechanism still remained. Significance Our data suggest that postsynaptic N-type and P/Q-type VGCCs mediate the DHPG-induced LTP at the trigeminal afferent synapses in the Vo.

Summary Voltage-dependent Ca2+ channels (VGCC) represent the principal source of Ca2+ ions driving evoked neurotransmitter release at presynaptic boutons. In mammals, presynaptic Ca2+ influx is mediated mainly via P/Q-type and N-type VGCC, which differ in their properties. Changes in their relative contributions tune neurotransmission both during development and in Hebbian plasticity. However, whether this represents a functional motif also present in other forms of activity-dependent regulation is unknown. Here, we study the role of VGCC in homeostatic plasticity (HSP) in mammalian hippocampal neurons using optical techniques. We find that changes in evoked Ca2+ currents specifically through P/Q-type, but not N-type, VGCC mediate bidirectional homeostatic regulation of both neurotransmitter release efficacy and the size of the major synaptic vesicle pools. Selective dependence of HSP on P/Q-type VGCC in mammalian terminals has important implications for phenotypes associated with P/Q-type channelopathies, including migraine and epilepsy., Graphical Abstract, Highlights • P/Q-type VGCC regulate homeostatic synaptic plasticity (HSP) in mammals • Changes in synaptic vesicle pool sizes during HSP are also mediated via P/Q-type VGCC • Expression of HSP is independent of N-type VGCC regulation • P/Q-type regulation of HSP may explain phenotypes of P/Q-type channelopathies, Jeans at al. show that both basal neurotransmission and synaptic vesicle pool sizes are specifically regulated by the presynaptic P/Q-type voltage-gated Ca2+ channel during HSP at mammalian hippocampal synapses. This may shed light on mechanisms underlying phenotypes associated with P/Q-type channelopathies, including migraine and epilepsy.

This study describes the expression of the voltage operated calcium channels (VOCCs) subunits α1A (typical of the P/Q family) and α1D (of the L family) in the bovine hypothalamus. The expression of both P/Q and L families has been characterized in the brain of adult mammals. However, their distribution and expression during foetal neuronal differentiation have not yet been determined. The expression profile of the α1A and α1D pore-forming subunits was investigated during four embryonic stages in bovine foetuses. Our data suggest that the expressions of α1A and α1D are correlated during development, with an increase only in males that peaks on the last period of gestation. Bovine male hypothalami showed significantly higher α1A and α1D expression values in comparison to female ones during the whole developmental period. In the females, the expression profiles of both genes were constant during all the developmental time. Immunohistochemical studies confirmed the presence of the α1A and α1D protein subunits in foetal hypothalamic neurones starting from the third foetal stage. Our data provide new information on the hypothalamic expression of α1A and α1D subunits during development in a mammal with a long gestation period and a large and convoluted brain.

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca 2+ -permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca 2+ -activated small-conductance type 2 (SK2)K + channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundantOCGABAinnervation is present along themammaliancochlea. The role of this neurotransmitter at theOCefferent innervation, however, is for the most part unknown.Weshow thatGABAfails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation ofOCefferent fibers activates presynapticGABA B(1a,2) receptors [GABAB (1a,2) Rs] that downregulate the amount of ACh released at the OC-hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA B Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA B1 -GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA B1a isoform selectively inhibits release at efferent cholinergic synapses. Fil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Zorrilla de San Martín, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ballestero, Jimena Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Torbidoni, Ana Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Fuchs, Paul A.. Johns Hopkins University; Estados Unidos Fil: Bettler, Bernhard. Universidad de Basilea; Suiza Fil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 μM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.

Nesfatin-1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin-1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin-1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin-1 elicits Ca²⁺ signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca²⁺ in cardiac pre-ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin-1 increases cytosolic Ca²⁺ concentration via a Gi/o-coupled mechanism. The nesfatin-1-induced Ca²⁺ rise is critically dependent on Ca²⁺ influx via P/Q-type voltage-activated Ca²⁺ channels. Repeated administration of nesfatin-1 leads to tachyphylaxis. Furthermore, nesfatin-1 produces a dose-dependent depolarization of cardiac vagal neurons via a Gi/o-coupled mechanism. In vivo studies, using telemetric and tail-cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin-1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin-1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus. Our results indicate that nesfatin-1, one of the most potent feeding peptides, increases cytosolic Ca²⁺ by promoting Ca²⁺ influx via P/Q channels and depolarizes nucleus ambiguus neurons; both effects are Gi/o-mediated. In vivo studies indicate that microinjection of nesfatin-1 into nucleus ambiguus produces bradycardia in conscious rats. This is the first report that nesfatin-1 increases the parasympathetic cardiac tone.

Calcium channels that mediate glutamate release (N-type and P/Q-type) are expressed in distinct populations of cerebrocortical nerve terminals in adult mice. mGlu7 receptors are exclusively expressed in nerve terminals containing N-type Ca(2+) channels, which are less tightly coupled to glutamate release than P/Q-type Ca(2+) channels. We recently reported that in addition to inhibit, mGlu7 receptors can also potentiate glutamate release via phosphatidyl inositol (4,5)-bisphosphate hydrolysis and activation of the non-kinase diacylglycerol binding protein Munc13-1, a protein that primes synaptic vesicles for exocytosis. Here, we assessed whether mGlu7 receptor-mediated potentiation of glutamate release is restricted to nerve terminals expressing N-type Ca(2+) channels to compensate for their weak coupling to release. In the hippocampus, mGlu7 receptors are expressed both in nerve terminals containing N-type Ca(2+) channels and in nerve terminals containing P/Q-type Ca(2+) channels. When analyzed, we observed potentiation of mGlu7 receptor mediated release in wild type hippocampal nerve terminals at physiological (1.3 mM) and low (0.1 mM) concentrations of external Ca(2+). By contrast, in nerve terminals from mice lacking the α1B subunit of N-type channels (Ca(v)2.2), in which evoked release is mediated by P/Q-type channels only, no release potentiation was observed at 1.3 mM Ca(2+). We conclude that release potentiation at 1.3 mM [Ca(2+)](e) occurs in nerve terminals expressing N-type channels, whereas that which occurs at low 0.1 mM [Ca(2+)](e) represents the release from nerve terminals containing P/Q-type Ca(2+) channels. Although, mGlu7 receptor mediated potentiation is independent of Ca(2+) channel activity, as it was induced by the Ca(2+) ionophore ionomycin, release potentiation is influenced by the Ca(2+) channel type and/or the associated release machinery.

Objective To investigate whether the normal expression of metastasis-associated protein 1 (MTA1) in Sertoli cells (SCs) is associated with adjacent germ cells (GCs) and to provide the functional relevance of MTA1 in this somatic cell. Methods The expression pattern of MTA1 in the SCs of impaired human spermatogenesis was determined using immunohistochemistry. The effect of the depletion of GCs on the expression of MTA1 in isolated SCs was evaluated using reverse transcriptase polymerase chain reaction in murine testes treated with busulphan. Finally, using multiple assays, the functional investigation of MTA1 by its specific knockdown was performed in SC-GC co-cultures. Results SCs were negatively immunolabeled in the tubules with impaired spermatogenesis. Depletion of murine GCs by treatment with busulphan resulted in a dramatic decrease of the MTA1 transcripts level in the isolated SCs on the 15th day of treatment and thereafter had totally abolished MTA1 expression by the 30th day of treatment, respectively. The addition of isolated round spermatids into SC culture could partially elevate MTA1 expression in the latter. Furthermore, MTA1 is crucial to maintain the GC nursery function and normal anchoring junction formation in SCs because ablation of MTA1 by siRNA induced extensive defects of genes related to SC homeostasis. Conclusion We propose a novel role for SC-expressing MTA1, which is determined by the presence of surrounding GCs, in mediating the crosstalk between SCs and GCs by influencing a broad spectrum of gene changes.

A 71-year-old chronic smoker had an 11-month history of monocular followed by binocular elliptical nystagmus and oscillopsia (video at [Neurology.org][1]). MRI brain showed extensive periventricular T2 signal changes (figure) and CSF showed elevated protein to 102 mg/dL. CSF and serum paraneoplastic panel revealed elevated serum titers of anti-P/Q-type voltage-gated calcium channel (VGCC) and anti-neuronal-type voltage-gated potassium channel antibodies. An underlying malignancy was not found after an extensive investigation. The patient was treated with carbamazepine for symptomatic control, followed by high-dose IV methylprednisolone, resulting in moderate improvement. Anti-VGCC antibodies have been implicated in paraneoplastic nystagmus and small cell lung cancer is the most common associated malignancy.1,2 [1]: http://neurology.org/lookup/doi/10.1212/WNL.0000000000003068

The repressor element-1 silencing transcription factor (REST) has emerged as a key controller of neuronal differentiation and has been shown to play a critical role in the expression of the neuronal phenotype; however, much has still to be learned about its role at specific developmental stages and about the functional targets affected. Among these targets, calcium signaling mechanisms are critically dependent on the developmental stage and their full expression is a hallmark of the mature, functional neuron. We have analyzed the role played by REST in GN11 cells, an immortalized cell line derived from gonadotropin hormone releasing hormone (GnRH) neurons at an early developmental stage, electrically non-excitable and with a strong migratory activity. We show for the first time that functional voltage-dependent calcium channels are expressed in wild type GN11 cells; down-regulation of REST by a silencing approach shifts these cells towards a more differentiated phenotype, increasing the functional expression of P/Q-type channels and reducing their migratory potential.

Background Paraneoplastic cerebellar degeneration is a rare neurological condition characterized by diffuse cerebellar dysfunction and magnetic resonance imaging evidence of progressive cerebellar atrophy. It has been associated with several autoantibodies and malignancies in adults. To date, only six cases have been described in male children. Patient Description We describe an eight-year-old girl with a prodrome of abdominal pain and vomiting followed by acute onset diplopia, dysarthria, dysmetria, and ataxia. She was found to have cerebellar degeneration in association with P/Q-type calcium channel antibodies. Conclusion This is the first child with documented paraneoplastic cerebellar degeneration in association with P/Q-type calcium channel antibodies.

The presence of P/Q type voltage gated calcium channel (VGCC) antibodies has been strongly correlated with Lambert Eaton Syndrome (LES), present in 90% of non-immunocompromised patients with LES. However, there have been case reports which have shown its association between paraneoplastic syndrome affecting both central nervous system and the peripheral nervous system causing encephalomyelitis and sensory neuronopathy/neuropathy. We present a case of a young man, who presented with encephalomyelitis, and was further noted to have superimposed cervical polyradiculopathy associated with P/Q type VGCC antibodies.

Calcium (Ca(2+)) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca(2+) concentration in the secretory vesicles was estimated to be in the range of ∼10 to 100 μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca(2+) dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca(2+) signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Ca(v)2.1) Ca(2+) channels. Blocking the p-type Ca(2+) channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca(2+) dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca(2+) indicator fluorescence. The results suggest that the free Ca(2+) content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process.

Voltage-gated Ca2+channels (VGCCs) of the P/Q-type, which are expressed at a majority of mammalian nerve terminals, show two types of Ca2+-dependent feedback regulation—inactivation (CDI) and facilitation (CDF). Because of the nonlinear relationship between Ca2+influx and transmitter release, CDI and CDF are powerful regulators of synaptic strength. To what extent VGCCs inactivate or facilitate during spike trains depends on the dynamics of free Ca2+([Ca2+]i) and the Ca2+sensitivity of CDI and CDF, which has not been determined in nerve terminals. In this report, we took advantage of the large size of a rat auditory glutamatergic synapse—the calyx of Held—and combined voltage-clamp recordings of presynaptic Ca2+currents (ICa(V)) with UV-light flash-induced Ca2+uncaging and presynaptic Ca2+imaging to study the Ca2+requirements for CDI and CDF.We find that nearly half of the presynaptic VGCCs inactivate during 100 ms voltage steps and require several seconds to recover. This inactivation is caused neither by depletion of Ca2+ions from the synaptic cleft nor by metabotropic feedback inhibition, because it is resistant to blockade of metabotropic and ionotropic glutamate receptors. Facilitation ofICa(V)induced by repetitive depolarizations or preconditioning voltage steps decays within tens of milliseconds. Since Ca2+buffers only weakly affect CDI and CDF, we conclude that the Ca2+sensors are closely associated with the channel. CDI and CDF can be induced by intracellular photo release of Ca2+resulting in [Ca2+]ielevations in the low micromolar range, implying a surprisingly high affinity of the Ca2+sensors.

The cytoskeletal matrix of the active zone and synaptic voltage-dependent calcium channels (VDCCs) are both necessary components for the organization and regulation of synaptic vesicle release. In this study, we report a novel interaction between the cytoskeletal matrix of the active zone protein, ELKS1b, and the VDCC subunit, β4, in the molecular layer of the cerebellum. We found that the two proteins coimmunoprecipitated using antibodies against each protein. Using fluorescent immunohistochemistry, we observed colocalization between ELKS1b and VDCC β4 in the molecular layer of the cerebellum, suggesting that these proteins are both present in molecular layer synapses. Analysis of a P/Q-type VDCC knockout mouse (Cacna1a(-/-)) revealed that the localization of the VDCC β4 subunit to the molecular layer was disrupted, although ELKS1b protein localization was not affected. These results demonstrate that these two proteins interact in vitro and colocalize in the cerebellum, and suggest that their interaction may play a role at the molecular layer synapses of the cerebellum.

The development of a neuronal dendritic tree is modulated both by signals from afferent fibers and by an intrinsic program. We have previously shown that chronic activation of either type 1 metabotropic glutamate receptors (mGluR1s) or protein kinase C (PKC) in organotypic cerebellar slice cultures of mice and rats severely inhibits the growth and development of the Purkinje cell dendritic tree. The signaling events linking receptor activation to the regulation of dendritic growth remain largely unknown. We have studied whether channels allowing the entry of Ca(2+) into Purkinje cells, in particular the type 3 transient receptor potential cation channels (TRPC3s), P/Q-type Ca(2+) channels, and T-type Ca(2+) channels, might be involved in signaling after mGluR1 or PKC stimulation. We show that the inhibition of dendritic growth seen after mGluR1 or PKC stimulation is partially rescued by pharmacological blockade of P/Q-type and T-type Ca(2+) channels, indicating that activation of these channels mediating Ca(2+) influx contributes to the inhibition of dendritic growth. In contrast, the absence of Ca(2+) -permeable TRPC3s in TRPC3-deficient mice or pharmacological blockade had no effect on mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. Similarly, blockade of Ca(2+) influx through glutamate receptor δ2 or R-type Ca(2+) channels or inhibition of release from intracellular stores did not influence mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. These findings suggest that both T-type and P/Q-type Ca(2+) channels, but not TRPC3 or other Ca(2+) -permeable channels, are involved in mGluR1 and PKC signaling leading to the inhibition of dendritic growth in cerebellar Purkinje cells.

Mutations in theCACNA1Agene are associated with neurological disorders, such as ataxia, hemiplegic migraine, and epilepsy. These mutations affect the pore-forming α1A-subunit of CaV2.1 channels and thereby either decrease or increase neuronal Ca2+influx. A decreased CaV2.1-mediated Ca2+influx has been shown to reduce the regularity of cerebellar Purkinje cell activity and to induce episodic cerebellar ataxia. However, little is known about how ataxia can be caused byCACNA1Amutations that increase the Ca2+influx, such as the S218L missense mutation. Here, we demonstrate that the S218L mutation causes a negative shift of voltage dependence of CaV2.1 channels of mouse Purkinje cells and results in lowered thresholds for somatic action potentials and dendritic Ca2+spikes and in disrupted firing patterns. The hyperexcitability ofCacna1aS218LPurkinje cells was counteracted by application of the activators of Ca2+-dependent K+channels, 1-EBIO and chlorzoxazone (CHZ). Moreover, 1-EBIO also alleviated the irregularity of Purkinje cell firing bothin vitroandin vivo, while CHZ improved the irregularity of Purkinje cell firingin vitroas well as the motor performance ofCacna1aS218Lmutant mice. The current data suggest that abnormalities in Purkinje cell firing contributes to cerebellar ataxia induced by the S218L mutation and they advocate a general therapeutic approach in that targeting Ca2+-dependent K+channels may be beneficial for treating ataxia not only in patients suffering from a decreased Ca2+influx, but also in those suffering from an increased Ca2+influx in their Purkinje cells.

Inherited loss of P/Q-type calcium channel function causes human absence epilepsy, episodic dyskinesia, and ataxia, but the molecular “birthdate” of the neurological syndrome and its dependence on prenatal pathophysiology is unknown. Since these channels mediate transmitter release at synapses throughout the brain and are expressed early in embryonic development, delineating the critical circuitry and onset underlying each of the emergent phenotypes requires targeted control of gene expression. To visualize P/Q-type Ca2+channels and dissect their role in neuronal networks at distinct developmental stages, we created a novel conditionalCacna1aknock-in mouse by inserting the floxed green fluorescent protein derivative Citrine into the first exon ofCacna1aand then crossed it with a postnatally expressing PCP2-Cre line for delayed Purkinje cell (PC) gene deletion within the cerebellum and sparsely in forebrain (purky). PCs inpurkymice lacked P/Q-type calcium channel protein and currents within the first month after birth, displayed altered spontaneous firing, and showed impaired neurotransmission. Unexpectedly, adultpurkymice exhibited the full spectrum of neurological deficits seen in mice with genomicCacna1aablation. Our results show that the ataxia, dyskinesia, and absence epilepsy caused by inherited disorders of the P/Q-type channel arise from signaling defects beginning in late infancy, revealing an early window of opportunity for therapeutic intervention.

Agonists at μ-opioid receptors (MORs) represent the gold standard for the treatment of severe pain. A key element of opioid analgesia is the depression of nociceptive information at the first synaptic relay in spinal pain pathways. The underlying mechanisms are, however, largely unknown. In spinal cord slices with dorsal roots attached prepared from young rats, we determined the inhibitory effect of the selective MOR agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) on monosynaptic Aδ- and C-fiber-evoked EPSCs in lamina I neurons. DAMGO depressed presynaptically Aδ- and C-fiber-mediated responses, indicating that MORs are expressed on central terminals of both fiber types. We next addressed the mechanisms of presynaptic inhibition. The effect of DAMGO at both Aδ- and C-fiber terminals was mainly mediated by an inhibition of N-type voltage-dependent Ca2+channels (VDCCs), and to a lesser extent of P/Q-type VDCCs. Inhibition by DAMGO was not reduced by K+channel blockers. The rate of miniature EPSCs was reduced by DAMGO in a dose-dependent manner. The opioid also reduced Ca2+-dependent, ionomycin-induced EPSCs downstream of VDCCs. DAMGO had no effect on the kinetics of vesicle exocytosis in C-fiber terminals, but decreased the rate of unloading of Aδ-fiber boutons moderately, as revealed by two-photon imaging of styryl dye destaining. Together, these results suggest that binding of opioids to MORs reduces nociceptive signal transmission at central Aδ- and C-fiber synapses mainly by inhibition of presynaptic N-type VDCCs. P/Q-type VDCCs and the transmitter release machinery are targets of opioid action as well.

Cortical axons contain a diverse range of voltage-activated ion channels, including Ca2+currents. Interestingly, Ca2+channels are not only located at presynaptic terminals, but also in the axon initial segment (AIS), suggesting a potentially important role in the regulation of action potential generation and neuronal excitability. Here, using two-photon microscopy and whole-cell patch-clamp recording, we examined the properties and role of calcium channels located in the AIS and presynaptic terminals of ferret layer 5 prefrontal cortical pyramidal cellsin vitro. Subthreshold depolarization of the soma resulted in an increase in baseline and spike-triggered calcium concentration in both the AIS and nearby synaptic terminals. The increase in baseline calcium concentration rose with depolarization and fell with hyperpolarization with a time constant of approximately 1 s and was blocked by removal of Ca2+from the bathing medium. The increases in calcium concentration at the AIS evoked by subthreshold or suprathreshold depolarization of the soma were blocked by the P/Q-channel antagonist ω-agatoxin IVA or the N-channel antagonist ω-conotoxin GVIA or both. The presence of these channels in the AIS pyramidal cells was confirmed with immunochemistry. Block of these channels slowed axonal action potential repolarization, apparently from reduction of the activation of a Ca2+-activated K+current, and increased neuronal excitability. These results demonstrate novel mechanisms by which calcium currents may control the electrophysiological properties of axonal spike generation and neurotransmitter release in the neocortex.

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca 2+ channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca 2+ channel β subunits (Ca v β), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca 2+ channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca v β but not the Gem/Ca v β interaction, and that Gem coimmunoprecipitates with the P/Q channel α 1 subunit (Ca v α 1 ) in a Ca v β-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca v α 1 critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca v α 1 abolishes Gem inhibition of P/Q channels and confers Ca v β-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca v α 1 that binds Ca v β). Our results challenge the prevailing view regarding the role of Ca v β in RGK inhibition of high voltage-activated Ca 2+ channels and prompt a paradigm in which Gem directly binds and inhibits Ca v β-primed Ca v α 1 on the plasma membrane.

The contributions of P- and R-type Ca2+ channels on glycinergic nerve endings (boutons) projecting to the rat spinal sacral commissural nucleus (SDCN) neurons are not understood. Thus, we investigated the functional role of P- and R-type Ca2+ channels by measuring the inhibitory postsynaptic currents (eIPSCs) evoked from individual nerve endings (boutons) by focal electrical stimulation. The current amplitude and failure rate (Rf) of glycinergic eIPSCs varied directly with changes in [Ca2+](o). Low concentration of omega-Aga IVA (P-type selective antagonist) suppressed eIPSCs as much as high concentration (both P- and Q-type selective) indicating little contribution of Q-type Ca2+ channels. Antagonism of R-type Ca2+ channels with SNX-482 and Ni2+ greatly decreased the current amplitude and increased failure rate (Rf) of glycinergic eIPSCs. Overall, our results suggest that the dominant control of glycine release depends on Ca2+ entry through P- and R-type Ca2+ channels that ubiquitously populate spinal glycine release sites.

Aspartate satisfies all the criteria normally required for identification of a CNS neurotransmitter. Nevertheless, little electrophysiological evidence supports the existence of aspartate transmission. In studies with rat hippocampal synaptosomes, chemically evoked aspartate release differed from glutamate release in its relative sensitivity to increased Ca2+ concentration outside the presynaptic active zones, inefficient coupling to P/Q-type Ca2+ channels, sensitivity to KB-R7943, and resistance to native Clostridial toxins. We took advantage of these differences to search for a potential aspartate-mediated response at Schaffer collateral synapses in organotypic hippocampal slice cultures. The slice cultures were pretreated with botulinum neurotoxin C (BoNT/C) to eliminate most of the glutamate release so that an expectedly smaller aspartate-like component of the compound EPSC could be detected by whole cell patch clamp recording. In control cultures, NMDA receptor activation accounted for only 18% of the evoked EPSC and an NR2B-selective antagonist reduced the NMDA receptor-mediated component by only 20%. Block of P/Q-type Ca2+ channels essentially eliminated the response and 0.1 μM KB-R7943 had no significant effect. In BoNT/C-pretreated cultures, however, NMDA receptor activation accounted for 77% of the evoked EPSC and an NR2B-selective antagonist reduced the NMDA receptor-mediated component by 57%. Block of P/Q-type Ca2+ channels reduced the response by only 28%, but 0.1 μM KB-R7943 reduced it by 45%. These results suggest that part of the Schaffer collateral synaptic response has pharmacological properties similar to those of synaptosomal aspartate release and may therefore be mediated at least partly by released aspartate.

Corticotropin-releasing factor (CRF) receptors have been demonstrated to be widely expressed in the central nervous system and in many peripheral tissues of mammalians. However, it is still unknown whether CRF receptors will function in cerebellar Purkinje neurons. In the present study, we investigated the expression profile of CRF receptors in rat cerebellum and identified a novel functional role of CRFR2 in modulating Purkinje neuron P-type Ca(2+) currents (P-currents). We found that CRFR2alpha mRNA, but not CRFR1 and CRFR2beta, was endogenously expressed in rat cerebellum. Activation of CRFR2 by UCN2 inhibited P-currents in a concentration-dependent manner (IC(50) approximately 0.07 microM). This inhibitory effect was abolished by astressin2B, a CRFR2 antagonist, and was blocked by GDP-beta-S, pertussis toxin, or a selective antibody raised against the G(o)alpha. Inhibition of phospholipase C (PLC) blocked the inhibitory action of UCN2. The application of diacylglycerol (DAG) antagonist, 1-hexadecyl-2-acetyl-sn-glycerol, as well as inhibition of either protein kinase C or its epsilon isoform (PKCepsilon) abolished the UCN2 effect while 1-oleoyl-2-acetyl-sn-glycerol (EI-150), a membrane-permeable DAG analogue, occluded UCN2-mediated inhibition. In addition, UCN2 significantly increases spontaneous firing frequency of Purkinje neurons in cerebellar slices. In summary, activation of CRFR2 inhibits P-currents in Purkinje neurons via G(o)alpha-dependent PLC/PKCepsilon pathway, which might contribute to its physiological functions in the cerebellum.

Blockage of GABA-A receptors in hippocampal neuronal cultures triggers synchronous bursts of spikes initiating neuronal plasticity, partly mediated by changes of gene expression. By using specific pharmacological blockers, we have investigated which sources of Ca2+ entry primarily control changes of gene expression induced by 20 microM gabazine applied for 30 min (GabT). Intracellular Ca2+ transients were monitored with Ca2+ imaging while recording electrical activity with patch clamp microelectrodes. Concomitant transcription profiles were obtained using Affymetrix oligonucleotide microarrays and confirmed with quantitative RT-PCR. Blockage of NMDA receptors with 2-amino-5-phosphonovaleric acid (APV) did not reduce significantly somatic Ca2+ transients, which, on the contrary, were reduced by selective blockage of L, N, and P/Q types voltage gated calcium channels (VGCCs). Therefore, we investigated changes of gene expression in the presence of blockers of NMDA receptors and L, N, and P/Q VGCCs. Our results show that: (i) among genes upregulated by GabT, there are genes selectively dependent on NMDA activation, genes selectively dependent on L-type VGCCs and genes dependent on the activation of both channels; (ii) the majority of genes requires the concomitant activation of NMDA receptors and Ca2+ entry through VGCCs; (iii) blockage of N and P/Q VGCCs has an effect similar but not identical to blockage of L-type VGCCs.

Migraine is a frequent and often disabling disease. Treatment is unsatisfactory in many patients. A disturbed dynamic balance between excitatory and inhibitory signal processing with enhanced cortical activity probably underlies common forms of migraine. Presynaptic voltage-gated Ca(2+) channels are critical determinants of neurotransmitter release and also contribute to trigeminovascular signal transduction. Because clinical evidence exists for migraine-prophylactic actions of Petasites hybridus extracts, we investigated whether petasins comprising the main constituents of the extract inhibit currents through presynaptic Ca(v)2.1 channels expressed in Xenopus laevis oocytes. P. hybridus extract (0.02 mg/ml), petasin, neopetasin, isopetasin, S-petasin, and iso-S-petasin (50 microM) were weak tonic blockers of Ca(v)2.1-mediated barium currents (I(Ba)) during infrequent depolarizations (0.1 Hz), but their inhibitory potency increased at higher stimulation rates (1 Hz), indicating preferential block of open and/or inactivated channels. Sulfur-containing compounds (S-petasin, Iso-S-petasin) were the most potent significantly promoting the accumulation of Ca(v)2.1 channel in inactivated states during pulse trains (I(Ba) decrease during 1-Hz pulse trains: control, 45%, S-petasin, 79%; iso-S-petasin, 80%). For the Eucalyptus williamsiania sesquiterpenes alpha- and gamma-eudesmol, a comparable use-dependent inhibition was found in addition to a tonic block component. Alpha-eudesmol and petasins accelerated the voltage-dependent inactivation of Ca(v)2.1 channels during depolarizations. We demonstrate that S-petasin, iso-S-petasin, and eudesmol are Ca(v)2.1 channel inhibitors preferentially acting as use-dependent channel blockers and with the sulfur-containing substituent in position 3 of the petasins serving as important functional feature. The Ca(v)2.1-inhibitory properties of these petasins may contribute to migraine-prophylactic properties described for P. hybridus extracts.

Release of acetylcholine (ACh) from preganglionic nerve terminals requires calcium entry through voltage-gated calcium channels. The calcium channel subtype required for ACh release varies depending on the particular ganglionic synapse. I have investigated the functional role of calcium channels in transmitter release from parasympathetic and sympathetic preganglionic terminals in pelvic and celiac ganglia of female mice. Single electrode voltage clamp was used to measure EPSC amplitude in the absence and presence of selective calcium channel antagonists. In pelvic ganglia ω- conotoxin GVIA, a selective N-type calcium channel antagonist, reduced the amplitude of EPSCs evoked by pelvic nerve stimulation by 46 ± 5% (n = 8, P = 0.015). In contrast, in the celiac ganglion, ω- conotoxin GVIA had no effect on the amplitude of EPSCs evoked by splanchnic nerve stimulation (P = 0.09, n = 7). EPSCs in both pelvic and celiac ganglia were resistant to the P-type calcium channel antagonist agatoxin (50 nM, n = 5 for both ganglia) and the R-type calcium channel antagonist SNX482 (100 nM, n = 4 for both ganglia). These results indicate that in female mice, release of ACh in sympathetic pathways to prevertebral ganglia does not require calcium entry from N-type calcium channels. However, release of ACh from sacral parasympathetic preganglionic neurons requires calcium entry from both N-type and toxin-resistant calcium channels.

Disorders affecting the postsynaptic side of the neuromuscular junction include autoimmune myasthenia gravis (MG) as well as some of the congenital myasthenic syndromes (CMS). Lambert-Eaton myasthenic syndrome (LEMS) is an acquired autoimmune neuromuscular disorder in which autoantibodies are directed against the presynaptic calcium channels. Here we describe two monozygous twin brothers: case 1 was diagnosed with an indeterminate form of acquired postsynaptic neuromuscular junction defect at age 32 and case 2 with LEMS at age 47. Case 1 presented clinically with mild generalized myasthenic weakness, neurophysiological examination revealed disturbed neuromuscular transmission along with probable myositis and serum analysis regarding antibodies against the acetylcholine receptor and muscle-specific tyrosine kinase was negative. Case 2 presented with proximal muscle fatigue accompanied by areflexia at rest and antibodies against the P/Q-type voltage-gated calcium channels were present. Neurophysiologically, case 2 had reduced baseline compound motor action potential amplitudes on neurography, decrement on low-frequency repetitive nerve stimulation (RNS) and pathological increment on high frequency RNS. To our knowledge this is the first case report of its kind and adds an intriguing contrast to the more common diagnosis of CMS in monozygous twins.

We investigated if stimulation of T-type Ca(2+) channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H(2)S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5-13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca(2+), and by zinc chloride at a concentration known to selectively inhibit Ca(v)3.2 isoform of T-type Ca(2+) channels, but not Ca(v)3.1 or Ca(v)3.3. Further, L-ascorbate, recently proven to selectively inhibit Ca(v)3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H(2)S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca(2+) channels, especially Ca(v)3.2.