Ryanodine receptors selectively contribute to the formation of taste-evoked calcium signals in mouse taste cells

The peripheral taste system uses multiple signaling pathways to transduce a stimulus into an output signal that activates afferent neurons. All of these signaling pathways depend on transient increases in intracellular calcium, but current understanding of these calcium signals is not well developed. Using molecular and physiological techniques, this study establishes that ryanodine receptors (RyRs), specifically isoform 1, are expressed in taste cells and that their physiological function differs among cell types employing different signaling pathways. RyR1 contributes to some taste-evoked signals that rely on calcium release from internal stores but can also supplement the calcium signal that is initiated by opening voltage-gated calcium channels. In taste cells expressing both signaling pathways, RyR1 contributes to the depolarization-induced calcium signal but not to the calcium signal that depends on calcium release from stores. These data suggest that RyR1 is an important regulator of calcium signaling and that its physiological role in taste cells is dictated by the nature of the calcium signaling mechanisms expressed.     

Abnormal neurogenesis in the dentate gyrus of adult mice lacking 1,25-dihydroxy vitamin D3 (1,25-(OH)2 D3)

In this study, we employed 1α-hydroxylase knockout (1α-(OH)ase(-/-) ) mice to investigate the influence of 1,25-dihydroxy vitamin D(3) (1,25-(OH)(2) D(3) ) deficiency on the adult neurogenesis in the hippocampal dentate gyrus (DG). The numbers of both 24-hr-old BrdU(+) cells and proliferating cell nuclear antigen positive cells in 8-week-old 1α-(OH)ase(-/-) mice increased approximately twofold compared with wild-type littermates. In contrast, the numbers of 7- and 28-day-old BrdU(+) cells in 1α-(OH)ase(-/-) mice decreased by 50% compared with wild-type mice, while the proportion of BrdU(+) /NeuN(+) cells in BrdU(+) population showed no difference between 1α-(OH)ase(-/-) and wild-type mice. Apoptotic cells in the subgranular zone (SGZ) of DG markedly increased in 1α-(OH)ase(-/-) mice. Replenishment of 1,25-(OH)(2) D(3) , but not correction of serum calcium and phosphorus levels, completely prevented changes in the neurogenesis in 1α-(OH)ase(-/-) mice. The absence of 1,25-(OH)(2) D(3) led to an increase in the expression of L-type voltage-gated calcium channel (L-VGCC) and a decrease in the nerve growth factor (NGF) mRNA level. Treatment with the L-VGCC inhibitor nifedipine blocked the increased cell proliferations by 1,25-(OH)(2) D(3) deficiency. Administration of NGF significantly attenuated the loss of newborn neurons in 1α-(OH)ase(-/-) mice.     

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses

It has been known that Ca²⁺ plays an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. Synaptotagmin 1, 2, and 9 were found to act as the Ca²⁺ sensors for synchronous release and synaptotagmin 7 and Doc‐2 were proposed as the Ca²⁺ sensors for asynchronous release. Comparatively, the Ca²⁺ sensor for spontaneous release remains a mystery. At the Calyx of Held synapse, the Ca²⁺sensor for spontaneous release was found not identical to the sensor for synchronous release, synaptotagmin 2. As Ca²⁺ sensors have different sensitivity to Sr²⁺ and Ca²⁺ and induce significantly different rate of vesicle release, Sr²⁺ is traditionally used as a tool to examine the intrinsic properties of different Ca²⁺ sensors. Here, we employed cell‐attached patch recording and presynaptic/postsynaptic whole‐cell recording at the Calyx of Held synapses of synaptotagmin 2 knock‐out mice to assay the Sr²⁺ and Ca²⁺ influx into the nerve terminal at resting potential and observed the effects of Ca²⁺ and Sr²⁺ on spontaneous neurotransmitter release. We found that the dwell time of single voltage gated Ca²⁺ channel opening increased around threefold for Sr²⁺ than Ca²⁺ with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to Sr²⁺ than Ca²⁺. Thus, our study reveals some of the intrinsic properties of Ca²⁺ sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.     

Calcium inflow-dependent protein kinase C activity is involved in the modulation of transmitter release in the neuromuscular junction of the adult rat

Using intracellular recording, we studied how protein kinase C activity affected miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in the neuromuscular junctions of the levator auris longus muscle from adult rats. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA, 10 nM) increased the quantal content by approximately 150% (P<0.05). On the other hand, the quantal content decreased by approximately 40% (P<0.05) for all the protein kinase C inhibitors tested (Calphostin-C, 10 microM; Chelerythrine, 1 microM; Staurosporine, 200 nM). These changes in acetylcholine release were maintained at plateau for 1 to 7 h. Moreover, none of the protein kinase C activators or inhibitors used could modify the spontaneous MEPP mean size (P>0.05). We reduced the calcium influx in nerve terminals using the P/Q-type channel blocker omega-Aga-IVA(100 nM) or with 5 mM magnesium in physiological solution. In neither situation was the quantal content modified by PMA or by CaC. However, when high Ca2+ (5 mM) was added to a preparation that was previously blocked with omega-Aga-IVA, PMA and CaC had their full effect. We conclude that under physiological conditions PKC is dependent on the calcium inflow through the P/Q-type voltage-dependent calcium channels during evoked activity and works near the maximum rate at normal external calcium concentration.     

Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage‐gated sodium channel Scn2a

More than 1,200 mutations in neuronal voltage‐gated sodium channel (VGSC) genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2a^Q54 transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2a^Q54 phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2a^Q54 mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2a^Q54 mice congenic on the C57BL/6J strain. Genetic mapping and RNA‐Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2a^Q54 phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage‐gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2a^Q54 seizure phenotype. Scn2a^Q54 mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.     

Mechanisms,pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors

Photoreceptors have depolarized resting potentials that stimulate calcium‐dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca²⁺ dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole‐cell recordings from light‐adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post‐synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca²⁺ influx by lowering extracellular Ca²⁺, by application of Cd²⁺ and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca²⁺. We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd²⁺ or by increased intracellular Ca²⁺ buffering in rods, but not in cones, that were voltage clamped at ?70 mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked excitatory post‐synaptic currents (EPSCs) suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca²⁺‐independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran‐conjugated pHrodo. Spontaneous release in rods occurred more frequently at non‐ribbon sites than evoked release events. The function of Ca²⁺‐independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise.     

A superfusion apparatus for the examination of neurotransmitter release from synaptosomes

A superfusion apparatus for examining the release of neurotransmitter from synaptosomes is described. The apparatus provides for accurate temperature control, rapid switching and continuous gassing of superfusing buffers, short (i.e. 1-s) pulses of high-potassium-containing buffer for examining depolarized release, rapid collection of superfusing eluate, and low tissue chamber dead volume. Depolarization (high-potassium) evoked release and to some degree spontaneous release of [3H]dopamine from rat striatal synaptosomes are dependent on buffer calcium concentration. The putative calcium channel blocker nickel reduces both spontaneous and high-potassium-evoked [3H]dopamine release.     

Upregulation of L‐type Cav1 channels in the development of psychological dependence

Although L‐type voltage‐dependent Ca²⁺ channels regulate activity‐dependent processes including synaptic plasticity and synapse formation, there are few data on the changes of Ca_v1 channel expression in psychological dependence. This study investigated the role of L‐type Ca_v1 channel expression in the brain of mouse that was psychologically dependent on methamphetamine (2 mg/kg, subcutaneous injection [s.c.]), cocaine (10 mg/kg, s.c.), and morphine (5 mg/kg, s.c.) with the conditioned place preference paradigm. Intracerebroventricular administration of nifedipine (3, 10, and 30 nmol/mouse) dose‐dependently reduced the development of methamphetamine‐, cocaine‐, and morphine‐induced rewarding effect. Under such conditions, protein levels of both Ca_v1.2 and Ca_v1.3 in the frontal cortex and the limbic forebrain were significantly increased on methamphetamine‐, cocaine‐, and morphine‐induced psychologically dependent mice. These findings suggest that the upregulation of Ca_v1.2 and Ca_v1.3 participated in the development of psychological dependence. Synapse 64:440–444, 2010. © 2010 Wiley‐Liss, Inc.     

Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage‐gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic–clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain‐dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T‐type calcium channel family, as an epilepsy modifier in the Scn2a^Q54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a^+/? Dravet phenotype. Scn1a^+/? mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia‐induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a^+/? epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.     

Amyotrophic lateral sclerosis: Serum factors enhance spontaneous and evoked transmitter release at the neuromuscular junction

Sera from 30 patients with sporadic amyotrophic lateral sclerosis (ALS) were tested to determine their effects at the neuromuscular junction. Spontaneous transmitter release was significantly increased, as evidenced by a 151% increase in MEPP frequency, by sera from 16 ALS patients. In addition, 16 patients' sera elevated EEP quantal content by an average of 89%. Eleven sera produced both effects. There was no consistent change in MEPP amplitude or time course, indicating the absence of a humoral effect on postjunctional ACh receptors or endplate membrane function. These results suggest that a portion of the sporadic ALS patient population possess serum factors that can alter presynaptic function of the motor nerve terminal. Evidence from the present experiments indicates that alterations at the neuromuscular junction are a result of a combination of increased Ca²⁺ influx into the cell and an independent increase in intracellular calcium concentration. © 1998 John Wiley & Sons, Inc. Muscle Nerve, 21: 81–90, 1998.     

A case of encephalomyeloradiculopathy in a non-carcinomatous patient associated with P/Q type voltage gated 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.     

两种钙通道拮抗剂抑制脑缺血复灌后转录因子c—Jun的表达

目的：研究两种钙离子通道：NMDA受体型和L－型电压门控钙通道（L－VGCC）在短暂全脑缺血复灌后海马c-Jun表达中的作用。方法：采用SD大鼠四动脉结扎全脑缺血模型，取缺血复灌不同时间（0,1,3,6,12,24和72h)以及对照组2D大鼠的海以，应用免疫印迹的方法来研究c-Jun的表达并观察几种钙通道拮抗剂对c-Jun表达的影响。结果：在假手术以及复灌的各个时间点均有表达，并在复灌6h达到高峰。氯胺酮（一种非竞争性NMDA受体拮抗剂）和硝苯吡啶（一种L－VGCC阻滞剂）抑制c-Jun表达的增加。而DNQX（一种AMPA／KA受体拮抗剂）则无抑制作用（数据未显示）。结论：缺血复灌后，c-Jun的表达增加了，这种增加与NMDA受体和L－VGCC这两种钙离子通道的开放有关。     

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals

Ca²⁺‐induced Ca²⁺ release (CICR) via type‐3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca²⁺ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular‐free Ca²⁺ ([Ca²⁺]_i: Ca²⁺ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation‐induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.     

Differential expression of proteoglycans at central and peripheral nodes of Ranvier

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage-gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan-3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan-3. We have examined the molecular composition of the PNS nodes in syndecan-3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan-3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.     

The action of Lambert-Eaton myasthenic syndrome immunoglobulin G on cloned human voltage-gated calcium channels

In the Lambert-Eaton myasthenic syndrome (LEMS), immunoglobulin G (IgG) autoantibodies to presynaptic voltage-gated calcium channels (VGCCs) at the neuromuscular junction lead to a reduction in nerve-evoked release of neurotransmitter and muscle weakness. We have examined the action of LEMS IgGs on cloned human VGCCs stably expressed in transfected human embryonic kidney (HEK293) cell lines: 10-13 (alpha(1A-2), alpha(2b)delta, beta(4a)) and C2D7 (alpha(1B-1), alpha(2b)delta, beta(1b)). All LEMS IgGs studied showed surface binding to [(125)I]-omega-CTx-MVIIC-labeled VGCCs in the alpha(1A) cell line and two of six IgGs showed surface binding to [(125)I]-omega-CTx-GVIA-labeled VGCCs in the alpha(1B) cell line. We next studied the effect of LEMS IgGs (2 mg/ml) on whole-cell calcium currents in the alpha(1A) and alpha(1B) cell lines. Overnight treatment of alpha(1A) (10-13) cells with LEMS IgGs led to a significant reduction in peak current density without alteration of the current-voltage relationship or the voltage dependence of steady-state inactivation. In contrast, LEMS IgGs did not reduce peak current density in the alpha(1B) cell line. Overall these data demonstrate the specificity of LEMS IgGs for the alpha(1A) cell line and suggest that LEMS IgGs bind to and downregulate VGCCs in this cell line. Although several LEMS IgGs can be shown to bind to the alpha(1B) (C2D7) cell line, no functional effects were seen on this channel.