Calmodulin activity regulates group I metabotropic glutamate receptor‐mediated signal transduction and synaptic depression

Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR 5 (mGluR1/5), are coupled to Gq and modulate activity‐dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation‐dependent long‐term depression (LTD). Although it has been established that intracellular Ca²⁺ and the Gq‐regulated signaling molecules are required for mGluR1/5 LTD, whether and how Ca²⁺ regulates Gq signaling and upregulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of the Ca²⁺ sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor N‐[4‐aminobutyl]‐5‐chloro‐2‐naphthalenesulfonamide hydrochloride (W13) suppressed the mGluR1/5‐stimulated activation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and p70‐S6 kinase 1 (S6K1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist‐induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca²⁺/CaM‐dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Furthermore, disruption of the CaM–CaMK–ERK1/2 signaling cascade suppressed the mGluR1/5‐stimulated upregulation of Arc expression. Altogether, our data suggest CaM as a new Gq signaling component for coupling Ca²⁺ and protein upregulation and regulating mGluR1/5‐mediated synaptic modification. © 2016 Wiley Periodicals, Inc.     

Store-operated calcium entry modulates neuronal network activity in a model of chronic epilepsy

Store-operated Ca²⁺ entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca²⁺ stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca²⁺ concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.     

Enhanced astroglial Ca2+ signaling increases excitatory synaptic strength in the epileptic brain

The fine‐tuning of synaptic transmission by astrocyte signaling is crucial to CNS physiology. However, how exactly astroglial excitability and gliotransmission are affected in several neuropathologies, including epilepsy, remains unclear. Here, using a chronic model of temporal lobe epilepsy (TLE) in rats, we found that astrocytes from astrogliotic hippocampal slices displayed an augmented incidence of TTX‐insensitive spontaneous slow Ca²⁺ transients (STs), suggesting a hyperexcitable pattern of astroglial activity. As a consequence, elevated glutamate‐mediated gliotransmission, observed as increased slow inward current (SICs) frequency, up‐regulates the probability of neurotransmitter release in CA3‐CA1 synapses. Selective blockade of spontaneous astroglial Ca²⁺ elevations as well as the inhibition of purinergic P2Y1 or mGluR5 receptors relieves the abnormal enhancement of synaptic strength. Moreover, mGluR5 blockade eliminates any synaptic effects induced by P2Y1R inhibition alone, suggesting that the Pr modulation via mGluR occurs downstream of P2Y1R‐mediated Ca²⁺‐dependent glutamate release from astrocyte. Our findings show that elevated Ca²⁺‐dependent glutamate gliotransmission from hyperexcitable astrocytes up‐regulates excitatory neurotransmission in epileptic hippocampus, suggesting that gliotransmission should be considered as a novel functional key in a broad spectrum of neuropathological conditions. GLIA 2015;63:1507–1521     

Modulation of intracellular calcium mobilization and GABAergic currents through subtype-specific metabotropic glutamate receptors in neonatal rat hippocampus

Group I metabotropic glutamate receptors (mGluRs) are coupled to phosphoinositide hydrolysis, and are thought to modulate neuronal excitability, by mobilizing intracellular Ca²⁺. Difference in Ca²⁺ mobilization among subclasses of the receptors has been reported, and regarded as a possible cause of variant neuronal modifications. In hippocampal interneurons, several subclasses of mGluRs including mGluR1 and mGluR5 have been immunohistochemically identified. The subclass-specific physiological effects of mGluRs on neuronal transmission in hippocampus, however, have not been fully elucidated. In the present study, effects of group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) on intracellular calcium concentration were examined in hippocampal interneurons. Application of DHPG increased fluorescence ratio in neonatal CA3 stratum oriens/alveus interneurons. The DHPG-induced calcium mobilization was markedly inhibited by mGluR1-specific antagonist, cyclopropan[b]chromen-1a-carboxylate (CPCCOEt). Inhibition of the calcium elevation by mGluR5-specific antagonist, 6-methyl-2-(phenylazo)-3-pyrindol (MPEP), was weaker than that of CPCCOEt. The fluorescence ratio was not significantly changed by application of mGluR5-specific agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG). DHPG induced calcium responses in CA1 interneurons as in CA3, and the responses were partially inhibited by MPEP treatment. Effects of group I mGluR agonist and antagonist were also investigated, on GABA_A receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in CA3 pyramidal neurons. The GABAergic sIPSCs were facilitated by DHPG perfusion, and the potentiation was reduced by CPCCOEt, and less distinctly by MPEP. The sIPSCs were not significantly potentiated by CHPG application. These results indicate that mGluR1 is functional in hippocampal interneurons, and DHPG exerts its effect mainly through this receptor at early developmental period.     

STIM1, STIM2, and Orai1 regulate store‐operated calcium entry and purinergic activation of microglia

Activation of microglia is the first and main immune response to brain injury. Release of the nucleotides ATP, ADP, and UDP from damaged cells regulate microglial migration and phagocytosis via purinergic P2Y receptors. We hypothesized that store‐operated Ca²⁺ entry (SOCE), the prevalent Ca²⁺ influx mechanism in non‐excitable cells, is a potent mediator of microglial responses to extracellular nucleotides. Expression analyses of STIM Ca²⁺ sensors and Orai Ca²⁺ channel subunits, that comprise the molecular machinery of SOCE, showed relevant levels of STIM1, STIM2, and Orai1 in cultured mouse microglia. STIM1 expression and SOCE were down‐regulated by treatment of microglia with lipopolysaccharide, suggesting that inflammation limits SOCE by lower STIM1 abundance. Ca²⁺ entry induced by cyclopiazonic acid, ATP, the P2Y₆ receptor agonist UDP, or the P2Y₁₂ receptor agonist 2‐methylthio‐ADP (2‐MeSADP) was clearly affected in microglia from Stim1^–/–, Stim2^–/–, and Orai1^–/– mice. SOCE blockers or ablation of STIM1, STIM2, or Orai1 severely impaired nucleotide‐induced migration and phagocytosis in microglia. Thus, this study assigns SOCE, regulated by STIM1, STIM2, and Orai1 an essential role in purinergic signaling and activation of microglia. GLIA 2015;63:652–663     

Deletion of Stim1 in Hypothalamic Arcuate Nucleus Kiss1 Neurons Potentiates Synchronous GCaMP Activity and Protects against Diet-Induced Obesity

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High-frequency firing of hypothalamic arcuate Kiss1 (Kiss1^ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1^ARH neurons to generate a slow EPSP mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1^ARH neurons. High-frequency optogenetic stimulation of Kiss1^ARH neurons also releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca²⁺ signaling and neuronal excitability through its interaction with plasma membrane (PM) calcium (e.g., TRPC) channels. Therefore, we hypothesized that deletion of Stim1 in Kiss1^ARH neurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that deletion of Stim1 in Kiss1 neurons significantly increased the amplitude and duration of the slow EPSP and augmented synchronous [Ca²⁺]i oscillations in Kiss1^ARH neurons. Deletion of Stim1 in Kiss1^ARH neurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD). Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1^ARH neurons, which ultimately affects the coordination between energy homeostasis and reproduction.SIGNIFICANCE STATEMENT Hypothalamic arcuate kisspeptin (Kiss1^ARH) neurons are essential for stimulating the pulsatile release of gonadotropin-releasing hormone (GnRH) and maintaining fertility. However, Kiss1^ARH neurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane (PM) calcium channels. We have conditionally deleted Stim1 in Kiss1^ARH neurons and found that it significantly increased the excitability of Kiss1^ARH neurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD).     

Neuron–astrocyte signaling is preserved in the aging brain

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca²⁺ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte–neuron signaling is derived from studies with young animals; however, the features of astrocyte–neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte–neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month‐old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter‐dependent intracellular Ca²⁺ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG‐induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte–neuron signaling in pathological conditions. Disruption of the astrocytic IP₃R2 mediated‐signaling, which is required for neurotransmitter‐induced astrocyte Ca²⁺ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte–neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca²⁺ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569–580     

Early-life seizures alter synaptic calcium-permeable AMPA receptor function and plasticity

Calcium (Ca²⁺)-mediated⁴ signaling pathways are critical to synaptic plasticity. In adults, the NMDA glutamate receptor (NMDAR) represents a major route for activity-dependent synaptic Ca²⁺ entry. However, during neonatal development, when synaptic plasticity is particularly high, many AMPA glutamate receptors (AMPARs) are also permeable to Ca²⁺ (CP-AMPAR) due to low GluA2 subunit expression, providing an additional route for activity- and glutamate-dependent Ca²⁺ influx and subsequent signaling. Therefore, altered hippocampal Ca²⁺ signaling may represent an age-specific pathogenic mechanism. We thus aimed to assess Ca²⁺ responses 48 h after hypoxia-induced neonatal seizures (HS) in postnatal day (P)10 rats, a post-seizure time point at which we previously reported LTP attenuation. We found that Ca²⁺ responses were higher in brain slices from post-HS rats than in controls and that this increase was CP-AMPAR-dependent. To determine whether synaptic CP-AMPAR expression was also altered post-HS, we assessed the expression of GluA2 at hippocampal synapses and the expression of long-term depression (LTD), which has been linked to the presence of synaptic GluA2. Here we report a decrease 48 h after HS in synaptic GluA2 expression at synapses and LTD in hippocampal CA1. Given the potentially critical role of AMPAR trafficking in disease progression, we aimed to establish whether post-seizure in vivo AMPAR antagonist treatment prevented the enhanced Ca²⁺ responses, changes in GluA2 synaptic expression, and diminished LTD. We found that NBQX treatment prevents all three of these post-seizure consequences, further supporting a critical role for AMPARs as an age-specific therapeutic target.     

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non‐cell‐autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1^G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1^G93A rats, we have demonstrated that the typical Ca²⁺ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca²⁺ concentration. Since the expression of protein kinase C epsilon isoform (PKC?) has been found to be considerably reduced in astrocytes from hSOD1^G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKC? expression was found to restore Ca²⁺ oscillations induced by mGluR5 activation in hSOD1^G93A‐expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKC? expression in astrocytes from wild‐type animals with specific PKC?‐shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake‐mediated by mGluR5 activation. These results suggest that PKC? is required to generate Ca²⁺ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKC? could impair this neuroprotective process and participate in the progression of ALS.     

Acute ethanol alters calcium signals elicited by glutamate receptor agonists and K depolarization in cultured cerebellar Purkinje neurons

The effect of acute ethanol on Ca²⁺ signals evoked by ionotropic (iGluR) and metabotropic (mGluR) glutamate receptor (GluR) activation and K⁺ depolarization was examined in cultured rat cerebellar Purkinje neurons to assess the ethanol sensitivity of these Ca²⁺ signaling pathways. Mature Purkinje neurons 3 weeks in vitro were studied. iGluRs were activated by (RS)-α-amino-3-hydroxyl-5 methyl-4-isoxazolepropionic acid (AMPA; 1 and 5 μM) and domoate (5 μM). mGluRs were activated by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 μM) and (R,S)-3,5-dihydroxyphenylglycine (DHPG; 200 μM). These agents and K⁺ (150 mM) were applied from micropipettes by brief (1 s) microperfusion pulses. Ca²⁺ levels were monitored at 2–3 s intervals during pre- and post-stimulus periods using microscopic digital imaging and the Ca²⁺ sensitive dye fura-2. iGluR and mGluR agonists and K⁺ produced abrupt increases in intracellular Ca²⁺ that slowly recovered to baseline resting levels. Acute exposure to ethanol at 33 mM (150 mg%) and 66 mM (300 mg%) significantly reduced the amplitude of the Ca²⁺ signals to iGluR agonists and K⁺ with little or no effect on Ca²⁺ signals to mGluR agonists. In contrast, acute ethanol at 10 mM (45 mg%) had no effect on the Ca²⁺ signals to the iGluR agonist AMPA but significantly enhanced the Ca²⁺ signals to the mGluR agonist DHPG. These results show that ethanol modulates Ca²⁺ signaling linked to GluR activation in a receptor subtype specific manner, and suggest that Ca²⁺ signaling pathways linked to GluR activation and membrane depolarization may be important mechanisms by which ethanol alters the transduction of excitatory synaptic signals at glutamatergic synapses and thereby affects intercellular and intracellular communication in the CNS.     

Tubular aggregate myopathy with features of Stormorken disease due to a new STIM1 mutation

STIM1 is a reticular Ca²⁺ sensor composed of a luminal and a cytosolic domain. Missense mutations in the luminal domain have been associated with tubular aggregate myopathy (TAM), while cytosolic mutations can cause Stormorken syndrome, a multisystemic disease associating TAM with asplenia, thrombocytopenia, miosis, ichthyosis, short stature and dyslexia. Here we present the case of a 41-year-old female complaining of exercise intolerance. Clinical examination showed short stature, scoliosis, proximal muscle weakness with lower limb predominance, and ophthalmoplegia. Laboratory tests revealed hypocalcemia, mild anemia and elevated creatine kinase (CK) levels. Whole-body muscle magnetic resonance imaging (MRI) revealed asplenia. Muscle biopsy was consistent with TAM. STIM1 gene analysis disclosed the novel c.252T>A, p.D84E missense mutation which was shown to induce constitutive STIM1 clustering in a functional study. This study reports a novel STIM1 mutation located in the Ca²⁺-binding EF domain causing TAM with features of Stormorken syndrome.     

Astrocyte dysfunction in Alzheimer disease

Astrocytes are glial cells that are distributed throughout the central nervous system in an arrangement optimal for chemical and physical interaction with neuronal synapses and brain blood supply vessels. Neurotransmission modulates astrocytic excitability by activating an array of cell surface receptors and transporter proteins, resulting in dynamic changes in intracellular Ca²⁺ or Na⁺. Ionic and electrogenic astrocytic changes, in turn, drive vital cell nonautonomous effects supporting brain function, including regulation of synaptic activity, neuronal metabolism, and regional blood supply. Alzheimer disease (AD) is associated with aberrant oligomeric amyloid β generation, which leads to extensive proliferation of astrocytes with a reactive phenotype and abnormal regulation of these processes. Astrocytic morphology, Ca²⁺ responses, extracellular K⁺ removal, glutamate transport, amyloid clearance, and energy metabolism are all affected in AD, resulting in a deleterious set of effects that includes glutamate excitotoxicity, impaired synaptic plasticity, reduced carbon delivery to neurons for oxidative phosphorylation, and dysregulated linkages between neuronal energy demand and regional blood supply. This review summarizes how astrocytes are affected in AD and describes how these changes are likely to influence brain function. © 2017 Wiley Periodicals, Inc.     

Suppression of presynaptic calcium influx by metabotropic glutamate receptor agonists in neonatal rat hippocampus

Mechanisms of presynaptic inhibition by metabotropic glutamate receptor (mGluR) agonists were investigated in neonatal rat hippocampal CA1 region using the optical recording technique recently developed. Following selective loading of presynaptic terminals with a fluorescent Ca²⁺ indicator dye rhod-2 AM, changes in Ca²⁺ signals and the corresponding field excitatory postsynaptic potentials (EPSPs) induced by single electrical stimuli to the Schaffer collateral-commissural (SCC) pathway were recorded simultaneously. Application of a mGluR agonist, 1 S,3 R-1-aminocyclopentane-1,3-dicar?ylic acid (1 S,3 R-ACPD; 100 μM) or (±)-1-aminocyclopentane-trans-1,3-dicar?ylic acid (trans-ACPD; 100 μM), reversibly reduced both the field EPSP and the presynaptic Ca²⁺ transient, and the quantitative relationship between them was quite similar to that observed during application of Cd²⁺, a non-selective Ca²⁺ channel blocker, or in a Ca²⁺-free solution. Application of 4-aminopyridine (4-AP; 1 mM), a blocker of certain subtypes of voltage-dependent K⁺ channels, significantly inhibited the 1 S,3 R-ACPD effect. Application of DCG-IV, a novel mGluR2/mGluR3-selective agonist, suppressed field EPSPs only slightly even at a high dose (3 μM). These results suggest that activation of presynaptic mGluR different from mGluR2/mGluR3 suppresses the action potential-triggered Ca²⁺ influx, probably via 4-AP-sensitive mechanisms, and thereby reduces glutamate release in neonatal rat hippocampal CA1 region.     

Roles of somatic A-type K~+ channels in the synaptic plasticity of hippocampal neurons

In the mammalian brain, information encoding and storage have been explained by revealing the cellular and molecular mechanisms of synaptic plasticity at various levels in the central nervous system, including the hippocampus and the cerebral cortices. The modulatory mechanisms of synaptic excitability that are correlated with neuronal tasks are fundamental factors for synaptic plasticity, and they are dependent on intracellular Ca²⁺-mediated signaling. In the present review, the A-type K⁺ (I _A) channel, one of the voltage-dependent cation channels, is considered as a key player in the modulation of Ca²⁺ influx through synaptic NMDA receptors and their correlated signaling pathways. The cellular functions of I _A channels indicate that they possibly play as integral parts of synaptic and somatic complexes, completing the initiation and stabilization of memory.     

Functional Integration of Calcium Regulatory Mechanisms at Purkinje Neuron Synapses

Cerebellar Purkinje neurons receive synaptic inputs from three different sources: the excitatory parallel fibre and climbing fibre synapses as well as the inhibitory synapses from molecular layer stellate and basket cells. These three synaptic systems use distinct mechanisms in order to generate Ca²⁺ signals that are specialized for specific modes of neurotransmitter release and post-synaptic signal integration. In this review, we first describe the repertoire of Ca²⁺ regulatory mechanisms that generate and regulate the amplitude and timing of Ca²⁺ fluxes during synaptic transmission and then explore how these mechanisms interact to generate the unique functional properties of each of the Purkinje neuron synapses.     

Protein kinase C modulates field-evoked transmitter release from cultured rat cerebellar granule cells via a dendrotoxin-sensitive K+ channel

The role of protein kinase C (PKC) in the control of neurotransmitter release from cultured rat cerebellar granule cells was investigated. Release of preloaded [³H]-d -aspartate which is incorporated into synaptic vesicles in this preparation was evoked by electrical field stimulation or elevated KCl. PKC activation by phorbol esters resulted in a large facilitation of field-evoked Ca²⁺-dependent [³H]-d -aspartate release and a lesser enhancement of KCl-stimulated release. Inhibition of PKC by Ro 31-8220 or staurosporine virtually abolished field-evoked release but had no effect on KCl-evoked release. Field-evoked, but not KCl-evoked, synaptic vesicle exocytosis monitored by the fluorescent vesicle probe FM2-10 was inhibited by staurosporine. PKC was not directly modulating neurite Ca²⁺ channels coupled to release, as Ro 31-8220 did not inhibit these channels. Activation or inhibition of PKC modulated field-evoked plasma membrane depolarization, but had no effect on KCl-evoked depolarization, consistent with a regulation of Na⁺ or K⁺ channels activated by field stimulation. No modulation of field-evoked neurite Na⁺ influx was seen using phorbol esters. Phorbol ester-induced facilitation of field-evoked [³H]-d -aspartate release and neurite Ca²⁺ entry was non-additive with that produced by the specific K⁺ channel antagonist dendrotoxin-1, suggesting that PKC modulates transmitter release from field-stimulated cerebellar granule cells by inhibiting a dendrotoxin-1-sensitive K⁺ channel.     

Somatic Ca2+ signaling in cerebellar Purkinje neurons

Activity‐driven Ca²⁺ signaling plays an important role in a number of neuronal functions, including neuronal growth, differentiation, and plasticity. Both cytosolic and nuclear Ca²⁺ has been implicated in these functions. In the current study, we investigated membrane‐to‐nucleus Ca²⁺ signaling in cerebellar Purkinje neurons in culture to gain insight into the pathways and mechanisms that can initiate nuclear Ca²⁺ signaling in this neuronal type. Purkinje neurons are known to express an abundance of Ca²⁺ signaling molecules such as voltage‐gated Ca²⁺ channels, ryanodine receptors, and IP3 receptors. Results show that membrane depolarization evoked by brief stimulation with K⁺ saline elicits a prominent Ca²⁺ signal in the cytosol and nucleus of the Purkinje neurons. Ca²⁺ influx through P/Q‐ and L‐type voltage‐gated Ca²⁺ channels and Ca²⁺‐induced Ca²⁺ release (CICR) from intracellular stores contributed to the Ca²⁺ signal, which spread from the plasma membrane to the nucleus. At strong K⁺ stimulations, the amplitude of the nuclear Ca²⁺ signal exceeded that of the cytosolic Ca²⁺ signal, suggesting the involvement of a nuclear amplification mechanism and/or differences in Ca²⁺ buffering in these two cellular compartments. An enhanced nuclear Ca²⁺ signal was more prominent for Ca²⁺ signals elicited by membrane depolarization than for Ca²⁺ signals elicited by activation of the metabotropic glutamate receptor pathway (mGluR1), which is linked to Ca²⁺ release from intracellular stores controlled by the IP3 receptor. © 2009 Wiley‐Liss, Inc.