Activation of ryanodine receptors is required for PKA‐mediated downregulation of A‐type K+ channels in rat hippocampal neurons

A‐type K⁺ channels (I_A channels) contribute to learning and memory mechanisms by regulating neuronal excitabilities in the CNS, and their expression level is targeted by Ca²⁺ influx via synaptic NMDA receptors (NMDARs) during long‐term potentiation (LTP). However, it is not clear how local synaptic Ca²⁺ changes induce I_A downregulation throughout the neuron, extending from the active synapse to the soma. In this study, we tested if two major receptors of endoplasmic reticulum (ER), ryanodine (RyRs), and IP₃ (IP₃R) receptors, are involved in Ca²⁺‐mediated I_A downregulation in cultured hippocampal neurons of rats. The downregulation of I_A channels was induced by doubling the Ca²⁺ concentration in culture media (3.6 mM for 24 hrs) or treating with glycine (200 μM for 3 min) to induce chemical LTP (cLTP), and the changes in I_A peaks were measured electrophysiologically by a whole‐cell patch. We confirmed that Ca²⁺ or glycine treatment significantly reduced I_A peaks and that their effects were abolished by blocking NMDARs or voltage‐dependent Ca²⁺ channels (VDCCs). In this cellular processing, blocking RyRs (by ryanodine, 10 μM) but not IP₃Rs (by 2APB, 100 μM) completely abolished I_A downregulation, and the LTP observed in hippocampal slices was more diminished by ryanodine rather than 2APB. Furthermore, blocking RyRs also reduced Ca²⁺‐mediated PKA activation, indicating that sequential signaling cascades, including the ER and PKA, are involved in regulating I_A downregulation. These results strongly suggest a possibility that RyR contribution and mediated I_A downregulation are required to regulate membrane excitability as well as synaptic plasticity in CA3‐CA1 connections of the hippocampus. © 2017 Wiley Periodicals, Inc.     

Synaptic Input and ACh Modulation Regulate Dendritic Ca2+ Spike Duration in Pyramidal Neurons,Directly Affecting Their Somatic Output

Nonlinear synaptic integration in dendrites is a fundamental aspect of neural computation. One such key mechanism is the Ca²⁺ spike at the apical tuft of pyramidal neurons. Characterized by a plateau potential sustained for tens of milliseconds, the Ca²⁺ spike amplifies excitatory input, facilitates somatic action potentials (APs), and promotes synaptic plasticity. Despite its essential role, the mechanisms regulating it are largely unknown. Using a compartmental model of a layer 5 pyramidal cell (L5PC), we explored the plateau and termination phases of the Ca²⁺ spike under input current perturbations, long-step current-injections, and variations in the dendritic high-voltage-activated Ca²⁺ conductance (that occur during cholinergic modulation). We found that, surprisingly, timed excitatory input can shorten the Ca²⁺ spike duration while inhibitory input can either elongate or terminate it. A significant elongation also occurs when the high-voltage-activated Ca²⁺ channels (Ca_HVA) conductance is increased. To mechanistically understand these phenomena, we analyzed the currents involved in the spike. The plateau and termination phases are almost exclusively controlled by the Ca_HVA inward current and the I_m outward K⁺ current. We reduced the full model to a single-compartment model that faithfully preserved the responses of the Ca²⁺ spike to interventions and consisted of two dynamic variables: the membrane potential and the K⁺-channel activation level. A phase-plane analysis of the reduced model provides testable predictions for modulating the Ca²⁺ spike and reveals various dynamical regimes that explain the robust nature of the spike. Regulating the duration of the Ca²⁺ spike significantly impacts the cell synaptic-plasticity window and, as we show, its input-output relationship.SIGNIFICANCE STATEMENT Pyramidal neurons are the cortex''s principal projection neurons. In their apical tuft, dendritic Ca²⁺ spikes significantly impact information processing, synaptic plasticity, and the cell''s input-output relationship. Therefore, it is essential to understand the mechanisms regulating them. Using a compartmental model of a layer 5 pyramidal cell (L5PC), we explored the Ca²⁺ spike responses to synaptic perturbations and cholinergic modulation. We showed a counterintuitive phenomenon: early excitatory input shortens the spike, whereas weak inhibition elongates it. Also, we demonstrated that acetylcholine (ACh) extends the spike. Through a reduced model containing only the membrane potential and the K⁺-channel activation level, we explained these phenomena using a phase-plane analysis. Our work provides new information about the robustness of the Ca²⁺ spike and its controlling mechanisms.     

Calcium Channel-Dependent Induction of Long-Term Synaptic Plasticity at Excitatory Golgi Cell Synapses of Cerebellum

Golgi cells, together with granule cells and mossy fibers, form a neuronal microcircuit regulating information transfer at the cerebellum input stage. Despite theoretical predictions, little was known about long-term synaptic plasticity at Golgi cell synapses. Here, we have used whole-cell patch-clamp recordings and calcium imaging to investigate long-term synaptic plasticity at excitatory synapses impinging on Golgi cells. In acute mouse cerebellar slices, mossy fiber theta-burst stimulation (TBS) could induce either long-term potentiation (LTP) or long-term depression (LTD) at mossy fiber-Golgi cell and granule cell-Golgi cell synapses. This synaptic plasticity showed a peculiar voltage dependence, with LTD or LTP being favored when TBS induction occurred at depolarized or hyperpolarized potentials, respectively. LTP required, in addition to NMDA channels, activation of T-type Ca²⁺ channels, while LTD required uniquely activation of L-type Ca²⁺ channels. Notably, the voltage dependence of plasticity at the mossy fiber-Golgi cell synapses was inverted with respect to pure NMDA receptor-dependent plasticity at the neighboring mossy fiber-granule cell synapse, implying that the mossy fiber presynaptic terminal can activate different induction mechanisms depending on the target cell. In aggregate, this result shows that Golgi cells show cell-specific forms of long-term plasticity at their excitatory synapses, that could play a crucial role in sculpting the response patterns of the cerebellar granular layer.SIGNIFICANCE STATEMENT This article shows for the first time a novel form of Ca²⁺ channel-dependent synaptic plasticity at the excitatory synapses impinging on cerebellar Golgi cells. This plasticity is bidirectional and inverted with respect to NMDA receptor-dependent paradigms, with long-term depression (LTD) and long-term potentiation (LTP) being favored at depolarized and hyperpolarized potentials, respectively. Furthermore, LTP and LTD induction requires differential involvement of T-type and L-type voltage-gated Ca²⁺ channels rather than the NMDA receptors alone. These results, along with recent computational predictions, support the idea that Golgi cell plasticity could play a crucial role in controlling information flow through the granular layer along with cerebellar learning and memory.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Decoding glutamate receptor activation by the Ca2+ sensor protein hippocalcin in rat hippocampal neurons

Hippocalcin is a Ca²⁺‐binding protein that belongs to a family of neuronal Ca²⁺sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically released glutamate can be decoded by hippocalcin translocation. Local AMPA receptor activation resulted in fast hippocalcin‐YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor‐dependent depolarization and following Ca²⁺influx via voltage‐operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin‐YFP translocation in a dendritic shaft at the application site due to direct Ca²⁺influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin‐YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed in response to a single synaptic vesicle release and to back‐propagating action potentials. The translocation to spines required Ca²⁺influx via synaptic NMDA receptors in which Mg²⁺ block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine heads and even closely (within 1–2 μm) located spines on the same dendritic branch signalled independently. Thus, we conclude that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site‐ and time‐specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.     

Modulation of presynaptic Ca2+ currents in frog motor nerve terminals by high pressure

Presynaptic Ca²⁺‐dependent mechanisms have already been implicated in depression of evoked synaptic transmission by high pressure (HP). Therefore, pressure effects on terminal Ca²⁺ currents were studied in Rana pipiens peripheral motor nerves. The terminal currents, evoked by nerve or direct stimulation, were recorded under the nerve perineurial sheath with a loose macropatch clamp technique. The combined use of Na⁺ and K⁺ channel blockers, [Ca²⁺]_o changes, voltage‐dependent Ca²⁺ channel (VDCC) blocker treatments and HP perturbations revealed two components of presynaptic Ca²⁺ currents: an early fast Ca²⁺ current (I_CaF), possibly carried by N‐type (Ca_V2.2) Ca²⁺ channels, and a late slow Ca²⁺ current (I_CaS), possibly mediated by L‐type (Ca_V1) Ca²⁺ channels. HP reduced the amplitude and decreased the maximum (saturation level) of the Ca²⁺ currents, I_CaF being more sensitive to pressure, and may have slightly shifted the voltage dependence. HP also moderately diminished the Na⁺ action current, which contributed to the depression of VDCC currents. Computer‐based modeling was used to verify the interpretation of the currents and investigate the influence of HP on the presynaptic currents. The direct HP reduction of the VDCC currents and the indirect effect of the action potential decrease are probably the major cause of pressure depression of synaptic release.     

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca²⁺ antagonist, cilnidipine, on voltage-dependent Ca²⁺ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca²⁺ channels at the low concentrations under 10⁻⁶ M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca²⁺ current (I_Ca) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I_Ca at the low concentrations under 10⁻⁷ and 10⁻⁶ M cilnidipine blocked not only L- but also N-type HVA I_Ca. At the high concentration over 10⁻⁶ M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca²⁺ channels. This is the first report that cilnidipine at lower concentration of 10⁻⁶ M blocks both L- and N-type HVA I_Ca in the hippocampal neurons.     

In vitro findings of alterations in intracellular calcium homeostasis in schizophrenia

The pathogenesis of schizophrenia involves several complex cellular mechanisms and is not well understood. Recent research has demonstrated an association between primary disturbances characteristic of the disease, including altered dopaminergic and glutamatergic neurotransmission, and impairments in neuronal calcium (Ca²⁺) homeostasis and signaling. Emerging Ca²⁺ hypothesis links and unifies various cellular processes involved in the pathogenesis of schizophrenia and suggests a central role of dysregulation of Ca²⁺ homeostasis in the etiology of the disease. This review explores the in vitro data on Ca²⁺ homeostasis and signaling in schizophrenia. Major limitation in this research is the lack of schizophrenia markers and validated disease models. As indicated in this review, one way to overcome these limitations may be analyses of Ca²⁺ signalosomes in peripheral cells from schizophrenia patients. Validation of animal models of schizophrenia may permit the application of advanced Ca²⁺ imaging techniques in living animals.     

Synaptic Contributions to Cochlear Outer Hair Cell Ca2+ Dynamics

For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca²⁺ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca²⁺ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca²⁺ influx have been poorly explored: voltage-gated L-type Ca²⁺ channels (VGCCs) at synapses with Type II afferent neurons, and α9α10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca²⁺ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca²⁺ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca²⁺-ATPases most likely present in nearby synaptic cisterns. Although Ca²⁺ currents in OHCs are small, VGCC Ca²⁺ signals were comparable in size to those elicited by α9α10 receptors, and were potentiated by ryanodine receptors (RyRs). In contrast, no evidence of potentiation by RyRs was found for MOC Ca²⁺ signals over a wide range of presynaptic stimulation strengths. Our study shows that despite the fact that these two Ca²⁺ entry sites are closely positioned, they differ in their regulation by intracellular cisterns and/or organelles, suggesting the existence of well-tuned mechanisms to separate the two different OHC synaptic functions.SIGNIFICANCE STATEMENT Outer hair cells (OHCs) are sensory cells in the inner ear operating under very special constraints. Acoustic stimulation leads to fast changes both in membrane potential and in the intracellular concentration of metabolites such as Ca²⁺. Tight mechanisms for Ca²⁺ control in OHCs have been reported. Interestingly, Ca²⁺ is crucial for two important synaptic processes: inhibition by efferent cholinergic neurons, and glutamate release onto Type II afferent fibers. In the current study we functionally imaged Ca²⁺ at these two different synapses, showing close positioning within the basolateral compartment of OHCs. In addition, we show differential regulation of these two Ca²⁺ sources by synaptic cisterns and/or organelles, which could result crucial for functional segregation during normal hearing.     

Erratum to: Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum: [Brain Research 903 (2001) 247–252]

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (α_1A), N-type (α_1B), and L-type (α_1C and α_1D) Ca²⁺ channels were observed in the hippocampus at days 1–5 after ischemic injury. However, increased expression of N-type Ca²⁺ channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca²⁺ channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca²⁺ channels may prove useful for future investigations.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

Dendritic mechanisms controlling the threshold and timing requirement of synaptic plasticity

Active conductances located and operating on neuronal dendrites are expected to regulate synaptic integration and plasticity. We investigate how Kv4.2‐mediated A‐type K⁺ channels and Ca²⁺‐activated K⁺ channels are involved in the induction process of Hebbian‐type plasticity that requires correlated pre‐ and postsynaptic activities. In CA1 pyramidal neurons, robust long‐term potentiation (LTP) induced by a theta burst pairing protocol usually occurred within a narrow window during which incoming synaptic potentials coincided with postsynaptic depolarization. Elimination of dendritic A‐type K⁺ currents in Kv4.2^?/? mice, however, resulted in an expanded time window, making the induction of synaptic potentiation less dependent on the temporal relation of pre‐ and postsynaptic activity. For the other type of synaptic plasticity, long‐term depression, the threshold was significantly increased in Kv4.2^?/? mice. This shift in depression threshold was restored to normal when the appropriate amount of internal free calcium was chelated during induction. In concert with A‐type channels, Ca²⁺‐activated K⁺ channels also exerted a sliding effect on synaptic plasticity. Blocking these channels in Kv4.2^?/? mice resulted in an even larger potentiation while by contrast, the depression threshold was shifted further. In conclusion, dendritic A‐type and Ca²⁺‐activated K⁺ channels dually regulate the timing‐dependence and thresholds of synaptic plasticity in an additive way. © 2010 Wiley‐Liss, Inc.     

Age-related change in short-term synaptic plasticity intrinsic to excitatory striatal synapses

Aging disrupts the expression of synaptic plasticity in many central nervous system (CNS) structures including the striatum. We found age differences in paired-pulse plasticity to persist at excitatory striatal synapses following block of gamma aminobutyric acid (GABA)_A and GABA_B receptors, a property that was independent of the number of afferents activated. High Mg²⁺/low Ca²⁺ artificial cerebral spinal fluid (ACSF) reduced release probability and consequently the size of the evoked excitatory post-synaptic potential (EPSP). High Mg²⁺/low Ca²⁺ ACSF also increased the expression of paired-pulse facilitation and eliminated the age difference seen previously in normal ACSF. These data suggest that age differences in paired-pulse plasticity reflect an alteration in release probability at excitatory striatal synapses. In support of this hypothesis, we found age differences in another presynaptic form of plasticity referred to as synaptic augmentation. Examination of the synaptic depression that developed during the conditioning tetanus also revealed an age-related increase in synaptic depression. These data indicate that age-related changes in facilitation may be due in part to a reduction in the readily releasable pool of synaptic vesicles. Dendritic structure (spine density and dendritic length) was correlated with short-term synaptic plasticity, but these relationships depended upon the variance associated with age (hierarchical regression). Post-hoc within-age group regressions demonstrated relationship between spine density and paired-pulse plasticity. No other age-specific correlations were found. These findings imply an age-dependent association between altered dendritic morphology and changes in synaptic plasticity. Synapse 27:57–68, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Expression of Ca channels from rat brain with model phenylketonuria in Xenopus oocytes

Ca²⁺ channels expressed in Xenopus oocytes by means of mRNA purified from the brain of the rats subjected to chronic treatment with l-phenylalanine in order to model conditions typical for the congenital disease called phenylketonuria (PKU) were studied using double microelectrode technique. The amplitude of Ca²⁺ channel currents (I_Ba, 40 mM Ba²⁺ as a charge carrier) directed in the oocytes by mRNA from the brain of the animals with model PKU was significantly smaller compared to the control animals (145±23 nA vs. 270±38 nA, p<0.025) while the voltage-dependence of both currents was similar and typical for that of high voltage-activated (HVA) Ca²⁺ channels. No evidence for the expression of low voltage-activated Ca²⁺ channels were found. The decrease of the overall HVA Ba²⁺ current under model PKU occurred primarily at the expense of the decaying, ω-conotoxin-sensitive component which accounted for about 64% of the total current amplitude in control, and apparently was associated with the activity of the expressed N-type Ca²⁺ channels. ω-Aga-IVA-sensitive, P/Q component of I_Ba that contributed not more than 10% to the total current in control showed no change under PKU conditions. In addition to the decreased amplitude, Ba²⁺ current from model PKU animals showed accelerated run-down during prolonged recording (50%/h compared to 15%/h in control). Our data suggest that hyperphenylalaninemic conditions affect the expression of preferentially N-type Ca²⁺ channels via the reduction of their specific mRNA content as well as influence the type and manner of channels regulation. The underexpression of N-type Ca²⁺ channels is consistent with the decrease in the overall number of synaptic contacts during PKU and may be one of the factors contributing to the severe damage of the brain function.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

N‐type calcium channels mediate a GABAB presynaptic modulation in the corticostriatal synapse in turtle's paleostriatum augmentatum

Spikes population evoked by a paired pulse protocol were used to assess the influence of GABA_A and GABA_B receptors agonists and antagonists on the synaptic potentials and in the S₂/S₁ ratio in a paired pulse (PP) protocol in the cortico‐paleostriatum augmentatum synapses of the turtle. GABA_A agonist, muscimol, decreased the amplitude of synaptic responses whereas the facilitation produced with the PP protocol did not change, suggesting a postsynaptic action for GABA_A receptors. GABA_B agonist, baclofen, enhanced paired pulse ratio indicating a presynaptic modulation through the GABA_B receptor. Selective antagonists for N‐ and P/Q‐type Ca²⁺‐channels also enhanced paired pulse ratio, suggesting that any of these channel types may be involved in neurotransmitter release. However, the strong paired pulse facilitation produced by baclofen was occluded by blocking the N‐type Ca²⁺ channels with ω‐conotoxin GVIA (1 μM), but not by the blockage of P/Q‐type Ca²⁺ channels with ω‐agatoxin TK (400 nM). These data suggest that N and P/Q channels participate in the neurotransmitter release, whereas only N‐type Ca²⁺ channels are involved in the presynaptic modulation of GABA_B in the corticostriatal synapse of the turtle. Synapse 63:855–862, 2009. © 2009 Wiley‐Liss, Inc.     

Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca_v2.2 and Ca_v2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals.