High sensitivity of spontaneous spike frequency to sodium leak current in a Lymnaea pacemaker neuron

The spontaneous rhythmic firing of action potentials in pacemaker neurons depends on the biophysical properties of voltage‐gated ion channels and background leak currents. The background leak current includes a large K⁺ and a small Na⁺ component. We previously reported that a Na⁺‐leak current via U‐type channels is required to generate spontaneous action potential firing in the identified respiratory pacemaker neuron, RPeD1, in the freshwater pond snail Lymnaea stagnalis. We further investigated the functional significance of the background Na⁺ current in rhythmic spiking of RPeD1 neurons. Whole‐cell patch‐clamp recording and computational modeling approaches were carried out in isolated RPeD1 neurons. The whole‐cell current of the major ion channel components in RPeD1 neurons were characterized, and a conductance‐based computational model of the rhythmic pacemaker activity was simulated with the experimental measurements. We found that the spiking rate is more sensitive to changes in the Na⁺ leak current as compared to the K⁺ leak current, suggesting a robust function of Na⁺ leak current in regulating spontaneous neuronal firing activity. Our study provides new insight into our current understanding of the role of Na⁺ leak current in intrinsic properties of pacemaker neurons.     

Voltage‐induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation‐secretion coupling is assumed to be dependent only on extracellular calcium ([Ca²⁺]_o). Ryanodine receptor (RyR)‐sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca²⁺]_i) that are voltage‐dependent. We hypothesised that voltage‐elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage‐gated calcium channels and/or from such voltage‐sensitive ryanodine‐mediated calcium stores. Increases in [Ca²⁺]_i upon depolarisation in the presence of voltage‐gated calcium channel blockers, or in the absence of [Ca²⁺]_o, still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca²⁺]_o, there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Furthermore, such depolarisations lead to increases in [Ca²⁺]_i. Pre‐incubation with BAPTA‐AM resulted in > 50% inhibition of peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Nifedipine but not nicardipine inhibited both the high K⁺ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage‐induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation‐induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine‐sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.     

Mechanism of the medium‐duration afterhyperpolarization in rat serotonergic neurons

Most serotonergic neurons display a prominent medium‐duration afterhyperpolarization (mAHP), which is mediated by small‐conductance Ca²⁺‐activated K⁺ (SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of Ca²⁺ which activates the mAHP channels in serotonergic neurons. In voltage‐clamp experiments, an outward current was recorded at ?60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or (‐)‐bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca²⁺ channel blocker Co²⁺ and was partially blocked by both ω‐conotoxin and mibefradil, which are blockers of N‐type and T‐type Ca²⁺ channels, respectively. Neither blockers of other voltage‐gated Ca²⁺ channels nor DBHQ, an inhibitor of Ca²⁺‐induced Ca²⁺ release, had any effect on the SK current. In current‐clamp experiments, mAHPs following action potentials were only blocked by ω‐conotoxin and were unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced to fire in an in vivo‐like pacemaker rate, only ω‐conotoxin was able to increase their firing rate (by ~30%), an effect identical to the one previously reported for apamin. Our results demonstrate that N‐type Ca²⁺ channels are the only source of Ca²⁺ which activates the SK channels underlying the mAHP. T‐type Ca²⁺ channels may also activate SK channels under different circumstances.     

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.     

Bombesin facilitates GABAergic transmission and depresses epileptiform activity in the entorhinal cortex

Bombesin and the bombesin‐like peptides including neuromedin B (NMB) and gastrin‐releasing peptide (GRP) are important neuromodulators in the brain. We studied their effects on GABAergic transmission and epileptiform activity in the entorhinal cortex (EC). Bath application of bombesin concentration‐dependently increased both the frequency and amplitude of sIPSCs recorded from the principal neurons in the EC. Application of NMB and GRP exerted the same effects as bombesin. Bombesin had no effects on mIPSCs recorded in the presence of TTX but slightly depressed the evoked IPSCs. Omission of extracellular Ca²⁺ or inclusion of voltage‐gated Ca²⁺ channel blockers, Cd²⁺ and Ni²⁺, blocked bombesin‐induced increases in sIPSCs suggesting that bombesin increases GABA release via facilitating extracellular Ca²⁺ influx. Bombesin induced membrane depolarization and slightly increased the input resistance of GABAergic interneurons recorded from layer III of the EC. The action potential firing frequency of the interneurons was also increased by bombesin. Bombesin‐mediated depolarization of interneurons was unlikely to be mediated by the opening of a cationic conductance but due to the inhibition of inward rectifier K⁺ channels. Bath application of bombesin, NMB and GRP depressed the frequency of the epileptiform activity elicited by deprivation of Mg²⁺ from the extracellular solution suggesting that bombesin and the bombesin‐like peptides have antiepileptic effects in the brain. © 2013 Wiley Periodicals, Inc.     

Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Voltage‐gated sodium channel Nav1.5 contributes to astrogliosis in an in vitro model of glial injury via reverse Na+/Ca2+ exchange

Astrogliosis is a prominent feature of many, if not all, pathologies of the brain and spinal cord, yet a detailed understanding of the underlying molecular pathways involved in the transformation from quiescent to reactive astrocyte remains elusive. We investigated the contribution of voltage‐gated sodium channels to astrogliosis in an in vitro model of mechanical injury to astrocytes. Previous studies have shown that a scratch injury to astrocytes invokes dual mechanisms of migration and proliferation in these cells. Our results demonstrate that wound closure after mechanical injury, involving both migration and proliferation, is attenuated by pharmacological treatment with tetrodotoxin (TTX) and KB‐R7943, at a dose that blocks reverse mode of the Na⁺/Ca²⁺ exchanger (NCX), and by knockdown of Na_v1.5 mRNA. We also show that astrocytes display a robust [Ca²⁺]_i transient after mechanical injury and demonstrate that this [Ca²⁺]_i response is also attenuated by TTX, KB‐R7943, and Na_v1.5 mRNA knockdown. Our results suggest that Na_v1.5 and NCX are potential targets for modulation of astrogliosis after injury via their effect on [Ca²⁺]_i. GLIA 2014;62:1162–1175     

Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca2+ homeostasis

Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 μm ) effects on SNc neurons in acute rat midbrain slices, using whole‐cell patch‐clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide‐sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca²⁺] ([Ca²⁺]_i) (73.8 ± 7.7 nm ) and intracellular [Na⁺] (3.1 ± 0.6 mm ) (all with 1 μm ). The outward current was not affected by a high ATP level (10 mm ) in the patch pipette but was decreased by Trolox. The [Ca²⁺]_i rise was abolished by removing extracellular Ca²⁺, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N‐(p‐amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine‐123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm ) that, by itself, did not evoke a [Ca²⁺]_i rise resulted in a large (46.6 ± 25.3 nm ) Ca²⁺ response when baseline [Ca²⁺]_i was increased by a ‘priming’ protocol that activated voltage‐gated Ca²⁺ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca²⁺]_i and the rotenone‐induced [Ca²⁺]_i rise. This correlation was not seen in non‐dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP‐gated K⁺ channels and TRPM2‐like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone‐induced [Ca²⁺]_i rise by a small increase in baseline [Ca²⁺]_i.     

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca²⁺ signaling was only examined in soma and thick primary processes of astrocytes where Ca²⁺‐sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca²⁺ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two‐photon imaging of Ca²⁺ transients in adult CA1 hippocampal astrocytes using a new single‐cell patch‐loading strategy to image Ca²⁺‐sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca²⁺ transients whereas astrocyte soma rarely showed spontaneous Ca²⁺ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L‐ or T‐type voltage‐gated calcium channels. Removal of extracellular Ca²⁺ almost completely and reversibly abolished the spontaneous signals while IP₃R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca²⁺. The Ca²⁺ influx dependency of the spontaneous signals in patch‐loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca²⁺ influx pathways in astrocytes. The mechanisms underlying these localized Ca²⁺ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093–2103     

Two types of Ca2+ channel linked to two endocytic pathways coordinately maintain synaptic transmission at the Drosophila synapse

Endocytosis at the presynaptic terminal is initiated by Ca²⁺ influx through voltage‐gated Ca²⁺ channels. At the Drosophila neuromuscular junction, we demonstrated two components of endocytosis linked to distinct Ca²⁺ channels. A voltage‐gated Ca²⁺ channel blocker, (R)‐(+)‐Bay K8644 (R‐BayK), selectively blocked one component (R‐BayK‐sensitive component) without affecting exocytosis, while low concentrations of La³⁺ preferentially depressed the other component (La³⁺ ‐sensitive component). In a temperature‐sensitive mutant, shibire^ts, at non‐permissive temperatures, dynamin clusters were found immunohistochemically at the active zone (AZ) during the R‐BayK‐sensitive endocytosis, while they were detected at the non‐AZ during the La³⁺‐sensitive endocytosis. Immunostaining of the Ca²⁺ channel α₂δ subunit encoded by straightjacket (stj) was found within the AZ, and a mutation in stj depressed the R‐BayK‐sensitive component but enhanced the La³⁺ ‐sensitive one, indicating that the α₂δ subunit is associated with the R‐BayK‐sensitive Ca²⁺ channel. Filipin bound to the non‐AZ membrane and inhibited the La³⁺ ‐sensitive component, but not the R‐BayK‐sensitive one. We concluded that the R‐BayK‐sensitive component of endocytosis occurred at the AZ and termed this AZ endocytosis. We also concluded that the La³⁺ ‐sensitive component occurred at the non‐AZ and termed this non‐AZ endocytosis. These two types of endocytosis were modulated by various drugs towards opposite directions, indicating that they were differentially regulated. During high‐frequency stimulation, AZ endocytosis operated mainly in the early phase, whereas non‐AZ endocytosis operated in the late phase. Thus, intense synaptic transmission is coordinately maintained by synaptic vesicle recycling initiated by Ca²⁺ influx through the two types of Ca²⁺ channel.     

Two distinct types of depolarizing afterpotentials are differentially expressed in stellate and pyramidal‐like neurons of entorhinal‐cortex layer II

Two types of principal neurons, stellate cells and pyramidal‐like cells, are found in medial entorhinal‐cortex (mEC) layer II, and are believed to represent two distinct channels of information processing and transmission in the entorhinal cortex–hippocampus network. In this study, we found that depolarizing afterpotentials (DAPs) that follow single action potentials (APs) evoked from various levels of holding membrane voltage (V_h) show distinct properties in the two cells types. In both, an evident DAP followed the AP at near‐threshold V_h levels, and was accompanied by an enhancement of excitability and spike‐timing precision. This DAP was sensitive to voltage‐gated Na⁺‐channel block with TTx, but not to partial removal of extracellular Ca²⁺. Application of 5‐μM anandamide, which inhibited the resurgent and persistent Na⁺‐current components in a relatively selective way, significantly reduced the amplitude of this particular DAP while exerting poor effects on the foregoing AP. In the presence of background hyperpolarization, DAPs showed an opposite behavior in the two cell types, as in stellate cells they became even more prominent, whereas in pyramidal‐like cells their amplitude was markedly reduced. The DAP observed in stellate cells under this condition was strongly inhibited by partial extracellular‐Ca²⁺ removal, and was sensitive to the low‐voltage‐activated Ca²⁺‐channel blocker, NNC55‐0396. This Ca²⁺ dependence was not observed in the residual DAP evoked in pyramidal‐like cells from likewise negative V_h levels. These results demonstrate that two distinct mechanism of DAP generation operate in mEC layer‐II neurons, one Na⁺‐dependent and active at near‐threshold V_h levels in both stellate and‐pyramidal‐like cells, the other Ca²⁺‐dependent and only expressed by stellate cells in the presence of background membrane hyperpolarization. © 2015 Wiley Periodicals, Inc.     

Dendritic excitability during increased synaptic activity in rat neocortical L5 pyramidal neurons

Recent years have seen increased study of dendritic integration, mostly in acute brain slices. However, due to the low background activity in brain slices the integration of synaptic input in slice preparations may not truly reflect conditions in vivo. To investigate dendritic integration, back‐propagation of the action potential (AP) and initiation of the dendritic Ca²⁺ spike we simultaneously recorded membrane potential at the soma and apical dendrite of layer 5 (L5) pyramidal neurons in quiescent and excited acute brain slices. After excitation of the brain slice the somatic input resistance decreased and the apparent passive space constant shortened. However, the back‐propagating AP and dendritic Ca²⁺ spike were robust during increased synaptic activity. The dendritic Ca²⁺ spike was suppressed by the ionic composition of the bath solution required for slice excitation, suggesting that Ca²⁺ spikes may be smaller in vivo than in the acute slice preparation. The results presented here suggest that, under the conditions of slice excitation examined in this study, the increased membrane conductance induced by activation of voltage‐gated channels during back‐propagation of the AP and dendritic Ca²⁺ spike initiation is sufficiently larger than the membrane conductance at subthreshold potentials to allow these two regenerative dendritic events to remain robust over several levels of synaptic activity in the apical dendrite of L5 pyramidal neurons.     

Repeated cocaine exposure decreases dopamine D2‐like receptor modulation of Ca2+ homeostasis in rat nucleus accumbens neurons

The nucleus accumbens (NAc) is a limbic structure in the forebrain that plays a critical role in cognitive function and addiction. Dopamine modulates activity of medium spiny neurons (MSNs) in the NAc. Both dopamine D₁‐like and D₂‐like receptors (including D1R or D_1,5R and D2R or D_2,3,4R, respectively) are thought to play critical roles in cocaine addiction. Our previous studies demonstrated that repeated cocaine exposure (which alters dopamine transmission) decreases excitability of NAc MSNs in cocaine‐sensitized, withdrawn rats. This decrease is characterized by a reduction in voltage‐sensitive Na⁺ currents and high voltage‐activated Ca²⁺ currents, along with increased voltage‐gated K⁺ currents. These changes are associated with enhanced activity in the D1R/cAMP/PKA/protein phosphatase 1 pathway and diminished calcineurin function. Although D1R‐mediated signaling is enhanced by repeated cocaine exposure, little is known whether and how the D2R is implicated in the cocaine‐induced NAc dysfunction. Here, we performed a combined electrophysiological, biochemical, and neuroimaging study that reveals the cocaine‐induced dysregulation of Ca²⁺ homeostasis with involvement of D2R. Our novel findings reveal that D2R stimulation reduced Ca²⁺ influx preferentially via the L‐type Ca²⁺ channels and evoked intracellular Ca²⁺ release, likely via inhibiting the cAMP/PKA cascade, in the NAc MSNs of drug‐free rats. However, repeated cocaine exposure abolished the D₂R effects on modulating Ca²⁺ homeostasis with enhanced PKA activity and led to a decrease in whole‐cell Ca²⁺ influx. These adaptations, which persisted for 21 days during cocaine abstinence, may contribute to the mechanism of cocaine withdrawal. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Calcium dependence of spontaneous neurotransmitter release

Spontaneous release of neurotransmitters is regulated by extracellular [Ca²⁺] and intracellular [Ca²⁺]. Curiously, some of the mechanisms of Ca²⁺ signaling at central synapses are different at excitatory and inhibitory synapses. While the stochastic activity of voltage‐activated Ca²⁺ channels triggers a majority of spontaneous release at inhibitory synapses, this is not the case at excitatory nerve terminals. Ca²⁺ release from intracellular stores regulates spontaneous release at excitatory and inhibitory terminals, as do agonists of the Ca²⁺‐sensing receptor. Molecular machinery triggering spontaneous vesicle fusion may differ from that underlying evoked release and may be one of the sources of heterogeneity in release mechanisms.     

Transforming growth factor‐β1 primes proliferating adult neural progenitor cells to electrophysiological functionality

The differentiation of adult neural progenitors (NPCs) into functional neurons is still a limiting factor in the neural stem cell field but mandatory for the potential use of NPCs in therapeutic approaches. Neuronal function requires the appropriate electrophysiological properties. Here, we demonstrate that priming of NPCs using transforming growth factor (TGF)‐β1 under conditions that usually favor NPCs' proliferation induces electrophysiological neuronal properties in adult NPCs. Gene chip array analyses revealed upregulation of voltage‐dependent ion channel subunits (Kcnd3, Scn1b, Cacng4, and Accn1), neurotransmitters, and synaptic proteins (Cadps, Snap25, Grik4, Gria3, Syngr3, and Gria4) as well as other neuronal proteins (doublecortin [DCX], Nrxn1, Sept8, and Als2cr3). Patch‐clamp analysis demonstrated that control‐treated cells expressed only voltage‐dependent K⁺‐channels of the delayed‐rectifier type and the A‐type channels. TGF‐β1‐treated cells possessed more negative resting potentials than nontreated cells owing to the presence of delayed‐rectifier and inward‐rectifier channels. Furthermore, TGF‐β1‐treated cells expressed voltage‐dependent, TTX‐sensitive Na⁺ channels, which showed increasing current density with TGF‐β1 treatment duration and voltage‐dependent (+)BayK8644‐sensitive L‐Type Ca²⁺ channels. In contrast to nontreated cells, TGF‐β1‐treated cells responded to current injections with action‐potentials in the current‐clamp mode. Furthermore, TGF‐β1‐treated cells responded to application of GABA with an increase in membrane conductance and showed spontaneous synaptic currents that were blocked by the GABA‐receptor antagonist picrotoxine. Only NPCs, which were treated with TGF‐β1, showed Na⁺ channel currents, action potentials, and GABAergic currents. In summary, stimulation of NPCs by TGF‐β1 fosters a functional neuronal phenotype, which will be of relevance for future cell replacement strategies in neurodegenerative diseases or acute CNS lesions. GLIA 2013;61:1767–1783     

Calcium electrogenesis in neocortical pyramidal neurons in vivo

Much of what is known about Ca²⁺ electrogenesis in neocortical cells has been derived from in vitro studies. Since Ca²⁺ currents are controlled by various modulators, comparing these findings to in vivo data is essential. Here, we analysed tetrodotoxin (TTX)-resistant, presumably Ca²⁺-mediated potentials in intracellularly recorded neocortical neurons in vivo. TTX was applied locally to block Na⁺ channels. Its effectiveness was demonstrated by the elimination of fast spikes and orthodromic responses. In response to depolarizing current pulses bringing the membrane potential beyond ≈–33 mV, 71% of neurons generated high-threshold Ca²⁺ spikes averaging 17 mV. This is in contrast with in vitro findings, where high-threshold spikes could only be elicited following the blockade of K⁺ conductances. Consistent with this, neurons dialysed with K⁺ channel blockers in vivo generated high-threshold spikes that had a lower threshold (≈–40 mV) and, with intracellular Cs⁺, a larger amplitude, indicating the presence of K⁺ currents opposing the activation of Ca²⁺ channels. Only 15% of cortical cells displayed low-threshold Ca²⁺ spikes. To compare high-threshold Ca²⁺ spikes evoked by synaptic stimuli or current injection, another group of cortical neurons was dialysed with QX-314 and Cs⁺, in the absence of extracellular TTX. Synaptic stimuli applied on a background of membrane depolarization elicited presumed Ca²⁺ spikes whose amplitude varied in a stepwise fashion. Thus, although there are numerous similarities between in vivo and in vitro data, some significant differences were found, which suggest that the high-voltage activated Ca²⁺ currents and/or the K⁺ conductances that oppose them are subjected to different modulatory influences in vivo than in vitro.     

Colonic inflammation up-regulates voltage-gated sodium channels in bladder sensory neurons via activation of peripheral transient potential vanilloid 1 receptors

Background Primary sensory neurons express several types of ion channels including transient receptor potential vanilloid 1 (TRPV1) and voltage‐gated Na⁺ channels. Our previous studies showed an increased excitability of bladder primary sensory and spinal neurons triggered by inflammation in the distal colon as a result of pelvic organ cross‐sensitization. The goal of this work was to determine the effects of TRPV1 receptor activation by potent agonists and/or colonic inflammation on voltage‐gated Na⁺ channels expressed in bladder sensory neurons. Methods Sprague–Dawley rats were treated with intracolonic saline (control), resiniferatoxin (RTX, 10^?7 mol L^?1), TNBS (colonic irritant) or double treatment (RTX followed by TNBS). Key Results TNBS‐induced colitis increased the amplitude of total Na⁺ current by two‐fold and of tetrodotoxin resistant (TTX‐R) Na⁺ current by 78% (P ≤ 0.05 to control) in lumbosacral bladder neurons during acute phase (3 days post‐TNBS). Instillation of RTX in the distal colon caused an enhancement in the amplitude of total Na⁺ current at ?20 mV from ?112.1 ± 18.7 pA/pF (control) to ?183.6 ± 27.8 pA/pF (3 days post‐RTX, P ≤ 0.05) without changes in TTX resistant component. The amplitude of net Na⁺ current was also increased by 119% at day 3 in the group with double treatment (RTX followed by TNBS, P ≤ 0.05 to control) which was significantly higher than in either group with a single treatment. Conclusions & Inferences These results provide evidence that colonic inflammation activates TRPV1 receptors at the peripheral sensory terminals leading to an up‐regulation of voltage gated Na⁺ channels on the cell soma of bladder sensory neurons. This mechanism may underlie the occurrence of peripheral cross‐sensitization in the pelvis and functional chronic pelvic pain.     

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.     

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca²⁺]_i, (2) a calcium and voltage dependent potassium channel (K_Ca). K_Ca channels had a conductance of (302±15) pS (n=5) and were activated by [Ca²⁺]_i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on K_Ca channels from rat cerebellum.     

The relationship between inflammation‐induced neuronal excitability and disrupted motor activity in the guinea pig distal colon

Background Colitis is associated with increased excitability of afterhyperpolarization neurons (AH neurons) and facilitated synaptic transmission in the myenteric plexus. These changes are accompanied by disrupted propulsive motility, particularly in ulcerated regions. This study examined the relationship between myenteric AH neuronal hyperexcitability and disrupted propulsive motility. Methods The voltage‐activated Na⁺ channel opener veratridine, the intermediate conductance Ca²⁺‐activated K⁺ channel inhibitor TRAM‐34 and the 5‐HT₄ receptor agonist tegaserod were used to evaluate the effects of neuronal hyperexcitability and synaptic facilitation on propulsive motility in normal guinea pig distal colon. Because trinitrobenzene sulfonic acid (TNBS)‐colitis‐induced hyperexcitability of myenteric afferent neurons involves increases in hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channel activity, the HCN channel inhibitors Cs⁺ and ZD7288 were used to suppress AH neuronal activity in TNBS‐colitis. Key Results In non‐inflamed preparations, veratridine halted propulsive motility (P < 0.001). The rate of propulsive motor activity was significantly reduced following addition of TRAM‐34 and tegaserod (P < 0.001). In TNBS‐inflamed preparations, in which motility was temporarily halted or obstructed at sites of ulceration, both Cs⁺ and ZD7288 normalized motility through the inflamed regions. Immunohistochemistry studies demonstrated that the proportion of AH neurons in the myenteric plexus was unchanged in ulcerated regions, but there was a 10% reduction in total number of neurons per ganglion. Conclusions and Inferences These findings support the concept that inflammation‐induced neuroplasticity in myenteric neurons, involving changes in ion channel activity that lead to enhanced AH neuronal excitability, can contribute to impaired propulsive colonic motility.