Effects of Growth Hormone-Releasing Peptide-2 (GHRP-2) on Membrane Ca2+ Permeability in Cultured Ovine Somatotrophs

The newly synthesized GH-releasing peptide, GHRP-2 (D-Ala-D-βNal-Ala-Trp-D-Phe-Lys-NH₂), was studied in somatotroph-enriched populations of ovine pituitary cells in primary culture. Nystatin-perforated whole-cell recordings were made on identified somatotrophs after 4–14 days of culture. Using a standard bath solution (containing Na⁺, Ca²⁺) and an electrode solution containing K⁺ in current-clamp recordings, GHRP-2 (10 nM) depolarized the membrane potential of the cells triggering a burst of action potentials. Voltage-clamp recordings indicated that GHRP-2 produced a slowly inactivated inward current with a slight reduction in outward current. The inward current was blocked by the Ca²⁺ channel blocker, Co²⁺ (0.5 mM). Ca²⁺ currents were then isolated using tetraethylammonium bath solution and an electrode solution containing Cs⁺. Ovine somatotrophs possess transient (T type) and long lasting (L type) Ca²⁺ currents. The L type current was abolished by addition of nifedipine (3 μM) to the bath solution and T type current was isolated on this basis. Current-voltage relationships indicated an increase in both T and L type Ca²⁺ currents in response to GHRP-2. The voltage-dependent inactivation curve for T type Ca²⁺ current was shifted towards a less negative level by the peptide. Intracellular free Ca²⁺ concentration ([Ca²⁺]i) in somatotroph-enriched populations was specifically increased by GHRP-2 but this effect was totally abolished by blockade of membrane Ca²⁺ channels. These data show that GHRP-2 causes an influx of Ca²⁺ leading to an increase in [Ca²⁺]i in ovine somatotrophs. The Ca²⁺ currents were both L type and T type with a shift in the inactivation curve of the latter by the releasing peptide.     

Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

Multiple Types of Ca2+ Channels in Mouse Motor Nerve Terminals

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca²⁺-channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω-Aga-IVA (0.3 μM), a blocker of P-type Ca²⁺channels] or ω-conotoxin MVIIC [ω-CTx-MVIIC (2 μM), a P-and Q-type Ca²⁺-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L-type Ca²⁺ channels) plus ω-conotoxin GVIA [ω-CTx-GVIA (2 μM), a blocker of N-type Ca²⁺ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca²⁺ signals, a small decrease in presynaptic Ca²⁺ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4-diaminopyridine (3, 4-DAP, a k⁺-channel blocker) or high Ca²⁺(10 mM) to accelerate and amplify the endplate potentials and Ca²⁺ currents. The endplate potentials amplified by 3, 4-DAP or by high Ca²⁺ correspondingly proved to be quite resistant to both ω-Aga-IVA and ω-CTx-MVIIC; ωAga-IVA exerted only a partial inhibitory effect on endplate potentials, and the ω-Aga-IVA-resistant component was further inhibited by ω-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca²⁺ channel blocker Cd²⁺ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na⁺ and K⁺ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca²⁺ currents in the triangularis sterni also revealed partial inhibition by ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA. Cd²⁺ (300 μM) abolished the toxin-resistant component of the Ca²⁺ current. In contrast, a combination of nifedipine (50 μM) with ω-CTx-GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca²⁺channels, i.e. ω-Aga-IVA-sensitive, ω-CTx-MVIIC-sensitive and toxin-resistant Ca²⁺ channels, coexist in mouse motor nerve terminals.     

Role of Voltage-gated Ca2+ Channels and Intracellular Ca2+ in Rat Sympathetic Neuron Survival and Function Promoted by High K+ and Cyclic AMP in the Presence or Absence of NGF

We have examined how NGF-dependent rat sympathetic neurons maintain Ca²⁺ homeostasis when challenged with high K⁺ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K⁺ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca²⁺ channels within 2 days. Although resting [Ca²⁺]_i was elevated by 1.6-fold, this was 50% less than the rise in [Ca²⁺]_i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca²⁺]_i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca²⁺ current, suggesting that voltage-sensitive Ca²⁺ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K⁺ medium with no significant rise in resting [Ca²⁺]_i, although neurites did not grow. Neither Ca²⁺ channel density nor resting [Ca²⁺]_i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca²⁺. However, the dihydropyridine antagonist nitrendipine blocked both high K⁺- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca²⁺]_i above resting levels to survive with either high K⁺ or CPTcAMP, but dihydropyridine-sensitive Ca²⁺ channel activity may be essential for their survival promoting actions.     

Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn2+ Quench of Fura 2 Fluorescence

In this study the rate of Mn²⁺ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca²⁺ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca²⁺ entry in isolation from effects on Ca²⁺ store release. The rate of fluorescence quench by 0.1 mM Mn²⁺ was markedly increased by 25 mM K⁺- evoked depolarization or by 200 μM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K⁺- or NMDA-evoked [Ca²⁺]_i response. The stimulatory effect of NMDA on Mn²⁺ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn²⁺ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca²⁺ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t_1/2 of quench was unaffected by α-amino-hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA- or K⁺-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca²⁺]_i elevations evoked by these agents are due to actions on Ca²⁺ stores. However, thapsigargin elevated [Ca²⁺Ii in the presence of normal [Ca²⁺]_i, but not in nominally Ca²⁺-free medium, indicating that it evokes Ca²⁺ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn²⁺ quench or to occur via Mn²⁺-impermeant channels.     

Opioid Inhibition of Ca2+ Channel Subtypes in Bovine Chromaffin Cells: Selectivity of Action and Voltage-dependence

Bovine chromaffin cells possess a mixture of high-voltage-activated Ca²⁺ channel subtypes: L-type, dihydropyridine-sensitive channels, and N-, P- and Q-types, ω-conotoxin MVIIC-sensitive channels. In these cells, we studied the reversible, naloxone-antagonized inhibition of Ba²⁺ currents by the opioid agonist met-enkephalin (IC₅₀= 272 nM). This inhibition could be resolved into a voltage-dependent and a voltage-independent component. The first was revealed by its slow Ba²⁺ current activation kinetics at 0 mV and by the current facilitation induced by short prepulses to +90 mV. The second was estimated as the residual inhibition persisting after the facilitation protocol. The two inhibitory components varied markedly from cell to cell and each contributed to about half of the total inhibition. Replacement of internal GTP by GDP-β-S or cell pretreatment with pertussis toxin completely abolished the voltage-dependent inhibition by opioids, partially preserving the voltage-independent component. The opioid-induced inhibition was not selective for any Ca²⁺ channel subtype, being not prevented after the addition of specific Ca²⁺ channel antagonists. However, when separately analyzing the contribution of each channel type to the voltage-dependent and voltage-independent modulation, a clear-cut distinction could be achieved. The voltage-independent inhibition was effective on all Ca²⁺ channel subtypes but predominantly on L-type Ca²⁺ channels. The voltage-dependent process was abolished by ω-conotoxin-MVIIC, but unaffected by nifedipine, and was thus sharply restricted to non-L-type channels (N-, P- and Q-types). Our data suggest a functionally distinct opioid receptor-mediated modulation of L- and non-L-type channels, i.e. of the two channel classes sharing major control of catecholamine secretion from bovine chromaffin cells.     

Ionic channel currents in cultured neurons from human cortex

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na⁺ current, two types of Ca²⁺ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K⁺ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca²⁺-activated K⁺ channels with slope conductances of 146 and 23 pS (K⁺ _iK⁺ _o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca²⁺. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K⁺ and Ca²⁺ channel currents, but Na⁺ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc.     

Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

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.     

Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

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.     

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells

Resting membrane potential (RMP), K⁺ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11–22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11–13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at ∼–70 mV and the other relatively less-polarized at ∼–40 mV. Ca²⁺_o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a ∼Nernstian relationship between RMP and [K⁺]_o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca²⁺_o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K⁺_o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU^–, tetanus toxin⁺ cells. Differentiating neurons were estimated to contain increased K⁺_i relative to less-polarized cells, coinciding with the developmental expression of Cs⁺/Ba²⁺-sensitive and Ca²⁺-dependent K⁺ channels. Both K⁺ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca²⁺_o-dependent and tetrodotoxin-insensitive to Na⁺_o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K⁺ channels and voltage-dependent Na⁺ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Functional Ca2+ and Na+ channels on mouse Schwann cells cultured in serum‐free medium: regulation by a diffusible factor from neurons and by cAMP

Regulation of expression of functional voltage-gated ion channels for inward currents was studied in Schwann cells in organotypic cultures of dorsal root ganglia from E19 mouse embryos maintained in serum-free medium. Of the Schwann cells that did not contact axons, 46.5% expressed T-type Ca²⁺ conductances (I_CaT). Two days or more after excision of the ganglia, and consequent disappearance of neurites, I_CaT were detectable in only 10.9% of the cells, and the marker 04 disappeared. On Schwann cells deprived of neurons, T- (but not L-) type Ca²⁺ conductances were re-induced by weakly hydrolysable analogues of cAMP, and by forskolin (an activator of adenylyl cyclase) after long-term treatment (4 days). With CPT cAMP (0.1–2 mm ), 8Br cAMP, db cAMP or forskolin (0.01 or 0.1 mm ), the proportion of cells with I_CaT was not significantly different from the proportion in the cultures with neurons. These agents also induced expression in some cells of tetrodotoxin-resistant Na⁺ currents, which were rarely induced by neurons, but 04 was not re-induced by cAMP analogue treatments that re-induced I_CaT. Inward currents (Ba²⁺ or Na⁺) were partly restored (P < 0.05) on Schwann cells cultured for 6–7 days beneath a filter bearing cultured neurons. In contrast, addition of neuron-conditioned medium was ineffective. The results suggest that neurons activate, via diffusible and degradable factors, a subset of Schwann cell cAMP pathways leading to expression of I_CaT, and activate additional non-cAMP pathways that lead to expression of 04.     

Interneuron-specific Ca2+ Responses Linked to Metabotropic -and lonotropic Glutamate Receptors in Rat Hippocampal Slices

Glutamate-mediated regulation of intracellular Ca²⁺ levels was examined in different populations of CA1 interneurons, using confocal microscopy and the Ca²⁺ indicator fluo 3-AM in rat hippocampal slices. Interneurons in basal [stratum oriendalveus (OA)] and apical [strata radiatum and lacunosum-moleculare (R/LM)] dendritic layers responded heterogeneously to glutamate. In control medium, OA interneurons responded mostly with oscillatory Ca²⁺ responses, which consisted of a large Ca²⁺ transient and successive smaller elevations. R/LM interneurons responded mostly with biphasic responses, characterized by an initial large transient and a secondary prolonged elevation. Other interneurons in both R/LM and OA responded with transient elevations in Ca²⁺ levels. lonotropic glutamate receptor antagonists (±)2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitro-quinoxaline-2,3-dione reduced peak Ca²⁺ responses in OA and R/LM cells, and blocked biphasic responses in R/LM interneurons. The metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine reduced peak Ca²⁺ responses only in OA interneurons, and prevented oscillatory responses. In low Ca²⁺ medium, peak responses were reduced in R/LM but not in OA interneurons, and oscillatory responses were absent. Combination of ionotropic and metabotropic receptor antagonists blocked all glutamate-evoked Ca²⁺ responses. Activation of different types of glutamate receptors may thus produce heterogeneous Ca²⁺ signals in subpopulations of CA1 interneurons. lonotropic receptors may generate biphasic responses in interneurons in apical dendritic layers, whereas combined activation of metabotropic and ionotropic receptors may trigger oscillatory responses in interneurons of basal dendritic layers. These heterogeneous Ca²⁺ responses indicate that glutamate-mediated Ca²⁺ processes and second messenger systems differ in subpopulations of hippocampal interneurons and suggest possible postsynaptic functional specialization of interneurons.     

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O₂) on the properties of K⁺ and Ca²⁺ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K⁺ current amplitudes were not affected by this period of hypoxia, but K⁺ current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K⁺ current density was separated into Ca²⁺-dependent and Ca²⁺-independent components by bath application of 200 μM Cd²⁺, which blocked Ca²⁺ currents and therefore, indirectly, Ca²⁺-dependent K⁺ currents. Ca²⁺-dependent K⁺ current density was not significantly different in control and chronically hypoxic type I cells. Cd²⁺-resistant (Ca²⁺-insensitive) K⁺ current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po₂ 15–22 mmHg) caused reversible, selective inhibition of Ca²⁺-dependent K⁺ currents in both groups of cells and Ca²⁺-insensitive K⁺ currents were unaffected by acute hypoxia. Ca²⁺ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca²⁺ channel current inhibition caused by nifedipine (5 μM). Acute hypoxia did not affect Ca²⁺ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca²⁺-insensitive, voltage-gated K⁺ currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.     

Membrane Properties of Identified Guinea-Pig Paraventricular Neurons and their Response to an Opioid μ-Receptor Agonist: Evidence for an Increase in K+ Conductance

Intracellular recordings were obtained from neurons in the paraventricular nucleus (PVN) of guinea-pig hypothalamic slices. Passive and active properties of the neurons were determined, and when possible, recorded neurons were injected with biocytin. The effects of a μ-receptor opioid agonist [D-Ala², Nme-Phe⁴, Gly⁵-ol]enkephalin (DAGO) were studied in order to determine which types of cells have μ receptors and to test the hypothesis that an increase in K⁺ conductance causes μ-receptor-mediated inhibition in the PVN. The recorded cells inside the PVN were divided into two groups, primarily on the basis of the presence or absence of a low threshold Ca²⁺ spike (LTS). In one group of neurons, LTS potentials could not be evoked (non-LTS cells, n = 42). In another group of neurons (n = 35), LTS potentials with one or two Na⁺ spikes could be initiated with depolarizing pulses superimposed on steady hyperpolarizing currents; however, these neurons did not fire robust bursts (i.e. non-bursting LTS cells). The mean time constant of non-LTS cells (19.9±1.6ms; mean ± SEM) was significantly shorter than that of non-bursting LTS cells (26.7 ± 2.1 ms). There were no differences in the mean resting membrane potential, spike amplitude, spike duration, input resistance, spike threshold and pattern of synaptic inputs between the two groups. Intracellular labeling with biocytin combined with cresyl violet counter-staining demonstrated that the two types of cells were located in the PVN. The mean diameters of non-LTS cells along their long axis (25.4 ± 1.7 μm) and short axis (17.5 ±2.2 μm, n = 7) were larger than those of non-bursting LTS cells (22.1 ±1.0 μm, 15.2 ± 1.1 μm, respectively, n = 12); this suggests that non-LTS cells are magnocellular neurons and non-bursting LTS cells are parvocellular neurons, as previously described in the rat. Bath application of DAGO at a concentration of 1 μM inhibited 7 of 25 (28%) non-LTS cells tested, and 4 of 21 (19%) non-bursting LTS cells. The main effect of DAGO on PVN cells was a hyperpolarization of membrane potential (4 to 15 mV) with a decrease of input resistance (20% to 38%). Quinine (100 μM to 1 mM), a K⁺ channel blocker, eliminated or reduced inhibitions mediated by DAGO. The apparent reversal potential of the hyperpolarization induced by the μ-receptor agonist was about ?85 mV. These data in the guinea-pig PVN suggest that non-LTS cells are putative magnocellular neurons, and non-bursting LTS cells are parvocellular neurons. The μ-receptor agonist, DAGO, directly hyperpolarizes approximately equal numbers of each of these cell types. Experiments with quinine and injected currents support the hypothesis that DAGO activation of μ receptors on neurons in the region of the PVN leads to an increase in K⁺ conductance.     

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.