Somatostatin-14 and somatostatin-28 inhibit calcium currents in rat neocortical neurons

The prosomatostatin-derived peptides, somatostatin-14 and somatostatin-28, are believed to function as neurotransmitters or neuromodulators in the cerebral cortex. To investigate the molecular mechanisms by which these peptides induce their physiological effects in the cerebral cortex, we have examined the effects of somatostatin-14 and somatostatin-28 on voltage-dependent Ca2+ currents in rat neocortical neurons in culture. Ca2+ currents were recorded using whole-cell patch-clamp techniques under conditions in which K+ and Na+ currents were blocked. Ca2+ currents were induced by depolarization from the holding potential of -80 mV. Somatostatin-14 (100 nM) and somatostatin-28 (100 nM) did not significantly affect low-voltage activated Ca2+ currents, but blocked high-voltage activated Ca2+ currents and slowed the activation of this current. The effects of both peptides were concentration-dependent and reversible. Furthermore, the effects of somatostatin-14 and somatostatin-28 on the high-voltage activated Ca2+ currents were not additive, suggesting that both peptides regulate this ionic current through similar cellular mechanisms. When patch pipettes used to record the Ca2+ currents contained 100 microM cAMP and 0.5 mM isobutylmethylxanthine, a phosphodiesterase inhibitor, somatostatin-14 and somatostatin-28 still inhibited Ca2+ currents, indicating that the effects of these peptides on the Ca2+ currents were cAMP-independent. Inclusion of the non-hydrolysable guanine triphosphate analogue, guanine triphos-somatostatin-14 or somatostatin-28, suggesting the involvement of guanine nucleotide binding proteins in the actions of the peptides on the Ca2+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+ and Ca2+ currents of acutely isolated adult rat nodose ganglion cells

The electrical properties of nodose ganglion cells acutely isolated from adult rats were studied using the whole-cell patch-clamp recording method. Current-clamp recordings revealed a mean resting membrane potential of -54.3 mV and an input resistance of 527 M omega. Depolarizing current steps evoked action potentials with the following properties (mean): amplitude 111 mV, threshold -36 mV, and rate of rise 117 V/s. Two types of action potentials were observed, short and long duration. These properties, with the exception of input resistance (527 M omega cf. 50 M omega), are similar to those reported previously using intracellular recording methods in intact nodose ganglia (11, 20, 28). Brief application of 10 microM 5-hydroxytryptamine resulted in a rapid depolarization and burst of action potentials in the majority of cells. With voltage-clamp recording, step depolarizations to potentials positive to -10 mV elicited a transient inward current that was followed by a sustained outward current. Inward Na+ current was isolated by ion substitution and pharmacological agents. Two types of Na+ current were observed. One current was completely abolished by 3-15 microM tetrodotoxin (TTX), had a rapid time course, activated over the potential range -60 to -10 mV, and attained half-maximal conductance at -30 mV. The other current persisted in the presence of 15 microM TTX, had a slower time course, activated over the potential range -30 to 0 mV, and attained half-maximal conductance at -15 mV. In addition, 500 microM Cd2+ and 5.0 mM Co2+ reduced the TTX-insensitive current to 53 and 42% of control, respectively. Inward Ca2+ current was isolated by ion substitution and pharmacological agents and was identified by a dependence on external Ca2+. Cd2+ (500 microM) and Co2+ (5 mM) reduced the maximal inward current to 5 and 20% of control, respectively. When Ba2+ was substituted for Ca2+ as the charge carrier, the maximal inward current increased to 175% of control. Some cells had two Ca2+ current components, an inactivating component that activated near -60 mV and a large sustained current that activated near -40 mV. The initial inactivating current appeared as a "hump" on the current-voltage (I-V) curve over the potential range of -60 to -30 mV. The results indicate that, following isolation of these adult mammalian neurons, the membrane surfaces are sufficiently clean to allow patch-clamp recording.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct inhibition of voltage-activated Ca2+ channels by delta-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents

Both mu- and delta-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca(2+) channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of delta-opioid agonists on voltage-activated Ca(2+) channel currents (I(Ca)) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B(4) (IB(4)). The delta-opioid agonists [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage-activated I(Ca) in IB(4)-negative than IB(4)-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type I(Ca) in IB(4)-negative than IB(4)-positive neurons. However, DPDPE had no significant effect on the R-type I(Ca) in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage-activated I(Ca) in all the DRG neurons with T-type I(Ca). Double immunofluorescence labeling showed that the majority of the delta-opioid receptor-immunoreactive DRG neurons had IB(4) labeling, while all DRG neurons immunoreactive to delta-opioid receptors exhibited Cav(3.2) immunoreactivity. Additionally, DPDPE significantly inhibited high voltage-activated I(Ca) in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that delta-opioid agonists have a distinct effect on voltage-activated Ca(2+) channels in different phenotypes of primary sensory neurons. High voltage-activated Ca(2+) channels are more sensitive to inhibition by delta-opioid agonists in IB(4)-negative than IB(4)-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca(2+) currents.     

Calbindin-D28K (CaBP) levels and calcium currents in acutely dissociated epileptic neurons

Summary Nerve cells that lack the cytoplasmic Ca²⁺ binding protein Calbindin-D_28K (CaBP) appear to be selectively vulnerable to Ca²⁺-related injury consistent with a postulated intraneuronal Ca²⁺-buffering role of CaBP. We have confirmed the selective loss of CaBP from the dentate gyrus during kindling-induced epilepsy in acutely dissociated granule cells (GCs) from kindled rats. Immunohistochemically stained kindled neurons showed a significant loss of CaBP when compared to controls (p < 0.001; ANOVA). The Ca²⁺-buffering role of CaBP was assessed in acutely dissociated control and kindled GCs by examining a physiological process highly sensitive to intracellular Ca²⁺-buffering: the Ca²⁺ -dependent inactivation of high-voltage activated (HVA or L-type) Ca²⁺ currents in the absence (or presence) of exogenous Ca²⁺-chelators. Whole-cell patch clamp recordings in kindled GCs demonstrated a markedly enhanced Ca²⁺-dependent inactivation of Ca²⁺-currents. After brief conditioning Ca²⁺ currents, in the absence of an exogenous intraneuronal Ca²⁺-chelator, subsequent test Ca²⁺ currents were inactivated by 58.3% in kindled GCs, a significant increase from the 37.4% inactivation observed in control GCs (p< 0.005; ANOVA). The differential Ca²⁺ current decay and Ca²⁺-dependent inactivation were prevented in both control and kindled GCs upon loading the neurons with the exogenous Ca²⁺-chelator BAPTA. These experiments demonstrate a high correlation between the loss of CaBP and changes in Ca²⁺ current inactivation and are consistent with the hypothesis that CaBP contributes to the physiological Ca²⁺-buffering in mammalian neurons.     

Transient outward currents in cochlear ganglion neurons of the chick embryo.

Cochlear ganglion neurons were isolated from chick embryos and membrane currents recorded using the patch-clamp technique. Depolarizing voltage steps elicited transient outward currents whose inactivation was best fitted by a double-exponential function with time constants < 30 ms and > 100 ms. The fast inactivating transient outward current (Ito,f) had a threshold for activation of -61 +/- 5.5 mV; steady-state inactivation was voltage-dependent between -90 and -60 mV, with half-inactivation near -75 mV. The slowly inactivating outward current (Ito,s) showed an activation threshold of 34 +/- 4 mV. Half-inactivation was at -67 +/- 3 mV. Ito,f was blocked by 4-aminopyridine which did not affect Ito,s. The effect was concentration- and voltage-dependent. Tetraethylammonium had no effect on either fast or slow transient currents but reduced the amplitude of the non-inactivating outward current in a dose-dependent manner. Ito,f was strongly inhibited by removing Ca2+ from the extracellular bathing solution. Cobalt ions inhibited Ito,f in a dose-dependent manner between 2 and 20 mM. The inhibitory effect of Co2+ was voltage-dependent, displaying a bell-shaped inhibition curve as a function of membrane voltage, maximal inhibition occurring between -20 and 0 mV. Ca2+ removal did not affect Ito,s and partially reduced the amplitude of the steady-state current. These results provide kinetic and pharmacological evidence for the presence of two distinct transient outward currents in cochlear neurons. These currents may play a role in the first synaptic relay of sound transmission.     

Axotomy- and autotomy-induced changes in Ca2+ and K+ channel currents of rat dorsal root ganglion neurons

Sciatic nerve section (axotomy) increases the excitability of rat dorsal root ganglion (DRG) neurons. The changes in Ca2+ currents, K+ currents, Ca2+ sensitive K+ current, and hyperpolarization-activated cation current (I(H)) that may be associated with this effect were examined by whole cell recording. Axotomy affected the same conductances in all types of DRG neuron. In general, the largest changes were seen in "small" cells and the smallest changes were seen in "large" cells. High-voltage-activated Ca2+ channel current (HVA-I(Ba)) was reduced by axotomy. Although currents recorded in axotomized neurons exhibited increased inactivation, this did not account for all of the reduction in HVA-I(Ba). Activation kinetics were unchanged, and experiments with nifedipine and/or omega-conotoxin GVIA showed that there was no change in the percentage contribution of L-type, N-type, or "other" HVA-I(Ba) to the total current after axotomy. T-type (low-voltage-activated) I(Ba) was not affected by axotomy. Ca2+ sensitive K+ conductance (g(K,Ca)) appeared to be reduced, but when voltage protocols were adjusted to elicit similar amounts of Ca2+ influx into control and axotomized cells, I(K,Ca)(s) were unchanged. After axotomy, Cd2+ insensitive, steady-state K+ channel current, which primarily comprised delayed rectifier K+ current (I(K)), was reduced by about 60% in small, medium, and large cells. These data suggest that axotomy-induced increases in excitability are associated with decreases in I(K) and/or decreases in g(K,Ca) that are secondary to decreased Ca2+ influx. Because I(H) was reduced by axotomy, changes in this current do not contribute to increased excitability. The amplitude and inactivation of I(Ba) in all cell types was changed more profoundly in animals that exhibited self-mutilatory behavior (autotomy). The onset of this behavior corresponded with significant reduction in I(Ba) of large neurons. This finding supports the hypothesis that autotomy, that may be related to human neuropathic pain, is associated with changes in the properties of large myelinated sensory neurons.     

Whole-cell voltage-clamp study of the fading of GABA-activated currents in acutely dissociated hippocampal neurons

The lability of the responses of mammalian central neurons to gamma-aminobutyric acid (GABA) was studied using neurons acutely dissociated from the CA1 region of the adult guinea pig hippocampus as a model system. GABA was applied to the neuronal somata by pressure ejection and the resulting current (IGABA) recorded under whole-cell voltage clamp. In initial experiments we examined several basic properties of cells in this preparation. Our data confirm that passive and active membrane properties are similar to those which characterize cells in other preparations. In addition, GABA-dependent conductance (gGABA), reversal potential (EGABA), and the interaction of GABA with pentobarbital and bicuculline all appeared to be normal. Dendritic GABA application could cause depolarizing GABA responses, and somatic GABA application caused hyperpolarizations due to chloride (Cl-) movements. Repetitive brief applications (5-15 ms) of GABA (10(-5) to 10(-3) M) at a frequency of 0.5 Hz led to fading of successive peaks of IGABA until, at a given holding potential, a steady state was reached in which IGABA no longer changed. Imposing voltage steps lasting seconds during a train of steady-state GABA responses led initially to increased IGABA that then diminished with maintenance of the step voltage. The rate of decrease of IGABA at each new holding potential was independent of the polarity of the step in holding potential but was highly dependent on the rate of GABA application. Application rates as low as 0.05 Hz led to fading of IGABA, even with activation of relatively small conductances (5-15 nS). Since IGABA evoked by somatic GABA application in these cells is carried by Cl-, the Cl- equilibrium potential (ECl) is equal to the reversal potential for IGABA, i.e., to EGABA. The fading of IGABA with changes in holding potential can be almost entirely accounted for by a shift in ECl resulting from transmembrane flux of Cl- through the GABA-activated conductance. Maneuvers that prevent changes in the intracellular concentration of Cl-ions, [Cl-]i, including holding the membrane potential at EGABA during repetitive GABA application or buffering [Cl-]i with high pipette [Cl-], prevent changes in EGABA. Desensitization of the GABA response (an actual decrease in gGABA) occurs in these neurons during prolonged application of GABA (greater than 1 s) but with a slower time course than changes in EGABA. Whole-cell voltage-clamp techniques applied to tissue-cultured spinal cord neurons indicated that rapid shifts in EGABA result from repetitive GABA application in these cells as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-activated outward currents in neostriatal neurons

Whole-cell voltage-clamp recordings of outward currents were obtained from acutely dissociated neurons of the rat neostriatum in conditions in which inward Ca2+ current was not blocked and intracellular Ca2+ concentration was lightly buffered. Na+ currents were blocked with tetrodotoxin. In this situation, about 53 +/- 4% (mean +/- S.E.M.; n = 18) of the outward current evoked by a depolarization to 0 mV was sensitive to 400 microM Cd2+. A similar percentage was sensitive to high concentrations of intracellular chelators or to extracellular Ca2+ reduction (<500 microM); 35+/-4% (n=25) of the outward current was sensitive to 3.0 mM 4-aminopyridine. Most of the remaining current was blocked by 10 mM tetraethylammonium. The results suggest that about half of the outward current is activated by Ca2+ entry in the present conditions. The peptidic toxins charybdotoxin, iberotoxin and apamin confirmed these results, since 34 +/- 5% (n = 14), 29 5% (n= 14) and 28 +/- 6% (n=9) of the outward current was blocked by these peptides, respectively. The effects of charybdotoxin and iberotoxin added to that of apamin, but their effects largely occluded each other. There was additional Cd2+ block after the effect of any combination of toxins. Therefore, it is concluded that Ca2+-activated outward currents in neostriatal neurons comprise several components, including small and large conductance types. In addition, the present experiments demonstrate that Ca2+-activated K+ currents are a very important component of the outward current activated by depolarization in neostriatal neurons.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons posses both “big” and “small” types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM ω-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 μM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM ω-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 μM ω-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM ω-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly.The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Identification of two calcium currents in acutely dissociated neurons from the rat lateral geniculate nucleus

1. Intracellular recording in the in vitro slice preparation and whole-cell, patch-clamp recording of acutely dissociated neurons from the rat lateral geniculate nucleus (LGN) were combined to study the Ca currents underlying their electrical responses. In slices from young animals (postnatal days 13-16), we found that dorsal LGN neurons have responses similar to those of adult preparations, including the presence of a low-threshold Ca spike (LTS). After enzymatic isolation of LGN neurons from the same animals, the firing properties appeared well preserved, as indicated by whole-cell, current-clamp recordings from dissociated multipolar cells (presumably geniculocortical relay neurons). 2. Two types of Ca currents were identified in voltage-clamped, isolated LGN neurons on the basis of their voltage dependency, pharmacology, and selectivity properties. These two currents resemble the low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca channels found in rat sensory neurons (9). 3. The LVA current component required negative potentials (less than -80 mV) to deinactivate completely, started to activate around -60 mV and reached a plateau level around -25 mV. It peaked within 30-6 ms and decayed with a single time constant of approximately 24 ms at -20 mV. Its inactivation curve ranged from -100 to -40 mV, with a half-inactivation near -60 mV. The HVA current component could be isolated by holding the membrane potential positive to -60 mV, activated at potentials positive to -30 mV and peaked around +5 mV. The time-to-peak ranged from 30 to 6 ms in the voltage range from -30 to +35 mV and decayed very slowly with sustained depolarizing pulses (time constant ranged between 1,600 and 40 ms over the same voltage range). 4. The inactivation of LVA Ca current during depolarizing voltage steps was consistent with a voltage-dependent process. The recovery from inactivation after short (100 ms), inactivating prepulses displayed two exponential phases. The slower phase was predominant under conditions that induce large current flow through the membrane, suggesting a Ca-mediated mechanism. 5. The LVA current was preferentially blocked by 50 microM Ni2+, leaving the HVA currents almost unaltered. Fifty micromolars Cd2+, in contrast, seemed more effective in blocking the HVA component of the Ca current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low- and high-voltage-activated Ca2+ conductances in electrically excitable growth cones of chick dorsal root ganglion neurons

Growth cone Ca2+ currents of chick dorsal root ganglion (DRG) neurons were recorded by a patch pipette located on the cell soma. Somatic and neuritic conductances were selectively blocked either with TTX and Cd2+ or by superfusing with isotonic sucrose using a laminar flow perfusion system. DRG growth cones were electrically excitable and growth cone Ca2+ currents were similar to Ca2+ currents described in DRG somata. In particular low-voltage-activated (LVA) Ca2+ conductances were well represented contrary to previous suggestions in other cell types.