Specificity in the interaction of HVA Ca2+ channel types with Ca2+-dependent AHPs and firing behavior in neocortical pyramidal neurons

Intracellular recordings and organic and inorganic Ca2+ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca2+ channels are differentially coupled to Ca2+-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca2+ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N-type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between approximately 80 and 90% of the slow AHP (sAHP) were Ca2+ dependent. Based on the effects of specific organic Ca2+ channel blockers (dihydropyridines, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca2+ current subtypes.     

Calcium channel subtypes in lamprey sensory and motor neurons

Pharmacologically distinct calcium channels have been characterized in dissociated cutaneous sensory neurons and motoneurons of the larval lamprey spinal cord. To enable cell identification, sensory dorsal cells and motoneurons were selectively labeled with fluorescein-coupled dextran amine in the intact spinal cord in vitro before dissociation. Calcium channels present in sensory dorsal cells, motoneurons, and other spinal cord neurons were characterized with the use of whole cell voltage-clamp recordings and specific calcium channel agonist and antagonists. The results show that a transient low-voltage-activated (LVA) calcium current was present in a proportion of sensory dorsal cells but not in motoneurons, whereas high-voltage-activated (HVA) calcium currents were seen in all neurons recorded. The different components of HVA current were dissected pharmacologically and similar results were obtained for both dorsal cells and motoneurons. The N-type calcium channel antagonist omega-conotoxin-GVIA (omega-CgTx) blocked >70% of the HVA current. A large part of the omega-CgTx block was reversed after washout of the toxin. The L-type calcium channel antagonist nimodipine blocked approximately 15% of the total HVA current. The dihydropyridine agonist (+/-)-BayK 8644 markedly increased the amplitude of the calcium channel current. The BayK-potentiated current was not affected by omega-CgTx, indicating that the reversibility of the omega-CgTx effect is not due to a blockade of L-type channels. Simultaneous application of omega-CgTx and nimodipine left approximately 15% of the HVA calcium channel current, a small part of which was blocked by the P/Q-type channel antagonist omega-agatoxin-IVA. In the presence of the three antagonists, the persistent residual current (approximately 10%) was completely blocked by cadmium. Our results provide evidence for the existence of HVA calcium channels of the N, L, and P/Q types and other HVA calcium channels in lamprey sensory neurons and motoneurons. In addition, certain types of neurons express LVA calcium channels.     

Multiple subtypes of voltage-gated calcium channel mediate transmitter release from parasympathetic neurons in the mouse bladder

Multiple subtypes of voltage-gated calcium channels are coupled to transmitter release from central neurons; however, only N-type channels have been shown to play a role in autonomic neurons. The aim of the present study was to investigate potential roles for other channel subtypes in transmitter release from parasympathetic neurons in the mouse bladder using calcium channel toxins alone and in combination. Transmitter release was measured indirectly by recording the contraction of bladder dome strips in response to electrical stimulation of the neurons by single pulses or trains of 20 pulses at 1-50 Hz. omega-Conotoxin-GVIA (GVIA) and omega-conotoxin-MVIIC (MVIIC) inhibited contractions in a concentration-dependent manner, with IC50 values of approximately 30 and 200 nM, respectively, at low stimulation frequencies. omega-Agatoxin-IVA (agatoxin) alone did not have any significant effect up to 300 nM. Cumulative addition of the toxins demonstrated that 300 nM agatoxin had a significant effect after N-type channels were blocked with 100 nM GVIA. MVIIC (3 microM) reduced the contraction amplitude further. Testing the toxins on the cholinergic or purinergic component of the contraction separately showed that acetylcholine release depends primarily on N-type channels and, to a lesser extent, on P- and Q-type channels, whereas ATP release involves predominantly P- and Q-type channels. In conclusion, parasympathetic neurons in the mouse bladder, like central neurons, use multiple calcium channel subtypes. Furthermore, the release of the two main transmitters in these neurons has differing dependencies on the calcium channel subtypes.     

Developmental features of human striatal tissue transplanted in a rat model of Huntington''s disease

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.     

Antagonists-resistant calcium currents in rat embryo motoneurons

Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5-20 mM Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed approximately 20% L-, approximately 45% N- and approximately 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20-200 nM omega-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nM in 5 mM Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 microM omega-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the alphaE channel. The current activates around -20 mV in 20 mM Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP-and omega-CTx-GVIA, and displays anomalous sensitivity to omega-Aga-IVA and omega-CTx-MVIIC.     

An electrophysiological study of the postnatal development of the corticospinal system in the macaque monkey

We characterized toxin-insensitive calcium currents expressed by acutely dissociated embryonic dorsal root ganglion neurons. In the presence of 3 microM omega-conotoxin-GVIA, 3 microM nitrendipine and either 500 nM omega-agatoxin-IVA or 500 nM omega-conotoxin-MVIIC to inhibit N-, L- and P/Q-type currents, respectively, all neurons expressed two residual currents: a T-type and another which we referred to as toxin-resistant current. The toxin-resistant current (i) consisted of an inactivating and a sustained components, (ii) had a threshold of activation and a steady-state inactivation comprised between that of the T-type current and that of the other high-voltage-activated currents, (iii) had the same permeability for barium and calcium used as charge carriers, (iv) was highly sensitive to both cadmium and nickel; and (v) was insensitive to 500 microM amiloride which abolished the T-type at this concentration. The properties of the toxin-resistant current are very similar to those of the currents expressed in oocytes following injection of alpha(1E) subunits which we demonstrated to be present in these neurons. Therefore a component of the toxin-resistant current calcium channels in sensory neurons may be closely related to those calcium channels formed by alpha(1E) subunits.     

Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurones from rat hippocampal slices

We have investigated the effects of protein tyrosine kinases (PTKs) inhibitors on high-threshold voltage activating (HVA) calcium currents in CA1 pyramidal neurones, whole-cell patch-clamp recorded from rat hippocampal slices. Genistein (100 microM) and tyrphostin B42 (100 microM), two PTKs inhibitors, reduced the steady-state barium current (IBa). On the other hand, daidzein and genistin (100 microM), two inactive analogues of genistein, had no effect on IBa amplitude. The inhibition induced by genistein was more pronounced at negative potentials. In order to characterize the calcium channels subtypes inhibited by PTKs inhibitors, we examined the effect of genistein in the presence of different calcium channel blockers. When L-type calcium channels were blocked by nifedipine, genistein induced a strong inhibition of the nifedipine-resistant IBa, suggesting an effect on non-L-type channels. Genistein did not antagonize the depressant effect of omega-Conotoxin-GVIA, a selective N-type calcium channel blocker, suggesting that N-type channels were not blocked by genistein. omega-Conotoxin-MVIIC (3-10 microM), a selective P/Q-type calcium channel blocker, greatly antagonized the depressant effect of genistein. Our results suggest that PTKs inhibitors reduce P-/Q-type, but not L- or N-types calcium currents in neurones of the CNS. The possible modulation of calcium channels by endogenous PTKs is discussed.     

Do we understand the evolution of genomic imprinting?

Voltage-gated calcium channels can be classified into high voltage activated (HVA) and low voltage activated (LVA or T-type) subtypes. The molecular diversity of HVA channels primarily results from different genes encoding their pore-forming alpha1 subunits. These channels share a common structure with an alpha1 subunit associated with at least two regulatory subunits (beta, alpha2-delta). Any of the six alpha1-related channels identified to date are regulated in their functional properties through an interaction with the ancillary beta-subunit. By contrast, the diversity and the molecular identity of LVA or T-type calcium channels have yet to be defined. Whether LVA channels are modulated by a beta-subunit, like HVA channels, is unknown. To address this issue, we have used an antisense strategy to inhibit beta-subunit expression in the NG 108-15 neuroblastoma cell line. Differentiated NG 108-15 cells express both LVA and HVA channels. We found that LVA currents were unaffected when cells were incubated with beta-antisense, while HVA currents were drastically decreased. Since LVA Ca channel currents in NG 108-15 cells are not regulated by beta-subunits, it is reasonable to postulate that the pore-forming subunit(s) of these channels lacks an interaction domain with a beta-subunit (AID). This molecular feature, which is common to various T-type channels, indicates further that LVA calcium channels belong to a channel family structurally distant from HVA channels.     

Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons

The dynamics of intracellular calcium concentration ([Ca2+]i) following activation of low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were studied in identified relay neurons and interneurons of the rat dorsal lateral geniculate nucleus (LGNd) in situ using Ca2+ imaging and patch-clamp techniques. In relay neurons, [Ca2+]i transients associated with the LVA Ca2+ current showed a fairly homogeneous somatodendritic distribution, whereas HVA transients significantly decreased to 65% of the somatic value at 60 microns dendritic distance. In interneurons, LVA transients significantly increased to 239% of the somatic value at 60 microns dendritic distance, whereas HVA transients were not significantly different in the soma and dendrites. These results indicate differences in [Ca2+]i dynamics, which may reflect a heterogeneous distribution of Ca2+ channels contributing to subcellular compartmentation in the two types of thalamic neurons.     

Calcium channel types contributing to excitatory and inhibitory synaptic transmission between individual hypothalamic neurons

The contribution of L-, N-, P- and Q-type Ca2+ channels to excitatory and inhibitory synaptic transmission and to whole-cell Ba2+ currents through Ca2+ channels (Ba2+ currents) was investigated in rat hypothalamic neurons grown in dissociated cell culture. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were evoked by stimulating individual neurons under whole-cell patch-clamp conditions. The different types of high-voltage-activated (HVA) Ca2+ channels were identified using nifedipine, omega-Conus geographus toxin VIA (omega-CTx GVIA), omega-Agelenopsis aperta toxin IVA (omega-Aga IVA), and omega-Conus magus toxin VIIC (omega-CTx MVIIC). N-, but not P- or Q-type Ca2+ channels contributed to excitatory as well as inhibitory synaptic transmission together with Ca2+ channels resistant to the aforementioned Ca2+ channel blockers (resistant Ca2+ channels). Reduction of postsynaptic current (PSC) amplitudes by N-type Ca2+ channel blockers was significantly stronger for IPSCs than for EPSCs. In most neurons whole-cell Ba2+ currents were carried by L-type Ca2+ channels and by at least two other Ca2+ channel types, one of which is probably of the Q-type and the others are resistant Ca2+ channels. These results indicate a different contribution of the various Ca2+ channel types to excitatory and inhibitory synaptic transmission and to whole-cell currents in these neurons and suggest different functional roles for the distinct Ca2+ channel types.     

Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex

A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 +/- 6.2 (SD) pF] than in neurons from layers II and III (8.6 +/- 2.8 and 7.9 +/- 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to -40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11-16 pA/pF at -30 mV, 2) inactivated rapidly with a time constant of approximately 22 ms at -30 mV, and 3) was completely steady-state inactivated at -60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.     

Conopressin affects excitability, firing, and action potential shape through stimulation of transient and persistent inward currents in mulluscan neurons

The molluscan vasopressin/oxytocin-related neuropeptide conopressin activates two persistent inward currents in neurons from the anterior lobe of the right cerebral ganglion of Lymnaea stagnalis that are involved in the control of male copulatory behavior. The low-voltage-activated (LVA) current is activated at a wide range of membrane potentials, its amplitude being only weakly voltage dependent. The high-voltage-activated (HVA) current is activated at potentials positive to -40 mV only and shows a steep voltage dependence. Occurrence of both currents varies from cell to cell, some expressing both and others only the HVA current. In most neurons that have the LVA current, a conopressin-independent persistent inward current (INSR) is found that resembles the HVA current in its voltage dependence. The functional importance of the LVA and HVA currents was studied under current-clamp conditions in isolated anterior lobe neurons. In cells exhibiting both current types, the effect of activation of the LVA current alone was investigated as follows: previously recorded LVA current profiles were injected into the neurons, and the effects were compared with responses induced by conopressin. Both treatments resulted in a strong depolarization and firing activity. No differences in firing frequency and burst duration were observed, indicating that activation of the LVA current is sufficient to evoke bursts. In cells exhibiting only the HVA current, the effect of conopressin on the response to a depolarizing stimulus was tested. Conopressin reversibly increased the number of action potentials generated by the stimulus, suggesting that the HVA current enhances excitability and counteracts accommodation. Conopressin enhanced action potential broadening during depolarizing stimuli in many neurons. Voltage-clamp experiments performed under ion-selective conditions revealed the presence of transient sodium and calcium currents. Using the action potential clamp technique, it was shown that both currents contribute to the action potential. The calcium current, which is activated mainly during the repolarizing phase of the action potential, is augmented by conopressin. Thus conopressin may directly modulate the shape of the action potential. In summary, conopressin may act simultaneously on multiple inward currents in anterior lobe neurons of Lymnaea to affect firing activity, excitability, and action potential shape.     

Voltage gated calcium channels in molluscs: classification, Ca2+ dependent inactivation, modulation and functional roles

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorous Conus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca2+ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca2+ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies, Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.     

Heart rate variability in patients with atrial fibrillation is related to vagal tone

A "reduced retina" preparation, consisting of the photoreceptor layer attached to the pigment epithelium in the eyecup, was used to study the pharmacology of the calcium channels controlling glutamate release by photoreceptors in Xenopus. Glutamate release was evoked either by dark adaptation or by superfusion with elevated (20 mM) potassium medium. Both darkness- and potassium-induced release were blocked by cadmium (200 microM). The N-type calcium channel blocker, omega-conotoxin GVIA (500 nM), the P-type calcium channel blocker, omega-agatoxin IVA (20 nM), and the P- and Q-type channel blocker omega-conotoxin MVIIC (1 microM) had no effect on glutamate release. In contrast, the dihydropyridines, nifedipine (10 microM) and nitrendipine (10 microM), which affect L-type calcium channels, blocked both darkness- and potassium-induced release. Bay K 8644 (10 microM), which promotes the open state of L-type calcium channels, enhanced glutamate release. These results indicate that photoreceptor glutamate release is controlled mainly by dihydropyridine-sensitive calcium channels. A dependence of glutamate release on L-type calcium channels also has been reported for depolarizing bipolar cells of a fish retina. Thus, it appears that non-inactivating L-type calcium channels are appropriate to mediate transmitter release in neurons whose physiological responses are sustained, graded potentials.     

Presynaptic GABAB autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons

GABA is the primary transmitter released by neurons of the suprachiasmatic nucleus (SCN), the circadian clock in the brain. Whereas GABAB receptor agonists exert a significant effect on circadian rhythms, the underlying mechanism by which GABAB receptors act in the SCN has remained a mystery. We found no GABAB receptor-mediated effect on slow potassium conductance, membrane potential, or input resistance in SCN neurons in vitro using whole-cell patch-clamp recording. In contrast, the GABAB receptor agonist baclofen (1-100 microM) exerted a large and dose-dependent inhibition (up to 100%) of evoked IPSCs. Baclofen reduced the frequency of spontaneous IPSCs but showed little effect on the frequency or amplitude of miniature IPSCs in the presence of tetrodotoxin. The activation of GABAB receptors did not modulate postsynaptic GABAA receptor responses. The depression of GABA release by GABAB autoreceptors appeared to be mediated primarily through a modulation of presynaptic calcium channels. The baclofen inhibition of both calcium currents and evoked IPSCs was greatly reduced (up to 100%) by the P/Q-type calcium channel blocker agatoxin IVB, suggesting that P/Q-type calcium channels are the major targets involved in the modulation of GABA release. To a lesser degree, N-type calcium channels were also involved. The inhibition of GABA release by baclofen was abolished by a pretreatment with pertussis toxin (PTX), whereas the inhibition of whole-cell calcium currents by baclofen was only partially depressed by PTX, suggesting that G-protein mechanisms involved in GABAB receptor modulation at the soma and axon terminal may not be identical. We conclude that GABAB receptor activation exerts a strong presynaptic inhibition of GABA release in SCN neurons, primarily by modulating P/Q-type calcium channels at axon terminals.     

Calcium oxalate crystal attachment to cultured kidney epithelial cell lines

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca2+-dependent K+ channels (KCa) activated by calcium influx through voltage-dependent channels during the action potential (Hill et al. 1992, Neuroreport, 3, 943-945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the KCa channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by omega-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2+/-14.9% (mean +/- SD). Application of the P/Q-type calcium channel blocker omega-agatoxin IVA reduced the amplitude of the AHP by 20.3+/-10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (+/-)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100-200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive KCa channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.     

Vasopressin/oxytocin-related conopressin induces two separate pacemaker currents in an identified central neuron of Lymnaea stagnalis

The molluscan vasopressin/oxytocin analogue Lys-conopressin excites neurons in the anterior lobe of the right cerebral ganglion of the snail Lymnaea stagnalis. Persistent inward currents that underlie the excitatory response were studied with the use of voltage-ramp protocols in the identified neuron RCB1 and other anterior lobe neurons. Under whole cell voltage-clamp conditions, two types of conopressin-activated current could be distinguished on the basis of their voltage dependence: 1) a pacemaker-like current that was activated at potentials above -40 mV (high-voltage-activated current, I(HVA)) and 2) an inward current that was activated at all potentials between -90 and +10 mV (low-voltage-activated current, I(LVA)). Ion substitution experiments indicate that sodium is the main charge carrier for I(HVA) and I(LVA). Both currents are differentially affected by cadmium. I(HVA) and I(LVA) differ in dose dependence, with median effective concentration values of 7.7 x 10(-8) M and 2.2 x 10(-7) M, respectively. Vasopressin and oxytocin act as weak agonists for the conopressin responses. The kinetics of desensitization and washout of I(HVA) and I(LVA) are different. The HVA response shows little desensitization, whereas the LVA response desensitizes within minutes (time constant 80 +/- 28 s, mean +/- SD). The time constant of washout on removal of conopressin is 159 +/- 63 s for I(HVA) and 36 +/- 13 s for I(LVA). These results suggest that two distinct conopressin receptors are involved in the activation of both currents. The conopressin-activated currents induce or enhance a region of negative slope resistance in the steady-state current-voltage relation. They differ from a third persistent inward current that is carried by calcium and completely blocked by cadmium. The presumed functional roles of these currents, possibly including autoregulation, are discussed.     

Calcium currents of rhythmic neurons recorded in the isolated respiratory network of neonatal mice

To obtain a quantitative characterization of voltage-activated calcium currents in respiratory neurons, we performed voltage-clamp recordings in the transverse brainstem slice of mice from neurons located within the ventral respiratory group. It is assumed that this medullary region contains the neuronal network responsible for generating the respiratory rhythm. This study represents one of the first attempts to analyze quantitatively the currents in respiratory neurons. The inward calcium currents of VRG neurons consisted of two components: a high voltage-activated (HVA) and a low voltage-activated (LVA) calcium current. The activation threshold of the HVA current was at -40 mV. It was fully activated (peak voltage) between -10 and 0 mV. The half-maximal activation (V50) was at -27. 29 mV +/- 1.15 (n = 24). The HVA current was inactivated completely at a holding potential of -35 mV and fully deinactivated at a holding potential of -65 mV (V50, -52.26 mV +/- 0.27; n = 18). The threshold for the activation of the LVA current was at -65 mV. This current had its peak voltage between -50 and -40 mV (mean, V50 = -59. 15 mV +/- 0.21; n = 15). The LVA current was inactivated completely at a holding potential of -65 mV and deinactivated fully at a holding potential of -95 mV (mean, V50 = -82.40 mV +/- 0.32; n = 38). These properties are consistent with other studies suggesting that the LVA current is a T-type current. The properties of these inward currents are discussed with respect to their role in generating Ca2+ potentials that may contribute to the generation of the mammalian respiratory rhythm.     

Modulation of high voltage-activated calcium channels by somatostatin in acutely isolated rat amygdaloid neurons

We investigated actions of somatostatin (Som) on voltagegated calcium channels in acutely isolated rat amygdaloid neurons. Somatostatin caused a dose-dependent inhibition of the high voltage-activated (HVA) Ca2+ current, with little or no effect on the low voltage-activated (LVA) current. Nifedipine (2-10 microM) reduced the peak current by approximately 15% without reducing inhibition of current by Som significantly, ruling out L-type channels as the target of modulation. The modulation appears to involve N- and P/Q-type calcium channels. After pretreatment with omega-conotoxin-GVIA (omega-CgTx) or omega-agatoxin-IVA, the inhibition was reduced but not abolished, whereas the combined application of both toxins nearly abolished the modulation. The Som analog BIM-23060 mimicked the effects of Som, whereas BIM-23058 had no effect, implicating Som type-2 receptors (SSTR-2). The inhibition was voltage-dependent, being minimal for small depolarizations, and was often accompanied by a slowing of the activation time course. Strong depolarizing prepulses partially relieved the inhibition and restored the time course of activation. Intracellular dialysis with GTP gamma S led to spontaneous inhibition and a slowing of the current like that with Som and occluded the effects of the peptide. Dialysis with GDP beta S also diminished the inhibition. A short preincubation with 50 microM of the alkylating agent N-ethylmaleimide (NEM) prevented the action of somatostatin. These results suggest a role for NEM-sensitive G-proteins in the Som inhibition. Application of 8-CPT-cAMP and IBMX did not mimic or prevent the effects of Som.     

Calcium currents in acutely isolated stellate and pyramidal neurons of rat entorhinal cortex

Calcium currents were studied in morphologically identified pyramidal and stellate neurons acutely isolated from layer II/III of rat entorhinal cortex, using the whole-cell patch-clamp configuration. The peak amplitude of high-voltage activated current (HVA) measured at +10 mV was not different in both neuron populations with 0.94+/-0.08 nA for pyramidal and 1.03+/-0.08 nA for stellate cells. Stellate neurons had a larger capacitance (14.4+/-1. 1 pF) than pyramidal neurons (9.6+/-0.8 pF), indicating a 50% larger cell surface. Most striking was the difference between the current density in stellate (79+/-8 pA/pF) versus pyramidal neurons (113+/-13 pA/pF). The potential of half maximal inactivation was not different: -37+/-2 mV (pyramidals) and -37+/-3 mV (stellates). Half of the cells contained a low-voltage activated calcium current (LVA) with a peak amplitude that was twice as large in stellate as in pyramidal neurons (0.21+/-0.04 nA resp. 0.11+/-0.03 nA; at -50 mV). In contrast to the HVA component, the current density of the LVA component was not different between cell types (13+/-3 pA/pF vs. 13+/-2 pA/pF). This implies that the relative abundance of LVA and HVA currents in stellate and pyramidal neurons is different which could result in different firing characteristics. The potential of half maximal LVA inactivation was -88+/-4 mV (pyramidals) and -85+/-3 mV (stellates). The slope of the voltage dependent steady state inactivation was steeper in stellate (7+/-1 mV) than in pyramidal cells (10+/-2 mV).