Mechanism of the medium‐duration afterhyperpolarization in rat serotonergic neurons

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

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

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

In vitro electrophysiology of rat subicular bursting neurons

Intracellular recordings were used to study the electrophysiological properties of rat subicular neurons in a brain slice preparation in vitro. Cells were classified as bursting neurons (n = 102) based on the firing pattern induced by depolarizing current pulses. The bursting response recorded at resting membrane potential (−66.1 ± 6.2 mV, mean ± SD n = 94) was made up of a cluster of fast action potentials riding on a slow depolarization and was followed by an afterhyperpolarization. Tonic firing occurred at a membrane potential of approximately −55 mV. A burst also occurred upon termination of a hyperpolarizing current pulse. Tetrodotoxin (TTX, 1 μM) blocked the burst and decreased or abolished the underlying slow depolarization. These effects were not induced by the concomitant application of the Ca²⁺ channel blockers Co²⁺ (2 mM) and Cd²⁺ (1 mM). Subicular bursting neurons displayed voltage- and time-dependent inward rectifications of the membrane during depolarizing and hyperpolarizing current pulses. The inward rectification in the depolarizing direction was abolished by TTX, while that in the hyperpolarizing direction was blocked by extracellular Cs⁺ (3 mM), but not modified by Ba²⁺ (0.5–1 mM), TTX, or Co²⁺ and Cd²⁺. Tetraethylammonium (10 mM)-sensitive, outward rectification became apparent in the presence of TTX. These results suggest that neurons in the rat subiculum can display voltage-dependent bursts of action potentials as well as membrane rectification in the depolarizing and hyperpolarizing directions. These results also indicate that activation of a voltage-gated Na⁺ conductance may be instrumental in the initiation of bursting activity. Hippocampus 7:48–57, 1997. © 1997 Wiley-Liss, Inc.     

Correlation of anoxic neuronal responses and calbindin-D28k localization in stratum pyramidale of rat hippocampus

Immunohistochemical staining for the calcium-binding protein calbindin-D_28k (CaBP) was combined with Lucifer Yellow (LY) identification and intracellular recording of changes in membrane parameters of pyramidal neurons in CA2, CA1, and the sebiculum of rat hippocampal slices during brief exposure (4.0 ± 0.19 min) to N₂. Anoxia evoked either a depolarization or hyperpolarization of membrane potential (V_M) (+21.5 ± 2.79 mV above V_M = ?70.5 ± 1.50 mV, n = 30 and ?7.2 ± 0.72 mV below V_M = ?68.2 ± 1.34 mV, n = 24, respectively) and a fall in membrane resistance of =20%. Differences in the response could be correlated with the presence or absence of CaBP and the localization of neurons in different layers of stratum pyramidale and sectors of the hippocampus. For neurons immunopositive for calbindin (CaBP(⁺)), depolarization was observed more frequently (83%) than hyperpolarization (17%); in contrast, 44% of responses of calbindin-negative (CaBP(^?)) neurons were depolarizing and 56% were hyperpolarizing. Depolarizations of CaBP(⁺) neurons were more gradual in slope, and more rapidly reached a plateau in comparison with those recorded in CaBP(^?) neurons. Responses of neurons in the superficial layer of stratum pyramidale (in which 79% of CaBP(⁺) pyramidal neurons were situated) were mainly depolarizing (91%), while for those in the deep layer (which contained 89% of the CaBP(^?) cells) such responses were observed less often (45%). Depolarization was also more common than hyperpolarization for cells located in CA2/CA1c/CA1b (63%) than in the CA1a/subicular region (37%). The depolarizing response of the majority of pyramidal neurons which are CaBP(⁺), superficial, and closer to CA3 may reflect an efficient buffering of intracellular Ca²⁺, which maintains a low [Ca²⁺]_i, steep gradient for Ca²⁺ influx and may facilitate the movement of Ca²⁺ away from points of entry. The neurons which are CaBP(^?), deep, and closer to subiculum and in which N₂ evokes hyperpolarization, on the other hand, may have a sustained elevation/accumulation of cytosolic Ca²⁺ which could activate K⁺ conductance, inhibit Ca²⁺ influx, and stabilize the membrane potential. These experiments provide a functional correlate for CaBP and suggest that it may have a significant role in Ca²⁺ homeostasis and the determination of selective neuronal vulnerability. © 1995 Wiley-Liss, Inc.     

Voltage Oscillations in Xenopus Spinal Cord Neurons: Developmental Onset and Dependence on Co-activation of NMDA and 5HT Receptors

The development of intrinsic, N-methyl-D-aspartate (NMDA) receptor-mediated voltage oscillations and their dependence on co-activation of 5-hydroxytryptamine (5HT) receptors was explored in motor neurons of late embryonic and early larval Xenopus laevis. Under tetrodotoxin, 100 μM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2–5 μM 5HT, oscillations were observed in 12% of embryonic and 70% of larval motor neurons. The voltage oscillations depended upon co-activation of NMDA and 5HT receptors since they were curtailed by selectively blocking NMDA receptors with D-2-amino-5-phosphonovaleric acid (APV) or by excluding Mg²⁺ from the experimental saline. 5HT applied in the absence of NMDA also failed to elicit oscillations. Oscillations could be induced by the non-selective 5HT_1a receptor agonist, 5-carboxamidotryptamine (5CT) and both 5HT- and 5CT-induced oscillations were abolished by pindobind-5HT₁, a selective 5HT_1a receptor antagonist. To test whether 5HT enables voltage oscillations by modulating the voltage-dependent block of NMDA channels by Mg²⁺, membrane conductance was monitored under tetrodotoxin. Although 5HT caused membrane hyperpolarization of 4–8 mV, there was little detectable change in conductance. NMDA application caused an approximate 20 mV depolarization and an ‘apparent’ decrease in conductance, presumably due to the conductance pulse bringing the membrane into a voltage region where Mg²⁺ blocks the NMDA ionophore. 5HT further decreased conductance, which we propose is due to its enhancement of the voltage-dependent Mg²⁺ block. When the membrane potential was depolarized by ～20 mV via depolarizing current injection (to mimic the NMDA-induced depolarization), 5HT increased rather than decreased membrane conductance. Furthermore, 5HT did not affect the increase in membrane conductance following NMDA applications in zero Mg²⁺ saline. The results suggest that intrinsic, NMDA receptor-mediated voltage oscillations develop in a brief period after hatching, and that they depend upon the co-activation of 5HT and NMDA receptors. The enabling function of 5HT may involve the facilitation of the voltage-dependent block of the NMDA ionophore by Mg²⁺ through activation of receptors with 5HT_1a-like pharmacology.     

Intracellular calcium responses of circadian pacemaker neurons measured with fura-2

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni²⁺. To test directly if an influx of Ca²⁺ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K⁺. Calcium levels in the BRNs remained elevated during treatments with 50 mM K⁺ lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca²⁺ (approx. 1.7 × 10⁻⁷ M) during depolarization blocked the rise in intracellular Ca²⁺, verifying that a Ca²⁺ influx is required. The sustained Ca²⁺ elevation during depolarization was also prevented with 50 mM Ni²⁺, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni²⁺, which does not block phase shifts. The initial rise in [Ca²⁺]_i in response to 50 mM K⁺ was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca²⁺ signal.     

Two distinct propagating regenerative potentials in a single ethanol-treated axon

Superfusion of the cockroach (Periplaneta americana) giant axon by ethanol (0.5–2%) produces conditions which allow the generation and active propagation of two kinds of regenerative potentials. (a) The classical action potential with a threshold at about 30 mV above resting potential, an amplitude of about 105 mV, a duration of 1–2 ms, and a conduction velocity of 3–6 m/s. (b) A smaller depolarizing potential (SDP) with a threshold of 5–15 mV above resting level, an amplitude of 10–30 mV, a duration of several hundred ms and a conduction velocity of 0.1–0.6 m/s. The SDP is associated with a small increase in conductance (10–15%), and is abolished by tetrodotoxin (10⁻⁶ M) or by removing extracellular sodium. The amplitude of the SDP slightly increases when the extracellular Ca²⁺ concentration is elevated and is reduced at low (Ca²⁺)₀ concentration; however, it is not blocked in a solution containing high Mg²⁺ (19 mM), low Ca²⁺ (1 mM) concentrations, indicating that inward Ca²⁺ current is not required for the generation of SDP.     

Stimulation of Ca2+-Activated Non-specific Cationic Channels by Phospholipase C-linked Glutamate Receptors in Synaptoneurosomes?

The regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) by glutamate metabotropic receptors (mGluR) was studied in 8-day-old rat forebrain synaptoneurosomes using spectrofluorimetric methods. Here we demonstrate that metabotropic glutamate agonists induce in rat brain synaptoneurosomes a Ca²⁺ influx largely dependent upon the presence of Ca²⁺ in the external medium. The pharmacological profile of this influx is strongly correlated with the pharmacological profile of the activation of phosphoinositide hydrolysis, i.e. quisqualic acid ～ 1S,3R-amino-1-dicarboxylate-1,3 cyclopentane ? glutamate. This metabotropic glutamate receptor-induced Ca²⁺ influx is insensitive to voltage-dependent Ca²⁺ channel antagonists and occurs through a Mn²⁺ impermeant pathway. The study of the rapid kinetics shows that this influx is triggered after a 300 ms delay compared with that elicited by depolarizing agents and Ca²⁺ ionophore A23187. In order to assess further if mGluR stimulate this influx through the recruitment of inositol triphosphate (IP3)-sensitive intracellular Ca²⁺ stores, we have tested the effect of thapsigargin on membrane potential and intracellular Ca²⁺ simultaneously. Thapsigargin induces a depolarization of the synaptoneurosomal membrane followed by a massive Ca²⁺ influx, occurring via a Mn²⁺ nonpermeant route. This depolarizing effect is sensitive to the presence of the intracellular Ca²⁺ chelator [1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetoxymethyl ester], and partially sensitive to extracellular Na⁺, but insensitive to the presence of extracellular Ca²⁺. Taken together, our data suggest that mGluR stimulate self-maintained increases of [Ca²⁺]_i in rat forebrain synaptoneurosomes via the activation of a multistep mechanism, sequenced in the following steps: (i) mGluR-induced IP3 synthesis; (ii) IP3-stimulated intracellular Ca²⁺ release; (iii) Ca²⁺-activated non-specific cation channel, leading to local depolarization and a Ca²⁺ influx; and (iv) activation of Ca²⁺-sensitive phospholipase C.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Two types of potassium channels in the PC12 cell line

Activation of K⁺ channels in the PC12 cell line was studied by comparing⁸⁶Rb⁺ efflux under depolarizing and non-depolarizing conditions. Evidence for both Ca²⁺-dependent and voltage-dependent K⁺ channels was obtained by studying depolarization-induced⁸⁶Rb⁺ efflux in solutions of varying Ca²⁺ concentration and in the presence of K⁺ and Ca²⁺ channel blocking agents.     

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.     

Studies on bursting pacemaker potential activity in molluscan neurons. I. Membrane properties and ionic contributions

Bursting pacemaker potential (BPP) activity of identified molluscan neurons has been studied using cells from Aplysia and Otala. The results presented in this paper indicate that (1) a potassium conductance mediates the hyperpolarizing phase of the BPP; (2) the BPP amplitude is directly dependent on [Na⁺]₀; (3) BPP activity requires the presence of divalent cations and is prevented by Co²⁺ and La³⁺, but not D-600; (4) the apparent increase in membrane resistance during the depolarizing phase of the BPP can be accounted for by the movement of the membrane potential along the non-linear portion of the I–V curve; and (5) non-linear I–V relations and a minimal effective membrane resistance are pre-requisite to BPP generation. Coupled with recent observations on the presence of an inward current in these cells, the results suggest that the mechanisms underlying the BPP are similar to those proposed to describe the myocardial pacemaker potential: the hyperpolarizing phase is due to activation of a potassium conductance which slowly inactivates, resulting in a gradual depolarization until a voltage-dependent inward current is activated which then leads to an increasingly rapid depolarization and initiation of the burst of spikes. It would appear that Na⁺ may play the major role in carrying the inward current, although a secondary role for divalent cations cannot be discounted.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Characterization of a Calcium-dependent Current Generating a Slow Afterdepolarization of CA3 Pyramidal Cells in Rat Hippocampal Slice Cultures

A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). Action potentials elicited by intracellular injection of short-lasting (50 – 100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50 – 100 ms) depolarizing voltage jumps from –60 mV to between –30 and 0 mV induced a slowly decaying outward aftercurrent (I_AHP) which was depressed by bath application of muscarine (0.5 μM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (I_ADP)in voltage-clamp mode. This current was also induced in the presence of trans(±)-1-aminc-1,3-cyclopenta-nedicarboxylic acid (t-ACPD, 5 μM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 μM), while both of these agonists depressed I_AHP. I_ADP was depressed by reducing the external Ca²⁺ concentration from 3.8 to 0.5 mM, by external Co²⁺ (1 mM) and by external Cd²⁺ (10 – 100 μM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca²⁺]_i using the Ca²⁺ indicator fura-2 revealed that the amplitude of I_ADP was correlated with the amplitude of depolarization-induced Ca²⁺ influx, I_ADP was absent at membrane potentials < –90 mV, and reached maximal amplitudes at ～–55 mV. Raising the extracellular K⁺ concentration from 2.7 to 13.5 mM increased the amplitude of I_ADP and resulted in a positively directed shift of the apparent reversal potential of I_ADP. When the external Na⁺ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external Cl^- concentration from 159 to 20 mM did not affect I_ADP. We conclude that I_ADP most likely represents a Ca²⁺-activated cation current, rather than a Ca²⁺ tail current, or an electrogenic Ca²⁺ extrusion current.     

The effects of catecholamines on electrical activity of neurons in the guinea pig supraoptic nucleus in vitro

The role of the central noradrenergic system in the supraoptic neuroendocrine regulation was investigated using slices of the guinea pig hypothalamus. Noradrenaline produced a complex membrane effect comprising two distinct depolarizations: one, associated with a moderate increase in input resistance and resulting in an augmentation of the spontaneous firing rate; the other, unaccompanied by a detectable change in input resistance and resulting in depression of the firing rate. The former depolarization was reproducible by applying specific α-agonist, phenylephrine, whereas the latter was induced by a β-adrenergic agonist, isoproterenol. The actions of phenylephrine and isoproterenol were blocked by phentolamine and propranolol, respectively. Amplitude of the phenylephrine-induced depolarization was voltage-dependent with the estimated reversal potential of about −115 mV and changed as a function of [K⁺]_o. On the contrary, amplitude of the isoproterenol-induced depolarization was voltage-independent and was insensitive to changes in external concentrations of K⁺, Na⁺, Cl⁻ and Ca²⁺. We conclude that catecholamines directly modulate the activity of supraoptic neurons through two functionally distinct adrenoceptive sites on neurosecretory cells. The activation of α-receptors may increase cellular excitability through suppression of membrane K⁺ conductance while the activation of β-receptors would depress neuronal firings, possibly through some mechanism which is not directly linked to ionic channels.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

A Study of the Barium-sensitive and -insensitive Components of the Action of Thyrotropin-releasing Hormone on Lumbar Motoneurons of the Rat Isolated Spinal Cord

The electrophysiological action of thyrotropin-releasing hormone (TRH) on rat spinal motoneurons was studied in vitro using single-electrode voltage- and current-clamp techniques. In current-clamp conditions TRH elicited a slowly developing depolarization, associated with a large input resistance increase and sustained neuronal firing; the primary metabolites of TRH were ineffective. Under voltage-clamp conditions in the presence of tetrodotoxin, TRH evoked a large inward current (I_TRH; peaking at approximately –40 mV) associated with a large input conductance fall. Only 44% of cells displayed I_TRH reversal; when the chord conductance values of these cells were plotted against membrane potential, a bell-shaped relation occurred, indicating voltage-dependent block by TRH of a persistent conductance active over a wide range of membrane potentials. I_TRH reversal values were shifted to more positive levels in high K⁺ solution in Nernstian fashion; hence a large proportion of the TRH response is suggested to be mediated by the block of a K⁺ conductance (I_K(T)). I_K(T) (and its voltage-dependent block by TRH) was resistant to certain K⁺ channel antagonists (tetraethylammonium, Cs⁺, 4-aminopyridine or apamin), but was depressed by Ba²⁺. The Ba²⁺-resistant fraction of I_TRH was attenuated by Cd²⁺, Mn²⁺ or Co²⁺, indicating that it probably involved a Ca²⁺-sensitive inward current. Concomitant application of Ba²⁺ and Cd²⁺ induced a near-total block of the response to TRH. It is suggested that suppression of I_K(T), associated with the onset of a Ca²⁺-sensitive current, can explain the excitatory effect of TRH on rat spinal motoneurons.     

Effect of D890 on membrane properties of neocortical neurons

D890, a derivative of the Ca²⁺ channel antagonist D600, was intracellularly applied from conventional microelectrodes into pyramidal neurons of neocortical slices. The effects of D890 were ascertained by evaluating alterations in membrane properties following drug administration and by comparing these neurons to untreated controls. The amplitude of action potentials (APs) evoked by depolarizing current pulses was attenuated by up to 30% within about 15 min after impalement with D890-containing electrodes. AP rate of rise was reduced by up to 80% and duration was increased. These effects were dependent upon the rate of stimulation. When depolarizing pulses were delivered at low rates of stimulation (e.g. 0.1 Hz), the overshoot of evoked APs declined by about 10%. At higher frequencies (2Hz) the AP overshoot decreased by up to 90%. These effects were mostly reversible on decreasing the frequency of stimulation. A half maximal effect was attained at about 1 Hz, when APs of control neurons were unaltered. In neurons impaled with D890-containing electrodes, depolarizing current pulses delivered in the presence of tetraethylammonium (TEA) and tetrodotoxin elicited high threshold calcium spikes which had a duration between 20 and 200 ms. In the early phase of D890 application, the duration of Ca²⁺ spikes decreased in a reversible frequency-dependent manner; after prolonged application, however, the recovery was incomplete. On the average, Ca²⁺ spike amplitude and duration decreased by 20% and 50%, respectively, suggesting that D890 usually produces an incomplete blockade of the underlying CA current. The duration of the slow envelope of orthodromically evoked epileptiform paroxysmal depolarizing shifts (DSs), induced by bath application of 10⁻⁵ M bicuculline, was frequency dependent and consistently increased from about 20 ms to 150 ms (half amplitude width) at frequencies above 0.5 Hz. In the presence of D890, the action potentials superimposed on the slow envelope of the DS were attenuated, but neither the amplitude nor the frequency-dependent progressive prolongation of the DS was altered. Application of TEA in the presence of bicuculline (10⁻⁵ M) increased the amplitude and duration of the DS in neurons impaled with D890-containing electrodes. Under these conditions, the durations of DSs evoked by low frequency orthodromic stimulation (0.5Hz) were still progressively prolonged, while, in the same neuron, directly evoked Ca²⁺ spikes progressively decreased in amplitude and duration. During DSs, the membrane potential depolarized to levels beyond those required for directly eliciting high threshold Ca²⁺ spikes, however, a Ca²⁺-spike component was not obvious during the DS. A high conductance state of the membrane at the peak of the DS may limit Ca²⁺ spike electrogenesis. The results suggest that D890 affects fast Na⁺ currents and the conventional Ca²⁺ conductance underlying the Ca²⁺ spike. The absence of effects of D890 on the DS slow envelope suggests that Ca²⁺ conductances do not make a large contribution to the latter in the neurons studied, or that Ca²⁺ flows through channels with different pharmacological properties during the DS and the Ca²⁺ spike.     

Bradykinin evoked depolarization of a novel neuroblastoma × DRG neurone hybrid cell line (ND723)

Application of bradykinin (Bk) to neuroblastoma × dorsal root ganglion (DRG) neurone hybrid cells (ND723) evoked an inward (depolarizing) current associated with an increase in membrane conductance. This response was antagonized byd-Arg⁰, Hyp³, Thi^5,8,d-Phe⁷-Bk, but was not mimicked by des-Arg⁹-Bk, indicating the involvement of B₂-receptors. The response was unaltered by replacement of extracellular Na⁺ byN-methylglucamine. Replacement of extracellular Cl⁻ by gluconate⁻ shifted the estimate reversal potential to a more positive value, while the use of potassium acetate filled recording electrodes shifted the reversal potential to a more negative value, and reduced the response amplitude, indicating the importance of Cl⁻ in the response. This response to Bk was mimicked by the calcium ionophore ionomycin. Bk stimulated the formation of inositol 1,4,5-trisphosphate (IP₃), and increased the release of arachidonic acid. In addition, Bk produced an increase in [Ca²⁺]_i, as determined by microspectrofluorimetry. This was due to the release of Ca²⁺ from intracellular stores, since the response was unaltered when the cells were bathed in Ca²⁺-free solution. In summary, Bk depolarizesND723 cells, probably through the activation of a chloride conductance. It seems likely that this is secondary to the rise in cytosolic Ca²⁺ concentration, due to the release of Ca²⁺ from internal stores by IP₃. This Ca²⁺-activated chloride response is present in some sensory neurones, although its role in the activation of sensory neurones by Bk is at present unclear.     

Ca2+‐dependent ion channels underlying spontaneous activity in insect circadian pacemaker neurons

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae (Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20–70 Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca²⁺ imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca²⁺ transients in circadian pacemakers accompany tetrodotoxin (TTX)‐blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX‐sensitive sustained Na⁺ channels. Instead, they are predominantly driven by mibefradil‐sensitive, low‐voltage‐activated Ca²⁺ channels and DK‐AH269‐sensitive hyperpolarisation‐activated, cyclic nucleotide‐gated cation channels. Rhythmic depolarisations activate voltage‐gated Na⁺ channels and nifedipine‐sensitive high‐voltage‐activated Ca²⁺ channels. Together with Ca²⁺ rises, the depolarisations open repolarising small‐conductance but not large‐conductance Ca²⁺‐dependent K⁺ channels. In contrast, we hypothesise that P/Q‐type Ca²⁺ channels coupled to large‐conductance Ca²⁺‐dependent K⁺ channels are involved in input‐dependent activity.