Depolarizing Pulse Research Articles

Glucocorticoid regulation of cardiac K+ currents and L-type Ca2+ current in neonatal mice.

Previous studies have reported that dexamethasone (Dex) prolongs cardiac action potential repolarization in mice and rats. However, the cellular mechanisms of this effect have not been addressed. Because action potential duration is influenced by a complex interplay of both inward and outward currents, this study evaluated the role of K+ currents and the L-type Ca2+ current in response to chronic in vivo Dex treatment. Accordingly, neonatal mice were randomly allocated to treatment with Dex (1 mg/kg per day) or placebo (saline) given subcutaneously for 5 days. At 14 to 15 days of age, the L-type Ca2+ current and K+ currents were recorded in ventricular myocytes using whole-cell patch-clamp techniques. The density of peak outward K+ currents was significantly decreased in the chronic Dex-treated group, but the current measured at the end of a 1-second depolarization pulse was similar in both groups. We further measured the magnitudes of the fast-inactivating (I(to)) and the slowly inactivating (I(slow)) currents that contribute to the peak outward K+ currents. I(to) was reduced from 17.5+/-3.0 pA/pF (control) to 10.6+/-2.5 pA/pF (Dex) at +50 mV (P<0.05), but I(slow) was not significantly different. These data suggest that downregulation of I(to) is responsible for the reduced peak outward current. Time courses of the onset and offset of in vivo Dex effects were also assessed. A period of 3 days of treatment was required to observe the Dex effect on peak outward K(+) currents, whereas a 7-day period after discontinuation of Dex was required to recover the baseline current density. Acute in vitro treatment with Dex (1 micromol/L) had no effect on K+ current densities. In addition, chronic Dex treatment significantly increased the density of the L-type Ca2+ current (I(Ca-L)) from -7.2+/-0.5 pA/pF of control to -8.9+/-0.6 pA/pF of Dex at +10 mV, P<0.05. In conclusion, chronic in vivo Dex treatment decreases I(to) and increases I(Ca-L) in neonatal mouse ventricular myocytes, both of which contribute to the prolongation of cardiac action potential repolarization induced by glucocorticoids.

Heterogeneous distribution of the two components of delayed rectifier K+ current: a potential mechanism of the proarrhythmic effects of methanesulfonanilideclass III agents.

To elucidate the regional difference of the K+ current blocking effects of methanesulfonanilide class III agents. Regional differences in action potential duration (APD) and E-4031-sensitive component (IKr) as well as -insensitive component (IKs) of the delayed rectifier K+ current (IK) were investigated in enzymatically isolated myocytes from apical and basal regions of the rabbit left ventricle using the whole-cell clamp technique. At 1 Hz stimulation, APD was significantly longer in the apex than in the base (223.1 +/- 10.6 vs. 182.7 +/- 14.5 ms, p < 0.05); application of 1 microM E-4031 caused more significant APD prolongation in the apex than in the base (32.5 +/- 6.4% vs. 21.0 +/- 8.8%, p < 0.05), resulting in an augmentation of regional dispersion of APD. In response to a 3-s depolarization pulse to +40 mV from a holding potential of -50 mV, both IK tail and IKs tail densities were significantly smaller in apical than in basal myocytes (IK: 1.56 +/- 0.13 vs. 2.09 +/- 0.21 pA/pF, p < 0.05; IKs: 0.40 +/- 0.15 vs. 1.43 +/- 0.23, p < 0.01), whereas IKr tail density was significantly greater in the apex than in the base (1.15 +/- 0.13 vs. 0.66 +/- 0.11 pA/pF, p < 0.01). The ratio of IKs/IKr for the tail current in the apex was significantly smaller than that in the base (0.51 +/- 0.21 vs. 3.09 +/- 0.89; p < 0.05). No statistical difference was observed in the voltage dependence as well as activation and deactivation kinetics of IKr and IKs between the apex and base. Isoproterenol (1 microM) increased the time-dependent outward current of IKs by 111 +/- 8% during the 3-s depolarizing step at +40 mV and its tail current by 120 +/- 9% on repolarization to the holding potential of -50 mV, whereas it did not affect IKr. The regional differences in IK, in particular differences in its two components may underlie the regional disparity in APD, and that methanesulfonanilide class III antiarrhythmic agents such as E-4031 may cause a greater spatial inhomogeneity of ventricular repolarization, leading to re-entrant arrhythmias.

Effects of propafenone on K currents in human atrial myocytes.

1. The class Ic anti-arrhythmic agent, flecainide is known to inhibit the transient outward K current (Ito) selectively in human atrium. We studied the effects of propafenone, another class Ic antiarrhythmic agent, on K currents in human atrial myocytes using a whole-cell voltage-clamp method. 2. Propafenone inhibited both Ito and the sustained or ultra-rapid delayed rectifier K current (Isus or Ikur) evoked by depolarization pulses. The concentration for half-maximal inhibition (IC50) was 4.9 microM for Ito and 8.6 microM for Isus. Propafenone blocked Ito and Isus in a voltage- and use-independent fashion and accelerated the inactivation time constant of Ito [from 28.3 to 6.7 ms at 10 microM propafenone]. 3. The steady-state inactivation curve for Ito was unaffected by propafenone. Propafenone did not affect the initial current at depolarizing potentials, but it did produce a block that increased as a function of time after depolarization (time constant of 3.4 ms). This suggests that propafenone preferentially blocked Ito in the open state. 4. Propafenone had no significant effect on the rate at which Ito recovered from inactivation at -80 mV suggesting that propafenone dissociates rapidly from the channel. 5. The steady-state activation curve for Isus was not affected by propafenone. Propafenone slowed the time course of the onset of the Isus tail current. This suggests that propafenone blocked Isus in the open state. 6. The present results suggest that, unlike flecainide, propafenone blocks both Ito and Isus in human atrial myocytes in the open state at clinically relevant concentrations.

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery.

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.

Voltage-activated K+ currents of hypoglossal motoneurons in a brain stem slice preparation from the neonatal rat.

Whole cell, patch-clamp recordings were performed on motoneurons of the hypoglossus nucleus in a brain stem slice preparation from the neonatal rat brain. The aim was to investigate transient outward currents activated by membrane depolarization under voltage clamp conditions. In a Ca2+-free medium containing tetrodotoxin and Cs+, depolarizing voltage commands from a holding potential of -50 mV induced slow outward currents (Islow) with 34 +/- 6 ms (SE) onset time constant at 0 mV and minimal decline during a 1 s pulse depolarization. When the depolarizing command was preceded by a prepulse to -110 mV, the outward current became biphasic as it comprised a faster component (Ifast), which could be investigated in isolation by subtracting the two sets of records. Ifast showed rapid kinetics (9 +/- 4 ms 10-90% rise time and 70 +/- 20 ms decay time constant at 0 mV) and strong voltage-dependent inactivation (half inactivation was at -92.9 +/- 0.2 mV) from which it readily recovered with a biexponential timecourse (4.4 +/- 0.6 and 17 +/- 2 ms time constants at -110 mV membrane potential). Islow was selectively blocked by TEA (10-30 mM) while Ifast was preferentially depressed by 2-3 mM 4-aminopyridine. Analysis of tail current reversal indicated that both Islow and Ifast were predominantly due to K+ with minor permeability to Na+ (92/1 and 50/1, respectively). These results suggest that membrane depolarization activated distinct K+ conductances that, in view of their largely dissimilar kinetics, are likely to play a differential role in regulating the firing behavior of hypoglossal motoneurons.

Properties of bipolar VIPergic interneurons and their excitation by pyramidal neurons in the rat neocortex.

In the rat neocortex, a subset of GABAergic interneurons express the neuropeptide vasoactive intestinal peptide (VIP). Previously, we demonstrated that a population of VIPergic interneurons could be accurately identified by their irregular spiking (IS) pattern and their bipolar morphology. IS interneurons were studied in neocortical slices from 16-22-day-old rats using whole-cell recordings, intracellular labelling and single-cell RT-PCR. In response to a depolarizing pulse, IS interneurons typically discharged a burst of action potentials followed by spikes emitted at an irregular frequency. Several seconds of depolarization, micromolar concentrations of 4-aminopyridine, and nanomolar concentrations of either dendrotoxin I or K converted this irregular pattern to a sustained discharge, suggesting the involvement of an ID-like K+ current. The main glutamate receptor subunits detected in IS cells were GluR1 flop and GluR2 flop, GluR5 and GluR6, and NR2B and NR2D for the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartic acid (NMDA) subtypes, respectively. Paired whole-cell patch-clamp recordings indicated that pyramidal neurons provide intracortical glutamatergic inputs onto IS interneurons. Most connections had high probabilities of response and exhibited frequency-dependent paired pulse depression. Comparison of the amplitude distribution of paired responses suggested that most of these connections consisted of multiple functional release sites. Finally, two discrete subpopulations of IS cells could be identified based on the duration of the initial burst of action potentials and the differential expression of calretinin and choline acetyltransferase.

A single pacemaker cell model based on the phase response curve.

A single pacemaker cell model and its response to repetitive external depolarization stimulations is described in this paper. This model is a simple model based on the two most important functional properties of the cardiac pacemaker cells. The first property is the intrinsic pacemaker cycle length, which is an 'internal' parameter of the cell, describing the most important feature of a pacemaker cell. The second functional property is the phase response curve (PRC), which is an 'overall collective' function: it contains all the 'information' about the possible interactions of the pacemaker cell with the outside world (external stimulus, interaction with surrounding cells, etc.). This study demonstrates that by representing the pacemaker cell only by two fundamental features, and by applying a simple physical-mathematical model, a global picture of the system can be achieved, allowing us to explore qualitatively various physiological phenomena related to the pacemaker function. For example, we demonstrated that the PRC is a crucial parameter in the prediction of the entrainment phenomena of a single pacemaker cell in response to a periodic train of depolarization pulses. Specifically, the PRC permits a quantitative determination of the 1:1 synchronization range for a single pacemaker cell and an external depolarization pulse. Moreover, we show that the PRC can be used to represent the type of external stimulus applied to the pacemaker (e.g. depolarization pulse) and its intensity. Therefore, the PRC emerges as an important determinant and a useful 'tool' for the understanding of the dynamic interaction of pacemaker cells with the outside world. As a result of our simulations, we unveil a new important parameter: the 'degree of influence', which determines the range of 1:1 synchronization between an external depolarization pulse and a pacemaker cell. This interaction parameter is a direct function of the PRC parameters. It appears to be a helpful 'tool' for the understanding of synchronization and mutual entrainment mechanisms between the pacemaker cell and an external stimulus, and therefore it supports the basic importance of the PRC in the description and determination of these mechanisms.

Excitatory Synaptic Transmission in the Inner Retina: Paired Recordings of Bipolar Cells and Neurons of the Ganglion Cell Layer

Properties of glutamatergic synaptic transmission were investigated by simultaneously voltage-clamping a pair of connected bipolar cells and cells in the ganglion cell layer (GLCs) in the newt retinal slice preparation. Activation of the Ca2+ current in a single bipolar cell was essential for evoking the glutamatergic postsynaptic current in the GLC. Depolarization for as short as 15 msec activated both NMDA and non-NMDA receptors. On the other hand, analysis of the spontaneous glutamatergic synaptic currents of GLCs revealed that these currents consisted of mainly non-NMDA receptor activation with little contribution from NMDA receptors. This suggests that non-NMDA receptors of GLCs are clustered in postsynaptic membrane regions immediately beneath the release sites of bipolar cells and that NMDA receptors have lower accessibility to the released transmitter than non-NMDA receptors. Glutamate that is spilled over from the release sites may activate the NMDA receptors. When a prolonged depolarizing pulse was applied to a bipolar cell, the response induced by non-NMDA receptors was limited greatly by their fast desensitization, whereas NMDA receptors were able to produce a maintained response. The relationship between the pulse duration applied to the bipolar cell and the integrated charge of the response evoked in the GLC was almost linear. Therefore, we propose that both non-NMDA and NMDA receptors cooperate to transfer the graded photoresponses of bipolar cells proportionally to GLCs.

In vivo whole-cell recording from neurons of the superior colliculus in fetal rats

In vivo whole-cell recording was made from neurons of the superior colliculus (SC) in rat fetuses which were connected with the dam by the umbilical cord. Fast action potentials could be generated by changing membrane potentials to depolarizing direction. The firing thresholds of fetal SC neurons appeared to be higher (between −50 and −35 mV) than those of brain neurons in later developmental stages. The action potentials of fetal SC neurons, which were smaller in amplitude and wider in duration as compared to mature brain neurons, revealed a small or no afterhyperpolarization. In addition to these fast action potentials, slow depolarizations of smaller amplitudes were evoked by intracellular injection of long depolarization pulses. Most neurons of the fetal SC (7/11) revealed linear current–voltage ( I– V) relations, while the remaining neurons displayed marked rectification. In some fetal SC neurons, presumed EPSPs and IPSPs occurred spontaneously. These presumed postsynaptic potentials showed temporal summation. These results suggest that rat SC neurons are functionally active even before birth, though the membrane properties remain immature.

Autaptic inhibitory currents recorded from interneurones in rat cerebellar slices.

1. While the presence of autapses in the brain is indicated by a large body of morphological evidence, the functional role of these structures has remained unclear. To probe for autaptic currents, we have recorded current responses following short somatic depolarizing pulses in Cl--loaded interneurones (stellate and basket cells) from rat cerebellar slices (animals aged 27-39 days). 2. In approximately 20 % of the recordings, fluctuating inward current transients were obtained with a latency of 1.15-2.45 ms (measured from the peak of the depolarization-induced Na+ current; n = 10). 3. These transients were blocked by bicuculline and were sensitive to the extracellular Ca2+ concentration. 4. Assuming low release probability, as suggested by the high failure rate (0.65-0.92, n = 10), quantal sizes ranging from 21 to 178 pA (-70 mV; n = 10) were calculated from a variance analysis of autaptic current amplitudes. 5. We conclude that approximately 20 % of interneurones have a functional autapse. Autaptic currents may inhibit firing of interneurones during high frequency discharges.

Ionic Dependence of the Axotomy-Induced Long-Lasting Firing in an Identified Crayfish Motoneuron

I have reported previously that axotomy of an identifiable anal motoneuron of crayfish Procambarus clarkii induces a long-lasting firing and that a prolonged depolarizing pulse to its cut end can induce a similar response. In this study, I confirmed that this stimulus is comparable to axotomy; the frequency of stimulus-induced firing increases linearly with the stimulus intensity and its firing pattern is the same as that following axotomy. Then, when the cut end was bathed for more than 1 hr in test solutions, it was examined whether the stimulus to the cut end induces or blocks the response. Na(+)-free saline (Tris(+) replaced Na(+)) or TTX (3 x 10(-7) M) reversibly blocked the response within 30 min. By contrast, Mn(2+) saline (40 mM Mn(2+) replaced Ca(2+)) or Ca(2+)-free salines (Mg(2+) or 1 mM EDTA replaced Ca(2+)) cannot block the response, but instead increased the firing frequency. These results obtained with stimulus were confirmed also by those with axotomy. I concluded that axotomy-induced firing, which occurs locally at its cut region, is primarily responsible for voltage-dependent Na(+) conductances, but not for Ca(2+) ones.

Voltage dependence of the pattern and frequency of discrete Ca2+ release events after brief repriming in frog skeletal muscle.

Applying a brief repolarizing pre-pulse to a depolarized frog skeletal muscle fiber restores a small fraction of the transverse tubule membrane voltage sensors from the inactivated state. During a subsequent depolarizing test pulse we detected brief, highly localized elevations of myoplasmic Ca2+ concentration (Ca2+ "sparks") initiated by restored voltage sensors in individual triads at all test pulse voltages. The latency histogram of these events gives the gating pattern of the sarcoplasmic reticulum (SR) calcium release channels controlled by the restored voltage sensors. Both event frequency and clustering of events near the start of the test pulse increase with test pulse depolarization. The macroscopic SR calcium release waveform, obtained from the spark latency histogram and the estimated open time of the channel or channels underlying a spark, exhibits an early peak and rapid marked decline during large depolarizations. For smaller depolarizations, the release waveform exhibits a smaller peak and a slower decline. However, the mean use time and mean amplitude of the individual sparks are quite similar at all test depolarizations and at all times during a given depolarization, indicating that the channel open times and conductances underlying sparks are essentially independent of voltage. Thus, the voltage dependence of SR Ca2+ release is due to changes in the frequency and pattern of occurrence of individual, voltage-independent, discrete release events.

Intracellular recordings from medial septal neurons during hippocampal theta rhythm.

The electrophysiological properties of neurons of the medial septal nucleus and the nucleus of the diagnonal band of Broca (MS/DB) were studied using intracellular methods in urethane-anesthetized rats. Three types of rhythmically bursting neurons were identified in vivo on the basis of their action potential shapes and durations, afterhyperpolarizations (AHPs), membrane characteristics, firing rates and sensitivities to the action of muscarinic antagonist: (1) Cells with short-duration action potentials and no AHPs (2 of 34 rhythmic cells, 6%) had high firing rates and extremely reliable bursts with 6-16 spikes per theta cycle, which were highly resistant to scopolamine action. (2) Cells with short-duration action potentials and short-duration AHPs (8 of 34 rhythmic cells, 24%) also had high firing rates and reliable bursts with 4-13 spikes per theta cycle, phase-locked to the negative peak of the dentate theta wave. Hyperpolarizing current injection revealed a brief membrane time constant, time-dependent membrane rectification and a burst of firing at the break. Depolarizing current steps produced high-frequency repetitive trains of action potentials without spike frequency adaptation. The action potential and membrane and characteristics of this cell type are consistent with those described for GABAergic septal neurons. Many of these neurons retained their theta-bursting pattern in the presence of muscarinic antagonist. (3) Cells with long-duration action potentials and long-duration AHPs (24 of 34 rhythmic cells, 70%) had low firing rates, and usually only 1-3 spikes per theta cycle, locked mainly to the positive peak of the dentate theta rhythm. Hyperpolarizing current injection revealed a long membrane time constant and a break potential; a depolarizing pulse caused a train of action potentials with pronounced spike frequency adaptation. The action potential and membrane properties of this cell type are consistent with those reported for cholinergic septal neurons. The theta-related rhythmicity of this cell type was abolished by muscarinic antagonists. The phasic inhibition of "cholinergic" MS/DB neurons by "GABAergic" MS/DB neurons, followed by a rebound of their firing, is proposed as a mechanism contributing to recruitment of the whole MS/DB neuronal population into the synchronized rhythmic bursting pattern of activity that underlies the occurrence of the hippocampal theta rhythm.

Dissociation of postnatal hippocampal neurons for short term culture

A method is described to isolate hippocampal neurons from postnatal day 7 to postnatal day 21 rats for use in short term cultures using a combination of enzymatic and mechanical dissociation. A significant fraction of the cells (37.5±2.3%, n=10 cultures) were labelled with anti-GABA antibodies and the neurons survived a minimum of 8 days in culture. Patch-clamp recording in the current clamp mode revealed an average membrane potential and input resistance of −47.6±2.5 mV and 737±147 MΩ, respectively, after 24 h in culture. The cells exhibited normal excitability and fired multiple action potentials in response to a depolarizing pulse. The technique described here provides a good alternative to either the use of embryonic brain tissue for cultures used in electrophysiological studies which may not exhibit the mature phenotype or use of acutely isolated cells which may be unstable or have their channels and receptors modified by enzymatic treatment.

Synchronous bursting can arise from mutual excitation, even when individual cells are not endogenous bursters.

Mutual excitation between two neurons is generally thought to raise the excitation level of each neuron or, if they are both bursty, to act to synchronize their bursts. If only one is bursty, it can induce synchronized bursts in the other cell. Here we show that two nonbursty cells can be induced to burst in synchrony by mutual excitatory synaptic connections, provided the presynaptic threshold for graded synaptic transmission at each synapse is at a different level. This mechanism may operate in a recently discovered network in the lobster Homarus gammarus. By a duality between presynaptic threshold and injected current, we also show that two identical, nonbursty, mutual excitatory cells could be induced to burst in synchrony by injecting differing amounts of current in the two cells. Finally we show that differential oscillations between two mutual excitatory cells could be stopped by a slow-tailed hyperpolarizing current pulse into one cell or a slow-tailed depolarizing pulse into the other.

Single versus repetitive spiking to the current stimulus of A-beta mechanosensitive neurons in the crotaline snake trigeminal ganglion.

1. Intrasomal recordings of potentials produced by current stimulation in vivo were made from 24 (A-beta) touch and 19 vibrotactile neurons in the trigeminal ganglion of 29 crotaline snakes, Trimeresurus flavoviridis. 2. Usually touch neurons responded with a single action potential at the beginning of a prolonged depolarizing pulse, whereas all vibrotactile neurons responded with multiple spikes. 3. The electrophysiological parameters examined were membrane potential, threshold current, input resistance and capacitance, time constant, rebound latency, and its threshold current. Touch neurons had higher input resistance (and lower input capacitance) than vibrotactile neurons. 4. In conclusion, current injection, which elicits a single or multiple spiking, seems a useful way to separate touch neurons from vibrotactile neurons without confirming the receptor response, and some membrane properties are also specific to the sensory modality.

Influence of the mucosa on the excitability of myenteric neurons.

Intracellular microelectrodes were used to examine the active and passive membrane properties of neurons in the myenteric plexus of the guinea-pig small intestine. Neurons of two types were examined: S neurons, which have prominent fast excitatory postsynaptic potentials and in which action potentials are not followed by long-lasting afterhyperpolarizations, and AH neurons, which have long-lasting afterhyperpolarizations following soma action potentials. In preparations in which the myenteric ganglia and longitudinal muscle, but no mucosa, were present, most S neurons (59/64) responded to intracellular depolarizing current with brief bursts of action potentials. Regardless of the strength of a depolarizing current of 500-ms duration, these neurons never fired action potentials beyond the first 250 ms. S neurons in this state were called rapidly accommodating. In contrast, within 600 microm circumferential to the intact mucosa, 26/58 S neurons fired action potentials for most or all of the period of a 500-ms insightful depolarizing pulse. S neurons in this state were called slowly accommodating. Depolarization of S neurons in the rapidly accommodating state caused a rapidly developing reduction in membrane resistance (outward rectification; onset about 7 ms). This rectification was absent from S neurons in the slowly accommodating state. Tetraethylammonium blocked the early rectification and the changed neuronal state from rapidly accommodating to slowly accommodating. Application of tetrodotoxin to neurons in the slowly accommodating state revealed the early rectification, indicating that its absence from these neurons before tetrodotoxin was applied had been due to ongoing activity in axons providing synaptic input to the neurons. After the mucosa was disconnected from the other layers and laid back in its original position, all S neurons close to the mucosa were in the rapidly accommodating state (17/17). Slow excitatory postsynaptic potentials, evoked by electrical stimulation of nerve tracts, converted 17 of 43 S neurons from rapidly accommodating to slowly accommodating and eliminated the early outward rectification in these neurons. These results indicate that the action potential firing properties of S neurons can be changed by external influences, including the activity of synaptic inputs that release a slowly acting transmitter. Spontaneous antidromic action potentials were recorded in 8/62 AH neurons within 600 microm circumferential to the intact mucosa. It is concluded that, when the mucosa is intact, a background firing of sensory neurons occurs which leads to a state change in many S neurons innervated by the active sensory neurons. We conclude that this state change is caused by the block of a voltage-sensitive outward rectification.

Slow inactivation of Na+ current and slow cumulative spike adaptation in mouse and guinea-pig neocortical neurones in slices.

1. Spike adaptation of neocortical pyramidal neurones was studied with sharp electrode recordings in slices of guinea-pig parietal cortex and whole-cell patch recordings of mouse somatosensory cortex. Repetitive intracellular stimulation with 1 s depolarizing pulses delivered at intervals of < 5 s caused slow, cumulative adaptation of spike firing, which was not associated with a change in resting conductance, and which persisted when Co2+ replaced Ca2+ in the bathing medium. 2. Development of slow cumulative adaptation was associated with a gradual decrease in maximal rates of rise of action potentials, a slowing in the post-spike depolarization towards threshold, and a positive shift in the threshold voltage for the next spike in the train; maximal spike repolarization rates and after-hyperpolarizations were unchanged. 3. The data suggested that slow adaptation reflects use-dependent removal of Na+ channels from the available pool by an inactivation process which is much slower than fast, Hodgkin-Huxley-type inactivation. 4. We therefore studied the properties of Na+ channels in layer II-III mouse neocortical cells using the cell-attached configuration of the patch-in-slice technique. These had a slope conductance of 18 +/- 1 pS and an extrapolated reversal potential of 127 +/- 6 mV above resting potential (Vr) (mean +/- S.E.M.; n = 5). Vr was estimated at -72 +/- 3 mV (n = 8), based on the voltage dependence of the steady-state inactivation (h infinity) curve. 5. Slow inactivation (SI) of Na+ channels had a mono-exponential onset with tau on between 0.86 and 2.33 s (n = 3). Steady-state SI was half-maximal at -43.8 mV and had a slope of 14.4 mV (e-fold)-1. Recovery from a 2 s conditioning pulse was bi-exponential and voltage dependent; the slow time constant ranged between 0.45 and 2.5 s at voltages between-128 and -68 mV. 6. The experimentally determined parameters of SI were adequate to simulate slow cumulative adaptation of spike firing in a single-compartment computer model. 7. Persistent Na+ current, which was recorded in whole-cell configuration during slow voltage ramps (35 mV s-1), also underwent pronounced SI, which was apparent when the ramp was preceded by a prolonged depolarizing pulse.

GnRH-induced cytosolic calcium oscillations in pituitary gonadotrophs: phase resetting by membrane depolarization

Cultured rat pituitary gonadotrophs under whole-cell voltage clamp conditions respond to the hypothalamic hormone GnRH with synchronized oscillatory changes in both cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]i-activated, apamin-sensitive K+ current (IK(Ca)). We found, and report here for the first time, that in GnRH-stimulated cells a brief depolarizing pulse can elicit a transient [Ca2+]i rise similar to the endogenous cycle. Furthermore, Ca2+ entry during a single depolarizing pulse was found to shift the phase of subsequent endogenous [Ca2+]i oscillations, which thereafter continue to occur at their previous frequency before the pulse. Application of two consecutive depolarizing pulses showed that the size of the [Ca2+]i rise evoked by the second pulse depended on the time lapsed between two consecutive pulses, indicating that each endogenous or evoked [Ca2+]i rise cycle leaves the Ca2+ release mechanism of the gonadotroph in a refractory state. Recovery from this condition can be described by an exponential function of the time lapsed between the pulses (time constant of ca. 1 s). We propose that the underlying mechanism in both refractoriness after endogenous cycles and phase resetting by a brief pulse of Ca2+ entry involves the InsP3 receptor-channel molecule presumed to be located on the cytosolic aspect of the endoplasmic reticulum membrane.

Modulation of adenosine triphosphate-sensitive potassium channel and voltage-dependent calcium channel by activin A in HIT-T15 cells.

The ATP-sensitive potassium channel (KATP channel) determines the membrane potential of pancreatic beta-cells and plays a critical role in the regulation of insulin secretion. The present study was conducted to investigate the effect of activin A, a member of the transforming growth factor-beta supergene family, on the KATP channel in HIT-T15 clonal hamster insulinoma cells. In an excised inside-out patch, ATP-sensitive currents with a single channel conductance of approximately 20 picosiemens were observed. In an outside-out patch, currents with identical unitary conductance were also observed. In either case, the currents were augmented by diazoxide and blocked by glibenclamide, verifying that they were KATP channel currents. When KATP channel currents were monitored in an outside-out patch, activin A added to the bath solution inhibited KATP channel currents. Upon removal of activin A, the KATP channel currents were restored, suggesting that the inhibition was not due simply to spontaneous disappearance of channel activity (run-down). The KATP channel activity was markedly reduced after the addition of activin A and was reversed by diazoxide. Besides the inhibition of KATP channel, activin A increased, in a perforated patch, the amplitude of the inward Ba2+ current in response to a depolarizing pulse from -40 to +10 mV. Under the current clamp condition, activin A induced gradual depolarization, followed by a burst of action potentials. Activin-mediated action potentials were accompanied by an elevation of the cytoplasmic free calcium concentration. These results indicate that activin A causes depolarization of the plasma membrane by inhibiting the activity of the KATP channel. In addition, activin A directly modulates the voltage-dependent calcium channel and augments calcium entry.