Fast delayed rectifier potassium current is required for circadian neural activity

In mammals, the precise circadian timing of many biological processes depends on the generation of oscillations in neural activity of pacemaker cells in the suprachiasmatic nucleus (SCN). The ionic mechanisms that underlie these rhythms are largely unknown. Using the mouse brain slice preparation, we show that the magnitude of fast delayed rectifier (FDR) potassium currents has a diurnal rhythm that peaks during the day. Notably, this rhythm continues in constant darkness, providing the first demonstration of the circadian regulation of an intrinsic voltage-gated current in mammalian cells. Blocking this current prevented the daily rhythm in firing rate in SCN neurons. Kv3.1b and Kv3.2 potassium channels were widely distributed within the SCN, with higher expression during the day. We conclude that the FDR is necessary for the circadian modulation of electrical activity in SCN neurons and represents an important part of the ionic basis for the generation of rhythmic output.     

BK calcium-activated potassium channels regulate circadian behavioral rhythms and pacemaker output

Spontaneous action potentials in the suprachiasmatic nucleus (SCN) are necessary for normal circadian timing of behavior in mammals. The SCN exhibits a daily oscillation in spontaneous firing rate (SFR), but the ionic conductances controlling SFR and the relationship of SFR to subsequent circadian behavioral rhythms are not understood. We show that daily expression of the large conductance Ca(2+)-activated K(+) channel (BK) in the SCN is controlled by the intrinsic circadian clock. BK channel-null mice (Kcnma1(-/-)) have increased SFRs in SCN neurons selectively at night and weak circadian amplitudes in multiple behaviors timed by the SCN. Kcnma1(-/-) mice show normal expression of clock genes such as Arntl (Bmal1), indicating a role for BK channels in SCN pacemaker output, rather than in intrinsic time-keeping. Our findings implicate BK channels as important regulators of the SFR and suggest that the SCN pacemaker governs the expression of circadian behavioral rhythms through SFR modulation.     

A Ba(2+)-sensitive K(+) current contributes to the resting membrane potential of neurons in rat suprachiasmatic nucleus

A Ba(2+)-sensitive K(+) current was studied in neurons of the suprachiasmatic nucleus (SCN) using the whole cell patch-clamp technique in acutely prepared brain slices. This Ba(2+)-sensitive K(+) current was found in approximately 90% of the SCN neurons and was uniformly distributed across the SCN. Current-clamp studies revealed that Ba(2+) (500 microM) reversibly depolarized the membrane potential by 6.7 +/- 1.3 mV (n = 22) and concomitantly Ba(2+) induced an increase in the spontaneous firing rate of 0.8 +/- 0.2 Hz (n = 12). The Ba(2+)-evoked depolarizations did not depend on firing activity or spike dependent synaptic transmission. No significant day/night difference in the hyperpolarizing contribution to the resting membrane potential of the present Ba(2+)-sensitive current was observed. Voltage-clamp experiments showed that Ba(2+) (500 microM) reduced a fast-activating, voltage-dependent K(+) current. This current was activated at levels below firing threshold and exhibited outward rectification. The Ba(2+)-sensitive K(+) current was strongly reduced by tetraethylammonium (TEA; 20 and 60 mM) but was insensitive to 4-aminopyridine (4-AP; 5 mM) and quinine (100 microM). A component of Ba(2+)-sensitive K(+) current remaining in the presence of TEA exhibited no clear voltage dependence and is less likely to contribute to the resting membrane potential. The voltage dependence, kinetics and pharmacological properties of the Ba(2+)- and TEA-sensitive K(+) current make it unlikely that this current is a delayed rectifier, Ca(2+)-activated K(+) current, ATP-sensitive K(+) current, M-current or K(+) inward rectifier. Our data are consistent with the Ba(2+)- and TEA-sensitive K(+) current in SCN neurons being an outward rectifying K(+) current of a novel identity or belonging to a known family of K(+) channels with related properties. Regardless of its precise molecular identity, the current appears to exert a significant hyperpolarizing effect on the resting potential of SCN neurons.     

Persistent calcium current in rat suprachiasmatic nucleus neurons

The suprachiasmatic nuclei contain the primary circadian clock, and suprachiasmatic nuclei neurons exhibit a diurnal modulation of spontaneous firing rate. The present study examined the voltage-gated persistent Ca(2+) current, in acutely isolated rat suprachiasmatic nuclei neurons using a ramp-type voltage-clamp protocol. Slow triangular voltage-clamp commands from a holding potential of -85 mV to +5 mV elicited inward current (100-400 pA) that was completely blocked by Cd(2+). This current showed little or no hysteresis, and was identified as persistent Ca(2+) current. The threshold for persistent Ca(2+) current ranged between -60 and -45 mV, and it was maximal at about -8 mV. Nifedipine at 10-20 microM blocked 80-100%. To assess the role of persistent Ca(2+) current in the generation of spontaneous action potentials in both acutely isolated and intact suprachiasmatic nuclei neurons, the effect of Cd(2+) and nifedipine on firing rate was studied using on-cell recording. Application of Cd(2+) exerted a weak excitatory effect and nifedipine had no significant effect on the spontaneous firing rate of isolated suprachiasmatic nuclei neurons. In all intact suprachiasmatic nuclei neurons in slice preparations (n=15), Cd(2+) slowly inhibited spontaneous firing; in high-frequency firing cells (four of 15), a transient increase of firing rate preceded inhibition. No significant effect of nifedipine on firing rate of intact suprachiasmatic nuclei neurons was found. Therefore, persistent Ca(2+) current itself (as carrier of charge) does not appear to contribute significantly to spontaneous firing of suprachiasmatic nuclei neurons. A slowly developing inhibitory effect of Cd(2+) on spontaneous firing of intact suprachiasmatic nuclei neurons in slice preparations may be due to penetration of Cd(2+) through Ca(2+) channels, and its subsequent effect on intracellular mechanisms, while the transient increase of firing rate in high-frequency firing neurons is probably due to inhibition of Ca(2+)-activated K(+) current.     

Circadian difference in firing rate of isolated rat suprachiasmatic nucleus neurons

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian clock in mammals. Dissociated SCN neurons in long-term culture exhibit a circadian modulation of spontaneous electrical activity. To evaluate the presence of circadian differences in spontaneous activity of isolated SCN neurons without synaptic connections, dissociated rat SCN neurons were studied with on-cell recording 3-4 days after preparation, before the formation of dendrites, axons and synapses. A day-night difference in spontaneous electrical firing rate was found in acutely dissociated SCN neurons. During the first subjective day, the average firing rate (0.87+/-0.12 Hz) was significantly higher than during the first subjective night (0.24+/-0.06 Hz), while the firing rate on the next day (0.68+/-0.11 Hz) was significantly higher than during the preceding night. These data suggest that populations of isolated SCN neurons with no synaptic interactions contain a functioning circadian clock, and are particularly amenable to biophysical experiments.     

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic-clonic seizures. IC neurons are known to have Ca(2+) channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca(2+) channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca(2+) channels were activated by depolarizing pulses from a holding potential of -90 mV in 10 mV increments using 5 mM barium (Ba(2+)) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca(2+) channel currents that could be blocked by 50 microM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 microM, L-type channel blocker), omega-conotoxin GVIA (1 microM, N-type channel blocker), and omega-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) omega-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca(2+) channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 microM nickel and inactivated with tau of 15-50 ms, was considered as R-type Ca(2+) channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca(2+) channel antibodies showed that IC neurons express Ca(2+) channel alpha(1A), alpha(1B), alpha(1C), alpha(1D), and alpha(1E) subunits. We conclude that IC neurons express functionally all members of HVA Ca(2+) channels, but only a subset of these neurons appear to have developed functional LVA channels.     

Excitatory mechanisms in the suprachiasmatic nucleus: the role of AMPA/KA glutamate receptors

A variety of evidence suggests that the effects of light on the mammalian circadian system are mediated by direct retinal ganglion cell projection to the suprachiasmatic nucleus (SCN). This synaptic connection is glutamatergic and the release of glutamate is detected by both N-methyl-D-asparate (NMDA) and amino-methyl proprionic acid/kainate (AMPA/KA) iontotropic glutamate receptors (GluRs). It is well established that NMDA GluRs play a critical role in mediating the effects of light on the circadian system; however, the role of AMPA/KA GluRs has received less attention. In the present study, we sought to better understand the contribution of AMPA/KA-mediated currents in the circadian system based in the SCN. First, whole cell patch-clamp electrophysiological techniques were utilized to measure spontaneous excitatory postsynaptic currents (sEPSCs) from SCN neurons. These currents were widespread in the SCN and not just restricted to the retino-recipient region. The sEPSC frequency and amplitude did not vary with the daily cycle. Similarly, currents evoked by the exogenous application of AMPA onto SCN neurons were widespread within the SCN and did not exhibit a diurnal rhythm in their magnitude. Fluorometric techniques were utilized to estimate AMPA-induced calcium (Ca(2+)) concentration changes in SCN neurons. The resulting data indicate that AMPA-evoked Ca(2+) transients were widespread in the SCN and that there was a daily rhythm in the magnitude of AMPA-induced Ca(2+) transients that peaked during the night. By itself, blocking AMPA/KA GluRs with a receptor blocker decreased the spontaneous firing of some SCN neurons as well as reduced resting Ca(2+) levels, suggesting tonic glutamatergic excitation. Finally, immunohistochemical techniques were used to describe expression of the AMPA-preferring GluR subunits GluR1 and GluR2/3s within the SCN. Overall, our data suggest that glutamatergic synaptic transmission mediated by AMPA/KA GluRs play an important role throughout the SCN synaptic circuitry.     

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and NS1619, a BK(Ca) channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 microM) increased opening activity of a Ca(2+)-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK(Ca) channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K(+) channel (A-current) blocker, in an IbTx-sensitive manner.These results indicate that IbTx-sensitive BK(Ca) channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK(Ca) channels. These results suggest that opening BK(Ca) channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.     

Circadian modulation of GABA function in the rat suprachiasmatic nucleus: excitatory effects during the night phase

Gramicidin-perforated patch-clamp recordings were made from slices of the suprachiasmatic nucleus (SCN) of adult rats to characterize the role of gamma-amino butyric acid (GABA) in the circadian timing system. During the day, activation of GABA(A) receptors hyperpolarized the membrane of SCN neurons. During the night, however, activation of GABA(A) receptors either hyperpolarized or depolarized the membrane. These night-restricted depolarizations in a large subset of SCN neurons were capable of triggering spikes and thus appeared to be excitatory. The GABA(A) reversal potentials of SCN neurons revealed a significant day-night difference with more depolarized GABA(A) reversal potentials during the night than during the day. The emergence of depolarizing GABA(A)-mediated responses in a subset of SCN neurons at night can be ascribed to a depolarizing shift in GABA(A) reversal potential. The GABA(A) receptor antagonist bicuculline (12.5 microM) increased the spontaneous firing rate of all SCN neurons during the day, indicating that spontaneous GABA(A)-mediated inputs inhibited the SCN neurons during this period. The effect of bicuculline (12.5 microM) on the spontaneous firing rate of SCN neurons during the night was heterogeneous due to the mixture of depolarizing and hyperpolarizing GABA(A)-mediated inputs during this period. We conclude that GABA uniformly acts as an inhibitory transmitter during the day but excites a large subset of SCN neurons at night. This day-night modulation of GABAergic neurotransmission provides the SCN with a time-dependent gating mechanism that may counteract propagation of excitatory signals throughout the biological clock at day but promotes it at night.     

Activation of metabotropic glutamate receptors inhibits high-voltage-gated calcium channel currents of chicken nucleus magnocellularis neurons

Using whole cell patch-clamp recordings, we pharmacologically characterized the voltage-gated Ca2+ channel (VGCC) currents of chicken nucleus magnocellularis (NM) neurons using barium as the charge carrier. NM neurons possessed both low- and high-voltage-activated Ca2+ channel currents (HVA I(Ba2+)). The N-type channel blocker (omega-conotoxin-GVIA) inhibited more than half of the total HVA I(Ba2+), whereas blockers of L- and P/Q-type channels each inhibited a small fraction of the current. Metabotropic glutamate receptor (mGluR)-mediated modulation of the HVA I(Ba2+) was examined by bath application of glutamate (100 microM), which inhibited the HVA I(Ba2+) by an average of 16%. The inhibitory effect was dose dependent and was partially blocked by omega-conotoxin-GVIA, indicating that mGluRs modulate N and other type HVA I(Ba2+). The nonspecific mGluR agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarbosylic acid (1S,3R-ACPD), mimicked the inhibitory effect of glutamate on HVA I(Ba2+). Group I-III mGluR agonists showed inhibition of the HVA current with the most potent being the group III agonist L(+)-2-amino-4-phosphonobutyric acid. 1S,3R-ACPD (200 microM) had no effect on K+ or Na+ currents. The firing properties of NM neurons were also not altered by 1S,3R-ACPD. We propose that the inhibition of VGCC currents by mGluRs limits depolarization-induced Ca2+ entry into these highly active NM neurons and regulates their Ca2+ homeostasis.     

Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties

This study investigates the firing properties of the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons located in the external formation of the nucleus ambiguus. The results showed that inspiratory-activated and inspiratory-inhibited neurons are distributed with different density and site preference in this area. Inspiratory-inhibited neurons exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential under current clamp. The afterhyperpolarization in inspiratory-activated neurons was blocked by apamin, a blocker of the small-conductance Ca(2+)-activated K(+) channels; and that in inspiratory-inhibited neurons by charybdotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) channels. Under voltage clamp, depolarizing voltage steps evoked tetrodotoxin-sensitive rapid inward sodium currents, 4-aminopyridine-sensitive outward potassium transients and lasting outward potassium currents. 4-Aminopyridine partially blocked the lasting outward potassium currents of inspiratory-activated neurons but was ineffective on those of inspiratory-inhibited neurons. These findings suggest that inspiratory-activated and inspiratory-inhibited neurons are differentially organized and express different types of voltage-gated ion channels.     

Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron.

The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long-afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)-dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half-width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 microM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.     

Calcium-dependent fast depolarizing afterpotentials in vasopressin neurons in the rat supraoptic nucleus

Oxytocin (OT) and vasopressin (VP) synthesizing magnocellular cells (MNCs) in the supraoptic nucleus (SON) display distinct firing patterns during the physiological demands for these hormones. Depolarizing afterpotentials (DAPs) in these neurons are involved in controlling phasic bursting in VP neurons. Our whole cell recordings demonstrated a Cs(+)-resistant fast DAP (fDAP; decay tau = approximately 200 ms), which has not been previously reported, in addition to the well-known Cs(+)-sensitive slower DAP (sDAP; decay tau = approximately 2 s). Immunoidentification of recorded neurons revealed that all VP neurons, but only 20% of OT neurons, expressed the fDAP. The activation of the fDAP required influx of Ca(2+) through voltage-gated Ca(2+) channels as it was strongly suppressed in Ca(2+)-free extracellular solution or by bath application of Cd(2+). Additionally, the current underlying the fDAP (I(fDAP)) is a Ca(2+)-activated current rather than a Ca(2+) current per se as it was abolished by strongly buffering intracellular Ca(2+) with BAPTA. The I-V relationship of the I(fDAP) was linear at potentials less than -60 mV but showed pronounced outward rectification near -50 mV. I(fDAP) is sensitive to changes in extracellular Na(+) and K(+) but not Cl(-). A blocker of Ca(2+)-activated nonselective cation (CAN) currents, flufenamic acid, blocked the fDAP, suggesting the involvement of a CAN current in the generation of fDAP in VP neurons. We speculate that the two DAPs have different roles in generating after burst discharges and could play important roles in determining the distinct firing properties of VP neurons in the SON neurons.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.     

Angiotensin II regulates the activity of mouse suprachiasmatic nuclei neurons

Neuropeptide signaling plays key roles in coordinating cellular activity within the suprachiasmatic nuclei (SCN), site of the master circadian oscillator in mammals. The neuropeptide angiotensin II (ANGII) and its cognate receptor AT1, are both expressed by SCN cells, but unlike other SCN neurochemicals, very little is known about the cellular actions of ANGII within this circadian clock. We used multi-electrode, multiunit, extracellular electrophysiology, coupled with whole-cell voltage and current clamp techniques to investigate the actions of ANGII in mouse SCN slices. ANGII (0.001-10 microM) dose dependently stimulated and inhibited extracellularly recorded neuronal discharge in many SCN neurons ( approximately 60%). Both actions were blocked by pre-treatment with the AT1 receptor antagonist ZD7155 (0.03 microM), while suppressions but not activations were prevented by pre-treatment with the GABA A receptor antagonist bicuculline (20 microM). AT1 receptor blockade itself suppressed discharge in a subset ( approximately 30%) of SCN neurons, and this action was not blocked by bicuculline. In voltage-clamped SCN neurons (-70 mV), AT1 receptor activation dose-dependently enhanced the frequency of action potential-driven, GABA A receptor-mediated currents, but did not alter their responses to exogenously applied GABA. In current-clamped SCN neurons perfused with tetrodotoxin, ANGII induced a membrane depolarization with a concomitant decrease in input resistance. In conclusion we show that AT1 receptor activation by ANGII depolarizes SCN neurons and stimulates action potential firing, leading to increased GABA release in the mouse SCN. Additionally we provide the first evidence that endogenous AT1 receptor signaling tonically regulates the activities of some SCN neurons.     

Dopamine D1/5 receptor-mediated long-term potentiation of intrinsic excitability in rat prefrontal cortical neurons: Ca2+-dependent intracellular signaling

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input-output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors (V(hold) = -65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2-50 microM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 microM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297-induced LTP-IE was significantly attenuated by Cd(2+), [Ca(2+)](i) chelation, by inhibition of phospholipase C, protein kinase-C, and Ca(2+)/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca(2+) channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca(2+) influx via non-L-type Ca(2+) channels, activation of PLC, intracellular Ca(2+) elevation, activation of Ca(2+)-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K(+) and Ca(2+) currents to dynamically regulate overall excitability of PFC neurons.     

Biophysical characterization of rat caudal hypothalamic neurons: calcium channel contribution to excitability

Neurons in the caudal hypothalamus (CH) are responsible for the modulation of various processes including respiratory and cardiovascular output. Previous results from this and other laboratories have demonstrated in vivo that these neurons have firing rhythms matched to the respiratory and cardiovascular cycles. The goal of the present study was to characterize the biophysical properties of neurons in the CH with particular emphasis in those properties responsible for rhythmic firing behavior. Whole cell, patch-clamped CH neurons displayed a resting membrane potential of -58.0 +/- 1.1 mV and an input resistance of 319.3 +/- 16.6 MOmega when recorded in current-clamp mode in an in vitro brain slice preparation. A large proportion of these neurons displayed postinhibitory rebound (PIR) that was dependent on the duration and magnitude of hyperpolarizing current as well as the resting membrane potential of the cell. Furthermore these neurons discharged tonically in response to a depolarizing current pulse at a depolarized resting membrane potential (more positive than -65 mV) but switched to a rapid burst of firing to the same stimulus when the resting membrane potential was lowered. The PIR observed in these neurons was calcium dependent as demonstrated by the ability to block its amplitude by perfusion of Ca(2+)-free bath solution or by application of Ni(2+) (0.3-0.5 mM) or nifedipine (10 microM). These properties suggest that low-voltage-activated (LVA) calcium current is involved in the PIR and bursting firing of these CH neurons. In addition, high-voltage-activated calcium responses were detected after blockade of outward potassium current or in Ba(2+)-replacement solution. In addition, almost all of the CH neurons studied showed spike frequency adaptation that was decreased following Ca(2+) removal, indicating the involvement of Ca(2+)-dependent K(+) current (I(K,Ca)) in these cells. In conclusion, CH neurons have at least two different types of calcium currents that contribute to their excitability; the dominant current is the LVA or T-type. This LVA current appears to play a significant role in the bursting characteristics that may underlie the rhythmic firing of CH neurons.     

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I(ADP) to -58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na(+) and K(+) to this current were estimated to be about 1:5. Substitution of external Na(+) with N-methyl D-glucamine blocked the current while replacement of internal Cl(-) with gluconate did not block the I(ADP). The I(ADP) was also inhibited when CsCl-filled patch pipettes were used. The I(ADP) was blocked or substantially decreased in amplitude in the presence of N-type Ca(2+) channel antagonists, omega-conotoxin GVIA and omega-conotoxin MVIIC, respectively, and was eliminated by external Cd(2+), indicating that it was dependent on Ca(2+) entry. The I(ADP) was also inhibited by ryanodine (10-20 microM), indicating that Ca(2+)-induced Ca(2+) release was involved in its activation. Niflumic acid consistently inhibited the I(ADP) with an IC(50) of 63 microM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca(2+) channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca(2+) channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca(2+)-channel blocker, is generated by the opening of Ca(2+)-activated non-selective cation channels following action potential-mediated Ca(2+) entry mainly through N-type Ca(2+) channels. Ca(2+) release from ryanodine-sensitive stores triggered by Ca(2+) entry contributes significantly to the activation of this current.     

Action potential bursts in central snail neurons elicited by d-amphetamine: roles of ionic currents

The roles of the ionic currents in the firing of potential bursts elicited by d-amphetamine in central snail neurons were studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. Oscillations of membrane potential bursts were elicited by d-amphetamine. The action potential bursts elicited by d-amphetamine decreased following intracellular injection of either EDTA or magnesium, or extracellular application of lanthanum. Voltage-clamped studies revealed that d-amphetamine decreased the fast Na(+), Ca(2+) and transient outward K(+) currents of the RP4 neuron. It also decreased the steady-state K(+) current and elicited a negative slope resistance in the steady-state I-V curve between -50 and -10 mV. The amplitude of negative slope resistance was decreased if either Na(+)-free saline or Co(2+)-substituted Ca(2+)-free saline was perfused. d-Amphetamine did not increase the amplitude of the slowly inactivating Ca(2+) current or the persistent Na(+) currents of RP4 neuron. Tetraethylammonium, a blocker of the delayed outward K(+) current, elicited action potential bursts and negative slope resistance in the RP4 neuron, while 4-aminopyridine, an inhibitor of transient outward K(+) current (I(A)), did not.These results demonstrate that the delayed outward K(+) current and the negative slope resistance in steady-state I-V curve elicited by d-amphetamine may be responsible for the action potential bursts in central snail neurons elicited by d-amphetamine.