Ionic basis of tonic firing in spinal substantia gelatinosa neurons of rat

Ionic conductances underlying excitability in tonically firing neurons (TFNs) from substantia gelatinosa (SG) were studied by the patch-clamp method in rat spinal cord slices. Ca(2+)-dependent K(+) (K(CA)) conductance sensitive to apamin was found to prolong the interspike intervals and stabilize firing evoked by a sustained membrane depolarization. Suppression of Ca(2+) and K(CA) currents, however, did not abolish the basic pattern of tonic firing, indicating that it was generated by voltage-gated Na(+) and K(+) currents. Na(+) and K(+) channels were further analyzed in somatic nucleated patches. Na(+) channels exhibited fast activation and inactivation kinetics and followed two-exponential time course of recovery from inactivation. The major K(+) current was carried through tetraethylammonium (TEA)-sensitive rapidly activating delayed-rectifier (K(DR)) channels with a slow inactivation. The TEA-insensitive transient A-type K(+) (K(A)) current was very small in patches and was strongly inactivated at resting potential. Block of K(DR) rather than K(A) conductance by 1 mM TEA lowered the frequency and stability of firing. Intracellular staining with biocytin revealed at least three morphological groups of TFNs. Finally, on the basis of present data, we created a model of TFN and showed that Na(+) and K(DR) currents are sufficient to generate a basic pattern of tonic firing. It is concluded that the balanced contribution of all ionic conductances described here is important for generation and modulation of tonic firing in SG neurons.     

Na(+) entry through AMPA receptors results in voltage-gated k(+) channel blockade in cultured rat spinal cord motoneurons

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rectification. This inward rectification was not a genuine property of AMPA receptor currents but was a result of a concomitant decrease in outward current at potentials positive to -40.5 +/- 1.3 mV. The AMPA receptor current itself was nearly linear (rectification index 0.91). The kainate-inhibited outward current had a reversal potential close to the estimated K(+) equilibrium potential and was blocked by 30 mM tetraethylammonium. When voltage steps were applied, it was found that kainate inhibited both the delayed rectifier K(+) current K(V) and the transient outward K(+) current, K(A). The kainate-induced inhibition of K(+) currents was dependent on ion flux through the AMPA receptor, because no change in the membrane conductance was noticed in the presence of LY 300164. Removing extracellular Ca(2+) had no effect, whereas replacing extracellular Na(+) or clamping the membrane close to the estimated Na(+) equilibrium potential during kainate application attenuated the inhibition of the K(+) current. Sustained Na(+) influx induced by application of the Na(+) ionophore monensin could mimic the effect of kainate on K(+) conductance. These findings demonstrate that Na(+) influx through AMPA receptors results in blockade of voltage-gated K(+) channels.     

Similar properties of transient, persistent, and resurgent Na currents in GABAergic and non-GABAergic vestibular nucleus neurons

Sodium currents in fast firing neurons are tuned to support sustained firing rates >50-60 Hz. This is typically accomplished with fast channel kinetics and the ability to minimize the accumulation of Na channels into inactivated states. Neurons in the medial vestibular nuclei (MVN) can fire at exceptionally high rates, but their Na currents have never been characterized. In this study, Na current kinetics and voltage-dependent properties were compared in two classes of MVN neurons with distinct firing properties. Non-GABAergic neurons (fluorescently labeled in YFP-16 transgenic mice) have action potentials with faster rise and fall kinetics and sustain higher firing rates than GABAergic neurons (fluorescently labeled in GIN transgenic mice). A previous study showed that these neurons express a differential balance of K currents. To determine whether the Na currents in these two populations were different, their kinetics and voltage-dependent properties were measured in acutely dissociated neurons from 24- to 40-day-old mice. All neurons expressed persistent Na currents and large transient Na currents with resurgent kinetics tuned for fast firing. No differences were found between the Na currents expressed in GABAergic and non-GABAergic MVN neurons, suggesting that differences in properties of these neurons are tuned by their K currents.     

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.     

Whole-cell currents in two subpopulations of cultured rat petrosal neurons with different tetrodotoxin sensitivities.

In this study we use whole-cell recording to characterize at least two distinct populations of cultured neurons from perinatal rat petrosal or petrosal/jugular ganglia based on differential sensitivity of the transient inward Na+ current to tetrodotoxin. These ganglia supply chemoreceptor and baroreceptor afferents which mediate several cardiovascular reflexes. Approximately 50% of the neurons sampled had Na+ currents that were virtually unaffected by bath addition of tetrodotoxin (0.5-2.0 microM) but were abolished by choline substitution for external Na+. The majority of the remaining neurons had Na+ currents that were rapidly and reversibly blocked by 500 nM tetrodotoxin. A few cells had both tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents. All neurons had similar voltage-activated Ca2+ and K+ currents. The inward Ca2+ current had no obvious fast transient or T-type component and appeared to be due mainly to the presence of long-lasting L-type Ca2+ channels. The outward currents consisted largely of a delayed rectifying K+ current (IKdr) and a Ca(2+)-activated K+ current (IKca), but no obvious fast transient K+ current (IA) was observed. Exposure to a chemosensory stimulus, hypoxia (PO2 approximately 20 Torr), had no effect on these neurons, in contrast to the pronounced decrease in K+ current it produces in cultured glomus cells, the presumed chemoreceptors and normal targets for a subset of petrosal neurons in vivo. Current-clamp recordings indicated that some neurons gave single spikes while others gave multiple spikes in response to long-depolarizing stimuli. No correlation between spiking behaviour and tetrodotoxin-sensitivity was observed. Thus, cultures enriched in petrosal neurons contain subpopulations with differential sensitivities to tetrodotoxin. Since many of these neurons innervate a single chemosensory target organ, the carotid body, it is of interest to know whether one or both subtypes can form functional synapses with glomus cells of the carotid body and mediate a chemoreceptor reflex.     

Differential contributions of Shaker and Shab K+ currents to neuronal firing patterns in Drosophila

Different K(+) currents participate in generating neuronal firing patterns. The Drosophila embryonic "giant" neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K(+) currents and to delineate the mutational effects of identified K(+) channels. Mutations of Sh and Shab K(+) channels removed part of inactivating I(A) and sustained I(K), respectively, and the remaining I(A) and I(K) revealed the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns were characterized by different profiles of K(+) current voltage dependence and kinetics and by differential mutational effects. Shab channels regulated membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons showed a gradual decline in repolarization during current injection and their spike activities became limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal I(A) by 4-aminopyridine converted the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab I(K) by quinidine mimicked the Shab phenotypes and converted tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K(+) current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, displayed different profiles of spike properties and K(+) current compositions, providing opportunities for mutational analysis in functionally specialized neurons.     

Computer simulations of N-methyl-D-aspartate receptor-induced membrane properties in a neuron model.

1. To evaluate the role of N-methyl-D-aspartate (NMDA) receptors in simulations of the lamprey spinal locomotor network, we developed a computer-simulated electrical model of a neuron that contains NMDA channels in addition to voltage-gated Na+, K+, and Ca2+ channels and Ca(2+)-activated K+ channels [K(Ca) channels]. 2. The voltage dependence of the Mg2+ block of the Na(+)-K+ current flow through the NMDA channel was modeled according to a scheme of open-channel block. To account for the regulation of K(Ca) channels by NMDA and membrane voltage, we modeled two separate Ca2+ pools that had different voltage dependencies and dynamics. 3. Pacemaker-like membrane potential oscillations could be elicited in the model neuron, which resembled those observed experimentally in the presence of bath-applied NMDA and tetrodotoxin. The effect of changing different channel parameters were tested to determine under which conditions such membrane potential oscillations could occur. 4. The oscillation amplitude was determined by the potential levels at which the NMDA channels and voltage-dependent K+ channels, respectively, were activated. The oscillation frequency and the relative durations of the de- and hyperpolarized phases of the oscillations were determined by the balance between the depolarizing (NMDA channels) and hyperpolarizing [K(Ca) channels] currents. 5. Simulated alterations of the Mg2+ concentration and the K+ conductance as well as injection of constant current caused changes of the oscillations corresponding to those observed experimentally. The de- and hyperpolarizing phases could be reset by brief current pulses. 6. We conclude that the present model can account for the effects of bath-applied NMDA on spinal neurons. This permits an incorporation of NMDA-receptor-mediated properties in simulation models of the lamprey locomotor network.     

The firing patterns of rat melanotrophs recorded using the patch clamp technique

Cell-attached and whole-cell recordings were made from adult rat melanotrophs maintained in vitro by standard cell culture techniques. In cell-attached recordings the cells showed small biphasic currents which reflected spontaneous cell firing. Single channel currents often had distinct relaxations and depolarizing currents through single channels could trigger the discharge of an action potential in the cell; both observations are consistent with the high input resistance (1-10 G omega) measured in the whole-cell configuration. The discharge of action potentials occurring either spontaneously or by current injection was eliminated by tetrodotoxin or by removing Na from the external medium. A Na-dependent plateau depolarization which activated near the spike threshold was also seen. In cells exposed to tetrodotoxin and K-channel blocking agents it was possible to evoke a long-lasting (up to 20 s) action potential which was enhanced and reduced, respectively, by Ba and Cd and thus appeared to reflect currents through voltage-activated Ca channels. Small amplitude Ca-dependent depolarizations could also be evoked at membrane potentials as low as -40 mV. In cell-attached and whole-cell recordings 10 mM Ba caused the discharge of tetrodotoxin-insensitive action potentials prior to a maintained depolarization of the membrane. The low threshold for Ca-dependent depolarizations suggest that Ca influx might occur in these cells even at the resting potential. Additionally, both a Ca current and the current underlying the Na-dependent plateau depolarization may influence the rate of cell firing and in doing so further increase Ca influx through voltage-activated channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+-activated K+ current contributes to postexcitatory hyperpolarization in neocortical intrinsically bursting neurons

The ionic mechanisms underlying the termination of action-potential (AP) bursts and postburst afterhyperpolarization (AHP) in intrinsically bursting (IB) neocortical neurons were investigated by performing intracellular recordings in thin slices of rat sensorimotor cortex. The blockade of Ca(2+)-activated K(+) currents enhanced postburst depolarizing afterpotentials, but had inconsistent and minor effects on the amplitude and duration of AHPs. On the contrary, experimental conditions resulting in reduction of voltage-dependent Na(+) entry into the cells caused a significant decrease of AHP amplitude. Slice perfusion with a modified artificial cerebrospinal fluid in which LiCl (40 mM) partially replaced NaCl had negligible effects on the properties of individual APs, whereas it consistently increased burst length and led to an approximately 30% reduction in the amplitude of AHPs following individual bursts or short trains of stimulus-induced APs. Experiments performed by partially replacing Na(+) ions with choline revealed a comparable reduction in AHP amplitude associated with an inhibition of bursting activity. Moreover, in voltage-clamp experiments carried out in both in situ and acutely isolated neurons, partial substitution of extracellular NaCl with LiCl significantly and reversibly reduced the amplitude of K(+) currents evoked by depolarizing stimuli above-threshold for Na(+)-current activation. The above effect of Na(+)-to-Li(+) substitution was not seen when voltage-gated Na(+) currents were blocked with TTX, indicating the presence of a specific K(+)-current component activated by voltage-dependent Na(+) (but not Li(+)) influx. The above findings suggest that a Na(+)-activated K(+) current recruited by the Na(+) entry secondary to burst discharge significantly contributes to AHP generation and the maintenance of rhythmic burst recurrence during sustained depolarizations in neocortical IB neurons.     

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.     

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.     

Voltage-gated sodium channels shape subthreshold EPSPs in layer 5 pyramidal neurons from rat prefrontal cortex

The role of voltage-dependent channels in shaping subthreshold excitatory postsynaptic potentials (EPSPs) in neocortical layer 5 pyramidal neurons from rat medial prefrontal cortex (PFC) was investigated using patch-clamp recordings from visually identified neurons in brain slices. Small-amplitude EPSPs evoked by stimulation of superficial layers were not affected by the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid but were abolished by the AMPA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione, suggesting that they were primarily mediated by AMPA receptors. AMPA receptor-mediated EPSPs (AMPA-EPSPs) evoked in the apical dendrites were markedly enhanced, or increased in peak and duration, at depolarized holding potentials. Enhancement of AMPA-EPSPs was reduced by loading the cells with lidocaine N-ethylbromide (QX-314) and by local application of the Na(+) channel blocker tetrodotoxin (TTX) to the soma but not to the middle/proximal apical dendrite. In contrast, blockade of Ca(2+) channels by co-application of Cd(2+) and Ni(2+) to the soma or apical dendrite did not affect the AMPA-EPSPs. Like single EPSPs, EPSP trains were shaped by Na(+) but not Ca(2+) channels. EPSPs simulated by injecting synaptic-like current into proximal/middle apical dendrite (simEPSPs) were enhanced at depolarized holding potentials similarly to AMPA-EPSPs. Extensive blockade of Ca(2+) channels by bath application of the Cd(2+) and Ni(2+) mixture had no effects on simEPSPs, whereas bath-applied TTX removed the depolarization-dependent EPSP amplification. Inhibition of K(+) currents by 4-aminopyridine (4-AP) and TEA increased the TTX-sensitive EPSP amplification. Moreover, strong inhibition of K(+) currents by high concentrations of 4-AP and TEA revealed a contribution of Ca(2+) channels to EPSPs that, however, seemed to be dependent on Na(+) channel activation. Our results indicate that in layer 5 pyramidal neurons from PFC, Na(+), and K(+) voltage-gated channels shape EPSPs within the voltage range that is subthreshold for somatic action potentials.     

mGluR1, but not mGluR5, mediates depolarization of spinal cord neurons by blocking a leak current

The modulation of neuronal excitability by group I metabotropic glutamate receptors (mGluRs) was studied in isolated lamprey spinal cord. At resting potential, application of the group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) slightly depolarized the cells. However, at depolarized membrane potentials, this agonist induced repetitive firing. When Na+ channels were blocked by TTX, DHPG induced a slight depolarization at rest that increased in amplitude as the neurons were held at more depolarized membrane potentials. In voltage-clamp conditions, DHPG application induced an inward current associated with a decrease in membrane conductance when cells were held at -40 mV. At resting membrane potential, no significant change in the current was induced by DHPG, although a decrease in membrane conductance was seen. The conductance blocked by DHPG corresponded to a leak current, since DHPG had no effect on the voltage-gated current elicited by a voltage step from -60 to -40 mV, when leak currents were subtracted. The leak current blocked by DHPG is mediated by fluxes of both K+ and Na+. The subtype of group I mGluR mediating the block of the leak current was characterized using specific antagonists for mGluR1 and mGluR5. The inhibition of the leak current was blocked by the mGluR1 antagonist LY 367385 but not by the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The DHPG-induced blockage of the leak current required phospholipase C (PLC)-activation and release of Ca2+ from internal stores as the effect of DHPG was suppressed by the PLC-blocker U-73122 and after depletion of intracellular Ca2+ pools by thapsigargin. Our results thus show that mGluR1 activation depolarizes spinal neurons by inhibiting a leak current. This will boost membrane depolarization and result in an increase in the excitability of spinal cord neurons, which could contribute to the modulation of the activity of the spinal locomotor network.     

Contributions of voltage- and Ca2+-activated conductances to GABA-induced depolarization in spider mechanosensory neurons

Activation of ionotropic gamma-aminobutyric acid type A (GABA(A)) receptors depolarizes neurons that have high intracellular [Cl(-)], causing inhibition or excitation in different cell types. The depolarization often leads to inactivation of voltage-gated Na channels, but additional ionic mechanisms may also be affected. Previously, a simulated model of spider VS-3 mechanosensory neurons suggested that although voltage-activated Na(+) current is partially inactivated during GABA-induced depolarization, a slowly activating and inactivating component remains and may contribute to the depolarization. Here, we confirmed experimentally, by blocking Na channels prior to GABA application, that Na(+) current contributes to GABA-induced depolarization in VS-3 neurons. Ratiometric Ca(2+) imaging experiments combined with intracellular recordings revealed a significant increase in intracellular [Ca(2+)] when GABA(A) receptors were activated, synchronous with the depolarization and probably due to Ca(2+) influx via low-voltage-activated (LVA) Ca channels. In contrast, GABA(B)-receptor activation in these neurons was previously shown to inhibit LVA current. Blockade of voltage-gated K channels delayed membrane repolarization, extending GABA-induced depolarization. However, inhibition of Ca channels significantly increased the amplitude of GABA-induced depolarization, indicating that Ca(2+)-activated K(+) current has an even stronger repolarizing effect. Regulation of intracellular [Ca(2+)] is important for many cellular processes and Ca(2+) control of K(+) currents may be particularly important for some functions of mechanosensory neurons, such as frequency tuning. These data show that GABA(A)-receptor activation participates in this regulation.     

Apamin reduces the late afterhyperpolarization of lamprey spinal neurons, with little effect on fictive swimming.

The role of the late afterhyperpolarization (late AHP) in the firing properties of lamprey spinal neurons was tested by bath application of apamin, a selective blocker of the sk calcium-dependent potassium current. Intracellular recordings of identified motoneurons and interneurons were made with micropipette electrodes in the isolated lamprey spinal cord. Apamin reversibly reduced the amplitude of the late afterhyperpolarization without affecting other aspects of the action potential or the resting potential. The firing frequencies of the neurons were enhanced by apamin over a range of depolarizing current pulse injections. The effect of apamin was also tested on fictive swimming, which was induced in the isolated spinal cord by bath application of an excitatory amino acid (D-glutamate or N-methyl-D,L-aspartate). A concentration of apamin (10 microM) sufficient to substantially reduce the late AHP had no significant effect on the ventral root burst rate, intensity, or phase lag during fictive swimming.     

Low-voltage-activated calcium current does not regulate the firing behavior in paired mechanosensory neurons with different adaptation properties

Low-voltage-activated Ca(2+) currents (LVA-I(Ca)) are believed to perform several roles in neurons such as lowering the threshold for action potentials, promoting burst firing and oscillatory behavior, and enhancing synaptic excitation. They also may allow rapid increases in intracellular Ca(2+) concentration. We discovered LVA-I(Ca) in both members of paired mechanoreceptor neurons in a spider, where one neuron adapts rapidly (Type A) and the other slowly (Type B) in response to a step stimulus. To learn if I(Ca) contributed to the difference in adaptation behavior, we studied the kinetics of I(Ca) from isolated somata under single-electrode voltage-clamp and tested its physiological function under current clamp. LVA-I(Ca) was large enough to fire single action potentials when all other voltage-activated currents were blocked, but we found no evidence that it regulated firing behavior. LVA-I(Ca) did not lower the action potential threshold or affect firing frequency. Previous experiments have failed to find Ca(2+)-activated K(+) current (I(K(Ca))) in the somata of these neurons, so it is also unlikely that LVA-I(Ca) interacts with I(K(Ca)) to produce oscillatory behavior. We conclude that LVA-Ca(2+) channels in the somata, and possible in the dendrites, of these neurons open in response to the depolarization caused by receptor current and by the voltage-activated Na(+) current (I(Na)) that produces action potential(s). However, the role of the increased intracellular Ca(2+) concentration in neuronal function remains enigmatic.     

Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine selectively reduces slow Ca2+- and Na+-mediated K+ currents in cat neocortical neurons

1. The effects of norepinephrine (NE) and related agonists and antagonists were examined on large neurons from layer V of cat sensorimotor cortex ("Betz cells") were examined in a brain slice preparation using intracellular recording, constant current stimulation and single microelectrode voltage clamp. 2. Application of NE (0.1-100 microM) usually caused a small depolarization from resting potential; hyperpolarizations were rare. Application of NE reversibly reduced rheobase and both the Ca2+- and Na+-dependent portions of the slow afterhyperpolarization (sAHP) that followed sustained firing evoked by constant current injection. The faster Ca2+-dependent medium afterhyperpolarization (mAHP), the fast afterhyperpolarization (fAHP), the action potential, and input resistance were unaffected. 3. The changes in excitability produced by NE application were most apparent during prolonged stimulation. The cells exhibited steady repetitive firing to currents that were formerly ineffective. The slow phase of spike frequency adaptation was reduced selectively and less habituation occurred during repeated long-lasting stimuli. The relation between firing rate and injected current became steeper if firing rate was averaged over several hundred milliseconds. 4. During voltage clamp in TTX, NE application selectively reduced the slow component of Ca2+-mediated K+ current. The faster Ca2+-mediated K+ current was unaffected, as were two voltage-dependent, transient K+ currents, the anomalous rectifier and leakage conductance measured at resting potential. Depolarizing voltage steps in the presence of Cd2+ revealed an apparent time- and voltage-dependent increase of the persistent Na+ current after NE application. The voltage-clamp results suggested ionic mechanisms for all effects seen during constant current stimulation except the depolarization from resting potential. The latter was insensitive to Cd2+ and TTX and occurred without a detectable change in membrane conductance. 5. NE application did not alter Ca2+ spikes evoked in the presence of TTX and 10 mM TEA. Inward Ca2+ currents examined during voltage clamp in TTX (with K+ currents reduced) became slightly larger after NE application. We conclude that NEs reduction of the slow Ca2+-mediated K+ current is not caused by reduction of Ca2+ influx. 6. Effects on membrane potential, rheobase, and the sAHP were mimicked by the beta-adrenergic agonist isoproterenol, but not by the alpha-adrenergic agonists clonidine or phenylephrine at higher concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)