Hypothalamic warm-sensitive neurons possess a tetrodotoxin-sensitive sodium channel with a high Q10

We have investigated the ionic current responses to temperature of dissociated cells from the preoptic and anterior hypothalamus (PO/AH) of rat, using the 'whole-cell' configuration. The majority of the recorded neurons showed a linear increase in a non-inactivating inward current during warming (30-40 degrees C), and the Q10 was about 2. However, about 24% of PO/AH neurons were markedly sensitive to warming and the increase in non-inactivating inward current to a rise in temperature in the hyperthermic range (35-40 degrees C) had a high Q10 (4.3-7.0). This increase in current in the hyperthermic range was reversibly blocked by tetrodotoxin (TTX). The inward current in neurons with a Q10 of 2 was not affected by TTX. The results show that some neurons in the PO/AH possess a non-inactivating sodium channel that is highly temperature-sensitive in the hyperthermic range. These neurons are presumably the 'primary' warm-sensitive neurons.     

Effects of GABAB-agonist on rat hypothalamic neurons: functional antagonism with mu-receptor agonist

Extracellular and whole-cell patch clamp recordings were made from neurons in slices of the preoptic area/anterior hypothalamus (PO/AH) of rats, to investigate the effects of the GABA(B)-receptor agonist baclofen on neuronal response characteristics, as well as its interactions with mu-opioid receptor agonist PL-017 on the level of central temperature controller. Baclofen decreased tonic activity (firing rate) in all types of neurons, but increased temperature sensitivity (temperature coefficient, TC) in warm-sensitive neurons. The decrease in firing rate during baclofen application was accompanied with significant membrane hyperpolarization and decrease of input resistances. The tonic activity (in all type of PO/AH neurons), as well as the temperature sensitivity (in warm-sensitive neurons), were inhibited by mu-opioid receptor agonist PL-017. Remarkably, the effect on temperature sensitivity was abolished and absence of synergism in regard to firing rate decrease occurred, when baclofen and PL-017 were applied simultaneously. Our results are step of understanding the complicated mechanisms of action of neurotransmitters and their interactions on the level of central temperature controller-the neurons of the PO/AH.     

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)     

Persistent Na+ current and Ca2+ current boost graded depolarization of rat retinal amacrine cells in culture

Retinal amacrine cells are depolarized by the excitatory synaptic input from bipolar cells. When a graded depolarization exceeds the threshold level, trains of action potentials are generated. There have been several reports that both spikes and graded depolarization are sensitive to tetrodotoxin (TTX). In the present study, we investigated the contribution of voltage-gated currents to membrane depolarization by using rat GABAergic amacrine cells in culture recorded by the patch-clamp method. Injection of a negative current induced membrane hyperpolarization, the waveform of which can be well fitted by a single exponential function. Injection of positive current depolarized the cell, and the depolarization exceeded the amplitude expected from the passive properties of the membrane. The boosted depolarization sustained after the current was turned off. Either 1 microM TTX or 2 mM Co2+ suppressed the boosted depolarization, and co-application of TTX and Co2+ blocked it completely. Under the voltage clamp, we identified a transient Na+ current (fast I(Na)), a TTX-sensitive persistent current that reversed the polarity near the equilibrium potential of Na+ (I(NaP)), and three types of Ca2+ currents (I(Ca)), L, N, and the pharmacological agent-resistant type (R type). These findings suggest that the I(NaP) and I(Ca) of amacrine cells boost depolarization evoked by the excitatory synaptic input, and they may aid the spread of electrical signals among dendritic arbors of amacrine cells.     

Cold transduction by inhibition of a background potassium conductance in rat primary sensory neurones

Transduction in cutaneous cold receptors is poorly understood at present. We have studied this question using dorsal root ganglion (DRG) neurones in primary culture as a model of the otherwise inaccessible receptor terminal. Whole-cell recordings during cooling from 32 to 20 degrees C revealed a large depolarization (>8mV) in 22 of 88 DRG neurones (25%), sometimes accompanied by action potentials. In cold-sensitive neurones cooling inhibited a time-independent background K+ current (Icold) which was resistant to tetraethylammonium and 4-aminopyridine. Ouabain elicited a substantially smaller depolarization than cooling, and no action potentials. We conclude that excitation by cooling in this model is primarily due to inhibition of Icold and that the previously suggested role of the Na+/K+ adenosine triphosphatase is secondary. We suggest that Icold may underlie cold transduction in cutaneous thermoreceptors.     

A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ.

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around -55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At -60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01-1 mM). Tetraethylammonium (0.1-30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials.This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.     

Electrophysiological properties and thermosensitivity of mouse preoptic and anterior hypothalamic neurons in culture

Responses of mouse preoptic and anterior hypothalamic neurons to variations of temperature are key elements in regulating the setpoint of homeotherms. The goal of the present work was to assess the relevance of culture preparations for investigating the cellular mechanisms underlying thermosensitivity in hypothalamic cells. Our working hypothesis was that some of the main properties of preoptic/anterior hypothalamic neurons in culture are similar to those reported by other authors in slice preparations. Indeed, cultured preoptic/anterior hypothalamic neurons share many of the physiological and morphological properties of neurons in hypothalamic slices. They display heterogenous dendritic arbors and somatic shapes. Most of them are GABAergic and their activity is synaptically driven by the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors. Active membrane properties include a depolarizing "sag" in response to hyperpolarization, and a low threshold spike, which is present in a majority of cells and is generated by T-type Ca2+ channels. In a fraction of the cells, the low threshold spike repeats rhythmically, either spontaneously, or in response to depolarization. The background synaptic noise in cultured neurons is characterized by the presence of numerous postsynaptic potentials which can be easily distinguished from the baseline, thus providing an opportunity for assessing their possible roles in thermosensitivity. An unexpected finding was that GABA-A receptors can generate both hyper- and depolarizing postsynaptic potentials in the same neuron. About 20% of the spontaneously firing preoptic/anterior hypothalamic neurons are warm-sensitive. Warming (32-41 degrees C) depolarizes some cells, a phenomenon which is Na+-dependent and tetrodotoxin-insensitive. The increased firing rate of warm-sensitive cells in response to warming can be prepotential and/or synaptically driven. Overall, our data suggest that a warm-sensitive phenotype is already developed in cultured cells. Therefore, and despite obvious differences in their networks, cultured and slice preparations of hypothalamic neurons can complement each other for further studies of warm-sensitivity at the cellular and molecular level.     

Hypoxia-activated Ca2+ currents in pacemaker neurones of rat rostral ventrolateral medulla in vitro.

We examined the effects of brief periods of hypoxia or application of cyanide on the discharge and membrane properties of medullary pacemaker neurones in slices of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata of rats. Stable intracellular recordings were obtained from seventy-nine neurones within the RVL which exhibited spontaneous rhythmic discharge in the absence of excitatory postsynaptic potentials (EPSPs). The membrane potential cycles of these neurones could be reset with an evoked spike without eliciting EPSPs or inhibitory postsynaptic potentials and hence met criteria of RVL pacemaker neurones. Hypoxia, produced by reducing O2 from 95 to 20% for 40 s or exposure to cyanide (30-300 microM for 40 s), reversibly increased neuronal discharge 1.6-fold (20% O2) or 2.6-fold (300 microM cyanide), respectively, in association with membrane depolarization and a significant fall in membrane resistance. The membrane responses to hypoxia and cyanide were observed in the presence of tetrodotoxin (TTX) at a concentration (10 microM) which eliminated spontaneous spikes or spikes evoked by intracellular depolarization. When recorded at a holding potential of -70 mV by single-electrode voltage clamp, hypoxia or cyanide (300 microM) elicited inward currents of 0.44 +/- 0.06 and 0.58 +/- 0.08 nA, respectively, which are attenuated by reducing the concentration of extracellular Ca2+ ions, and abolished by 2 mM CoCl2 and 100 microM NiCl2, but not affected by 50 microM CdCl2, replacement of 83% extracellular Na+, or adenosine deaminase (2U ml-1). We conclude that hypoxia and cyanide directly excite RVL pacemaker neurones in vitro by a common mechanism: activation of Ca2+ channel conductance.     

Reduced low-voltage activated K+ conductances and enhanced central excitability in a congenitally deaf (dn/dn) mouse

We have investigated changes in the neuronal excitability of the auditory brainstem in a congenitally deaf mouse ( deafness dn/dn ). Whole cell patch recordings from principal neurones of the medial nucleus of the trapezoid body (MNTB) showed strikingly enhanced excitability in the deaf mice when compared to control CBA mice at 12–14 days postnatal. MNTB neurones in normal CBA mice showed the phenotypic single action potential response on depolarization in current clamp; however, recordings from CBA mice carrying the homozygous deafness mutation fired trains of action potentials on depolarization. We show here that these changes are associated with reduced functional expression of dendrotoxin-sensitive Kv1 potassium channels. In contrast, no differences were found in voltage-gated calcium currents between control and deaf mice. These results reveal that loss of hair cell function in the cochlea leads to changes in ion channel expression in the central nervous system and suggests that this deafness model will be an important tool in understanding central changes occurring in human congenital deafness and in exploring activity-dependent regulation of ion channel expression.     

Biophysical characterization of whole-cell currents in O2-sensitive neurons from the rat glossopharyngeal nerve

In this study we use nystatin perforated-patch and conventional whole-cell recording to characterize the biophysical properties of neuronal nitric oxide synthase (nNOS)-expressing paraganglion neurons from the rat glossopharyngeal nerve (GPN), that are thought to provide NO-mediated efferent inhibition of carotid body chemoreceptors. These GPN neurons occur in two populations, a proximal one near the bifurcation of the GPN and the carotid sinus nerve, and a more distal one located further along the GPN. Both populations were visualized in whole mounts by vital staining with the styryl pyridinium dye, 4-Di-2-ASP (D289). Following isolation in vitro, proximal and distal neurons had similar input resistances (mean: 1.5 and 1.6 GOmega, respectively), input capacitances (mean: 25.0 and 27.4 pF, respectively), and resting potentials (mean: -53.9 and -53.3 mV, respectively). All neurons had similar voltage-dependent currents composed of: tetrodotoxin (TTX)-sensitive Na+ currents (IC50 approximately 0.2 microM), prolonged and transient Ca2+ currents, and delayed rectifier-type K+ currents. Threshold activation for the Na+ currents was approximately -30 mV and they were inactivated within 10 ms. Inward Ca2+ currents consisted of nifedipine-sensitive L-type, omega-agatoxin IVA-sensitive P/Q-type, omega-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni2+-sensitive, but SNX-482-insensitive, T-type channels. The voltage-dependent outward K+ currents were sensitive to tetraethylammonium (TEA; 10 mM) and 4-aminopyridine (4-AP; 2 mM). Exposure to a chemosensory stimulus, hypoxia (PO2 range: 80-5 Torr), caused a dose-dependent decrease in K+ current which persisted in the presence of TEA and 4-AP, consistent with the involvement of background K+ channels. Under current clamp, GPN neurons generated TTX-sensitive action potentials, and in spontaneously active neurons, hypoxia caused membrane depolarization and an increase in firing frequency. These properties endow GPN neurons with an exquisite ability to regulate carotid body chemoreceptor function during hypoxia, via voltage-gated Ca2+-entry, activation of nNOS, and release of NO.     

Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.     

Cellular mechanisms of orexin actions on paraventricular nucleus neurones in rat hypothalamus

Using whole-cell patch clamp techniques we have examined the cellular mechanisms underlying the effects of orexin A (OX-A) on electrophysiologically identified magnocellular and parvocellular neurones in the rat hypothalamic paraventricular nucleus (PVN). The majority of magnocellular neurones (67 %) showed concentration-dependent, reversible depolarizations in response to OX-A. These effects were abolished in tetrodotoxin (TTX), suggesting them to be indirect effects on this population of neurones. OX-A also caused increases in excitatory postsynaptic current (EPSC) frequency and amplitude in magnocellular neurones. The former effects were again blocked in TTX while increases in mini-EPSC amplitude remained. Depolarizing effects of OX-A on magnocellular neurones were also found to be abolished by kynurenic acid, supporting the conclusion that these effects were the result of activation of a glutamate interneurone. Parvocellular neurones (73 % of those tested) also showed concentration-dependent, reversible depolarizations in response to OX-A. In contrast to magnocellular neurones, these effects were maintained in TTX, indicating direct effects of OX-A on this population of neurones. Voltage clamp analysis using slow voltage ramps demonstrated that OX-A enhanced a non-selective cationic conductance with a reversal potential of -40 mV in parvocellular neurones, effects which probably explain the depolarizing effects of this peptide in this subpopulation of PVN neurones. These studies have identified separate cellular mechanisms through which OX-A influences the excitability of magnocellular and parvocellular PVN neurones.     

Chick spinal accessory lobes contain functional neurons expressing voltagegated sodium channels generating action potentials

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological evidence has shown that neurons are present in AL and behavioral evidence suggests that AL acts as a sensory organ of equilibrium during bipedal walking. However, there is little functional evidence to indicate that cells in AL have neuronal functions. To elucidate this point, we developed a method to dissociate cells from chick AL and made electrophysiological recordings with the whole-cell patch clamp technique. Cells dissociated by enzymatic digestion from chick AL contained two major types of cells. One was round with clear cytosol and the other had a round cell body, rich cytosolic structures and some processes. Rapidly activating inward currents and slowly activating outward currents were recorded in response to depolarizing pulses to -10 mV under the voltage clamp configuration only from the latter type of cells. TTX at 100 nM inhibited the inward current by 85%, indicating the functional expression of TTX-sensitive voltage-gated Na(+) channel (VGSC). Activation and inactivation kinetics of the inward currents in AL cells were similar to those of mammalian VGSC. The VGSC-expressing AL cells generated action potentials in response to depolarization under the current clamp configuration. These results clearly indicate that functional neurons expressing fast inactivating and TTXsensitive VGSC which generate action potentials exist in the AL of the chick. These lines of cellular evidence clearly indicate that functional neurons exist in ALs and further support the proposal that the chick ALs function as the sensory organ of equilibrium.     

Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons.

1. Intracellular recordings were made from neurons in the dorsal motor nucleus of the vagus (DMV) in transverse slices of rat medulla maintained in vitro at 30 degrees C. Neurons had a resting potential of -59.8 +/- 1.4 (SE) mV (n = 39) and input resistance of 293 +/- 23 M omega (n = 44). 2. Depolarization elicited overshooting action potentials that were blocked by tetrodotoxin (TTX; 1 microM). In the presence of TTX, two types of action potentials having low and high thresholds could be elicited. The action potentials were blocked by cobalt (2 mM) indicating they were mediated by calcium currents. 3. Under voltage clamp, depolarization of the cell from membrane potentials negative of the resting potential activated a transient potassium current. This current was selectively blocked by 4-aminopyridine (4-AP) (5 mM) and catechol (5 mM) indicating that it is an A-type current. This current inactivated with a time constant of 420 ms and recovered from inactivation with a time constant of 26 ms. 4. When calcium currents were blocked by cadmium or cobalt, the rate of action potential repolarization was slower. In the presence of tetraethylammonium (TEA; 200-400 microM) or charybdotoxin (CTX; 30 nM) a small "hump" appeared on the repolarizing phase of the action potential that was abolished by addition of cadmium. These results indicate that a calcium-activated potassium current (IC) contributes to action potential repolarization. 5. Actions potentials elicited from hyperpolarized membrane potentials repolarized faster than those elicited from resting membrane potential. This effect could be blocked by catechol, indicating that voltage-dependent potassium currents (IA) can also contribute to action-potential repolarization. In the presence of catechol and calcium channel blockers, action potentials still had a significant early afterhyperpolarization suggesting that another calcium independent outward current is also active during repolarization. This fast afterhyperpolarizations (AHP) was not blocked by TEA. 6. Action potentials were followed by prolonged AHPs, which had two phases. The initial part of the AHP was blocked by apamin (100 nM) indicating that it results from activation of SK type calcium-activated potassium channels. The slow phase was selectively blocked by catechol suggesting that it is due to activation of IA. 7. It is concluded that a TTX-sensitive sodium current and two calcium currents contribute to the action potential in rat DMV neurons. At least three different currents contribute to action-potential repolarization: IC, IA, and a third unidentified calcium-insensitive outward current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sodium influx blockade and hypoxic damage to CA1 pyramidal neurons in rat hippocampal slices.

We studied the effects of lidocaine and tetrodotoxin (TTX) on hypoxic changes in CA1 pyramidal neurons to examine the ionic basis of neuronal damage. Lidocaine (10 and 100 microM) and TTX (6 and 63 nM) delayed and attenuated the hypoxic depolarization and improved recovery of the resting and action potentials after 10 min of hypoxia. Lidocaine (10 and 100 microM) and TTX (63 nM) reduced the number of morphologically damaged CA1 cells and improved protein synthesis measured after 10 min hypoxia. Lidocaine (10 microM) attenuated the increase in intracellular sodium (181 vs. 218%) and the depolarization (-21 vs. -1 mV) during hypoxia but did not significantly attenuate the changes in ATP, potassium, or calcium measured at 10 min of hypoxia. Lidocaine (100 microM) attenuated the changes in membrane potential, sodium, potassium, ATP, and calcium during hypoxia. TTX (63 nM) attenuated the changes in membrane potential (-36 vs. -1 mV), sodium (179 vs. 226%), potassium (78 vs. 50%), and ATP (24 vs. 11%) but did not significantly attenuate the increase in calcium during hypoxia. These data indicate that the primary blockade of sodium channels can secondarily alter other cellular parameters. The hypoxic depolarization and the increase in intracellular sodium appear to be important triggers of hypoxic damage independent of their effect on cytosolic calcium; a treatment that selectively blocked sodium influx (lidocaine 10 microM) improved recovery. Our data indicate that selective blockade of sodium channels with a low concentration of lidocaine or TTX improves recovery after hypoxia by attenuating the rise in cellular sodium and the hypoxic depolarization. This blockade improves the resting and action potentials, histologic state, and protein synthesis of CA1 pyramidal neurons after 10 min of hypoxia to rat hippocampal slices. A higher concentration of lidocaine, which also improved ATP, potassium, and calcium concentrations during hypoxia was more potent. In conclusion, the depolarization and increased sodium concentration during hypoxia account for a portion of the neuronal damage after hypoxia independent of changes in calcium.     

Hypoxic augmentation of fast-inactivating and persistent sodium currents in rat caudal hypothalamic neurons

Previous work from this laboratory has indicated that TTX-sensitive sodium channels are involved in the hypoxia-induced inward current response of caudal hypothalamic neurons. Since this inward current underlies the depolarization and increased firing frequency observed in these cells during hypoxia, the present study utilized more detailed biophysical methods to specifically determine which sodium currents are responsible for this hypoxic activation. Caudal hypothalamic neurons from approximately 3-wk-old Sprague-Dawley rats were acutely dissociated and patch-clamped in the voltage-clamp mode to obtain recordings from fast-inactivating and persistent (noninactivating) whole cell sodium currents. Using computer-generated activation and inactivation voltage protocols, rapidly inactivating sodium currents were analyzed during normal conditions and during a brief (3-6 min) period of severe hypoxia. In addition, voltage-ramp and extended-voltage-activation protocols were used to analyze persistent sodium currents during normal conditions and during hypoxia. A polarographic oxygen electrode determined that the level of oxygen in this preparation quickly dropped to 10 Torr within 2 min of initiation of hypoxia and stabilized at <0.5 Torr within 4 min. During hypoxia, the peak fast-inactivating sodium current was significantly increased throughout the entire activation range, and both the activation and inactivation values (V(1/2)) were negatively shifted. Furthermore both the voltage-ramp and extended-activation protocols demonstrated a significant increase in the persistent sodium current during hypoxia when compared with normoxia. These results demonstrate that both rapidly inactivating and persistent sodium currents are significantly enhanced by a brief hypoxic stimulus. Furthermore the hypoxic-induced increase in these currents most likely is the primary mechanism for the depolarization and increased firing frequency observed in caudal hypothalamic neurons during hypoxia. Since these neurons are important in modulating cardiorespiratory activity, the oxygen responsiveness of these sodium currents may play a significant role in the centrally mediated cardiorespiratory response to hypoxia.     

Primary cold-sensitive neurons in acutely dissociated cells of rat hypothalamus

Local warming or cooling of the preoptic and anterior hypothalamus (PO/AH) areas evokes various thermoregulatory responses in mammals. We have hypothesized that warm- and cold-sensitive neurons recorded in the PO/AH are multiple thermostats that regulate the core temperature against heat and cold, respectively. However, since the proportion of cold-sensitive neurons is low, it is still controversial whether primary cold-sensitive neurons exist in the PO/AH. To answer this question, we investigated cold-sensitive neurons with Ca(2+) imaging in acutely dissociated PO/AH cells. Their threshold temperatures were 27.3+/-0.44 degrees C (mean+/-SEM, n=55). In extracellular recordings cooling evoked discharges in these cold-sensitive neurons. We conclude that primary cold-sensitive neurons with low threshold temperatures exist in PO/AH.     

Patch-clamp study of postnatal development of CA1 neurons in rat hippocampal slices: membrane excitability and K+ currents.

1. The postnatal development of membrane properties and outward K+ currents in CA1 neurons in rat hippocampal slices was studied with the use of whole-cell patch-clamp techniques. 2. Neurons at all postnatal ages (2-30 days; P2-30) were capable of generating tetrodotoxin (TTX)-sensitive action potentials in response to intracellular injection of depolarizing current pulses. There was a gradual increase in the amplitude and a decrease in the duration of these action potentials with age. Stable values for spike duration were reached by P15, whereas spike amplitude increased until P20-25. In P2-5 neurons, the duration of action potentials was greatly prolonged by depolarization from the resting membrane potential, indicating a weak spike repolarizing mechanism at depolarized potentials. In contrast, the duration of spikes evoked in P20-30 neurons was not affected by similar changes in the membrane potential. 3. Application of tetraethylammonium (TEA, 10 mM) had no effect on the duration of spikes in P3-5 neurons, whereas application of 4-aminopyridine (4-AP, 2 mM) produced large increases in spike duration. In contrast, the duration of spikes in P26 neurons was greatly increased after TEA application, whereas 4-AP had smaller effects on spike duration in these neurons. 4. The input resistance and membrane time constant decreased with age from P2 to P15. The values for both parameters were considerably greater than those reported with conventional intracellular recording electrodes in the immature hippocampus. The resting membrane potential became more hyperpolarized with age. When the recording pipettes contained KCl (140 mM), the resting potential of P3-4 neurons was 34 mV depolarized compared with resting potentials observed with potassium gluconate-filled pipettes. Only a 13-mV change in resting potential was observed during similar comparisons in P27-28 neurons. 5. Outward currents activated by depolarization were examined with the use of voltage-clamp techniques in P2-30 neurons. In P2-5 cells, a small, slowly inactivating outward current was evoked with depolarizing commands from holding potentials near -50 mV. By preceding the depolarizing commands with a hyperpolarizing prepulse, an additional early transient outward current was evoked. The sustained and transient outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), TEA, and 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic receptor activation induces a prolonged post-stimulus afterdepolarization with a conductance decrease in guinea-pig olfactory cortex neurones in vitro.

The persistent excitation of guinea-pig olfactory cortical neurones in vitro by the muscarinic agonist oxotremorine-M (OXO-M) was investigated. In OXO-M (10-20 microM), a slowly-decaying afterdepolarization (sADP) accompanied by sustained repetitive firing was induced following a long depolarizing stimulus. The corresponding slow inward current (IADP) revealed under voltage clamp behaved like a K(+)-mediated tail current, but was associated with a decreased membrane conductance. IADP was insensitive to tetrodotoxin (TTX), Ba2+, Cs+, or 4-aminopyridine (4-AP), but was blocked by 500 microM TEA or TBA (tetrabutylammonium). The OXO-M response and IADP were also reduced by Cd2+ or Ca(2+)-free solution, suggesting a dependence on Ca(2+)-entry. We propose that OXO-M induces a novel outward K+ current that can be slowly de-activated by Ca(2+)-entry during a depolarizing stimulus. Summation of IADP tail currents could contribute to the sustained muscarinic excitation of mammalian cortical neurones.     

Properties of a persistent inward current in normal and TEA-injected motoneurons

1. Membrane currents of normal and TEA-injected cat lumbar motoneurons were investigated using the technique of somatic voltage clamp. 2. The current-voltage (I-V) relation of healthy motoneurons contains a region of negative slope conductance caused by a persistent inward current component (Ii). In the most striking examples, Ii is net inward at some potentials between 10 and 30 mV positive to resting potential. 3. Near its activation threshold (greater than or equal to 10 mV positive to rest), Ii does not decrement during prolonged voltage steps and, in most cells, activates very slowly. Ii amplitude increases and time to peak Ii decreases with further small increments of depolarization, and Ii decrements during sustained voltage steps. Maximum Ii amplitude occurs 20--30 mV positive to rest in most cells. Ii is not visible at sufficiently large depolarizations. 4. Ii appears to be mixed with potassium current components at nearly every potential where it is visible. These include a slow outward current first activated near Ii activation threshold, a fast outward current additonally activated at larger depolarizing potentials, and a fast, transient outward current that obscures the true onset of Ii at nearly every potential. 5. Ii is not carried by sodium entering via the fast, transient channels and is present after pharmacological blockage of sodium currents. It is proposed that Ii is predominantly carried by calcium ions. 6. The presence of inward tail currents after repolarization from potentials that activate a steady outward current suggest that Ii remains present but hidden at large depolarizations. Ii inactivation was further investigated in TEA-injected motoneurons since Ii and the tail currents are more prominent in these cells. 7. Conventional recordings from TEA-injected motoneurons suggest that a prolonged, postspike plateau potential is maintained by a persistent inward current. Voltage-clamp data can account for the principal features of the plateau potential. 8. Voltage-clamp results in TEA-injected motoneurons suggest that Ii is subject to little or no inactivation at potentials less than or equal to 30 mV positive to rest and to partial inactivation, at most, at higher potentials during steps lasting less than or equal to 100 ms. The apparent decay of Ii during sustained depolarization is caused by the development of a larger outward current. 9. Ii is similar in several ways to a persistent calcium current observed in some molluscan neurons. Theoretical and experimental results suggest that Ii is generated predominantly in a local region under voltage control and that the observed membrane currents govern somatic membrane potential and cell behavior.