Glycine-activated chloride currents of neurons freshly isolated from the ventral tegmental area of rats

Properties of whole-cell glycine currents (I_Gly) of ventral tegmental area (VTA) neurons from 3- to 7-day old Sprague–Dawley rats were investigated with the patch-clamp technique. Ninety-three percent of the 126 neurons examined produced I_Gly in response to glycine. For 70% of these neurons, I_Gly did not decay in response to a threshold concentration of glycine (1–5 μM). At elevated glycine concentrations, I_Gly consistently decayed from a peak to a steady state (SS). I_Gly increased in amplitude sigmoidally as a function of the concentration of agonist with an EC₅₀ of 32 μM. Strychnine (STR), when co-applied with glycine after a prepulse of STR, suppressed both the peak and SS I_Gly noncompetitively. In the absence of a prepulse, STR had a smaller effect on peak I_Gly while increasing its decay rate; the SS amplitude decreased. These STR effects were concentration dependent with an IC₅₀ of 31 nM and 184 nM STR for the peak and SS I_Gly, with prepulse, respectively, and 732 nM and 193 nM for the peak and SS I_Gly, respectively, without prepulse. Picrotoxin (PTX) co-applied with glycine suppressed both the peak and the SS I_Gly with an IC₅₀ of 25 μM. In contrast to STR, 1 min preincubation with PTX had no effect on I_Gly. Thus, PTX acts on the open channel. The inhibitory effects of both STR and PTX on I_Gly did not depend on the membrane potential.     

The effect of graded hypertonia on interstitial volume, tissue resistance and synaptic transmission in rat hippocampal tissue slices

Rat hippocampal slices were exposed for 30 min to each of three levels of increased osmolarity (π_o), achieved by adding 25, 50 or 100 mM mannitol to the bathing solution. The interstitial volume (ISV) determined as the relative volume of dilution of the probe ion, tetra-methyl-ammonium (TMA⁺), increased markedly, indicating cell shrinkage. Tissue resistance (R_o) decreased only slightly with increasing π_o. The discrepancy between ISV increase and R_o decrease suggests increased electrical resistance of cell membranes. TMA⁺ dilution appears to be a more reliable measure of ISV than is R_o During recovery from hypertonic treatment the previously expanded ISV frequently shrank, suggesting post-hypertonic cell swelling. Hypertonic treatment significantly depressed orthodromically transmitted population spikes and extracellular synaptic potentials (fEPSPs), and the degree of depression varied with the increase in π_o. Changing recording condition due to reduced R_o could not account for the depression of population spikes and fEPSPs. Following return to normal π_o, orthodromic population spikes frequently overshot initial control amplitude. An isolated episode of spreading depression occurred in about half of the slices following exposure to the most severely hypertonic solution. At the end of 2.5 h recovery, orthodromic spikes did not significantly differ from those of untreated control slices observed for the same length of time. We conclude that synaptic transmission is depressed by elevation of π_o and the depression is concentration dependent and reversible.     

Low external NaCl concentration and low osmolarity enhance voltage-gated Ca currents but depress K currents in freshly isolated rat hippocampal neurons

In previous experiments, reducing bath osmolarity (pi o) or external NaCl concentration ([NaCl]o) caused an increase in synaptic currents recorded in whole-cell configuration from pyramidal cells of CA1 region of hippocampal slices. Slow inward current surges, assumed to be calcium currents, were also enhanced. This contrasted with the strong, reversible, generalized depression of voltage-dependent ion currents in isolated neurons, caused by sudden, brief exposure to very low pi o. I have now recorded voltage-dependent whole-cell Na-, K- and Ca-currents from freshly isolated hippocampal CA1 pyramidal neurons during more gradual lowering of pi o or [NaCl]o. Changes in cell size were determined from image areas and changes in intracellular Ca2+ activity were measured as the ratio of the fluorescences of fluo-3/fura-red by confocal microscopy. Iso-osmotic substitution of 40 or 60 mM NaCl by mannitol or sucrose for 5-7 min, or reducing pi o by deleting NaCl from the bath (osmolarity decreased by 69 or 108 mosM/kg) depressed K currents. Na currents were also strongly depressed, but this is in part attributable to reduced driving potential and ionic conductance. The depression of IK varied widely and it was inversely correlated with the degree of hypotonic swelling, suggesting that reduced ion channel conductance also reduces permeability to water. Reducing [NaCl]o by 60 mM, or pi o by 105 mosM/kg consistently and reversibly increased Ca currents. Intracellular Ca2+ level also increased, but the changes of [Ca2+]i and ICa were not correlated. Facilitation of the Ca influx into presynaptic terminals could explain the increase of synaptic currents. Depression of outward currents could also contribute to the irritability of the central nervous system typical of clinical hyponatremia.     

Hypotonic exposure enhances synaptic transmission and triggers spreading depression in rat hippocampal tissue slices

Low extracellular osmotic pressure (π_o) is known to enhance CNS resposiveneess and the chance of seizures, but the mechanism of the hyperexcitability is not clear. We recorded evoked potentials in st. radiatum and st. pyramidale of CA1. Tissue electrical resistance (R_o) was determined from the voltage drop (V_Ro) evoked by constant current pulses. Lowering of π_o by reducing [NaCl] caused a concentration-dependent increase of amplitude and duration of extracellular excitatory postsynaptic potentials (fEPSPs). fEPSPs increased much more than didV_Ro, but antiddromic population spikes increased in proportion toV_Ro. fEPSP increased also in isosmotic low NaCl (fructose or mannitol substituted) olutions, but not as much as in low π_o. In moderately hypotonic solutions orthodromic population spikes increased as expected from the augmented fEPSP, but in strong hypotonia input-output curves shifted to the left and single stimuli evoked multiple population spikes, indicating lowering of threshold of postsynaptic neurons. Blocking N-methyl-d-aspartate (NMDA) receptors did not diminish the enhancement of fEPSP amplitude. Spreading depression (SD) erupted in most slices in very low π_o, but not in isosmotic low [NaCl] solutions. We conclude that the hypotonic enhancement of EPSPs depends, in part, on the lowering of [Na⁺]_o and/or of [Cl⁻]_o, and it may be augmented by dendritic swelling favoring electrotonic spread of EPSPs from dendrites to somata, and buildup of transmitter concentration due to swelling of perisynaptic glia. SD can be initiated by cell swelling, but the depolarization associated with SD is probably not caused by the opening of stretch-gated ion channels.     

Effects of insulin-like growth factor 1 on synaptic excitability in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory and neuroendocrine actions, and it also prevents beta amyloid-induced death of hippocampal neurons. However, its functions in the synaptic excitability remain uncertain. Here we investigated the effects of IGF-1 on synaptic excitability in cultured rat hippocampal neurons using whole-cell patch clamp recordings. Incubation the hippocampal neurons with different concentrations of IGF-1 for 24 h or 30 min significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on the frequency of miniature EPSCs (mEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs). The mean amplitudes, rise, and decay kinetics of sEPSCs, mEPSCs, and sIPSCs were not significantly affected by IGF-1, indicating that IGF-1 increased the probability of neurotransmitter release but did not modulate postsynaptic receptors. The effects of IGF-1 were mediated by mitogen-activated protein kinase (MAPK). IGF-1 activated the ERK1/2 signaling pathway in cultured hippocampal neurons, and the inhibitor PD98059 blocked the enhancement of sEPSCs induced by IGF-1. These results demonstrated the regulatory function of IGF-1 on synaptic excitability in hippocampal neurons and its underlying signaling mechanism.     

Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells

Summary. Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca²⁺ imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na⁺ current could be fitted by the Hill equation with a half-blocking concentration of 0.56mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca²⁺]_i response of ORCs, suggesting that linalool suppresses Ca²⁺ currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells.     

Orthodromic synaptic activation of rat olfactory bulb mitral cells in isolated slices

Axons of olfactory receptor neurons terminate in the glomerular layer of the olfactory bulb, where they synapse with the apical dendrites of mitral cells. Although the mitral cell and its excitation by the olfactory nerve have been the subject of numerous experimental investigations, in vitro studies of these neurons have primarily used nonmammalian preparations. We have recorded the responses of rat olfactory bulb mitral cells to stimulation of the olfactory nerve layer in vitro using extracellular and whole cell patch techniques. Olfactory bulbs were cut into 400-μm thick slices in approximately horizontal section and submerged in a recording chamber. Patch clamp electrodes were guided into the mitral cell layer, which was visible under a dissecting microscope. A stimulating electrode was placed onto the olfactory nerve layer (ONL) rostral to the recording electrode. In extracellular recordings, mitral cells typically responded to ONL stimulation with a prolonged excitation lasting 1 s or longer. With whole cell patch recordings, membrane resistances (mean 272 MΩ) were substantially higher than those reported in previous intracellular studies that used sharp electrodes. Small spontaneous excitatory potentials were present in some mitral cells. ONL stimulation caused a prolonged depolarization comparable to the duration of the period of excitation observed in extracellular recordings. At membrane potentials near −55 mV, ONL stimulation evoked a train of spikes. All but the first of these spikes were blocked by hyperpolarization of the membrane to −65 mV.     

Effects of insulin-like growth factor 1 on voltage-gated ion channels in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory, and neuroendocrine actions, and it is also prevents amyloid beta-induced death of hippocampal neurons. However, its functions on the voltage-gated ion channels in hippocampus remain uncertain. In the present study, we investigated the effects of IGF-1 on voltage-gated potassium, sodium, and calcium channels in the cultured rat hippocampal neurons using the whole-cell patch clamp recordings. Following incubation with different doses of IGF-1 for 24 h, a block of the peak transient A-type K+ currents amplitude (IC50: 4.425 ng/ml, Hill coefficient: 0.621) was observed. In addition, after the application of IGF-1, the amplitude of high-voltage activated Ca2+ currents significantly increased but activation kinetics did not significantly alter (V1/2: -33.45 +/- 1.32 mV, k = 6.16 +/- 1.05) compared to control conditions (V1/2: -33.19 +/- 2.28 mV, k = 7.26 +/- 1.71). However, the amplitude of Na+, K+, and low-voltage activated Ca2+ currents was not affected by the application of IGF-1. These data suggest that IGF-1 inhibits transient A-type K+ currents and enhances high-voltage-activated Ca2+ currents, but has no effects on Na+ and low-voltage-activated Ca2+ currents.     

Calcium spikes and calcium currents in neurons from the medial preoptic nucleus of rat

Ca²⁺ spikes, their contribution to firing patterns, and the underlying Ca²⁺ currents in neurons of the medial preoptic nucleus of rat were investigated by tight-seal whole-cell recordings in a slice preparation. Two different types of spikes were recorded: Low-threshold spikes were generated from membrane potentials <−75 mV. High-threshold spikes were recorded when K⁺ currents were reduced, and were readily evoked from membrane potentials near −40 mV. Both types of spikes were blocked by substitution of Co²⁺ for Ca²⁺ in the external medium, but were insensitive to 2.0 μM TTX. Under voltage-clamp conditions, two main types of Ca²⁺ currents were characterized: low-threshold currents that activated at membrane potentials >−60 mV, and high-threshold currents that activated at potentials >−30 mV. The low-threshold current and the low-threshold spike were more sensitive to block by external Ni²⁺ than to block by Cd²⁺, whereas the high-threshold current and the high-threshold spike were more sensitive to block by external Cd²⁺ than to block by Ni²⁺. Significant fractions of the high-threshold currents were blocked by 10 μM nifedipine, 1.0 μM ω-conotoxin GVIA, 50 nM ω-agatoxin IVA and 1.0 μM ω-conotoxin MVIIC, suggesting the presence of L-, N-, P- and Q-type Ca²⁺ channels. There were also a high-threshold current component insensitive to the above mentioned toxins. It is proposed that the low-threshold current serves as a trigger for short bursts of fast spikes from hyperpolarized levels, whereas the high-threshold current is involved in the Cd²⁺-sensitive burst firing seen in relatively depolarized neurons.     

Imbalance between excitation and inhibition among synaptic connections of CA3 pyramidal neurons in cultured hippocampal slices

A fundamental property of small neuronal ensembles is their ability to be selectively activated by distinct stimuli. One cellular mechanism by which neurons achieve this input selectivity is by modulating the temporal dynamics of excitation and inhibition. We explored the interplay of excitation and inhibition in synapses between pyramidal neurons of cornu ammonis field 3 of the hippocampal formation (CA3) in cultured rat hippocampal slices, where activation of a single excitatory cell can readily recruit local interneurons. Simultaneous whole-cell recordings from pairs of CA3 pyramidal neurons revealed that the strength of connections was neither uniform nor balanced. Rather, stimulation of presynaptic neurons elicited distinct combinations of excitatory postsynaptic current–inhibitory postsynaptic current (EPSC–IPSC) amplitudes in the postsynaptic neurons. EPSC–IPSC sequences with small EPSCs had large IPSCs and sequences that contained large EPSCs had small IPSCs. In addition to differences in the amplitudes of the responses, the kinetics of the EPSCs were also different, creating distinct temporal dynamics of excitation and inhibition. Weaker EPSCs had significantly slower kinetics and were efficiently occluded by IPSCs, thereby further limiting their contribution to depolarizing the postsynaptic membrane. Our data suggest that hippocampal pyramidal cells may use an imbalance between excitation and inhibition as a filter to enhance selectivity toward preferential excitatory connections.     

Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices

Hippocampal slices exposed to brief anoxia combined with elevated glucose exhibit greater postanoxic recovery of synaptic transmission. Glucose may have improved recovery of synaptic transmission by enhancing the production of metabolic energy during and after anoxia. This enhancement should provide more ATP for energy-requiring ion transport processes, and lead (1) to a delayed onset of complete depolarization of CA1 pyramidal cells during anoxia (anoxic depolarization) and (2) to greater ion transport activity following anoxia. A delay in anoxic depolarization would protect neurons from damage if the duration of anoxic depolarization was shortened. Greater postanoxic ion transport would allow the re-establishment of ion gradients supportive of neuronal and synaptic excitability. The effects of glucose and anoxia on ion homeostasis and synaptic transmission were examined in rat hippocampal slices exposed to different glucose concentrations (5–20 mM). The duration of anoxic depolarization was held constant so that postanoxic damage related to this duration was controlled. We found that K⁺ transport and recovery of synaptic transmission after anoxia in hippocampal slices improved as glucose concentration increased. Also, anoxic depolarization was delayed as glucose concentration increased. Thus, added glucose may improve postanoxic recovery of synaptic transmission by better supporting ion transport.     

Cocaine and lidocaine have additive inhibitory effects on the GABAA current of acutely dissociated hippocampal pyramidal neurons

Inhibition mediated by γ-aminobutyric acid (GABA) is a major target for the central actions of cocaine and lidocaine, which can result in seizures, especially when these drugs are abused in combination. In the present study, we investigated how cocaine and lidocaine interact to depress GABA current (I_GABA), recorded by the whole-cell technique in freshly isolated rat hippocampal neurons. Cocaine depressed I_GABA in a concentration dependent manner, such that cocaine was more potent against lower than higher GABA concentrations: the cocaine IC₅₀ was 0.13, 0.62 and 1.2 mM for GABA at 2, 10 and 100 μM, respectively. Cocaine depressed I_GABA to the same extent in the absence and presence of 1 μM tetrodotoxin, indicating that cocaine inhibition of I_GABA is distinct from its Na⁺ channel blocking action. Lidocaine reversibly depressed I_GABA evoked by 10 μM GABA, with an IC₅₀ of 9.8 mM. In the presence of 3 mM lidocaine, 0.3 mM cocaine depressed I_GABA (10 μM GABA) to 30±7%. The significantly greater depression by the combined agents (p<0.05) indicates additive effects on the GABA receptor/channel complex, which are likely to contribute to the additive convulsant effects noted when these drugs are abused in combination.     

Analysis of the function of GABA(B) receptors on inhibitory afferent neurons of Purkinje cells in the cerebellar cortex of the rat

Purkinje cells, the output neurons of the cerebellar cortex, receive inhibitory input from basket, stellate and neighbouring Purkinje cells. The aim of the present study was to clarify the role of GABAB receptors on neurons giving inhibitory input to Purkinje cells. In sagittal slices prepared from the cerebellar vermis of the rat, the GABAB receptor agonist baclofen lowered the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded in Purkinje cells. These effects were prevented by the GABAB receptor antagonist CGP 55845. Two mechanisms were involved in the depression of the inhibitory input to Purkinje cells. The first mechanism was suppression of the firing of basket, stellate and Purkinje cells. The second mechanism was presynaptic inhibition of GABA release from terminals of the afferent axons. This was indicated by the finding that baclofen decreased the amplitude of IPSCs occurring in Purkinje cells synchronously with action potentials recorded in basket cells. A further support for the presynaptic inhibition is the observation that baclofen decreased the amplitude of autoreceptor currents which are due to activation of GABAA autoreceptors at axon terminals of basket cells by synaptically released GABA. The presynaptic inhibition was partly due to direct inhibition of the vesicular release mechanism, because baclofen lowered the frequency of miniature IPSCs recorded in Purkinje cells in the presence of cadmium and in the presence of tetrodotoxin plus ionomycin. The results show that activation of GABAB receptors decreased GABAA receptor-mediated synaptic input to cerebellar Purkinje cells both by lowering the firing rate of the inhibitory input neurons and by inhibiting GABA release from their axon terminals with a presynaptic mechanism.     

Modulation of Ca2+ currents in rat thalamocortical relay neurons by activity and phosphorylation

Rhythmic low and high frequency activity in thalamocortical networks depend critically on activation of low- and high-voltage-activated (LVA, HVA) Ca2+ currents. In order to test whether Ca2+ currents are modified during repetitive activation, acutely isolated thalamocortical relay neurons of rats, at postnatal days 12 (P12) to P20, were investigated using patch-clamp, Ca2+ imaging and Western blot techniques. High-voltage-activated, but not LVA Ca2+ currents were reduced significantly during 2 Hz stimulation. Ca2+ imaging experiments demonstrated a close correlation between the increase in intracellular Ca2+ levels and the decrease in HVA Ca2+ current amplitudes. Further examination of HVA Ca2+ currents revealed a 'U-shaped' inactivation curve and a time-dependent inactivation process that could be described by a two-exponential function. The 'U-shape' was significantly reduced, current amplitude was increased significantly and time-dependent inactivation revealed a one-exponential decline with Ba2+ as the charge carrier, following activation of the cAMP/PKA pathway, and following application of phosphatase inhibitors (ascomycin, calyculin A). Western blot analysis and the effect of ascomycin indicated an involvement of calcineurin in the inactivation process. Isolation of HVA Ca2+ current components by subtype-specific blockers revealed that changes in time-dependent inactivation, inactivation curve and current amplitude were carried mainly by L-type and N-type Ca2+ currents. Furthermore, Ca2+-dependent inactivation was operative during stimulation protocols mimicking tonic action potential firing. These data indicate a modulation of L- and N-type Ca2+ channels by phosphorylation, resulting jointly in an increased intracellular Ca2+ influx during activity of the ascending brainstem system, the latter occurring during states of wakefulness.     

Effects of extracellular delta-aminolaevulinic acid on sodium currents in acutely isolated rat hippocampal CA1 neurons

The effects of delta-aminolaevulinic acid (ALA) on voltage-gated sodium channel (VGSC) currents (I(Na)) in acutely isolated hippocampal CA1 neurons from 10- to 12-day-old Wistar rats were examined by using the whole-cell patch-clamp technique under voltage-clamp conditions. ALA from 0.01 microm to 20 microm was applied to the recorded neurons. Low concentrations of ALA (0.01-1.0 microM) increased I(Na) amplitude, whereas high concentrations of ALA (5.0-20.0 microM) decreased it. The average I(Na) amplitude reached a maximum of 117.4 +/- 3.9% (n = 9, P < 0.05) with 0.1 microM ALA, and decreased to 78.1 +/- 3.8% (n = 13, P < 0.05) with 10 microm ALA. ALA shifted the steady-state activation and inactivation curves of I(Na) in the hyperpolarizing direction with different V0.5, suggesting that ALA could depress the opening threshold of the voltage-gated sodium channel (VGSC) and thus increase the excitability of neurons through facilitating the opening of VGSC. The time course of recovery from inactivation was significantly prolonged at both low and high concentrations of ALA, whereas either low or high concentrations of ALA had no significant effect on the attenuation of I(Na) during stimulation at 5 Hz, indicating that the effect of ALA on VGSC is state-independent. Furthermore, we found that application of ascorbic acid, which blocks pro-oxidative effects in neurons, could prevent the increase of I(Na) amplitude at low concentrations of ALA. Baclofen, an agonist of GABAb receptors, induced some similar effects to ALA on VGSC, whereas bicuculline, an antagonist of GABAa receptors, could not prevent ALA-induced effects on VGSC. These results suggested that ALA regulated VGSC mainly through its pro-oxidative effects and GABAb receptor-mediated effects.     

Presynaptic and postsynaptic modulation of glutamatergic synaptic transmission by activation of α1‐ and β‐adrenoceptors in layer V pyramidal neurons of rat cerebral cortex

Adrenergic agonists have different modulatory effects on excitatory synaptic transmission depending on the receptor subtypes involved. The present study examined the loci of α₁‐ and β‐adrenoceptor agonists, which have opposite effects on excitatory neural transmission, involved in modulation of glutamatergic transmission in layer V pyramidal cells of rat cerebral cortex. Phenylephrine, an α₁‐adrenoceptor agonist, suppressed the amplitude of AMPA receptor‐mediated excitatory postsynaptic currents evoked by repetitive electrical stimulation (eEPSCs, 10 pulses at 33 Hz). The coefficient of variation (CV) of the 1st eEPSC amplitude and paired‐pulse ratio (PPR), which were sensitive to extracellular Ca²⁺ concentration, were not affected by phenylephrine. Phenylephrine suppressed miniature EPSC (mEPSC) amplitude without changing its frequency. In contrast, isoproterenol, a β‐adrenoceptor agonist, strongly increased the amplitude of the 1st eEPSC compared with that of the 2nd to 10th eEPSCs, which resulted in a decrease in PPR. Isoproterenol‐induced enhancement of eEPSC amplitude was accompanied by a decrease in CV. Isoproterenol increased the frequency of mEPSCs without significant effect on amplitude. Phenylephrine suppressed inward currents evoked by puff application of glutamate, AMPA, or NMDA, whereas isoproterenol application was not accompanied by significant changes in these inward currents. These findings suggest that phenylephrine decreases eEPSCs through postsynaptic AMPA or NMDA receptors, while the effects of isoproterenol are mediated by facilitation of glutamate release from presynaptic terminals without effect on postsynaptic glutamate receptors. These two different mechanisms of modulation of excitatory synaptic transmission may improve the “signal‐to‐noise ratio” in cerebral cortex. Synapse 63:269–281, 2009. © 2008 Wiley‐Liss, Inc.     

Effects of ATP and GTP on voltage-gated K+ currents in glandular and muscular sympathetic neurons

This study assesses the effects of ATP and GTP on the kinetic properties of voltage-gated K+ currents in anatomically identified postganglionic sympathetic neurons innervating the submandibular gland and the masseter muscle in rats. Three types of K+ currents were isolated: the I(Af) steady-state inactivating at more hyperpolarized potentials, I(As) steady-state inactivating at less hyperpolarized potentials than I(Af) and the I(K) current independent of membrane potential. The kinetic properties of these currents were tested in neurons with ATP (4 mM) and GTP (0.5 mM) or without ATP and GTP in the intracellular solution.In glandular and muscular neurons in the absence of ATP and GTP in the intracellular solution, the current density of I(Af) was significantly larger (142 pA/pF and 166 pA/pF, respectively) comparing to cells with ATP and GTP (96 pA/pF and 100 pA/pF, respectively). The I(As) was larger only in glandular neurons (52 pA/pF vs. 37 pA/pF).Conversely, I(K) current density was smaller in glandular and muscular neurons without ATP and GTP (17 pA/pF and 31 pA/pF, respectively) comparing to cells with ATP and GTP (57 pA/pF and 58 pA/pF, respectively). In glandular (15.5 nA/ms vs. 6.9 nA/ms) and muscular (10.9 nA/ms vs. 7.5 nA/ms) neurons, the I(Af) activated faster in the absence of ATP and GTP. Half inactivation voltage of I(Af) in glandular (-110.0 mV vs. -119.7 mV) and muscular (-108.4 vs. -117.3 mV) neurons was shifted towards depolarization in the absence of ATP and GTP. We suggest that the kinetic properties of K+ currents in glandular and muscular sympathetic neurons change markedly in the absence of ATP and GTP in the cytoplasm. Effectiveness of steady-state inactivated currents (I(Af) and I(AS)) increased, while effectiveness of steady-state noninactivated currents decreased in the absence of ATP and GTP. The effects were more pronounced in glandular than in muscular neurons.     

Pacemaker activity of locus coeruleus neurons: whole-cell recordings in brain slices show dependence on cAMP and protein kinase A

Noradrenergic neurons of the rat locus coeruleus (LC) are endogenous pacemakers that exhibit slow, tonic firing even in the complete absence of synaptic inputs. In the present study a time-dependent decline in LC spontaneous firing activity was found on intracellular dialysis during whole-cell recording with low-resistance patch electrodes; this decline was accentuated by a specific inhibitor of cAMP-dependent protein kinase (PKI5-24). Conversely, the inclusion of cAMP, 8-Br-cAMP, or the catalytic subunit of cAMP-dependent protein kinase (PKAcat) in the patch pipettes dose-dependently increased firing rate; intracellular PKI5-24 blocked both 8-Br-cAMP and PKAcat-induced firing in LC neurons. These results indicate that endogenous cAMP, via a phosphorylation-dependent route, drives tonic pacemaker activity in LC neurons.     

Effects of hemoglobin and its breakdown products on synaptic transmission in rat hippocampal CA1 neurons

During head injuries and hemorrhagic stroke, blood is released into the extravascular space. The pooled erythrocytes get lysed and hemoglobin is released into the intracranial cavities. Therefore, neurons may be exposed to hemoglobin and/or its breakdown products, hemin and iron, for long periods of time. In this study, the electrophysiological actions of these agents on synaptic transmission in rat hippocampal CA1 pyramidal neurons were studied using extracellular field- and whole cell patch-recordings. Previously our laboratory reported that commercially available hemoglobin produced a dose dependent suppression of synaptic transmission in hippocampal CA1 neurons. In the present study, however, we found that this depression was caused by impurities present in the hemoglobin samples. Commercially available hemoglobin and methemoglobin did not have a significant effect on synaptic transmission. Although, reduced-hemoglobin prepared using a method described by Martin et al. [J. Pharm. Exp. Ther. 232 (1985) 708], produced a significant depression of synaptic transients, these effects were due to contamination with bisulfite that was present due to the reducing procedure. Therefore, the technique of Martin et al. was inadequate in removing the reducing agents or their breakdown products. A number of studies in literature used commercial samples of hemoglobin or reduced hemoglobin prepared using the method of Martin et al. Our observations indicate that it would be important to determine if contaminants, rather than hemoglobin, are responsible for the observed effects in these studies. Unlike hemoglobin, its breakdown products, ferrous chloride and hemin, produced an irreversible and significant depression of field excitatory postsynaptic potentials. The relevance of these effects in neurological complications that follow head injuries and hemorrhagic stroke awaits further investigation.     

Effects of Ca antagonists and aminoglycoside antibiotics on Ca current in isolated outer hair cells of guinea pig cochlea

The effects of various Ca²⁺ antagonists and aminoglycoside antibiotics on the Ca²⁺ channel in isolated outer hair cells of the guinea pig were investigated using a whole-cell patch-clamp technique. The inhibitory action was in the order of La³⁺Cd²⁺Ni²⁺Co²⁺ for inorganic Ca²⁺ antagonists, and flunarizine = nicardipine > ω-Conotoxin > methaxyverapamil = diltiazem amiloride for orgaini ones. Aminoglycoside antibiotics also had antagonistic effects on the Ca²⁺ channel.