Kainate/glutamate-induced changes in intracellular calcium and pH in leech glial cells.

Kainate evokes a non-desensitizing membrane depolarization in neuropile glial cells of the leech Hirudo medicinalis. We measured membrane potential and intracellular pH, pH(i), using double-barrelled pH-sensitive microelectrodes, and intracellular calcium, Ca2+i, using the change in fluorescence ratio of intracellularly injected fura-2, in these glial cells in situ. 20-50 microM kainate produced a depolarization of 18-28 mV and a decrease of pH(i) by 0.27 +/- 0.07 pH units. Ca2+i increased by 306 +/- 128 nM upon kainate, which could be inhibited by the non-NMDA antagonist CNQX. Glutamate (0.1 mM) also produced a fall in pH(i) and a rise in Ca2+i, which were however, much smaller. Quisqualate and N-methyl-D-aspartate had only small or no effects on membrane potential, pH(i) or Ca2+i. It is concluded that leech neuropile glial cells have a kainate-type glutamate receptor, which mediate significant transients of intracellular H+ and Ca2+.     

Dendritic calcium transients in the leech giant glial cell in situ.

Glial cells have been shown to respond to neuronal activity with changes in the membrane potential and the intracellular Ca2+ concentration. In order to get closer to glial structures associated with neuronal synapses, we have now looked at Ca2+ signalling in the glial processes ("glial dendrites") in response to neurotransmitters and neuronal activity. Single giant glial cells in situ of isolated ganglia of the leech Hirudo medicinalis were filled iontophoretically with the Ca(2+)-sensitive dyes Oregon green 488 BAPTA-1 or Fluo-3. Relative Ca(2+)-dependent fluorescence changes in response to bath and focal application of the ionotropic glutamate receptor agonist kainate (50 microM) and of 5-hydroxytryptamine (5-HT, 100 microM) were recorded in glial dendrites, using confocal laser scanning microscopy. The amplitudes of the [Ca2+]i transients in the dendritic processes were 2-4 times larger than those recorded in the cell body. Electrical stimulation of a nerve root (20 Hz for 15 s) elicited [Ca2+]i transients in glial dendrites (n = 32) that were reduced by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; n = 14). The results demonstrate that neuronal activity can evoke [Ca2+]i transients not only in glial cell bodies but also in glial dendrites, where these transients display regional variation. This may reflect local release of neurotransmitters like glutamate and 5-hydroxytryptamine and/or regional differences in the density of glial receptors.     

Glutamate and kainate increase intracellular sodium activity in leech neuropile glial cells

Na+-selective, double-barrelled microelectrodes were used to measure intracellular Na+ activity (aiNa) and membrane potential (Em) in neuropile glial cells of isolated segmental ganglia in the leech Hirudo medicinalis. Bath application of glutamate (10(-3) M) resulted in membrane depolarizations of about 5 mV and a concomitant increase of aiNa by between 2 and 10 mM. Kainate (10(-4) M) elicited depolarizations of up to 40 mV amplitude followed by a prominent after hyperpolarization. During kainate, aiNa increased by 7 to 25 mM. In contrast to glutamate, an initial decrease of aiNa was detected during the action of kainate. N-methyl-D-aspartate (NMDA, 10(-5)-10(-3) M) had no effect of Em and aiNa. The results indicate that leech glial cells have a kainate-preferring non-NMDA glutamate receptor.     

Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in rat spinal dorsal horn

In a rat spinal slice preparation the participation of excitatory amino acid (EAA) receptors in the responses of deep dorsal horn neurons to repetitive stimulation of lumbar dorsal roots was investigated using 3 EAA receptor antagonists, kynurenic acid, D-(-)-2-amino-4-phosphonovaleric acid (D-APV) and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) and current-clamp and voltage-clamp techniques. We found that the slow excitatory synaptic response evoked by 10-20 Hz electrical stimulation of primary afferent fibers consisted of two depolarizing components: an initial component lasting 1-5 s and a late one of 1-3 min duration. The initial and late components of the slow excitatory synaptic response can also be distinguished on the basis of their voltage-dependence and sensitivity to Mg2+ ions, kynurenate, D-APV and CNQX. In the presence of Mg2+, the initial component of the slow excitatory synaptic response increased with membrane hyperpolarization, whereas the late component decreased in most of the cells examined. In a zero-Mg2+ medium, the initial component was potentiated, but the late component was reduced. In both transverse and longitudinal spinal cord slices perfused with 1.2 mM Mg(2+)-containing medium, bath application of kynurenic acid (0.1-0.5 mM), D-APV (0.05-0.1 mM) and CNQX (5-7 microM) caused a reversible reduction of the peak amplitude of the initial slow depolarizing component that was greater in transverse (kynurenic acid: by 92.6 +/- 5.0%; D-APV: by 69.1 +/- 7.8%; CNQX: by 76.6 +/- 9.8%) than in longitudinal slices (kynurenic acid: by 53.3 +/- 1.3%; D-APV: by 31.5 +/- 9.1%; CNQX: by 35.3 +/- 11.1%). In contrast, all 3 antagonists of EAA receptors produced no consistent change in the peak amplitude or half-duration of the late depolarizing component of the slow excitatory synaptic response. Our results obtained with EAA receptor antagonists, at resting membrane potentials, in the absence and presence of Mg2+ and synaptic inhibition, indicate that the synaptic activation of the NMDA- and non-NMDA-receptor systems of deep spinal dorsal horn neurons by repetitive stimulation of primary afferent fibers may be selectively involved in the mediation of the initial, but not the late depolarizing component of the slow excitatory synaptic response.     

Effects of a nitric oxide synthase inhibitor on non-cholinergic junction potentials in the circular muscle of the guinea pig ileum.

Intracellular microelectrodes were used to record junction potentials from the circular muscle cells of the guinea pig ileum in vitro at 37 degrees C in a modified Krebs solution containing nifedipine (1-2 microM) and hyoscine (1 microM). Transmural nerve stimulation, using volleys of three pulses at 50 Hz, produced a complex response consisting of an inhibitory junction potential (IJP) followed by a prolonged depolarization. Following the addition of the nitric oxide synthase inhibitor NG-nitro-L-arginine (NOLA, 100 microM) the amplitude of the IJP (recorded 10 mm aboral to the stimulating electrodes) was increased by approx. 10% (n = 4). The further addition of apamin (250 nM) abolished the IJP revealing a non-cholinergic excitatory junction potential (EJP). In other experiments (n = 8), preparations were treated with apamin then subjected to substance P desensitization (500 nM, > 20 min). Transmural nerve stimulation now produced a triphasic response (recorded 1 mm aboral to the stimulating electrodes) consisting of: (a) an initial hyperpolarization (approx. 5 mV) lasting about 1 s; followed by (b) a depolarization reaching a peak (approx. 7 mV less negative than the RMP) approx. 2 s after nerve stimulation; and finally (c) a small (approx. 3 mV) hyperpolarization. The addition of NOLA reduced all three phases by 80-90% (n = 8). The subsequent addition of L-arginine (5 mM) partially reversed these effects (n = 3). Conditioning hyperpolarization up to 20 mV increased the amplitude of the NOLA-sensitive IJP and EJP. Further conditioning hyperpolarization reduced the amplitude of the IJP and enhanced the amplitude of the EJP. Large conditioning hyperpolarizations (> 60 mV) reduced the amplitude of both the IJP and EJP. An estimation of the membrane conductance changes occurring during the initial hyperpolarization and depolarization suggest that it was either unchanged or increased. During large conditioning hyperpolarizations in the absence of nerve stimulation, the membrane potential was unstable and began to show spontaneous oscillations (up to 30 mV, every 4-5 s) resembling slow waves. These experiments indicate that NO, or a related compound, appears to mediate the nerve induced apamin-resistant IJP and substance P- and hyoscine-resistant EJP in the circular muscle of the guinea pig ileum.     

Peptide-mediated glial responses to Leydig neuron activity in the leech central nervous system

Neuronal activity may lead to a variety of responses in neighbouring glial cells; in general, an ensemble of neurons needs to be active to evoke a K+- and/or neurotransmitter-induced glial membrane potential change. We have now detected a signal transfer from a single neuromodulatory Leydig neuron to the giant neuropil glial cells in the central nervous system of the leech Hirudo medicinalis. Activation of a Leydig neuron, two of which are located in each segmental ganglion, elicits a hyperpolarization in the giant neuropil glial cells. This hyperpolarization could be mimicked by bath application of the peptide myomodulin A (1 nM-1.0 microM). Myomodulin-like immunoreactivity has recently been found to be present in a set of leech neurons, including Leydig neurons (Keating & Sahley 1996, J. Neurobiol., 30, 374-384). The glial responses to Leydig neuron stimulation persisted in a high-divalent cation saline, when polysynaptic pathways are suppressed, indicating that the effects on the glial cell were direct. The glial responses to myomodulin A application persisted in high-Mg2+/low-Ca2+ saline, when chemical synaptic transmission is suppressed, indicating a direct effect of myomodulin A on the glial membrane. The glial hyperpolarization evoked by myomodulin A was dose dependent (EC50 = 50 nM) and accompanied by a membrane conductance increase of approximately 25%. Ion substitution experiments indicated that myomodulin A triggered a Ca2+-independent K+ conductance. Thus, our results suggest, for the first time, direct signal transmission from an identified modulatory neuron to an identified glial cell using a myomodulin-like peptide.     

Mechanism of NMDA receptor contribution to axon-to-glia signaling in the crayfish medial giant nerve fiber

Electrical stimulation of crayfish giant axons at high frequency activates group II metabotropic and NMDA glutamate receptors on adjacent glial cells via release of N-acetylaspartylglutamate and glutamate formed upon its hydrolysis. This produces a transient depolarization followed by a prolonged hyperpolarization of glial cells that involves nicotinic acetylcholine receptor activation. The hyperpolarization is nearly completely blocked by antagonists of metabotropic glutamate receptors but only slightly reduced by inhibition of NMDA receptors. We report that the NMDA-induced hyperpolarization of glial cells is reduced by decreased calcium in the solution bathing the giant nerve fiber, while removal of sodium ions or block of voltage-dependent calcium channels completely prevents the glial response to NMDA. Inhibition of nicotinic acetylcholine receptors or removal of extracellular Cl(-) converts the glial response from a hyperpolarization to a depolarization that is sensitive to NMDA receptor antagonist. We propose that NMDA receptor activation by glutamate, formed from extracellular N-acetylaspartylglutamate during nerve stimulation, contributes to glial hyperpolarization by increasing intracellular Ca(2+) via opening of voltage-sensitive Ca(2+) channels. Based on our previous work, we propose further that the added Ca(2+) supplements that produced by N-acetylaspartylglutamate and glutamate acting on group II metabotropic glutamate receptors to cause an increased release of acetylcholine and a larger hyperpolarization.     

Glucose regulation of synaptic transmission in the dorsolateral septal nucleus of the rat.

Intracellular recordings were made from neurons in the dorsolateral septal nucleus (DLSN) of rat brain slices. Lowering the concentration of extracellular glucose resulted in a concentration-dependent membrane hyperpolarization associated with a cessation of spontaneous firing. The amplitude of the excitatory postsynaptic potential (EPSP), inhibitory postsynaptic potential (IPSP), and late hyperpolarizing potential (LHP) evoked by a single stimulus applied to the fimbrial/fornix pathway was decreased when the concentration of glucose was reduced to 0-2 mM. Substitution of glucose with 2-deoxy-D-glucose (11 mM), an antimetabolite of glucose substrate, mimicked the effects of glucose depletion. Mannoheptulose (10-20 mM), a potent hexokinase blocker, and dinitrophenol (50 microM), a potent inhibitor of oxidative phosphorylation, produced both the hyperpolarization and inhibition of postsynaptic potentials, even in the presence of 11 mM glucose. The sulphonylureas, glibenclamide (10 microM) and tolbutamide (1 mM), did not antagonize the hyperpolarization and the inhibition of the postsynaptic potentials produced by glucose depletion. The amplitude of membrane depolarizations produced by pressure application of glutamate (10 mM) and the membrane hyperpolarizations produced by pressure application of either muscimol (1 mM) or baclofen (1 mM) were almost unchanged, even when glucose was reduced to 1-2 mM. These results indicate that intracellular glucose metabolism regulates the function of septal neurons, not only by changing the resting membrane potential, but also by presynaptically affecting neurotransmission between the hippocampal formation and the lateral septum.     

Intracellular acidification of the leech giant glial cell evoked by glutamate and aspartate

Glutamate is an excitatory receptor agonist in both neurones and glial cells, and, in addition, glutamate is also a substrate for glutamate transporter in glial cells. We have measured intracellular and extracellular pH changes induced by bath application of glutamate, its receptor agonist kainate, and its transporter agonist aspartate, in the giant neuropile glial cell in the central nervous system of the leech Hirudo medicinalis, using double-barrelled pH-sensitive microelectrodes. The giant glial cells responded to glutamate and aspartate (100–500 μM), and kainate (5–20 μM) with a membrane depolarization or an inward current, and with a distinct intracellular acidification. Glutamate and aspartate (both 500 μM) evoked a decrease in intracellular pH (pH_i) by 0.187 ± 0.081 (n = 88) and 0.198 ± 0.067 (n = 86) pH units, respectively. With a resting pH_i of 7.1 or 80 nM H⁺, these acidifications correspond to a mean increase of the intracellular H⁺ activity by 42 nM and 45 nM. Kainate caused a decrease of pH_i by 0.1 − 0.35 pH units (n = 15). The glutamate/aspartate-induced decrease in pH_i was not significantly affected by the glutamate receptor blockers kynurenic acid (1 mM) and 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX, 50–100 μM), which greatly reduced the kainate-induced change in pH_i. Extracellular alkalinizations produced by glutamate and aspartate were not affected by CNQX. Reduction of the external Na⁺ concentration gradually decreased the intracellular pH change induced by glutamate/aspartate, indicating half maximal activation of the acidifying process at 5–10 mM external Na⁺ concentration. When all external Na⁺ was replaced by NMDG⁺, the pH_iresponses were completely suppressed (glutamate) or reduced to 10% (aspartate). When Na⁺ was replaced by Li⁺, the glutamate- and aspartate-evoked pH_i responses were reduced to 18% and 14%, respectively. Removal of external Ca²⁺ reduced the glutamate- and aspartate-induced pH_i responses to 93 and 72%, respectively. The glutamate/aspartate-induced intracellular acidifications were not affected by the putative glutamate uptake inhibitor amino-adipidic acid (1 mM). DL-aspartate-β-hydroxamate (1 mM), and dihydrokainate (2 mM), which caused some pH_i decrease on its own, reduced the glutamate/aspartate-induced pH_i responses by 40 and 69%, respectively. The putative uptake inhibitor DL-threo-β-hydroxyaspartate (THA, 1 mM) induced a prominent intracellular acidification (0.36 ± 0.05 pH units, n = 9), and the pH_i change evoked by glutamate or aspartate in the presence of THA was reduced to less than 10%. The results indicate that glutamate, aspartate, and kainate produce substantial intracellular acidifications, which are mediated by at least two independent mechanisms: 1) via activation of non-NMDA glutamate receptors and 2) via uptake of the excitatory amino acids into the leech glial cell. © 1997 Wiley-Liss Inc.     

Cerebellar macroneurons in microexplant cell culture. Postsynaptic amino acid pharmacology

Cerebellar neurons derived from 17- to 19-day-old fetal rats have been grown in a monolayer in microexplant cell culture, and intracellular recording coupled with iontophoresis of amino acid neurotransmitters has been employed to characterize their amino acid chemosensitivity. Although these cultures contain at least 3 different neuronal cell types, intracellular recordings were obtained from large neurons (diameter greater than 15 microns) with 1-5 dendritic shafts and fine dendritic arborizations and which could, on morphological grounds, be identified as Purkinje cells. All neurons with resting membrane potentials greater than 25 mV and with action potentials evoked by intracellular stimulation, responded to iontophoretically applied glutamate and GABA. There was essentially no chemosensitivity to glycine, beta-alanine or taurine. Aspartate application evoked only small responses at high iontophoretic currents. GABA reversibly increased membrane conductance and produced hyperpolarization at resting membrane potential with reversal potentials between -50 and -40 mV (5-10 mV more negative than resting membrane potential). Glutamate reversibly increased membrane conductance and produced depolarizing responses with extrapolated reversal potentials between 0 and -10 mV. Aspartate augmented glutamate responses at low iontophoretic currents which did not directly alter membrane potential or conductance. Thus Purkinje cells grown in the absence of parallel fiber and climbing fiber input develop autonomous neuropharmacologic specificity similar to that of Purkinje cells in vivo.     

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons.

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity-dependent slow hyperpolarization in cat sensorimotor cortex in vitro

The synaptic regulatory mechanism of resting membrane potential of layer III and V pyramidal neurons was analyzed intracellularly in the slice preparation of cat sensorimotor cortex. During the tetanic stimulation of white matter, subthreshold membrane depolarization was induced, and after that, a slowly developing hyperpolarization was induced in the normal solution. When the membrane potential showed a slow change, spike duration and input resistance did not change and evoked single synaptic response did not reveal the enhancement of slow IPSPs. However, afterhyperpolarization following action potential was enhanced. The slow hyperpolarization and the enhancement of afterhyperpolarization were not observed in the cells treated with an NMDA receptor antagonist or a calcium channel blocker Ni(2+) (50-100 microM), or the cells hyperpolarized more than -80 mV before the tetanic stimulation.     

Pharmacological modification of the light-induced responses of Müller (glial) cells in the amphibian retina

The light-evoked responses of Müller (glial) cells were monitored by intracellular recording in the isolated, superfused retina of Xenopus laevis. Müller cells had dark resting potentials of -88.5 +/- 6.9 mV and small 1-2 mV light responses of variable waveform in normal Ringer's solution. Exposure to picrotoxin (0.5-1.0 mM) greatly enhanced the light response which then consisted of depolarizing transients (Vmax 5-15 mV) at stimulus onset and offset. GABA (5-10 mM) antagonized the picrotoxin effect and suppressed the light response, whereas 2-amino 4-phosphonobutyrate (0.10-0.15 mM) blocked selectively the 'on' transient. None of these agents appreciably modified the glial cells resting potential level. On the other hand, veratrine (6-9 micrograms/ml) depolarized the Müller cell by 4-13 mV and slowed and greatly reduced the light response. These effects were antagonized by tetrodotoxin (1-4 microM) which itself reduced the light response by 30-50% without altering its shape. On the basis of these findings, we suggest that alterations in the activity of the inner retinal neurons, i.e. amacrine and ganglion cells, are primarily responsible for the drug-induced changes in the membrane potential and light-evoked responses of the Müller cell.     

Glutamine currents in hippocampal neurons are attributable to contaminating glutamate

Physiological responses of cultured rat hippocampal neurons to exogenously applied glutamine were studied using tight-seal, whole-cell voltage clamp techniques in nominal extracellular magnesium. Four mM glutamine induced peak inward currents which were 4.6 +/- 1.7% (+/- 1 S.D.) of current responses to 100 microM glutamate. Ten microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 1 mM magnesium, 20 microM MK-801, and 200 microM D-aminophosphonovalerate (APV) blocked these responses in a pattern similar to N-methyl-D-aspartate antagonism. Current-voltage plots produced by 4 mM glutamine and 10 microM glutamate were identical. Even in the absence of extracellular magnesium, 4 mM glutamine was not toxic to our cultured neurons. The data suggest that physiological responses to glutamine are attributable to low concentrations of glutamate in commercial preparations and that glutamine alone has insignificant physiological and toxic effects.     

Modulation by hyperpolarization-activated cationic currents of voltage responses in human rods

We used the whole-cell patch-clamp recording technique on surgically excised human retina to examine whether human rod photoreceptors express hyperpolarization-activated cationic currents (I(h)) and to analyze the effects of I(h) on rod's voltage responses. Hyperpolarizing voltage steps from a holding potential of -60 mV evoked a slow inward-rectifying current in both rods in retinal slices and isolated rods. The slow inward-rectifying currents induced by hyperpolarization were markedly reduced by 3 mM Cs(+) (a blocker of I(h)) in the bath, but not by 3 mM Ba(2+) (an anomalous rectifier K(+) current blocker) or 1 mM SITS (a Cl(-) current blocker). A concentration-response curve for block by Cs(+) of the inward currents could be fitted by the Hill equation with a half-blocking concentration (IC(50)) of 41 microM and a Hill coefficient of 0.91. The time course of the inward current activation was well described at all recorded voltages by the sum of two exponentials. Under current-clamp conditions, injection of steps of current, either hyperpolarizing or depolarizing, elicited an initial rapid voltage change that was followed by a gradual decay in the voltage response. The decay in the voltage responses was eliminated by bath application of 3 mM Cs(+). The voltage dependence, pharmacology, and kinetics of the slow inward-rectifying currents described above suggest that human rods express I(h). We suggest that I(h) becomes activated in the course of large hyperpolarizations generated by bright-light illumination and may modify the waveform of the photovoltage in human rods.     

Quantitative physiological characterization of a quinoxalinedione non-NMDA receptor antagonist

The effects of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, or FG 9065) on excitatory amino acid responses in cultured neurons from rat hippocampus were studied using tight-seal whole-cell recording techniques. CNQX reduced the magnitude of peak inward currents produced by exogenously applied kainate, quisqualate, and N-methyl-D-aspartate (NMDA) with Ki's of 2.5, 3.5, and 96 microM, respectively. The antagonism was competitive against kainate and quisqualate, but noncompetitive against NMDA. Glycine markedly reduced CNQX antagonism of NMDA responses. The same recording technique using pairs of monosynaptically connected neurons demonstrated reversible diminution of excitatory postsynaptic potentials in 7 of 7 pairs, using CNQX at concentrations as low as 10 microM. CNQX applied alone did not evoke inward or outward currents at membrane potentials near the resting membrane potential and did not affect the current-voltage relationship at membrane potentials between -90 and -30 mV. These observations represent the first quantitative characterization of glutamate receptor antagonism by CNQX with respect to physiological rather than biochemical parameters and demonstrate that CNQX is far more potent and more selective than currently available non-NMDA antagonists. The results suggest that CNQX will be a useful pharmacologic tool for the study of synaptic transmission in a variety of systems in which glutamate or related excitatory amino acids are involved.     

Electrical properties and synaptic potentials of rabbit pancreatic neurons

Pancreatic ganglia receive innervation from a wide variety of extrinsic nerves and supply the predominant innervation to pancreatic acini, islets, and ducts. This study used intracellular recordings to investigate the electrical properties and synaptic potentials of rabbit pancreatic neurons. Neurons had a mean resting membrane potential of -54+/-0.4 mV and generated action potentials with a mean overshoot of 10+/-0.4 mV and a mean after-spike hyperpolarization (ASH) of 11+/-0.5 mV with duration of 210+/-19 ms. Action potentials exhibited a high threshold (-15+/-1 mV) for intracellular stimulation and a phasic firing pattern was observed in response to prolonged depolarizing currents. Stimulation of attached nerve bundles evoked multiple fast excitatory postsynaptic potentials (fEPSPs) which were abolished by hexamethonium in 75% of neurons, while a non-cholinergic fEPSP was observed in 25% of the neurons. Repetitive stimulation (3-30 Hz) evoked muscarinic slow EPSPs with a mean amplitude of 8+/-2 mV and duration of 5+/-1 s in a small subset (21%) of neurons. Exogenous muscarine evoked a mean slow depolarization of 10+/-1 mV amplitude in 22% of neurons tested. Following repetitive nerve stimulation non-cholinergic late, slow EPSPs with a mean amplitude of 4.3+/-0.4 mV were recorded in 32% of neurons. Nicotinic transmission was subject to inhibition mediated by presynaptic muscarinic receptors at low (0.5 Hz) stimulus frequencies in 80% of neurons. At higher frequencies (> or =1 Hz), either facilitation or depression of nicotinic transmission was observed depending on the ganglion studied. A population (9%) of neurons exhibited spontaneous, low-amplitude pacemaker-like potentials. Spontaneous fEPSPs and action potentials were also observed and these occasionally occurred in rhythmically timed bursts. Thus, distinct subpopulations of pancreatic neurons could be identified on the basis of both their intrinsic electrical properties and the receptors mediating and/or modulating synaptic transmission. These neurons function as critical sites of integration for synaptic input from extrinsic pancreatic nerves and thereby determine the postganglionic firing patterns presented to the pancreatic exocrine and endocrine secretory cells.     

Pre- and postsynaptic actions of serotonin on rat suprachiasmatic nucleus neurons

Serotoninergic transmission is implicated in the photic and non-photic regulation of circadian rhythms. 5-HT (1-100 microM), carboxamidotryptamine (5-CT 0.1-10 microM) and (+)-8-hydroxy-dipropylaminotetraline (8-OH-DPAT, 1-30 microM) dose-dependently activated an outward current (5-100 pA) in 30% of neurons voltage-clamped at -60 mV in the suprachiasmatic nucleus (SCN) in vitro slice. EC(50) values were 7.0 microM for 5-HT and 0.2 microM for 5-CT. Serotonin-induced outward current was associated with an increase in input conductance, and the current was blocked by Ba(2+) (1 mM). The amplitude of the current was enhanced by depolarization, reduced by hyperpolarization, and reversed its polarity during a hyperpolarization beyond the potassium equilibrium potential. Mean amplitudes of the 5-HT outward current changed with time of the subjective circadian day. The value near CT2 (23.8 pA) was about 4 times greater than that around CT14 (6.7 pA). Cells that responded with an outward current showed four types of morphology: monopolar, simple bipolar, curly bipolar and radial shaped; they were localized in all parts of the SCN. The EPSC evoked by retino-hypothalamic-tract (RHT) stimulation was inhibited 26% but the inward current induced by exogenously applied glutamate or NMDA was not affected by serotonin agonists. Focal stimulation-induced and spontaneous IPSC but not the exogenous GABA-induced outward current were inhibited by 5-HT agonists in a subpopulation of cells. In conclusion, 5-HT regulates SCN neurons by both pre- and post-synaptic inhibitory mechanisms; the latter may play a key role in modulating SCN circadian rhythm by activation of 5-HT receptors and opening of a potassium channel.     

Altered electrical activity in colonic smooth muscle cells from dystrophic (mdx) mice

Because the colon from dystrophic (mdx) mice shows an altered motor pattern, probably due to neural disorders, our aim was to examine the electrophysiological properties of muscle cells and the functionality of nitrergic transmission in circular muscle from normal and mdx colon. Normal colonic cells (resting membrane potential [RMP] about -50 mV) showed spontaneous hyperpolarizations (inhibitory junction potentials; IJPs) and cyclic slow depolarizations were sometimes recorded. Mdx colon had a depolarized RMP (about -36 mV) and spontaneous IJPs, but the cyclic activity was never observed. In the normal colon, Nomega-nitro-L-arginine methyl ester (L-NAME) induced depolarization and abolished the cyclic activity. In the mdx colon, L-NAME caused a slight depolarization. Both preparations displayed the same value of RMP in the presence of L-NAME. In normals, neural stimulation induced nonadrenergic, noncholinergic IJPs composed of fast hyperpolarizations followed by a nitrergic slow hyperpolarization, selectively abolished by L-NAME. In the mdx colon the evoked IJPs were composed only of the initial fast hyperpolarization, the nitrergic component being absent. The hyperpolarization to sodium nitroprusside was not significantly different in both preparations. We conclude that the colon from animals lacking in dystrophin displays different electrophysiological features because of an impairment of nitric oxide function.     

Cholinergic excitation of mammalian hippocampal pyramidal cells

Responses of CA1 pyramidal neurons to ACh were recorded with intracellular microelectrodes utilizing the in vitro guinea pig hippocampal slice preparation. ACh was delivered by drop or iontophoretic application to stratum oriens or stratum radiatum. Threshold dose for drop application was 1 mM. An initial hyperpolarization of 3.1 +/- 1.8 (S.D.) mV associated with a decrease in membrane input resistance (RN) of 21 +/- 9% (S.D.) occurred in about half the cells. This result is consistent with a presynaptic action of ACh mediated through excitation of inhibitory interneurons. This interpretation was supported by recordings of cholinergic excitatory responses from presumed interneurons, and repetitive spontaneous IPSPs from pyramidal neurons during the hyperpolarization. ACh evoked a slow depolarization (14.3 +/- 10.8 (S.D.) mV) accompanied by a peak increase in apparent input resistance (Ra) of about 60% in the majority of cells. Large increases in spike frequency were associated with these events but action potential shape was unchanged. Plots of Ra versus membrane potential following ACh application revealed that Ra increases were proportionately higher at depolarized membrane potential levels (less than or equal to -70 mV) in some neurons. In these cells Ra was increased significantly at -60 mV (28%), but only 6% at -75 mV. These results are consistent with the conclusion that ACh reduces a voltage-dependent gK, distinct from delayed rectification. ACh also induced a non-voltage-dependent increase in Ra in some cells. ACh-evoked changes in Ra were long-lasting and gave rise to alterations in firing mode, with development of burst generation. ACh also transiently blocked after hyperpolarizations which followed spike trains in pyramidal neurons and presumed interneurons, an action which may be related to effects on a Ca2+-activated gK.