Evidence that glutamate mediates axon-to-Schwann cell signaling in the squid

High-frequency stimulation (100 Hz) of isolated giant axons of the small squid Alloteuthis subulata and the large squid Loligo forbesi caused the periaxonal Schwann cell resting potential (Em = -40 mV) to hyperpolarize up to 11 mV in direct proportion to train duration and action potential amplitude. In both species, the Schwann cell also hyperpolarized up to 17 mV with the application of L-glutamate (10(-9) to 10(-6) M), in a dose-dependent manner. By contrast, in the presence of 10(-8) M d-tubocurarine (d-TC) to block the cholinergic component of the Schwann cell response, Schwann cells depolarized 8-9 mV during electrical stimulation of the axon or application of L-glutamate. In the presence of 10(-5) M 2-amino-4-phosphonobutyrate (2-APB), the hyperpolarization to glutamate and to axon stimulation was blocked, whereas the cholinergic (carbachol-induced) hyperpolarization was unaffected. In experiments with Alloteuthis, L-aspartate (10(-7) M) also caused a Schwann cell hyperpolarization, but this was not blocked by 2-APB. In tests with glutamate receptor agonists and antagonists, quisqualate (10(-5) M) produced a hyperpolarization blocked by 10(-4) M L-glutamic acid diethylester (GDEE), which also blocked the response to axonal stimulation. Kainic acid (10(-4) M) also caused a hyperpolarization, but n-methyl-D-aspartate (NMDA; 10(-4) M), ibotenate (10(-5) M), alpha-amino-3-hydroxy-5-methyl-isoxazole proprionate (AMPA; (10(-4) M), and isethionate (10(-5) M) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Quantification of angiotensin iontophoresis

A method of value for studying the effects of angiotensin II (AII) and angiotensin III (AIII) on the brain is microiontophoresis combined with single unit recording. The purpose of this study was to quantitate the release of angiotensins under various experimental conditions thus providing a firm basis for the iontophoretic application of angiotensins. Quantification of release was accomplished by adding the appropriate [3H]angiotensin to 1 X 10(-3) M solutions of AII and AIII and then measuring the counts released from the tip of the microiontophoretic pipette in vitro into a small volume of Ringer solution. Although both AII and AIII were released by diffusion from micropipettes, this release could all but be eliminated with a retaining current of 20 nA. The release of AII and AIII was linear with respect to the amount of ejecting current applied up to 60 nA, the highest current examined. Angiotensin II at pH 4.5 and AIII at pH 3.5 were released at similar rates of 41.5 and 45.1 fmol/min/nA respectively. Raising the pH of the AII solution to 4.5 reduced the rate of release to 17.9 fmol/min/nA. Transport numbers were determined as follows: AII pH 3.5-0.115; AII pH 4.5-0.035, and AIII pH 4.5-0.145. It can be concluded that angiotensins are readily released by microiontophoresis, the response is linear with respect to application current, and that with the use of the appropriate pH the rate of release of AII and AIII are comparable.     

Bradykinin induces hyperpolarizations in rat glioma cells and in neuroblastoma × glioma hybrid cells

The effect of the nonapeptide bradykinin on the membrane potential of permanent cell lines from neural origin was studied. A hyperpolarizing response of 10–30 s duration was produced when bradykinin was iontophoretically applied onto polyploid rat glioma cells (clone C6-4-2). Starting from the resting membra potential the peak value of the hyperpolarizing response was reached within 0.5–1.5 s. Then the potential returned more slowly to the origin value. The hyperpolarization was associated with an approximately 50% decrease in membrane resistance. Neither Na⁺ nor Cl⁻ seemed to be important for the hyperpolarizing response, since bradykinin elicited similar hyperpolarizations in cells exposed to media in which Na⁺ or Cl⁻ were replaced by choline or isethionate, respectively. Ca²⁺ fluxes are unlikely to be involved, since the addition of D600 did not affect the hyperpolarizations induced by bradykinin. However, a 10-fold increase in the concentration of K⁺ in the medium reduced the amplitude of the hyperpolarization by 40 mV. Thus, the hyperpolarization induced by bradykinin is associated with a decrease in membrane resistance which is likely to be caused by an increased K⁺-conductance. The glioma cells showed a desensitization upon repeated application of bradykinin. However, the sensitivity of the cells to bradykinin was restored after 3–8 min of incubation in the absence of bradykinin. Since an antagonist of bradykinin is not known, the specificity of the action of bradykinin is difficult to assess. Nevertheless, the hyperpolarizing response to bradykinin appears to be specific insofar as other peptides, i.e. lutoliberin, thyroliberin, neurotensin, substance P and apamin, exerted no effect on the membrane potential of the glioma cells. Bradikynin-elicited hyperpolarizations with characteristics similar to those described above could also be demonstrated in neuroblastoma × glioma hybrid cells, but not in multinucleated fibroblast cells.     

Slow inhibitory postsynaptic potentials and hyperpolarization evoked by noradrenaline in the neurones of mammalian sympathetic ganglion

Slow IPSPs evoked in the neurones of rabbit isolated superior cervical ganglion by repetitive orthodromic stimulation, and a response evoked in the neurones of this ganglion by perfusion of noradrenaline, were studied using intracellular microelectrodes. Slow IPSPs were observed in 36% of neurones studied, and when investigated after treatment with D-tubocurarine and neostigmine, had a mean amplitude of 4.4 +/- 0.2 mV (mean +/- S.E.) and duration of 5 sec to 1.5 min. Two types of slow IPSPs occurring in different neurones were found. The slow IPSP of the first type was followed by a decrease in cell input resistance, was increased by depolarization and decreased by hyperpolarization of the membrane, with the reversal potential, if estimated by extrapolation method, equal to -77.8 +/- 3.3 mV. The slow IPSP of the second type was not followed by any change in cell input resistance, was increased by hyperpolarization and decreased by depolarization. The slow IPSP of the second type was reversibly blocked by phentolamine (1.4 X 10(-4) M). Noradrenaline (1 X 10(-4) M) evoked hyperpolarization or hyperpolarization followed by depolarization in 55% of the neurones studied. Hyperpolarization evoked by noradrenaline had a mean amplitude to 5.0 +/- 0.2 mV, was not followed by any change in cell input resistance, was reversibly blocked by phentolamine (1.4 X 10(-4) M), and was decreased by both depolarization and hyperpolarization of the cell membrane. It has been concluded that there are two groups of neurones in superior cervical ganglia, different with respect to the ionic mechanisms underlying the slow IPSP. In the first group of neurones the slow IPSP is probably due to an increase in potassium permeability of the membrane. The ionic mechanisms underlying the slow IPSP in the second group of neurones of noradrenaline-induced hyperpolarization remain unclear.     

Adrenergic Receptor Activation Hyperpolarizes the Caudal Neurosecretory Cells of the Flounder, Platichthys flesus

The physiological factors that govern activity of the caudal neurosecretory system in teleost fish are poorly understood. Immunocytochemical evidence indicates that the neurosecretory Dahlgren cells are innervated by descending monoaminergic fibres. Using intracellular recording techniques in an isolated preparation of the posterior spinal cord of the flounder (Platichthys flesus) we have demonstrated that superfusion of adrenaline or noradrenaline (10^?7–10^?3 M) causes hyperpolarization of Dahlgren cells (up to ?30 mV). This hyperpolarization is likely to reflect an inhibitory effect of noradrenergic nerves on the neurosecretory system in vivo, reducing the rate of hormone release. Fluctuations in the input resistance and membrane time constant suggest involvement of a multiplicity of cellular mechanisms, including the opening and closing of populations of ion-selective channels. Superfusion with dopamine (10^?7–10^?3 M) had no effect. Superfusion with the β-adrenoreceptor agonist, isoprenaline, caused hyperpolarization but to a markedly lesser extent than the maximum effect of adrenaline or noradrenaline, suggesting that their effects are mediated, only in part, by a β-adrenoreceptor subtype. Superfusion of the preparation with a membrane permeable, non-hydrolysable cyclic AMP analogue (8-[4-chlorophenylthio]-cAMP) resulted in a slight hyperpolarization which was accompanied by a small, but significant, increase in input resistance. These data are consistent with at least part of the β-adrenoreceptor mediated effect involving closure of cAMP-sensitive ion channels. Superfusion with the β-adrenoreceptor agonist, phenylephrine, had no effect on any electrophysiological parameter studied. However, the α₂-adrenoreceptor agonist, clonidine, caused hyperpolarization which again failed to reach the maximum level produced by adrenaline or noradrenaline. Together, these data suggest that the adrenergic inhibition of Dahlgren cell activity is mediated by both α₂-and β-adrenoreceptor subtypes.     

Electrical responses of the muscularis externa to distension of the isolated guinea-pig distal colon

Abstract Enteric reflex pathways were studied in isolated segments of guinea-pig distal colon by recording the electrical responses to distension from the muscularis externa with suction electrodes. The end of the electrode wire were in the circular muscle and thus the recordings discussed below are deduced to be primarily from this layer. Moreover, intracellular microelectrodes in circular muscle cells and suction electrodes recorded similar events. Spontaneous activity consisted of myogenic slow waves at about 25 min ^-1 and transient biphasic potentials at about 6 min^-1 and 3-sec duration which were dependent on a stimulus from the enteric nervous system as they were blocked by tetrodotoxin (0.5 μM), d-tubocurarine (30 μM) and hexa-methonium (100 μM). Atropine (0.8 μM) blocked the depolarizing part of the biphasic potentials and unmasked transient spontaneous inhibitory junction potentials (IJPs) (～2-sec duration) which appeared to be responsible for the hyperpolarizing part of the biphasic potential. Three different responses were observed at sites oral to distension of the colon: a transient depolarizing response that was cholinergic (blocked by atropine (0.8 μM); ascending cholinergic excitation) and, after atropine, a transient IJP (ascending inhibition) which was followed by a transient non-cholinergic depolarizaton (ascending non-cholinergic excitation) that was sometimes followed by several cycles of slow wave activity. The oral responses to anal distension were also blocked by the nicotinic antagonists and were similar to the neurogenic spontaneous events, which also appeared to originate from activity in ascending nervous pathways. Four different responses were observed following distension of the oral end of the segment: an IJP followed by a prolonged phase of hyperpolarization that lasted for the duration of the distension (descending inhibition); a burst of depolarizing potentials (for up to 30 sec) that followed the termination of distensions up to 25 sec and was blocked by atropine (0.8 μM) (delayed cholinergic excitation), and a transient non-cholinergic response that immediately followed the termination of distension (non-cholinergic ‘off’ response). Apamin (0.5 μM) reduced the amplitude of the spontaneous IJPs and evoked IJPs. After apamin, distension evoked a small transient hyperpolarization at oral sites, which was similar to spontaneous events, and the prolonged hyperpolarization at anal sites. A second distension given within 20 sec of the first evoked an IJP of reduced amplitude at oral sites in every preparation. In contrast, the amplitudes of the oral Cholinergic excitation and descending inhibition were relatively unaffected by reducing the interval between distensions. Thus distension stimulates excitatory and inhibitory motor neurons supplying the circular muscle both oral and anal to the stimulus. The polarity of the reflex relies in part on the differences in timing and duration of responses as well as the transmission characteristics of the nervous pathways.     

The influence of dopamine of epileptiform burst activity in hippocampal pyramidal neurons

Dopamine (DA) application to guinea pig hippocampal CA1 neurons in vitro causes hyperpolarization of the resting potential, increase in conductance, and increase in amplitude and duration of the afterhyperpolarization (AHP). Since these changes could influence repetitive firing, we performed experiments to determine whether DA-induced effects would suppress epileptogenesis in the hippocampus. Epileptiform bursts were induced by adding penicillin (3.4 mM) to the perfusion medium. Focal application of DA (40-160 microns) onto CA1 cells (n = 15) produced a hyperpolarization averaging 4.5 mV beginning in 5-20 s and lasting up to 3 min. DA also caused an increase in the amplitude and duration of slow AHPs. The frequency of spontaneous epileptiform events however was not affected. CA3 neurons (n = 6) responded to DA application with an initial 1-3 mV depolarization beginning within 5-30 s and lasting 1-2 min. In 3 cases a small hyperpolarization lasting several minutes subsequently developed. AHP duration increased 70% and amplitude increased 35% (n = 4). Along with these membrane changes the frequency of epileptiform bursting in CA3 cells slowed for 1-3 min. We added DA (10-80 microM) to the perfusion medium to see whether a significant decrease in epileptiform burst frequency might occur in the follower CA1 region if greater numbers of pacemaker CA2 and CA3 cells were exposed to DA. Spontaneous CA1 bursting was reversibly slowed, the interburst interval became variable and increased from a mean of 4 to a mean of 5-7 s (n = 6). These results suggest that DA may play a role in decreasing the incidence or frequency of epileptogenic discharges in vivo.     

Multiple actions of acetylcholine at a muscarinic receptor studied in the rat hippocampal slice

Responses of hippocampal pyramidal cells to topical application of acetylcholine (ACh) were measured in the in vitro hippocampal slice preparation.ACh but not cyclic GMP produced a short-latency hyperpolarization associated with a decrease in input resistance. This was followed by a long-latency but long duration depolarization associated in some cells with an increase in input resistance. This change in resistance followed the depolarization and outlasted it by 5–20min, until complete recovery. During the depolarization there was a reduction in magnitude of EPSPs produced by activation of the Schaffer collateral excitatory afferents. The reversal potential for the hyperpolarization was about —95 mV, and it was blocked by 4-aminopyridine. The depolarization, but not the hyperpolarization was markedly attenuated in slices maintained in low (25°C) temperature. The responses to ACh were blocked by atropine but not byd-tubocurarine. The hyperpolarization as well as the depolarization were present in slices treated with tetrodotoxin (TTX)., but were reduced in slices superfused with a low Ca²⁺-high Mg²⁺ medium, and in slices treated with Mn²⁺ and Co²⁺ ions. It is suggested that ACh causes a fast increase in gK⁺, followed by along-lasting energy-dependent depolarization associated with action potential discharges, a decrease in conductance and a suppression of EPSPs.     

Effects of IL-1β on neuronal activities in the dorsal motor nucleus of the vagus in rat brain slices

Effects of recombinant human interleukin-1β (IL-1β) on the neuronal activities in the rat dorsal motor nucleus of the vagus (DMV) were investigated by extra- and intracellular recordings in slice preparations. Twelve (52%) of 23 spontaneously firing neurons recorded extracellularly, 7 of which were electrophysiologically identified as vagal motoneurons, were inhibited by a bath application of IL-1β at a dose of either 5.8 × 10⁻⁸ or 5.8 × 10⁻⁹ M. The duration of the responses ranged widely from about 10 min to more than 2 h. Two (9%) of the 23 neurons were excited, whereas the remaining 9 (39%) were not affected by IL-1β. Of 42 DMV neurons recorded intracellularly, 19 (45%) showed a hyperpolarization following an application of 5.8 × 10⁻⁸ M IL-1β, which still persisted in a TTX-containing solution. Two (5%) displayed depolarization and 21 (50%) were unaffected. The hyperpolarization in 16 of the 19 neurons (84%) ranged from −5 to −10 mV and lasted for more than 30 min without changing the input resistance. The IL-1β-induced hyperpolarization was completely blocked by concurrent perfusion with sodium salicylate. The remaining three neurons showed a short-lasting (5–14 min) hyperpolarization (ranging from −6 to −15 mV) with a decrease in the input resistance. These findings indicate that IL-1β mainly inhibits the vagal motoneurons in the DMV, at least partly through prostaglandin synthesis. This provides a mechanism that could account for the central action of IL-1β on visceral processes such as the inhibition of gastric acid secretion.     

Inhibition of energy metabolism by 3-nitropropionic acid activates ATP-sensitive potassium channels.

3-Nitropropionic acid (1 mM), which inhibits succinate dehydrogenase activity and reduces cellular energy, produces in the pyramidal cell layer of the hippocampal region CA1 a hyperpolarization for variable lengths of time before evoking an irreversible depolarization. Hyperpolarization is caused by an increased potassium conductance that is attenuated by glibenclamide (1-10 microM), a selective antagonist of ATP-sensitive potassium channels; in contrast, diazoxide (0.5 mM), an agonist at this channel, induces a hyperpolarization in CA1 neurons of rat hippocampal slices. The transient hyperpolarization after prolonged (ca. 1 h) application of 3-NPA is followed by a depolarization that is incompletely reversed by brief application of the glutamate antagonists (D-2-amino-5-phosphonopentanoic acid (APV), 6,7-dichloroquinoxaline-2,3-dione (CNQX), 3-(+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), 7-chloro-kynurenic acid (7Cl-KYN)). Early application of glibenclamide (within the initial 5 min) blocked or reduced hyperpolarization and accelerated the depolarization. These data suggest that metabolic inhibition by 3-NPA initially activates ATP-sensitive potassium channels. Events other than activation of glutamate receptors participate in the final depolarization resulting from uncoupling of oxidative phosphorylation.     

Characterization of Rebound Depolarization in Neurons of the Rat Medial Geniculate Body <Emphasis Type="Italic">In Vitro</Emphasis>

Rebound depolarization (RD) is a response to the offset from hyperpolarization of the neuronal membrane potential and is an important mechanism for the synaptic processing of inhibitory signals. In the present study, we characterized RD in neurons of the rat medial geniculate body (MGB), a nucleus of the auditory thalamus, using whole-cell patch-clamp and brain slices. RD was proportional in strength to the duration and magnitude of the hyperpolarization; was effectively blocked by Ni²⁺ or Mibefradil; and was depressed when the resting membrane potential was hyperpolarized by blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with ZD7288 or by activating G-protein-gated inwardly-rectifying K⁺ (GIRK) channels with baclofen. Our results demonstrated that RD in MGB neurons, which is carried by T-type Ca²⁺ channels, is critically regulated by HCN channels and likely by GIRK channels.     

An analysis of histamine-induced inhibitory response in molluscan neurons

Histamine elicited depolarization (excitation) in some neurons and hyperpolarization (inhibition) in other neurons of the central nervous system of the marine mollusc, Onchidium verruculatum. The histamine sensitive region was along the axon at some distance from the soma. H1-receptor blockers (SA-97 and mepyramine) suppressed the excitatory (H1) response without affecting the inhibitory (H2) response, while H2-receptor blockers (burimamide and metiamide) suppressed the H2-response without affecting the H1-response. The H1-response was associated with a marked increase in membrane conductance and was blocked by removal of the external Na. The H2-response consisted of a hyperpolarization without much change in conductance, compared with the hyperpolarization of same amplitude produced by glutamate in the same neuron. Passive polarization of the membrane and reduction of Cl concentrations to 1/5-1/25 caused no significant change in H2-response. The H2-response was slightly suppressed in K-free saline. Thus, it seems difficult to account for the hyperpolarization only by an increase in K or Cl conductance. Complete removal of Na and addition of ouabain blocked the H2-response, suggesting a contribution of an electrogenic Na-pump to the hyperpolarization. However, in 20 mM Na saline with or without K, histamine still caused clear hyperpolarization. In this solution, the histamine response was not affected by ouabain. Although it is difficult to exclude the possibility that an increase in K conductance may be responsible for the hyperpolarization, it is tentatively proposed as a hypothesis that the H2-response involved some active transport mechanism, different from a ouabain-sensitive electrogenic Na-pump.     

Diphenylhydantoin facilitation of labile, protein-independent memory

Diphenylhydantoin (DPH 10⁻⁴M) administered subcutaneously to chicks 5 min after a one-trial passive avoidance learning task successfully counteracted anmesia induced by pretreatment 5 min before learning with ouabain or cycloheximide (CXM). Biochemical assays confirmed that in chick forebrain homogenate DPH at concentrations of 1 and 5 × 10⁻⁴M enhanced Na⁺/K⁺ ATP'ase activity. Since both DPH and ouabain inhibit post-tetanic potentiation, the results support the hypothesis of an initial labile phase of memory based on sodium pump (Na⁺/K⁺ ATP'ase) activity. DPH was less effective in counteracting ouabain-induced amnesia if administered later than 10 min after learning and CXM-induced anmesia if administered later than 30 min after learning. This suggests that the effect of DPH on CXM-induced anmesia is through enhancement of Na⁺/K⁺ ATP'ase activity. It was suggested that the possible hyperpolarization of membrane potential associated with sodium pump activity may serve to mark the labile memory trace, enabling formation of a more permanent trace through protein synthesis.     

Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca²⁺/high Mg²⁺ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinicreceptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the γ-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.     

Associative mossy fibre LTP induced by pairing presynaptic stimulation with postsynaptic hyperpolarization of CA3 neurons in rat hippocampal slice

Whole cell recordings of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) evoked by minimal stimulation of commissural-associative (CF) and mossy fibre (MF) inputs were performed in CA3 pyramidal neurons. Paired responses (at 50 ms intervals) were recorded before, during and after hyperpolarization of the postsynaptic membrane (20-30 mV for 15-35 min). Membrane hyperpolarization produced a supralinear increase of EPSPs/EPSCs amplitude in MF-inputs. Synaptic responses remained potentiated for the rest of the recording period (up to 40 min) after resetting the membrane potential to control level (221 +/- 60%, n = 15 and 219 +/- 61%, n = 11 for MF-EPSP and MF-EPSC, respectively). We shall refer to this effect as hyperpolarization-induced LTP (HI-LTP). In the absence of afferent stimulation, membrane hyperpolarization was unable to produce HI-LTP. In contrast to MF-EPSPs, the mean amplitude of CF-EPSPs did not increase significantly after hyperpolarization relative to controls (138 +/- 29%, n = 22). HI-LTP was associated with modifications of classical indices of presynaptic release: paired-pulse facilitation, failures rate, coefficient of variation of EPSP amplitudes and quantal content. The induction of HI-LTP was NMDA independent but was dependent on metabotropic glutamate receptors (mGluRs) activation and calcium release from inositol 1,4,5-triphosphate (IP3)-sensitive intracellular stores: it was prevented by mGluR antagonist, intracellular heparin and BAPTA. We conclude that while the induction of HI-LTP was postsynaptic, its expression was presynaptic.     

Correlation of anoxic neuronal responses and calbindin-D28k localization in stratum pyramidale of rat hippocampus

Immunohistochemical staining for the calcium-binding protein calbindin-D_28k (CaBP) was combined with Lucifer Yellow (LY) identification and intracellular recording of changes in membrane parameters of pyramidal neurons in CA2, CA1, and the sebiculum of rat hippocampal slices during brief exposure (4.0 ± 0.19 min) to N₂. Anoxia evoked either a depolarization or hyperpolarization of membrane potential (V_M) (+21.5 ± 2.79 mV above V_M = ?70.5 ± 1.50 mV, n = 30 and ?7.2 ± 0.72 mV below V_M = ?68.2 ± 1.34 mV, n = 24, respectively) and a fall in membrane resistance of =20%. Differences in the response could be correlated with the presence or absence of CaBP and the localization of neurons in different layers of stratum pyramidale and sectors of the hippocampus. For neurons immunopositive for calbindin (CaBP(⁺)), depolarization was observed more frequently (83%) than hyperpolarization (17%); in contrast, 44% of responses of calbindin-negative (CaBP(^?)) neurons were depolarizing and 56% were hyperpolarizing. Depolarizations of CaBP(⁺) neurons were more gradual in slope, and more rapidly reached a plateau in comparison with those recorded in CaBP(^?) neurons. Responses of neurons in the superficial layer of stratum pyramidale (in which 79% of CaBP(⁺) pyramidal neurons were situated) were mainly depolarizing (91%), while for those in the deep layer (which contained 89% of the CaBP(^?) cells) such responses were observed less often (45%). Depolarization was also more common than hyperpolarization for cells located in CA2/CA1c/CA1b (63%) than in the CA1a/subicular region (37%). The depolarizing response of the majority of pyramidal neurons which are CaBP(⁺), superficial, and closer to CA3 may reflect an efficient buffering of intracellular Ca²⁺, which maintains a low [Ca²⁺]_i, steep gradient for Ca²⁺ influx and may facilitate the movement of Ca²⁺ away from points of entry. The neurons which are CaBP(^?), deep, and closer to subiculum and in which N₂ evokes hyperpolarization, on the other hand, may have a sustained elevation/accumulation of cytosolic Ca²⁺ which could activate K⁺ conductance, inhibit Ca²⁺ influx, and stabilize the membrane potential. These experiments provide a functional correlate for CaBP and suggest that it may have a significant role in Ca²⁺ homeostasis and the determination of selective neuronal vulnerability. © 1995 Wiley-Liss, Inc.     

Characterization of a Barium-sensitive Outward Current following Glutamate Application on Rat Midbrain Dopaminergic Cells

Using intracellular electrophysiological recordings in dopaminergic (principal) neurons of the rat mesencephalon maintained in vitro, we studied a postexcitatory amino acid response (PEAAR). Under current-clamp mode, bath application of glutamate produced a depolarization that was followed by a hyperpolarization when the perfusion of the excitatory amino acid was discontinued. Under single-microelectrode voltage-clamp mode, an outward current followed the glutamate-induced inward current. The PEAAR was associated with an increase in membrane conductance and reversed polarity at about -85 mV (2.5 mM extracellular K⁺). The null potential for the PEAAR was independent of the intracellular loading of chloride ions and was shifted towards less negative values (?23 mV) by increasing extracellular K⁺ from 2.5 to 8.5 mM. The PEAAR was present in neurons treated with tetraethylammonium (5–10 mM), apamin (1 μM) or glibenclamide (1–300 μM). However, it was strongly depressed or blocked by extracellular barium (300 μM to 1 mM), by low-calcium (0.5 mM) plus cadmium (100 μM) or magnesium (10 mM), and by low-sodium solutions. An outward response was also generated after an inward current induced by the perfusion of the specific agonists for the ionotropic excitatory amino acid receptors NMDA, a-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) and kainate. The PEAAR was not affected by tetrodotoxin (1 μM), saclofen (100–300 μM), bicuculline (30 μM), sulpiride (1 μM) or strychnine (1 μM). In addition, the inhibition of the ATP-dependent Na⁺-K⁺ pump by ouabain and strophanthidin (1–10 μM) prolonged the glutamate-induced membrane depolarization/inward current while the subsequent PEAAR was reduced or not observed. Our data indicate that the PEAAR mainly results from the activation of a barium-sensitive potassium current. This response might limit the excitatory and eventually neurotoxic effects of glutamate.     

Effect of butyrate on membrane potential, ionic currents and intracellular Ca2+ concentration in cultured rat myenteric neurones

Myenteric neurones from 1-10-day-old rats were isolated from the small and large intestine by enzymatic digestion with collagenase. Single cells were collected and kept in culture for up to 1 week. After 1-5 days in culture, membrane potential and ionic currents were measured with the whole-cell patch-clamp technique. The intracellular Ca2+ concentration was measured with the fura-2 method. The short-chain fatty acid butyrate (50 mmol L-1) induced a reversible hyperpolarization of the myenteric neurones by about 10 mV. This hyperpolarization was concomitant with an inhibition of a TTX-sensitive Na+ current. The hyperpolarization could be suppressed by intracellular application of Cs+, a nonselective K+ channel blocker. Fura-2 experiments revealed that butyrate induced an increase of the intracellular Ca2+ concentration. The butyrate response was suppressed by thapsigargin, indicating that butyrate stimulates the release of intracellular Ca2+. This release is responsible for the voltage response, because intracellular chelation of Ca2+ inhibited the butyrate induced hyperpolarization. Consequently, butyrate acts on enteric neurones by releasing Ca2+ from intracellular stores with the consequence of the activation of K+ channels, followed by a hyperpolarization.     

Inhibitory potentials in neurons of the deep layers of the in vitro neocortical slice

Neocortical neurons in slices of the rat sensorimotor region maintained in vitro generate postsynaptic potentials (PSPs) in response to focal extracellular stimulation. These PSPs are mainly depolarizing at the resting membrane potential (Vm) but a sequence of depolarizing-hyperpolarizing potentials is often disclosed by depolarizing the Vm. The stimulus-induced hyperpolarization can last up to 1000 ms and show two components: the early one (peak latency 10-20 ms), is inverted by diffusion of Cl- into the cell; the late one is diminished by augmenting [K+]o. The membrane conductance is increased throughout the stimulus-induced hyperpolarization, mainly during the first 10-60 ms. A decrease in excitability results from both the hyperpolarizing trend and the conductance increase. The latter is more effective in decreasing depolarizing than hyperpolarizing pulses of current injected intracellularly.