Selective inhibition of the slow K current at motor nerve ending by plasma from a myasthenia gravis patient

The effect of plasma from a myasthenia gravis (MG) patient, containing anti-presynaptic membrane receptor (PsmR) antibody on the membrane currents of motor nerve ending was investigated in mouse intercostal nerve triangularis sterni preparations by perineurial recording. After inhibition of both the fast K⁺ current and Ca²⁺-dependent K⁺ current by 30 mM Tetraethyl-ammonium (TEA) unmasked the voltage dependent fast Ca²⁺ current and the “Ca plateau”, which was contributed by the voltage-dependent slow Ca²⁺ current and slow K⁺ current. Application of the MG plasma caused further prolongation and increase of the Ca plateau, due to blockage of the slow K⁺ current. This effect was observed immediately after the application and could be partially reversed by washing, whereas no change was found by addition of the plasma from healthy persons. When K⁺ current was completely blocked by 30 mM TEA and 300 μM 3,4-diaminopyridine (3,4-DAP), the fast Ca²⁺ current and the slow Ca²⁺ current were revealed. Neither the fast nor the slow Ca²⁺ current could be affected by the MG plasma; It was also shown that the MG plasma was devoid of noticeable effect on the voltage dependent Na⁺ current, fast K⁺ current as well as the Ca²⁺-dependent K⁺ current. So the effect of the MG plasma with antibody to PsmR was concluded to inhibit the slow K⁺ current selectively. As we knew, the β-bungarotoxin binding protein was a kind of K⁺ channel, these results further confirmed that the β-bungarotoxin binding protein should be the target of the antibody to PsmR found in the plasma of some patients suffering from MG.     

Analysis of potential shifts associated with recurrent spreading depression and prolonged unstable spreading depression induced by microdialysis of elevated K in hippocampus of anesthetized rats

The potential shifts (ΔV_o) associated with spreading depression (SD) were analysed with the help of multiple extracellular recording and ion-selective microelectrodes in the CA1 region of the dorsal hippocampus of anesthetized rats. Recurrent waves of SD were induced by perfusing high K⁺ solution through microdialysis probes. SD-related ΔV_o had a composite wave shape, consisting of an early, rapidly shifting part (phase I) followed by a slower shift to a second negative maximum (phase II). ΔV_o shifts in stratum radiatum usually started earlier, always lasted longer and had lartger amplitude than those recorded in stratum pyramidale. The ΔV_o responses in stratum radiatum had an inverted saddle shape created by a transient relatively positive “hump” interposed between phases I and II. During this “hump”, the potentials in the two layers transiently approached one another. During continuous high K⁺ dialysis, successive ΔV_o waves episodes evolved according to a consistent pattern: while phase I remained unchanged, phase II increased in amplitude and duration with each episode. Eventually, a depressed state developed which lasted for many minutes, termed here prolonged unstable spreading depression. During phase I, ΔV_o and extracellular K ([K⁺]_o) changes were correlated. During phase II, [K⁺]_o decreased even as ΔV_o continued to increase. During SD, [Ca²⁺]_o decreased to <0.01 mM. During phases I and II, both [Ca²⁺]_o and [Na⁺]_o remained low. the recoverries of [Ca²⁺]_o and [Na⁺]_o had an initial fast and a later much slower phase and took several minutes longer than the recoveries of [K⁺]_o and ΔV_o. Depth profiles of ΔV_o and Δ[K⁺]_o revealed strikingly steep gradients early and late during a wave; but voltage and ion gradients were not precisely correlated either in time or in space. We conclude that ΔV_o of phases I and II are generated by different processes. Membrane ion currents cannot fully explain the ΔV_o responses. The possible contributions by ion diffusion and by active ion transport are discussed. The extremely low level to which [Ca²⁺]_o sinks during SD, and its two-phase recovery, indicate intracellular sequestration or binding of substantial amounts of Ca²⁺ ions. The residual deficit of [Ca²⁺_o following recovery of SP shifts may account for the persistent depression of synaptic transmission after repolarization of neurons.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

The antiepileptic drug losigamone decreases the persistent Na current in rat hippocampal neurons

The tetronic acid derivative losigamone is a new anticonvulsant drug with a mechanism of action that was previously unknown. The drug decreases the frequency of spontaneous action potentials and suppresses repetitive firing of neurons. Here we tested the hypothesis that losigamone suppresses the persistent Na⁺ current (I_NaP) in hippocampal neurons of rat brain slices and in cultured hippocampal neurons. Whole-cell voltage clamp recordings from neurons of juvenile rats (P15–P25) were performed with pipettes filled with Cs-gluconate or CsF. After pharmacological block of K⁺ and Ca²⁺ currents I_NaP was revealed by applying slow depolarizing voltage ramps from −70 to 0 mV. Losigamone (100–200 μM) was dissolved in DMSO (0.1%) and was applied by bath application or local pressure application. Losigamone induced a decrease in amplitude of I_NaP at depolarized membrane potentials which was reversible in cultured neurons. When tetrodotoxin (TTX) was added to the bath, I_NaP was blocked and only a residual non-specific outward cation current (I_cat) remained. Losigamone had no obvious effect on responses to voltage ramps under these conditions. Thus, losigamone did not affect I_cat or induce any additional currents. The data suggest that losigamone decreases neuronal excitability via a decrease in I_NaP.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

GABAergic synaptic current in dissociated nucleus basalis of Meynert neurons of the rat

γ-Aminobutyric acid (GABA)-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were recorded from dissociated rat nucleus basalis of Meynert neurons which still had their synaptic boutons attached. The membrane currents were recorded by the whole-cell patchcalmp technique. Elevated extracellular K⁺ concentration and the addition of the calcium ionophore, A23187, enhanced the amplitude and frequency of spontaneous IPSCs. Ryanodine and Ca²-free external solution containing EGTA or BAPTA markedly decreased the spontaneous IPSC activities. Spontaneous IPSC activities were reversibly reduced by baclofen and increased by phaclofen, indicating that the GABA_B receptor regulates the release of GABA from nerve terminals and acts as a negative autoreceptor.     

Novel Ca currents in mammalian CNS neurons

Akaike, Norio, Hisashi Yamanaka and Mitsutoshi Munakata: Novel Ca²⁺ Currents in Mammalian CNS Neurons. Prog. Neuro-Psychopharmacol. & Biol. Psychiatry. 1992, 16(6): 943–957.

1. 1. Voltage-dependent Ca²⁺ currents (I_Ca) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly dissociated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca²⁺ current (termed ‘TTX-I_Ca’). The TTX-I_Ca showed a heterogeneous distribution, preferentially in the dorsal site of CA1 region.

2. 2. Activation and inactivation processes of the TTX-I_Ca were highly potential-dependent, and the latter was fitted by a double exponential function. The TTX-I_Ca was activated at a threshold potential of about −55 mV and reached full activation at −30 mV. The steady-state inactivation of TTX-I_Ca could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of −72.5 mV.

3. 3. When the peak amplitudes of TTX-I_Ca were plotted as a function of extracellular Ca²⁺ concentration [Ca²⁺]_o), the current amplitude increased linearly without showing any saturation.

4. 4. The ratio of peak amplitude in the individual I-V relationships of Ca²⁺, Sr²⁺ and Ba²⁺ currents assing through the TTX-sensitive Ca²⁺-conducting channel was 1 : 0.33 : 0.05, although the current kinetics were much the same.

5. 5. TTX inhibited the TTX-I_Ca in time- and concentration-dependent manner without affecting the current kinetics. Lignocaine inhibited the TTX-I_Ca in a second in a concentration-dependent manner, with accelerating the inactivation process. The concentrations of half-inhibition (IC₅₀) were 3.5 × 10⁻⁹ M for TTX and 3.6 × 10⁻⁴ M for lignocaine.

6. 6. Scorpion toxin prolonged the inactivation phase of TTX-I_Ca in a time- and concentration-dependent manner. In the toxin-treated neurons, both the slow time constant of inactivation (τ_is) and its functional contribution to the total current increased with increasing the toxin concentration.

An adrenal slice preparation for the study of chromaffin cells and their cholinergic innervation

Thin slices (200–300 μm) of adrenal glands were prepared from Wistar rats. Patch-clamp recordings were made from visually identified chromaffin cells using the whole-cell and amphotericin B perforated-patch techniques. Electrophysiological properties of chromaffin cells in slices were similar to those in cultured cells. Catecholamine release from single chromaffin cells or cell clusters in slices was also measured by amperometry. Immunostaining of slices with an antineurofilament antibody revealed the presence of neuronal fibers. Acetylcholine release was stimulated either by raising external [K⁺] or by focally applying voltage pulses. Nicotinic excitatory postsynaptic currents (EPSCs) were detected, ranging from 20 pA to several hundreds of pA. Amplitude distributions of spontaneous EPSCs revealed clear equidistant peaks, supporting a quantal model for acetylcholine release onto chromaffin cells. The adrenal slice preparation therefore appears to be an excellent model for studying both the cholinergic innervation of chromaffin cells as well as catecholamine release from these cells.     

Calcium uptake related to K-depolarization and Na/Ca exchange in sheep brain synaptosomes

The uptake of Ca²⁺ by synaptosomes induced by K⁺-depolarization andby Na⁺/Ca²⁺ exchange was studied in synaptosomes in which the internal Na⁺ and K⁺ contents were varied by prolonged incubation at 30 °C or by inhibiting the Na⁺, K⁺-ATPase with 1 mM ouabain. Increased Na⁺ content of the synaptosomes is associated with an increase in Ca²⁺ uptake when the synaptosomes are placed in depolarizing K⁺ media. Furthermore, reduction in the [Na⁺]_o, when the [K⁺]_o is increased, in substitution for [Na⁺]_o, to depolarize the membrane, further increases the Ca²⁺ uptake. Under these conditions, Ca²⁺ entry probably occurs through voltage-sensitive channels and through the Na⁺/Ca²⁺ exchanger. Destruction of the Na⁺ gradient by monensin, or preloading the synaptosomes with K⁺, completely inhibits the Ca²⁺ uptake in a K⁺-depolarizing medium. It is shown that if the Na⁺ gradient is maintained constant during K⁺-depolarization, the Ca²⁺ uptake is very low and that most of the Ca²⁺ uptake is correlated with the Na⁺ gradient. Evidence is presented that K⁺ may stimulate the Na⁺/Ca²⁺ exchange mechanism. Furthermore, divalent cations, Mg²⁺, Mn²⁺ and Zn²⁺, known to block Ca²⁺ channels, also inhibit Na⁺/Ca²⁺ exchange.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

-Receptor and cholinergic receptor-linked changes in cytosolic Ca and membrane potential in primary rat astrocytes

Both phenylephrine and carbachol caused a sustained increase in Ca²⁺ influx and intracellular free Ca²⁺ of primary astrocytes as measured with ⁴⁵Ca²⁺ and fura-2. The responses to phenylephrine and carbachol were additive, suggesting that they use different releasable pools of Ca²⁺. If extracellular Ca²⁺ was removed by EGTA only a transient rise in cytosolic Ca²⁺ was seen upon application of the agonists. Both compounds caused depolarization of the astrocyte membrane as determined with the optical probe 3,3-diethylthiadicarboxyamineiodide. Activation of protein kinase C with 12-tetradecanoylphorbol myristate acetate (TPA) or the diacylglycerol analogue dioctanoylglycerol (DiC₈) also depolarized the cells. A prior activation of protein kinase C with TPA or DiC₈ abolished the depolarizing effect of phenylephrine suggesting that they act through the same mediators. If the cells were made ideally permeable to K⁺ with the ionophre valinomycin, or the K⁺ channels had been blocked with Ba²⁺, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neither TPA nor phenylephrine had any effect on the ⁸⁶Rb⁺ equilibrium potential across the cell membrane. The results suggest that the depolarizing effect of these substances could be through a blocking of K⁺ channels.     

Voltage-gated Currents in Rabbit Retinal Astrocytes

The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was −70 ± 4.8 mV (mean ± SEM; n = 54), and the input resistance and cell capacitance were 558 ± 3.6 MΩ and 19.5 ± 1.8 pF respectively. Depolarization to potentials positive to −50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na⁺ in the bathing solution, and reduced by 50% in the presence of 1 μM tetrodotoxin. The time-to-peak of the Na⁺ current was more than twice that for the Na⁺ current found in retinal neurons. The glial Na⁺ current was half-inactivated at −55 mV. A transient component of the outward K⁺ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between −150 and −50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.     

Function of serotonin in physiologic secretion of growth hormone and prolactin: Action of 5,7-dihydroxytryptamine, fenfluramine and p-chlorophenylalanine

The effect of 4-aminopyridine (4-AP) on the release of labeled transmitters in mouse brain synatosomes was studied in a superfusion system. 4-AP at μM concentrations notably stimulated the spontaneous release of labeled GABA and glutamate, and of acetylcholine (ACh) derived from tritiated choline. No effects on the release of labeled -aminoisobutyric acid were observed. The stimulation of GABA and ACh release was dependent on the presence of Ca²⁺ in the superfusion media, whereas the effect on glutamate release was more variable and no clear Ca²⁺-dependence was observed. In contrast to these results, 4-AP did not have any effect on the release of the above transmitters by K⁺-depolarization in the presence of Ca²⁺. These results are discussed in terms of the possible participation of Ca²⁺ in the action of 4-AP on spontaneous transmitter release in isolated nerve endings.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

Reflections of low calcium epileptiform activity from area CA1 into dentate gyrus in the rat hippocampal slice

Lowering of [Ca²⁻]₀ induces epileptiform activity in hippocampal area CA1, characterized by slow negative field potentials with superimposed trains of population spikes and by rises in [K⁺]₀. In dentate gyrus slow positive field potentials occur simultaneously with the activity in area CA1. The accompanying small rises in [K⁺]₀ may stem from spatial K⁺ redistribution through glial cells from area CA1.     

F11 neuroblastoma × DRG neuron hybrid cells express inhibitory μ- and δ-opioid receptors which increase voltage-dependent K currents upon activation

The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express μ- and δ-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of μ and δ, but not κ, receptor expression in F11 cells. Radioligand binding assaysshow that F11 cell membranes bind the μ- and δ-opioid receptor agonists, DAGO and DPDPE with K_d = 4.5 and 4.9 nM and B_max = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K⁺ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the κ-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, ß-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K⁺ current. The opioid-induced increase in voltage-dependent membrane K⁺ current in F11 cells resembles the inhibitory effect elicited by μ- and δ-opioid agonists in primary cultures of mouse DRG neurons.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.     

Effects of micromolar and nanomolar calcium concentrations on non-synaptic bursting in the hippocampal slice

To determine how [Ca²⁺]₀ affects non-synaptic epileptogenesis in the CA1 area of hippocampal slices, we compared the extracellularly recorded hyperactivity induced by ACSF containing either micromolar (‘low’-Ca²⁺, LC-ACSF) or nanomolar concentrations of Ca²⁺ (‘zero’-Ca²⁺, ZC-ACSF). Both solutions effectively blocked chemical synaptic transmission but spontaneous bursts developed more quickly and consistently in ZC-ACSF and were longer in duration and more frequent than those recorded in LC-ACSF. Antidromically evoked bursts were less epileptiform, i.e., they exhibited fewer population spikes (PSs), in ZC-ACSF. Increasing [Mg²⁺]₀ or decreasing [K⁺]₀ suppressed spontaneous LC-ACSF bursting but only decreased the intensity and frequency of bursting in ZC-ACSF. Either manipulation increased the epileptiform nature of the antidromically evoked field potential, thereby mimicking the effect of increasing [Ca²⁺]₀ from nanomolar to micromolar levels. Bath application of 250–500 μM GABA commonly arrested spontaneous bursting in LC-ACSF. In ZC-ACSF, GABA decreased the burst frequency but paradoxically superimposed high amplitude PSs on each burst. These effects were reversed by the GABA_A receptor antagonists bicuculline methiodide or picrotoxin (50–100 μM). These results indicate that simply lowering [Ca²⁺]₀ from micromolar to nanomolar concentrations increases the burst propensity and intensity of the CA1 population and can dramatically alter responses to pharmacological agents.     

Inhibition of the Met-enkephalin-induced K current in B-cluster neurons of Aplysia by nitric oxide donor

The effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on a methionine-enkephalin (Met-E)-induced K⁺ current recorded from B-cluster neurons in Aplysia cerebral ganglion were investigated with voltage-clamp and pressure ejection techniques. Bath-applied SNP (10–25 μM) reduced the Met-E-induced K⁺ current in the neurons without affecting the resting membrane conductance and holding current. The inhibitory effects of SNP were reversible. Pretreatment with methylene blue (10 μM), a non-specific inhibitor of guanylate cyclase, and hemoglobin (50 μM), a NO scavenger, decreased the SNP-induced inhibition of the Met-E-induced current. Intracellular injection of 1 mM guanosine 3′,5′-cyclic monophosphate (cGMP) or bath-applied 3-isobutyl-1-methylxanthine (IBMX; 50 μM), a nonspecific phosphodiesterase inhibitor, inhibited the Met-E-induced current. Furthermore, 1H-[1,2,4] oxadiazolo[4,3- a]quinoxalin-1-one (ODQ, 1 μM), a more specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Met-E-induced current. These results suggest that SNP induces suppression of the Met-E-induced K⁺ current recorded from B-cluster neurons of Aplysia cerebral ganglion via stimulation of cGMP formation.     

Spatiotemporal pH dynamics following insertion of neural microelectrode arrays

Insertion trauma is a critical issue when assessing intracortical electrophysiological and neurochemical recordings. Previous reports document a wide variety of insertion techniques with speeds ranging from 10 μm/s to 10 m/s. We hypothesize that insertion speed has an effect on tissue trauma induced by implantation of a neural probe. In order to monitor the neural interface during and after probe insertion, we have developed a silicon-substrate array with hydrous iridium oxide microelectrodes for potentiometric recording of extracellular pH (pH_e), a measure of brain homeostasis. Microelectrode sites were sensitive to pH in the super-Nernstian range (−85.9 mV/pH unit) and selective over other analytes including ascorbic acid, Na⁺, K⁺, Ca²⁺, and Mg²⁺. Following insertion, arrays recorded either triphasic or biphasic pH_e responses, with a greater degree of prolonged acidosis for insertions at 50 μm/s than at 0.5 mm/s or 1.0 mm/s (p < 0.05). Spatiotemporal analysis of the recordings also revealed micro-scale variability in the pH_e response along the array, even when using the same insertion technique. Implants with more intense acidosis were often associated histologically with blood along the probe tract. The potentiometric microsensor array has implications not only as a useful tool to measure extracellular pH, but also as a feedback tool for delivery of pharmacological agents to treat surgical brain trauma.