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).     

Potassium and sodium channels in human malignant glioma cells

Human malignant glioma cells from 5 different cell lines were voltage clamped and examined for the presence of depolarization-activated ion channels. Outward K-currents were elicited at membrane potentials > 40 mV, which had two main components, one which was delayed and blocked by externally applied tetraethylammonium (TEA, 10 mM), and another which was instantaneous and insensitive to TEA in the outside solution. The proportion of the two K-current components varied between cell lines. An increase in [Ca²⁺] in the range 0–4 mM, decreased the leak conductance and shifted the activation of the instantaneous outward K-current towards more positive potenttials. Mg²⁺, Zn²⁺ and Co²⁺ had qualitatively similar effects. Patch recordings with 150–160 mM K⁺-solution on both sides of the membrane revealed that the delayed outward K-current was carried through large conductance (250–300 pS) channels. Changes in free [Ca²⁺]_i from 0 to 2 × 10⁻⁸ M increased the activation of the large conductance K-channel. Small Na-currents were identified in cells from one cell line (Tp-378MG). The Na-conductance rangedfrom 0.5 to 7.5 nS in 25% of the cells, and was less than 0.5 nS in 75%. The Na-channels were activated and inactivated at 30–40 mV more positive potentials than in the mammalian peripheral nerve. Tetrodotoxin (100 mM) blocked g_Na almost completely.     

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.     

Protein phosphatase inhibitors prolong Ca-transients and divalent cation-dependent action potentials in leech Retzius cells

Elevation of [K⁺]_o for 30 s from 4 to 120 mM produced a fast and reversible depolarization and transient increase in [Ca²⁺]_i in fura-2 loaded Retzius cells of the leech. The protein phosphatase inhibitor, okadaic acid, significantly slowed the return of [Ca²⁺]_i toward baseline without affecting the amplitude of depolarization or rate of repolarization. Furthermore, okadaic acid and another phosphatase inhibitor, calyculin A, prolonged Ba²⁺-dependent action potentials. These results suggest that the kinetics of Ca²⁺ influx may be regulated by the activity of phosphatases PP-1 and/or PP-2A.     

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.     

Dependence of the acid-sensitive ion channel, ASIC1a, on extracellular Ca(2+) ions

The early H⁺-induced current in the embryonic spinal cord neurone depends on extracellular Ca²⁺ for its function. We have studied the Ca²⁺-dependence of homo- and heteromultimeric acid-sensing ion channels (ASICs) expressed in Cos cells. It was found that single-channel conductance of both the ASIC2a and the ASIC1a channel is reduced at membrane potentials more negative than −40 mV by elevated extracellular Ca²⁺. Due to this effect on unitary currents, the macroscopic ASIC2a peak current at −60 mV decreases gradually with rising extracellular Ca²⁺ concentration. In addition, the macroscopic ASIC1a current is very small at low extracellular Ca²⁺ and increases with rising Ca²⁺ up to 5 mM before decreasing again at still higher concentrations of extracellular Ca²⁺.     

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.     

Modification of the Na,K-pump of glial cells within cobalt-induced epileptogenic cortex of rat

The characteristics of a glial Na⁺,K⁺-pump dependent on extracellular K⁺ within epileptogenic cortex were studied electrophysiologically, biochemically and histochemically in vitro using slices from cobalt-induced epileptogenic cortex of rat. When the extracellular K⁺ concentration ([K⁺]_o) was varied between 4 and 40 mM, the mean slope of membrane potential plotted against [K⁺]_o was about 57 mV in glia from the normal cortex (tissue A) and about 44 mV in glia from the epileptogenic cortex (tissue B); whereas no significant difference in the resting membrane potential of these tissues was observed. In glia from tissue B, a marked transient hyperpolarization above control level was caused by replacement of elevated [K⁺]_o with the normal medium. Ouabain abolished these phenomena observed in glia from tissue B, but had no effect on the membrane potential during normal [K⁺]_o. Reduction of extracellular Na⁺, Ca²⁺ and Cl⁻ did not significantly affect the membrane potential of glia from either tissue. In tissue A, the cells marked by intracellular injection of horseradish peroxidase after intracellular recording were protoplasmic astrocytes; in tissue B, fibrous astrocytes with abnormal processes predominated. K⁺-dependent stimulation of Na⁺,K⁺-ATPase activity of the astrocyte-enriched fraction and its membrane preparation from tissue B was much larger than that from tissue A. A certain amount of the reaction product of K⁺-pNPPase activity was seen on glial plasma membrane within tissue B but not on that from tissue A. The above findings suggest that a glial Na⁺,K⁺-pump within actively firing epileptogenic cortex may be modified to increase in its activity.     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

Independent regulation of activation and inactivation phases in non-contractile Ca transients by nicotinic receptor at the mouse neuromuscular junction

Non-contractile Ca²⁺ mobilization (not accompanied by muscle contraction) occurs by the prolonged activation of nicotinic acetylcholine receptor in mouse diaphragm muscles treated with anticholinesterase. To elucidate the regulation properties of non-contractile Ca²⁺ mobilization by nicotinic receptor, the modes of action of competitive and depolarizing neurmuscular blockers were investigated. (+)-Tubocurarine (0.07–0.1 μM), pancuronium (0.05 μM) and -bungarotoxin (0.03–0.06 μM) decreased decay time (T₂, duration of inactivation phase) without changes in rise time (T₁, duration of activation phase) of non-contractile Ca²⁺ transients. These competitive antagonists also suppressed their peak amplitude at higher concentrations than those affectingT₂. Contractile Ca²⁺ transients were not inhibited by these antagonists at the concentrations used. Decamethonium (1 μM), a depolarizing blocker, suppressed the peak amplitude of non-contractile Ca²⁺ transients without affecting their duration. In contrast, succinylcholine (0.3 μM) suppressed both peak amplitude andT₁ without changingT₂, presumably via the receptor desentization. Succinylcholine but not decamthonium inhibited contractile Ca²⁺ transients at the concentrations used. These results demonstrate that the activation and inactivation phase in non-contractile Ca²⁺ transients are independently regulated by nicotinic acetylcholine receptor.     

Dopaminergic regulation of GABA release from the intact goldfish retina

The rate of release of [³H]GABA from intact goldfish retinas was studied using a modified superfusion technique. Small, significant increases in the rate of GABA release were observed when the retinas were exposed to dopamine (DA) (100–1000 μM); however, when free Ca²⁺ was removed from the medium, the basal rate of GABA release was increased and DA became inhibitory. Forskolin, a non-specific stimulator of adenylate cyclase in intact cells, also inhibited GABA release in the absence of Ca²⁺. There was no significant effect of forskolin in the presence of Ca²⁺; however, (+)-butaclamol, a dopamine antagonist, increased basal GABA release under these conditios. l-glutamic acid (l-Glu) (1–10 mM) causes up to a 10-fold increase in GABA release. In the presence of Ca²⁺, DA did not significantly alter the effects of l-Glu; however, in the absence of Ca²⁺ a significant inhibition of the effects of l-Glu by DA was observed. Forskolin, on the other hand, inhibited the effects of l-Glu both in the presence and absence of Ca²⁺. Finally, EGTA (0.3–1 mM) produced a large release of GABA: this release was inhibited by DA, forskolin, theophylline and 8-bromo cyclic AMP. These results suggest a model wherein DA stimulates Ca²⁺-dependent GABA release from one site and inhibits Ca²⁺-independent GABA release from another site via cyclic AMP-mediated event.     

Inward rectifying potassium channels in human malignant glioma cells

Human glioma cells obtained from established cell lines (Tp-276MG, Tp-301MG, Tp-378MG, Tp-483MG and U-251MG) were analyzed for the presence of ion channels with the tight-seal voltage clamp technique. The current-voltage relation revealed a marked inward rectification at hyperpolarizing voltages, due to the presence of inward rectifying K-channels in cells from all studied cell lines. These channels were conducting when the membrane potential was more negative than the K-equilibrium potential. The slope conductance for the inward K-currents (g_Ki) was affected both by [K⁺]_i and [K⁺]₀. g_Ki was proportional to [K⁺]₀ raised to 0.35 or 0.50, of which the larger value was measured in the presence of low [K⁺]_i (25mM). The rectification was not significantly different in cells perfused with Mg-free EDTA-buffered internal solution. Tl⁺ was 3.5 times more permaant than K⁺. g_ki was blocked by Cs⁺ (1 mM) in a voltage-dependent way (more effective in the hyperpolarized membrane), and by Na⁺ (154 mM) depending on voltage and time. From measurements of unitary current events in membrane patches (outside out or cell attached) the conductance of the single inward rectifying channel was estimated to be 27 ± 7 pS. This type of ion channel may be important for K-uptake by glial cells and hence for the K-homeostasis in the brain.     

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.     

-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.     

Excitation of locus coeruleus neurons by vasoactive intestinal peptide: evidence for a G-protein-mediated inward current

Vasoactive intestinal polypeptide (VIP) caused a reversible increase in the firing rate of locus coeruleus (LC) neurons. Voltage-clamp at −60 mV revealed that VIP induced an inward current, associated with a small increase in conductance. The inward current persisted in the presence of Co²⁺ (to block Ca²⁺ channels) or tetrodotoxin (to block fast voltage-dependent Na⁺ channels). Substitution (80%) of Na⁺ with choline or Tris reduced the VIP-elicited inward current by approximately 75%. Changing external K⁺ concentrations did not alter the effect of VIP. The inward current induced by VIP became irreversible after the intracellular administration of GTPγS, a hydrolysis-resistant analog of GTP which can cause a prolonged activation of G-proteins. The intracellular application of GDPβS, which can interfere with G-protein activation, attenuated the effect of VIP. Pertussis toxin, an inactivator of certain G-proteins, did not block the effect of VIP. We conclude that VIP directly excites LC neurons by inducing a largely Na-dependent inward current. As this effect became irreversible in the presence of intracellular GTPγS, was attenuated by GDPβS, and was not eliminated by pertussis toxin, mediation through a pertussis toxin-insensitive G-protein is suggested.     

Ionic currents in cultured supraoptic neurons: Actions of peptides and transmitters

The hypothalamo-neurohypophysial system has proved an excellent model for peptidergic neurons in the central nervous system. Electrophysiological studies using in vivo and in vitro preparations with extracellular and intracellular recording techniques have determined some of the intrinsic and extrinsic mechanisms that generate the striking firing patterns that the neurons exhibit. We have developed a dissociated cell preparation of these neurons and used patch clamp recording techniques to enable detailed studies of membrane properties underlying such activities. Cultured neonatal supraoptic neurons fired spontaneous action potentials which in some cells were distinctively patterned. Under voltage clamp, voltage-activated Na⁺, K⁺, and Ca²⁺ currents were recorded. K⁺ and Ca²⁺ currents were modulated by application of -adrenergic agonists, and Ca²⁺ currents were also modulated by κ-opioid agonists. The neurons were also sensitive to γ-aminobutyric acid which acted directly on Cl-channels. Spontaneous, patterned activity, the presence of functional receptors for neurotransmitters and the ability to study the neurons under voltage clamp suggest that this is an excellent model system for studying these peptidergic 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.