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.     

The role of cyclic AMP in chemoreception in the rabbit caroid body

The present study identified physiological factors which influence the generation (and degradation) of cyclic AMP (cAMP) in the arterial chemoreceptor tissue of the mammalian carotid body. Experiments established a 3-way correlation between cAMP generation, neurotransmitter release from chemoreceptor cells, and carotid sinus nerve (CNS) activity. Incubation of carotid bodies in vitro for 10 min in media equilibrated with different low O₂ (‘hypoxic’) gas mixtures (5% O₂ or 10% O₂, balance N₂) elevated basal cAMP levels (100% O₂ media) in proportion to the stimulus intesity. Similar experiments using nodose sensory ganglia showed that low O₂ stimulation did not alter cAMP levels in this non-chemosensory tissue. However, the adenylate cyclase (AC) activator, forskolin (10 μM), evoked large increases in the cyclic nucleotide content in both carotid bodies and nodose ganglia. After chronic (10 days) CSN denervation or synpathectomy, the basal levels of cAMP in the carotid body were elevated; the cAMP response to low O₂ media (stimulus minus control) was increased after CSN denervation but remained unaltered after sympathectomy. The effects of zero Ca²⁺ media on cAMP generation was examined in order to assess whether feedback from released neurotransmitters acting on known (presynaptic) type I cell receptors could have contributed to the observed changes in cAMP. Basal levels of cAMP were increased 2.8-fold, and the response to hypoxic stimulation was elevated 5-fold, in the absence of extracellular Ca²⁺. Forskolin (10 μM) did not alter basal release of [³H]-catecholamines ([³H]CA: synthesized from [³H]tyrosine, or resting CSN discharge; however, stimulus-evoked [³H]CA release and CSN discharge were potentiated in the presence of forskolin. This increased release was primarily due to enhanced efflux of dopamine (DA). At increasing stimulus strengths, however, the relative effect of forskolin on [³H]CA release was diminished. The data suggest that the chemoreceptor type I cells in the carotid body generate cAMP in their transductive response to hypoxia, but that the net levels of cAMP in the tissue are also regulated by both feedback actions of released neurotransmitters and by the sympathetic and sensory innervation to the organ. The effects of forskolin on [³H]CA release and CSN activity, combined with the finding that hypoxia increases the cAMP content of the carotid body, suggest the immediate invlovement of this classical second messenger in chemotransduction and chemotransmission of natural carotid body stimuli.     

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.     

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).     

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.     

Influence of cations on the electrical activity of neuroblastoma × glioma hybrid cells

Electrical excitability is one of the various neuronal properties of neuroblastoma × glioma hybrid cells. At a Ca²⁺ concentration of 1.8mM the action potential is inhibited by tetrodotoxin, suggesting that the inward current is carried by Na⁺ ions. In contrast, at a Ca²⁺ concentration of 20–36mM and even in the absence of Na⁺, spikes (sometimes repetitive) with strong hyperpolarizing afterpotential occur, which are no longer affected by tetrodotoxin. They are, however, blocked by antagonists of Ca²⁺ like La³⁺, Co²⁺, Mn²⁺, and the synthetic compounds D-600 and BAY a-1040. This seems to indicate that at high concentrations of Ca²⁺, the inward current of the action potential is essentially carried by Ca²⁺. Sr²⁺, but not Mg²⁺ can effectively substitute for Ca²⁺. It shows down the time course of the action potential. Ba²⁺ depolarizes the membrane gradually. If Ca²⁺ is also present, Ba²⁺ causes a reduced depolarization and spontaneous action potentials with no hyperpolarizing after-potential are observed.     

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.     

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.     

Prejunctional modulation of nitroxidergic nerve function in canine cerebral arteries

Modulation by acetylcholine, VIP, clonidine, ω-conotoxin and Mg²⁺ of the relaxant response to electrical and chemical stimulations of nitroxidergic nerves, in which nitric oxide (NO) acts as a neurotransmitter, was investigated in isolated canine cerebral arteries. Acetylcholine attenuated the response, the inhibition being reversed by atropine; however, physostigmine failed to reduce the response. VIP in submaximal doses did not alter the neurally induced relaxation. The same was true with clonidine, morphine and naloxane. Treatment with ω-conotoxin depressed the response to electrical nerve stimulation but did not influence the nicotine-induced relaxation. Mg²⁺ inhibited the relaxation caused by nerve stimulation and Ca²⁺ reversed the response. It is concluded that activation of prejunctional muscarinic receptors seems to inhibit the synthesis or release of NO from nerve terminals but endogenous acetylcholine from cholinergic nerve does not play a role in inhibiting the nitroxidergic nerve function. Prejunctional VIP, ₂ adrenergic and opioid receptors are not likely to participate in the regulation of nerve function. Ca²⁺ responsible for NO synthase activation would be introduced into nerve terminals via N-type Ca²⁺ channels when electrically stimulated and via non-N-, non-L-type channels when stimulated by nicotine. Mg²⁺ and Ca²⁺ counteract in the neurally induced relaxation, although the underlying mechanism was not determined.     

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.     

Chronic depolarization induced by veratridine increases the survival of rat retinal ganglion cells ‘in vitro’

During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na⁺ channels opened) induces an increase in neuronal survival. In the present work we show that 3 μM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 μM amiloride (an inhibitor of Ca²⁺ channels), 25 μM benzamil (an inhibitor of Na⁺ channels), 30 μM dantrolene and 7.5 μM caffeine (both inhibitors of Ca²⁺ release from the endoplasmatic reticulum) and 10 μM BAPTA-AM (an intracellular Ca²⁺ chelator). However, 5 μM nifedipine (a selective inhibitor of voltage-dependent -type Ca²⁺ channels) and 100 μM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 μM genistein (an inhibitor of tyrosine kinase enzymes), 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 μM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na⁺ influx, intracellular Ca²⁺ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.     

Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages

The effects of the opioid peptide dynorphin A (DynA) on phagocytosis in peritoneal macrophages was examined by flow cytometry (FCM). DynA enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), β-neo-endorphin (βNeo-End), DynA(9–17) and DynA(13–17) had no such activity, -Neo-endorphin ( Neo-End), dynorphin B (DynB), DynA(l–13) and DynA(6–17) enhanced phagocytosis less effectively than DynA. Naloxone did not inhibit the enhancement of phagocytosis induced by DynA. Unstimulated control phagocytosis was partially suppressed in Ca²⁺-free EGTA-containing solution and even in this solution DynA enhanced phagocytosis. However, the enhancement by DynA was suppressed in EGTA- and BAPTA-AM-containing Ca²⁺-free solution. The present study showed that enhancement of phagocytosis by DynA was independent of extracellular Ca²⁺ ([Ca²⁺]_o) and dependent on intracellular Ca²⁺ ([Ca²⁺]_i). The present results support DynA being one of the mediators from the nervous system that modulates the immune system.     

Properties of PAF-acether-induced platelet aggregation and secretion. Studies in gel-filtered human platelets

Human platelets rapidly lose their responsiveness to PAF-acether after blood collection. We collected blood from fasting donors and prepared gel-filtered platelets that remained responsive to PAF-acether for about 6 hours. Log-dose response studies showed bi-phasic aggregation between 20 and 100 nM PAF-acether with secretion of dense-, - and lysosomal granule contents during the second wave of aggregation. Between 0.2 and 10 nM PAF-acether aggregation was weak and no secretion occurred whereas 300 nM PAF-acether or more induced maximal aggregation and secretion. Secretion, however, was never more than 70, 55, and 30% of maximal secretable amount of 5HT, βTG and βN, respectively. Aggregation and secretion were enhanced by fibrinogen (optimal concentration 0.3–0.7 g.l⁻¹), required Ca²⁺ or Mg²⁺ but were inhibited when Mg²⁺ or Ca²⁺ were present at a concentration of 2 mM or more. These date show that human platelets are almost equally sensitive to PAF-acether as rabbit platelets, and respond with incomplete secretion of dense-, - and lysosomal granule contents.     

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.     

Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn²⁺, verapamil (40 μM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg²⁺, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca²⁺ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca²⁺ (nominally Ca²⁺-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn²⁺ and Mg²⁺, as well as Ca²⁺, enter the nerve terminal through Ca²⁺ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca²⁺ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na⁺ channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.     

The neuroleptic-like peptide desenkephalin-γ-endorphin does not antagonize the dopamine receptor agonist-induced inhibition of the release of [H]dopamine from rat nucleus accumbens slices in vitro

In rats, the non-opioid β-endorphin (βE) fragment desenkephalin-γ-endorphin (DEγE, βE_6–17) antagonizes the hypomotility induced by a small dose of dopamine (DA) receptor agonists. It has been suggested that DEγE might act in this respect by a direct or indirect blockade of presynaptically located DA receptors in the nucleus accumbens, thereby causing an increase of DA release. Therefore in the present study the effect of DEγE was examined on DA receptor agonist-induced inhibition of the electrically evoked release of previously accumulated [³H]DA from rat nucleus accumbens slices in vitro. The DA receptor agonists apomorphine, LY 171555 andn,n-di-n-propyl-7-hydroxy-2-aminotetralin (DP-7-AT) inhibited in a concentration-dependent manner the electrically evoked release of [³H]DA. The selective D₂ receptor antagonist (−)-sulpiride blocked the effects of apomorphine, corroborating that the DA receptor involved is of a D₂ type. DEγE was tested at several concentrations (10⁻⁹–10⁻⁶) and under various experimental conditions. DEγE, by itself, did not affect either the electrically stimulated or the basal release of [³H]DA. The inhibiting effect of DA receptor agonists was slightly reduced by DEγE, but this effect was present in some experiments only. It is concluded that DEγE does not function as an antagonist for the DA receptor mediating DA release and that the interaction observed in behavioural experiments between DA agonists and DEγE does not occur at the level of this receptor.     

Effects of d-aspartate on excitatory amino acid-induced release of [H]GABA from goldfish retina

The rate of release of [³H]GABA from isolated intact goldfish retinas was studied. Release of [³H]GABA is markedly stimulated by the inclusion in the incubation medium of the photoreceptor neurotransmitter candidates l-glutamate (l-Glu) and l-aspartate (l-Asp), and the glutamate analogs, kainate and quisqualate. At micromolar concentrations, kainate and quisqualate are effective releasers of [³H]GABA, whereas millimolar concentrations of l-Glu and l-Asp are required to release comparable amounts of [³H]GABA. The d-isomers of aspartate (d-Asp) and glutamate (d-Glu) are able to release [³H]GABA, but only when applied at high concentrations (3–30 mM). In the presence of 5 mM d-Asp, the effect of l-Glu in releasing [³H]GABA was markedly potentiated. This dose-response curve of l-Glu was shifted to the left in the presence of d-Asp, although the maximal amount of release was unchanged. d-Asp at 5 mM only slightly increased the GABA release induced by quisqualate, and it did not increase the GABA release induced by kainate. Finally, low concentrations of l-Asp were potentiated by d-Asp, but higher concentrations of l-Asp (3–10 mM) were clearly inhibited by this agent. This biphasic effect of d-Asp on l-Asp-induced release of [³H]GABA is a possible explanation for previously conflicting reports of d-Asp's effect on l-Asp action^2,8,29. Our data suggest that d-Asp has both pre- and postsynaptic sites of action.     

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.

Phenytoin and transmitter release at the neuromuscular junction of the frog

The effects of phenytoin (diphenylhydantoin, DPH) on transmitter release were studied at the frog neuromuscular junction. It was found that in Ringer's solutions containing a normal concentration of Ca²⁺ ions, DPH (1−2 × 10⁻⁴ M) depresses neurally evoked transmitter release, whereas in Ca²⁺-deficient Ringer's solutions it produces an increase in evoked release. Spontaneous transmitter liberation is augmented by DPH under all the above conditions. An abrupt disappearance of the evoked response occasionally occured with stimulation at 0.5 Hz, but a normal response could be elicited by a second stimulus delivered shortly after the first. At 100–200 Hz, DPH regularly induced a partial block in synaptic transmission. In8mM MgCl₂, this phenomenon appeared at 50 Hz and developed into a total neuromuscular blockade.     

Development of GABAA receptors on medial septum/diagonal band (MS/DB) neurons after postnatal ethanol exposure

The impact of ‘binge-like' ethanol exposure on postnatal days (PD) 4–9 was examined on development of γ-aminobutyric acid type A receptors (GABA_AR) during the first month of life in the rat. Whole-cell patch-clamp recordings in acutely isolated medial septum/diagonal band (MS/DB) neurons were used to define effects of rapidly applied ethanol and other allosteric modulators on bicuculline-sensitive GABA currents. Three age groups were examined including ‘pups' (PD 4–10), ‘juveniles' (PD 11–16) and ‘young adults' (PD 25–35). In untreated neurons, maximum responses to GABA and the apparent GABA EC₅₀ increased 2-fold during the first month of life. Potentiation of GABA responses by pentobarbital, midazolam, and loreclezole all increased with age, while Zn²⁺ inhibition declined. Initial inhibition by ethanol switched to potentiation of GABA responses during this time. In vivo, binge-like ethanol treatment (4.5 g kg⁻¹ day⁻¹ divided into two doses, 2 h apart on PD 4–9) reduced both the GABA maximal response and GABA EC₅₀ measured on PD 11–16. These measures returned to control levels by PD 25–35. After binge-like postnatal ethanol exposure, age-dependent loss of Zn²⁺ inhibition of GABA responses was increased, while potentiating actions of in vitro ethanol were blocked. GABA_AR modulation by other drugs was unaffected. These data suggest that early postnatal ethanol exposure disrupts the expected developmental pattern of GABA_AR function in MS/DB neurons, an action that could contribute to neurobehavioral deficits associated with the fetal alcohol syndrome. Whether these changes are due to cellular damage, delayed gene expression or post-translational modification needs to be determined.