Modulating effect of anticonvulsive agents on Ca2+ dependent membrane fusion in cell-free model of neurosecretion

The effect of antiepileptic drug ethosuximide and sodium valproat on fusion of synaptic vesicles with synaptosomal plasma membranes was studied in cell-free system. It was shown that ethosuximide and sodium valproat increases the rate of Ca(2+)-dependent fusion reaction in vitro. We have found that convulsant drugs pentylenetetrazol and picrotoxin did not fuse membrane components of the model system. Ethosuximide- and sodium valproat-provoked fusion of synaptic vesicles and plasma membranes of synaptosomes were suppressed by convulsant drugs pentylenetetrazol and picrotoxin.     

Cytomorphological alterations of mossy fiber boutons of the hippocampus produced by 3-acetylpyridine,methoxypyridoxine, reserpine,and iproniazid

Summary The hippocampal mossy fiber boutons of the rabbit were studied with phase and electron microscopy. The injection of 3-acetylpyridine, methoxypyridoxine, and reserpine diminishes the conspicuous osmiophilic density of the mossy fiber boutons in comparison to similar regions from nontreated animals as observable in phase microscopy. However, electron micrographs of the same samples show little or no diminution in the number of those synaptic vesicles consisting of a clear homogeneous center (Type I). Treatment with monoamine liberator, reserpine, results in the same cytomorphological appearance of the boutons as with convulsant agents. The number of synaptic dense-core vesicles (Type II) is not altered after treatment with the convulsant agents or reserpine.A certain extra-vesicular substance and a certain granular component of the vesicular membranes of Type I vesicles is progressively reduced after treatment with all of these drugs. It is suggested that this accounts for the decreased density by phase microscopy.The monoamine oxidase inhibitor, iproniazid, increases the density of the extra-vesicular substance as well as the particles attached to the vesicular membranes of Type I vesicles.It is suggested that these osmiophilic particles contain the biogenic monoamines (in this instance probably serotonin and/or histamine) and that in acute experiments the liberation of these neurotransmitters is not related to a disappearence of dense-core vesicles concommitant with a depletion of neurotransmitters but is from particles in the extra-vesicular substance and the granular component of the vesicular of the Type I vesicles.Furthermore, the functional role of zinc in the synaptic vesicles of mossy fiber boutons of the hippocampus is discussed in regard to a possible storage mechanism for biogenic monoamines.This study was partly supported by USPHS Grant 5 P10 ESOO159.     

Differential Effects of Tetanus Toxin on Inhibitory and Excitatory Neurotransmitter Release from Mammalian Spinal Cord Cells in Culture

The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.     

Quinolinic Acid-induced Seizures Stimulate Glutamate Uptake into Synaptic Vesicles from Rat Brain: Effects Prevented by Guanine-based Purines

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5′-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80–100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.     

An analysis of the in vivo interactions between chemical convulsants and anticonvulsants

The interactions between pentylenetetrazol (PTZ), picrotoxin (PIC), or bicuculline (BIC) and diazepam, phenobarbital, or valproate were subjected to Schild plot analysis. Log dose-probit response curves for minimal clonic seizures were determined for three chemical convulsants in the absence and in the presence of various concentrations of three anticonvulsants. The calculated median convulsant doses were subjected to Schild plot analysis and the pA2 values determined. A comparison of the pA2 values for the various convulsant/anticonvulsant combinations suggested the following conclusions: (i) the sequence of events leading to minimal clonic seizures evoked by PTZ or PIC involves a common receptor, (ii) BIC acts through a different receptor, and (iii) Schild plot analysis of the antagonism between convulsant and anticonvulsant is in agreement with their antagonism in vitro studies. Thus, Schild plot analysis can be useful in the evaluation of anticonvulsant activity in vivo and may offer some insight into the potential clinical usefulness of anticonvulsant substances.     

Comparison of the distribution of convulsant/barbiturate and benzodiazepine receptors using light microscopic autoradiography

Some convulsant drugs elicit CNS excitation by blocking neuronal activity at GABAergic synapses whereas depressant compounds may result in the enhancement of GABAergic transmission. These effects are thought to involve drug actions at a multireceptor complex involving a benzodiazepine receptor, GABA receptor, picrotoxin receptor and a chloride ionophore. A radiolabeled convulsant, [35S]t-butylbicyclophosphorothionate [( 35S]-TBT) has been developed and used to characterize the binding to the "picrotoxin" or convulsant/barbiturate site. The microscopic distribution of the convulsant/barbiturate sites are reported in this communication, as demonstrated by receptor autoradiography after labeling tissue sections with [35S]-TBT. Comparison of the distribution of these sites with those of the benzodiazepine receptors show a close regional correlation in many areas. The convulsant/barbiturate sites and the benzodiazepine receptors, however, are unevenly distributed in the rat cerebellum and exist in separate lamina.     

Interaction of barbiturates with dihydropicrotoxinin binding sites related to the GABA receptor-ionophore system.

Barbiturate drugs of diverse chemical structure inhibited the binding of [³H] α-dihydropicrotoxinin to rat brain membranes. This biologically active analoque of picrotoxin labels membrane sites related to the convulsant action of these drugs in inhibiting GABA postsynaptic receptor-ionophore function at a site distinct from the GABA receptor. Depressant barbiturates such as pentobarbital inhibited dihydropicrotoxinin binding competitively at therapeutic concentrations (IC₅₀ = 50 μM) whereas the drug does not alter GABA receptors, uptake, or release at this concentration. Antiepileptics such as phenobarbital (IC₅₀=400 μM), were weaker inhibitors of binding. Convulsant barbiturates, however, such as dimethylbutylbarbiturate (IC₅₀=0.05 μM) and cyclohexylidene-ethyl barbiturate (IC₅₀=0.7 μM), were potent inhibitors. The displacement of radioactive dihydropicrotoxinin binding by the convulsant barbiturates had different slopes and Hill numnbers (0.4) compared to displacement by depressant barbiturates and picrotoxinin itself (Hill numbers = 1.0), indicating heterogeneity of binding sites or negative cooperativity. These potent intractions of barbiturates with dihydropicrotoxinin binding sites are consistent with neurophysiological evidence that depressant or convulsant action of barbiturates may involve modulation of CNS inhibitory synaptic transmission at the level of the postsynaptic GABA receptor-ionophores.     

Dissociation of [35S]t-Butylbicyclophosphorothionate Binding Differentiates Convulsant and Depressant Drugs that Modulate GABAergic Transmission

The dissociation of [35S]t-butylbicyclophosphorothionate ([35S]TBPT) from binding sites on membranes from rat cerebral cortex, after addition of saturating concentrations of convulsant and depressant drugs, was studied. The addition of unlabeled TBPT, picrotoxinin, or pentamethylenetetrazol resulted in dissociation patterns that were monophasic and not distinguishable, suggesting that these convulsants bind competitively to the same (convulsant) sites. In contrast, gamma-aminobutyric acid (GABA) greatly facilitated [35S]TBPT dissociation by binding allosterically to the GABA recognition site of the receptor-ionophore complex. TBPT dissociation was similarly accelerated by the depressants etazolate, (+)-etomidate, and barbiturates. The convulsant and depressant S(+) and R(-) stereoisomers of N-methyl-5-phenyl-5-propyl-barbituric acid displayed large stereoselectivity in the acceleration of TBPT dissociation. These results suggest that depressants bind to sites different from the convulsant sites of the allosteric GABA receptor complex, or the binding of depressants to the same population of sites elicits negative cooperativity and dissociates the convulsants.     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.     

Citrate synthase activity increases in homogenates of the cerebral cortex from rats treated with the convulsant 3-mercaptopropionic acid

The administration of the convulsant 3-mercaptopropionic acid (150 mg/kg i.p.) increased the respiratory capacity of mitochondria isolated from rat cerebral cortex. This increase was observed when pyruvate-malate were used as substrates, but oxygen uptake was not activated with succinate, glutamate-malate or α-ketoglutarate. Citrate synthase activity in rat brain homogenates increased (about 40%) after the administration of convulsant doses of 3-mercaptopropionic acid (50 and 150 mg/kg). This effect was found after seizures but not during seizures or after a dose that did not produce convulsions (20 mg/kg). The enhancement of citrate synthase activity was observed at various oxaloacetate concentrations, with an increase in V_max. The enhancement was still evident after incubation and removal of the soluble phase by centrifugation, but not after freeze-thawing.     

Changes in Polyamine Levels in Rat Brain After Systemic Kainic Acid Administration: Relationship to Convulsant Activity and Brain Damage

We have examined the effects of systemic kainic acid (KA) administration (9 mg/kg, i.p.) on rat behavior, brain damage, and polyamine levels and the action of the specific ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) on these effects. KA elicited convulsant activity in 63% of the animals. In the acute convulsant phase (1-3 h after KA), a rapid decline (-39% at 3 h) of spermidine content in frontal cortex was found. After the acute convulsant phase, levels of hippocampal spermidine and spermine were reduced (-70 and -66%, respectively, at 8 h). A dramatic increase of putrescine content (68.1, 1,382, and 336% at 8 h, 24 h, and 9 days, respectively, after KA) was found, associated with histological signs of cortical brain damage (ischemia and necrosis). There was a close relationship between the concentration of putrescine and signs of delayed toxicity (body weight losses) 24 h and 9 days after KA. DFMO partially antagonized the convulsant activity and reduced the increased putrescine levels to approximately 50% of values in KA-treated animals at 24 h but did not change the pattern of histological damage. The role of polyamines in the early and late phases of KA-induced neurotoxicity is discussed.     

Structure and Location of a Gabaa Receptor Complex in the Central Nervous System

Abstract

GABA-gated chloride channels in the central nervous system contain a regulatory site, the benzodiazepine receptor, through which drugs can modulate the efficiency of GABAergic synaptic transmission and thereby affect the degree of anxiety, muscle tension, vigilance and convulsions. The biochemical analysis of the purified receptor complex with monoclonal antibodies shows a heterooligomeric composition of two glycosylated subunits (α,β). The immunoprecipitated complex contains the binding sites for GABA, benzodiazepines and the convulsant TBPS. The receptor complex was located, immuno-cytochemically, in synapses of brain regions rich in GABAergic nerve terminals.     

Properties of synaptic transmission at the neuromuscular junction of the squid, Loligo opalescens

1. Spontaneous and evoked synaptic activity were recorded from the muscles of squid fin and mantle. These spontaneous synaptic potentials were large (up to 30 mV) and pleomorphic. Their amplitudes were not normally distributed, nor did they appear to be clustered in integral multiples of some "unit" event size. 2. Electrical stimulation of the nerve resulted in muscle twitches when the bath calcium concentration was a third normal or higher. The frequency of spontaneous synaptic events was unaffected by low calcium. 3. The large size of spontaneous events may mean that the synchronized release of only a few such "quanta" are sufficient to cause muscle action potentials and contraction. 4. The shapes of spontaneous events correlated poorly with their amplitudes, which is consistent with release from multiple synaptic sites with distinct properties.     

A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells

Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.     

CHANGES IN THE FINE STRUCTURE OF THE NEUROMUSCULAR JUNCTION OF THE FROG CAUSED BY BLACK WIDOW SPIDER VENOM

Application of black widow spider venom to the neuromuscular junction of the frog causes an increase in the frequency of miniature end-plate potentials (min.e.p.p.) and a reduction in the number of synaptic vesicles in the nerve terminal. Shortly after the increase in min.e.p.p. frequency, the presynaptic membrane of the nerve terminal has either infolded or "lifted." Examination of these infoldings or lifts reveals synaptic vesicles in various stages of fusion with the presynaptic membrane. After the supply of synaptic vesicles has been exhausted, the presynaptic membrane returns to its original position directly opposite the end-plate membrane. The terminal contains all of its usual components with the exception of the synaptic vesicles. The only other alteration of the structures making up the neuromuscular junction occurs in the axon leading to the terminal. Instead of completely filling out its Schwann sheath, the axon has pulled away and its axoplasm appears to be denser than the control. The relation of these events to the vesicle hypothesis is discussed.     

The formation of synapses in the central nervous system

Interneuronal synapses are specialized contact zones formed between the transmitting pole of one neuron, usually an axon, and the receptive pole of another nerve cell, usually a dendritic process or the soma. The formation of these synaptic contacts is the result of cellular events related to neurite elongation, the establishment of polarity, axon guidance, and target recognition. A series of morphological rearrangements takes place once synaptic targets establish their initial contact. These changes include the clustering of synaptic vesicles in the presynaptic element and the formation of a specialized area capable of signal transduction at the postsynaptic target. The present review discusses the role of different synaptic proteins in the cellular events leading to the formation of synapses among neurons in the central nervous system.     

γ-Aminobutyric Acid Type A (GABAA) Receptor α Subunits Play a Direct Role in Synaptic Versus Extrasynaptic Targeting

GABA(A) receptors (GABA(A)-Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of γ2 and δ subunits in synaptic versus extrasynaptic targeting of GABA(A)-Rs. Here, we demonstrate differential function of α2 and α6 subunits in guiding the localization of GABA(A)-Rs. To study the targeting of specific subtypes of GABA(A)-Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA(A)-R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by α2β3γ2 receptors were very fast (rise time ～2 ms), whereas events mediated by α6β3δ receptors were very slow (rise time ～20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by α6β3 or α6β3γ2 receptors were significantly slower than those mediated by α2β3 or α2β3γ2 receptors, suggesting a differential role of α subunit in receptor targeting. This was confirmed by differential targeting of the same δ-γ2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assembled with the α2 or α6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of α6 and δ subunits brought α6β3δ receptors closer to synaptic sites. Therefore, the α subunits, together with the γ2 and δ subunits, play a critical role in governing synaptic versus extrasynaptic targeting of GABA(A)-Rs, possibly through differential interactions with gephyrin.     

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.     

Mykhailo F. Shuba

The spike activity of neurons of the sensorimotor cortex was analyzed in cats before, during, and after iontophoretic application of substances influencing synaptic transmission. It was shown that, in addition to glutamate ionotropic receptors, glutamate metabotropic receptors, as well as adrenergic and dopaminergic receptor systems, are involved in plastic rearrangements of synaptic connections among neocortical neurons. The pattern of aftereffects allows us to suppose that potentiation of synaptic events during conditioned reflex-related learning can be maintained for 10 to 20 min, an interval sufficient for consolidation of a memory track.