A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus.

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg(2+)-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg(2+)-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Synaptotagmins in membrane traffic: which vesicles do the tagmins tag?

The aim of this review is to give a broad picture of what is actually known about the synaptotagmin family. Synaptotagmin I is an abundant synaptic vesicle and secretory granule protein in neurons and endocrine cells which plays a key role in Ca(2+)-induced exocytosis. It belongs to the large family of C2 domain-proteins as it contains two internal repeats that have homology to the C2 domain of protein kinase C. Eleven synaptotagmin genes have been described in rat and mouse. Except for synaptotagmin I, and by analogy synaptotagmin II, the functions of these proteins are unknown. In this review we focus on data obtained on the various isoforms without exhaustively discussing the role of synaptotagmin I in neurotransmission. Numerous in vitro interactions of synaptotagmin I with key components of the exocytosis-endocytosis machinery have been reported. Additional data concerning the other synaptotagmins are now becoming available and are reviewed here. Only interactions which have been described for several synaptotagmins, are mentioned. It is unlikely that a single isoform displays all of these potential interactions in vivo and probably the subcellular distribution of the protein will favor some of them and preclude others. Therefore, to discuss the putative role of the various synaptotagmins we have examined in detail published data concerning their localization.

Synaptotagmin I and IV define distinct populations of neuronal transport vesicles.

Mammalian synaptotagmins constitute a multigene family of at least 11 membrane proteins. We have characterized synaptotagmin IV using antibodies directed against the C2A domain of the protein. Antibodies reacted specifically with a protein band that migrated as a 41-44 kDa doublet. Synaptotagmin IV expression was regulated throughout development. A strong decrease in the amount detected by Western blotting occurred between postnatal day 5 and adulthood, in agreement with studies on the expression of synaptotagmin IV transcripts. In subcellular fractionation, synaptotagmin IV was not detected in the synaptic vesicle-enriched fraction. Immunofluorescence microscopy was concordant with this finding. In 6-day-old rat cerebellum and cultured hippocampal neurons the subcellular distribution of synaptotagmin IV was clearly different from that of synaptotagmin I. Synaptotagmin IV displayed a punctate non-polarized distribution on neuronal extensions, whereas synaptotagmin I staining was essentially synaptic. Synaptotagmin IV staining was also observed in the soma in strong perinuclear fluorescent puncta superimposed on that of Golgi/TGN markers. Furthermore, synaptotagmin IV was seen in the proximal part of the growth cone domain and not in the microfilament-rich region which includes filopodia. Co-localizations with the adhesion molecules vinculin and zyxin at the proximal part of growth cones were observed. Synaptotagmin IV may thus be involved in the regulation of specific membrane-trafficking pathways during brain development.

Gamma-Hydroxybutyrate inhibits excitatory postsynaptic potentials in rat hippocampal slices.

Gamma-Hydroxybutyrate (GHB) has been shown to mimic different central actions of ethanol, to suppress alcohol withdrawal syndrome, and to reduce alcohol consumption both in rats and in humans. The aim of the present study was to determine if GHB shared with alcohol the ability to inhibit glutamate action at both NMDA and AMPA/kainate receptors. The NMDA or the AMPA/kainate receptors-mediated postsynaptic potentials were evoked in CA1 pyramidal neurons by stimulation of Schaffer-collateral commissural fibers in the presence of CGP 35348, bicuculline to block the GABA(B) and GABA(A) receptors, and 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 30 microM DL-2-amino-5-phosphonovalerate (d-APV) to block AMPA/kainate or NMDA receptors, respectively. GHB (600 microM) produced a depression of both NMDA and AMPA/kainate receptors-mediated excitatory postsynaptic potentials with recovery on washout. The GHB receptors antagonist, NCS-382, at the concentration of 500 microM had no effect per se on these responses but prevented the depressant effect of GHB (600 microM) on the NMDA and AMPA/kainate-mediated responses. In the paired-pulse experiments, GHB (600 microM) depressed the amplitude of the first and the second evoked AMPA/kainate excitatory postsynaptic potentials, and significantly increased the paired-pulse facilitation (PPF). These results suggest that GHB inhibits excitatory synaptic transmission at Schaffer-collateral commissural-pyramidal neurons synapses by decreasing the probability of release of glutamate.

Modulation of mice anxiety in response to cat odor as a consequence of predators diet.

The effectiveness of predator odours as repellents was assessed, and the behavioral antipredatory responses were characterized. Mice had free access to an unfamiliar runway containing different olfactory stimuli: modelling clay, or feces of a cat subjected either to a vegetarian or a carnivorous diet. The first experiment revealed various indices of a spontaneous behavioral pattern that included exploratory activity, different kinds of emotionality, and a range of active or passive defensive reactions until the appearance of absence of risk assessment strictly related to presence or absence of anxiety. These reactions differ with larger responses to feces resulting from a carnivorous as opposed to vegetarian diets. In the second experiment, chlordiazepoxide (0, 2.5, 5, or 7.5 mg/kg) had a dose-related anxiolytic effect on exploration in mice of both vegetarian and carnivorous groups but could not totally reverse the strong anxiogenic effect of carnivorous stimulus on defensive mechanisms. These differences are related to the nature of the mammalian cues. This paradigm may be a fear-motivated model of animal anxiety.

Acamprosate enhances N-methyl-D-apartate receptor-mediated neurotransmission but inhibits presynaptic GABA(B) receptors in nucleus accumbens neurons.

Acamprosate (calcium acetylhomotaurine) is used therapeutically in Europe to reduce relapse in weaned alcoholics. However, the mechanisms of acamprosate action in the central nervous system are still obscure, although early studies suggested an action on GABA receptors. The nucleus accumbens (NAcc) is a brain region thought to underlie ethanol reinforcement. Recent studies from our laboratory have demonstrated that ethanol inhibits both N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamatergic synaptic transmission in the NAcc. In the present study, we used voltage- and current-clamp intracellular recording of NAcc core neurons in a slice preparation to examine acamprosate actions on resting membrane properties and pharmacologically isolated synaptic responses. We isolated NMDA and non-NMDA receptor-mediated excitatory postsynaptic potentials or currents (EPSP/Cs) with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovalerate (d-APV), respectively. Bicuculline was also included to block GABA(A) receptors. Superfusion of acamprosate (5, 50, and 300 microM) did not alter the resting membrane properties of NAcc neurons. However, 300 microM acamprosate significantly increased the NMDA receptor-mediated components of EPSP/Cs (NMDA-EPSP/Cs) with recovery on washout. In contrast, 300 microM acamprosate had no significant effect on the non-NMDA receptor component of the EPSP/Cs (non-NMDA-EPSP/Cs). To test acamprosate actions on the GABA system, we superfused 60 microM d-APV and 20 microM CNQX to block glutamatergic transmission and evoked monosynaptic GABA(A) receptor-mediated synaptic responses within the NAcc. Acamprosate (300 microM) did not change these monosynaptic GABA(A)-IPSCs. We also used a paired-pulse paradigm to test whether acamprosate could act on presynaptic GABA(B) autoreceptors, in the presence of d-APV and CNQX to block glutamatergic transmission. Like 0.5 microM CGP 34358 (a GABA[B] receptor blocker), acamprosate significantly decreased the paired-pulse inhibition (PPI) of GABA(A)-IPSCs at short interstimulus intervals (ISIs). Thus, acamprosate may concomitantly enhance NMDA-EPSP/Cs while blocking presynaptic GABA(B) receptor-mediated inhibition of GABA release. These results suggest that acamprosate's clinical efficacy in preventing relapse in weaned alcoholics could derive from its interactions with both the glutamatergic and GABAergic systems in the NAcc.

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.

Developmental regulation of synaptotagmin I, II, III, and IV mRNAs in the rat CNS.

Synaptotagmin I is an abundant synaptic vesicle protein that has an essential function in mediating Ca2+-triggered neurotransmitter release. We have analyzed the distribution of four neural synaptotagmin isoforms during postnatal development of the rat CNS by in situ hybridization. Synaptotagmin I, II, III, and IV genes have distinct patterns of spatiotemporal expression except in cerebellum granule cells, where the four transcripts were detected during the formation of parallel fiber/Purkinje cell synapses. Throughout development synaptotagmin I mRNAs were widely expressed in brain, whereas synaptotagmin II transcripts were predominant in spinal cord. At all stages synaptotagmin III mRNAs were expressed uniformly in most neurons examined, although at a low level. Synaptotagmin I, II, and III gene expressions mainly increased during development and persisted in adulthood, mirroring neuronal differentiation. Conversely, synaptotagmin IV transcripts were predominant during perinatal development in a heterogeneous population of neurons and subsequently were expressed uniformly at a low level. Intense labeling was observed in the hippocampal CA3 field and in the subiculum, but not in the CA1 field, of the newborn rat. In cerebral cortex, lamina-specific labeling was detected with a high expression in cell layer V. Only a small number of Purkinje cell clusters were labeled in the flocculus and paraflocculus of the cerebellum. Heterogeneous sets of neurons expressing synaptotagmin IV gene also were observed in spinal cord. We thus speculate that synaptotagmin IV may a play a role in the development of the mammalian nervous system.

Allosteric modulation by single enantiomers of a C3-chiral 1,4-benzodiazepine of the gamma aminobutyric acid type A receptor channel expressed in Xenopus oocytes.

Xenopus laevis oocytes injected with Poly(A)(+)-RNA isolated from neuronal tissue express membrane proteins peculiar to the origin of mRNA. The translation of gamma aminobutyric acid type A (GABAA) receptors has been shown by dose/ response behavior of GABA and the reversible blockade of the GABA-induced current by picrotoxin. This current was analyzed quantitatively under two electrode voltage-clamp conditions. This methodology has been applied for the first time to study the functional properties of the receptor as a function of the stereochemistry of the ligands. The (+)-S and (-)-R enantiomers of a water-soluble benzodiazepine derivative, 7-chloro-1,3-dihydro-3-hemisuccinyloxy-5-phenyl-1,4-benzodiazep in-2-one (OXHEM), obtained by preparative high performance liquid chromatographic (HPLC) resolution on chiral stationary phase, act as agonists in the in vitro modulation of the chloride channel. The (+)-S-OXHEM enantiomer was the more active.

Naloxone blocks long-term depression of excitatory transmission in rat CA1 hippocampus in vitro.

In rat hippocampal slices, high intensity tetanic stimulation (two 1 s trains of 100 Hz separated by 20 s, 3-5X intensity of the test stimulus) of the Schaffer collateral-commissural (SCC) fibers induced a long-term depression (LTD) of the negative field excitatory postsynaptic potentials (fEPSP) in stratum radiatum of the CA1 region. The initial slope of the fEPSP, evoked by a single test shock applied to the SCC fibers, was depressed for a period longer than 40 min following such high intensity tetanic stimulation to this fiber system. However, the same tetanic stimulation delivered at low (test) intensity induced long-term potentiation (LTP) of the fEPSPs. Thus, similar patterns of stimulation can induce either LTP or LTD, depending on whether low- or high-intensity tetanic stimuli are delivered. The LTD induced by high strength tetanic stimulation was clearly blocked by the opioid antagonist naloxone (1 microM); however, the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate (AP5; 50 microM) had no effect on the LTD. Our data suggest that the strong stimulation used for LTD induction may have activated other afferent fiber systems and/or local interneurons in addition to SCC fibers, such as the enkephalin-containing terminals of the perforant path (PP) projecting to the stratum lacunosum moleculare or opioid peptide-containing interneurons. Thus, the resulting release of endogenous opioid peptides could play a role in the cellular mechanisms involved in some forms of long-term synaptic depression.

Further pharmacological validation of the BALB/c neophobia in the free exploratory paradigm as an animal model of trait anxiety.

The present experiments were aimed at investigating the ability of established or putative anxiolytics to reduce the neophobia exhibited by BALB/c mice in the free exploratory paradigm. Results confirm the anxiolytic effects of the benzodiazepine receptor full agonist chlordiazepoxide (2.5-7.5 mg/kg), of meprobamate (15-60 mg/kg) and of ethanol (0.5-1.5 g/kg) and extend the pharmacological action of these compounds to a test situation devoid of anxiogenic components, that is to trait anxiety. The non-competitive NMDA antagonist MK 801 (0.04-0.16 mg/kg) elicited very similar behavioural effects. However, the alpha 2-adrenoceptor antagonists yohimbine (0.5-2 mg/kg) and idazoxan (0.3-2.7 mg/kg), the barbiturate pentobarbital (3.75-30 mg/kg), the mixed 5HT2 receptor antagonist ritanserin (0.25-4 mg/kg) and the D2 dopaminergic antagonist sulpiride (8-32 mg/kg) failed to decrease neophobia in BALB/c mice. The discussion focuses on the adequacy of this animal model of human pathology. The BALB/c neophobia may not model panic attacks because of the absence of worsening by the panic-provoking agent yohimbine and the lack of attenuation by CCK-B receptor antagonists. Because of its chronicity, this paradigm may model generalized anxiety, a pathology that has been suggested to overlap trait anxiety.

Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.

Modulation of electrically elicited blink reflex components by visual and acoustic prestimuli in man.

The effects of a prestimulus on the electrically elicited blink reflex components were investigated in 20 healthy subjects. In the first group of 10 subjects (warned group), electric shocks were delivered in isolation or preceded, at an interstimulus interval (ISI) of 0.1 s, 1 s, or 10 s, by a visual or acoustic warning stimulus. In the second group of 10 subjects (unwarned group), the electric shocks were delivered either in isolation or preceded, at the same ISI, by visual or acoustic stimuli having no warning value. The modulation of the three blink reflex components was then analysed. Compared to the baseline condition, the R1 oligosynaptic component was enhanced at 0.1 s and 1 s ISI, in the warned group with the visual prestimulus, but only at 0.1 s after a visual and acoustic prestimulus in the unwarned group. On the contrary, the polysynaptic responses showed a different course: R2 was significantly reduced at the 0.1 s interval in the warned group with both the prestimuli, and only with the visual prestimulus in the unwarned group. The R3 was inhibited at all three intervals with the visual prestimulus, and at the 0.1 s and 1 s with the acoustic one in the warned group, and only at 0.1 s in the unwarned group, both after visual and acoustic prestimuli. The decrement in R2 and R3 observed with the shortest interval was probably related to the prepulse inhibition of startle reflex. Furthermore, only R3 was still inhibited at longer intervals, when the sustained processes of attention may have influenced this component. Perhaps this combination of events represents, in the warned group, the best preparation for voluntary reflex reaction.

The beta-carboline derivative DMCM decreases gamma-aminobutyric acid responses and Ca(2+)-mediated K(+)-conductance in rat neocortical neurons in vitro.

Electrophysiological recordings from neurons of rat frontal neocortical slices have been used to investigate the action of the beta-carboline methyl-6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylate (DMCM), on responses to gamma-aminobutyric acid (GABA) and on the excitability of the neurons. Iontophoretic application of GABA close to the intracellularly recorded cells (resting membrane potential -74 +/- 0.9 mV) elicited a depolarization associated with a decrease of input resistance, mediated by GABAA receptors. Bath application of DMCM (0.1-1 microM) reduced these GABA responses decreasing the affinity of the receptors for GABA. This effect was blocked by the benzodiazepine receptor (BZR) antagonist ZK 93426 (1 microM). DMCM (0.1 microM) also decreased the hyperpolarization that followed a train of action potentials (AHP), mediated by Ca(2+)-dependent K+ conductance, and increased the duration of Ca(2+)-dependent action potentials recorded after blockade of Na+ and K+ conductances. Neither effect was blocked by BZR antagonists. These results indicate that DMCM increases the excitability of neurons not only by reducing the gain of the GABAA/BZR complex, but also by modulating intrinsic membrane mechanisms.

Isolated blunt liver trauma: is nonoperative treatment justified?

During the past 8 years 13 children with isolated blunt liver trauma were managed nonoperatively. All patients selected for this management were hemodynamically stable after initial resuscitation and were without signs of other associated intraabdominal injuries on ultrasonogram and/or computed tomography. Patients were observed in an intensive care unit for at least 48 hours with repeated clinical assessments, laboratory studies, and bed rest. One patient with type 3 injury was operated on 8 days after injury because of sudden intraperitoneal bleeding on ambulation. Five patients required blood transfusions of not more than 300 mL per patient. Laboratory values returned to normal from 7 to 21 days after injury. Resolution of hepatic injury on ultrasonogram took from 1 to 3 months. Complete bed rest was prescribed for at least 10 days depending on the type of injury, with restricted activities up to 3 months postinjury. No complications were seen in this series.

Change in neuronal excitability in hippocampal sections of rats isolated after the destruction of the medial septal area.

The influence of chronic destruction of the medial septal area (MSA) on focal potentials (FP) of field CA1 evoked by stimulation of Schaffer collaterals was studied in experiments on viable sections of the hippocampus of rats. The preparation, incubation, and pickup of FP of the sections of the control and experimental groups were carried out in identical conditions using traditional methods. The amplitude-temporal of population EPSP and population spikes, their interdependence, and their dependence on the strength of the stimulating current were assessed using automated data analysis. It was demonstrated that coagulation of the MSA is accompanied by an increase in the excitability of neuronal complexes of the lateral portion of field CA1 which is manifested by the generation of multiple rhythmic discharges and the development of spike activity of the cells at a lower intensity of the synaptic inflow. It is hypothesized that destruction of the MSA leads to a relative attenuation of inhibitory control in the neuronal systems of field CA1, the elements of which are depolarized with anomalous synchronicity and generate rhythmic impulse trains.

[Epileptiform activity in the rat hippocampal slices isolated after local lesion of the septum by ibotenic acid]. / Epileptiformnaia aktivnost' v srezakh gippokampa krys, izolirovannykh posle lokal'nogo povrezhdeniia septuma ibotenovoi kislotoi.

Extra- and intracellular responses were recorded in the CA3 region of hippocampal slices of rats 1-4 weeks after intraseptal injection of ibotenic acid. Then they were compared with those from unoperated controls. In the slices from operated animals spontaneous and evoked synchronous epileptic field discharges were preserved invariably associated with paroxysmal depolarization shifts (PDSs) and bursts of action potentials of the cells. Both the field discharges and PDSs in the CA3 had an "all-or-nothing" character. There were no consistent effects of septal lesion on the mean resting potential or input resistance of the CA3 pyramidal neurons. Histological examination revealed a cell loss in the dorsal septum, whereas cholinergic neurons of the medial septum were not significantly damaged. The results suggest that intrinsic septal circuitry is critically involved in synchronization of CA3 neurons and control of their excitability.

Long-term Potentiation of NMDA Receptor-mediated EPSP in Guinea-pig Hippocampal Slices.

Hippocampal slices from guinea-pigs were used to examine the long-term potentiation (LTP) of the N-methyl-d-aspartate (NMDA)-mediated excitatory postsynaptic potential (EPSP). Intracellular recordings were performed from CA1 pyramidal neurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 - 10 microM) and picrotoxin (50 microM). In these experimental conditions test stimuli applied at low frequency (0.1 Hz) to the Schaffer collateral - commissural pathway evoked a prolonged EPSP (150 - 200 ms). To obtain this CNQX-resistant EPSP, stimulus intensities had to be raised above the level required to evoke an EPSP of comparable amplitude in physiological solution. Tetanic stimulation (two trains of 100 Hz, 1 s every 20 s) led to a potentiation of the CNQX-resistant EPSP, and this potentiated response was abolished with d-(-)-2-amino-5-phosphonovaleric acid (50 microM). The potentiation of the NMDA receptor-mediated EPSP was more pronounced for strong than for weak test stimuli, and was suppressed when test EPSPs were evoked during membrane hyperpolarization. These results suggest that NMDA receptor-mediated responses can undergo LTP, and hence can contribute to the maintenance of LTP.