Phorbol esters are hypothesised to produce a protein kinase C (PKC)-dependent increase in the probability of transmitter release
via two mechanisms: facilitation of vesicle fusion or increases in ...synaptic vesicle number and replenishment. We used a combination
of electrophysiology and computer simulation to distinguish these possibilities. We constructed a stochastic model of the
presynaptic contacts between a pair of hippocampal pyramidal cells that used biologically realistic processes and was constrained
by electrophysiological data. The model reproduced faithfully several forms of short-term synaptic plasticity, including short-term
synaptic depression (STD), and allowed us to manipulate several experimentally inaccessible processes. Simulation of an increase
in the size of the readily releasable vesicle pool and the time of vesicle replenishment decreased STD, whereas simulation
of a facilitation of vesicle fusion downstream of Ca 2+ influx enhanced STD. Because activation of protein kinase C with phorbol ester enhanced STD of EPSCs in rat hippocampal slice
cultures, we conclude that an increase in the sensitivity of the release process for Ca 2+ underlies the potentiation of neurotransmitter release by PKC.
Alpha-latrotoxin (LTX) causes massive release of neurotransmitters via a complex mechanism involving (i) activation of receptor(s) and (ii) toxin insertion into the plasma membrane with (iii) ...subsequent pore formation. Using cryo-electron microscopy, electrophysiological and biochemical methods, we demonstrate here that the recently described toxin mutant (LTXN4C) is unable to insert into membranes and form pores due to its inability to assemble into tetramers. However, this mutant still binds to major LTX receptors (latrophilin and neurexin) and causes strong transmitter exocytosis in synaptosomes, hippocampal slice cultures, neuromuscular junctions, and chromaffin cells. In the absence of mutant incorporation into the membrane, receptor activation must be the only mechanism by which LTXN4C triggers exocytosis. An interesting feature of this receptor-mediated transmitter release is its dependence on extracellular Ca2+. Because Ca2+ is also strictly required for LTX interaction with neurexin, the latter might be the only receptor mediating the LTXN4C action. To test this hypothesis, we used conditions (substitution of Ca2+ in the medium with Sr2+) under which LTXN4C does not bind to any member of the neurexin family but still interacts with latrophilin. We show that, in all the systems tested, Sr2+ fully replaces Ca2+ in supporting the stimulatory effect of LTXN4C. These results indicate that LTXN4C can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.
In hippocampal pyramidal cells (HPCs), Dopamine (DA) application (1 microM) produced, in 50% of recorded cells, an hyperpolarization of the resting membrane potential (r.m.p.) and an increase of the ...afterhyperpolarization (AHP) amplitude and duration in 79% of recorded cells. DA-induced effects on both the r.m.p. and AHP were mimicked by bath application of a D-1 selective agonist, SKF 38393 (20 microM). In addition, we have observed that a D-1 selective antagonist such as SCH 23390 (1 microM) abolished the action of both DA and SKF 38393. In contrast, the activation of D-2 receptors through LY 171555 (10 microns) produced, in 50% of cells, a depolarization of the r.m.p. and a depression of the AHP in 67% of recorded cells. These results suggest that the effects observed in hippocampal pyramidal neurons after DA application of micromolar concentration are mediated by D-1 subtype of receptors.
Miniature, gamma-aminobutyric acid A receptor mediated inhibitory postsynaptic currents (mIPSCs) were recorded from CA3 pyramidal cells in hippocampal slice cultures using whole-cell techniques in ...the presence of tetrodotoxin. The kinetics and amplitudes of the mIPSCs were analyzed with the aim of determining whether subclasses of events arising from distinct populations of presynaptic interneurons could be distinguished. Histograms of mIPSC amplitude, rise time constant, and decay time constant were all positively skewed, but discrete subsets of events could not be distinguished. The positive skew did not appear to result from electrotonic filtering of distal synaptic currents because there was no correlation among mIPSC amplitudes and the kinetic parameters. Analysis of the intervals between mIPSCs indicated that each event occurred independently. The analysis of spontaneous mIPSCs does not provide evidence of the innervation of pyramidal cells by heterogeneous interneurons.
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.
Acamprosate (calciumacetylhomotaurinate) is used therapeutically against relapse in weaned alcoholics. In the present study, the mechanism of action was investigated by making intracellular in vitro ...and extracellular in vivo recordings from rat neocortical neurons. Acamprosate (0.1-1 mM) added to the perfusion fluid in vitro reduced excitatory and inhibitory postsynaptic potentials and the depolarizing responses evoked by iontophoretic application of the excitatory amino acids, L-glutamate, L-aspartate, L-homocysteate and N-methyl-D-aspartate, but did not alter the responses to gamma-aminobutyric acid. Acamprosate decreased electrical excitability without apparently changing membrane potential, input resistance, afterhyperpolarization, or threshold and amplitude of the action potential. In vivo iontophoretic application of acamprosate reduced the extracellularly recorded unit activity elicited by iontophoretically applied L-glutamate, whereas spontaneous discharges remained unaffected. These data suggest that acamprosate reduces the postsynaptic efficacy of excitatory amino acid neurotransmitters and lowers neuronal excitability in the neocortex of the rat.
Objective To study the correlation between 2D and 3D uterine flow indexes and the presence or the absence of antinuclear antibodies (ANA) in women with unexplained recurrent miscarriage (uRM). ...Methods Fifty-two subjects (26 uRM and 26 control women) underwent 2D Doppler measurement of pulsatility index and resistance index of the uterine arteries in both the follicular and midluteal phase of the cycle. Additionally, 3D ultrasonography determination of vascularisation index, flow index, and vascularisation flow index was carried out with the aid of the VOCAL technique. Serum assay for the presence of ANA was performed in all women. Results Pulsatility index of ANA+ uRM women was higher than that of ANA- uRM women and control ANA+ and ANAwomen, both in the follicular and in the midluteal phase of the cycle. Vascularisation index in ANA- uRM women was significantly higher than that in ANA+ control women. Flow index in uRM ANA+ women was significantly lower than that of each of the other groups. Conclusion ANA might be involved in uRM by determining an impairment in uterine blood flow hemodynamic, particularly in uterine blood flow intensity and uterine artery impedance.
alpha -Latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (1) receptor binding-stimulation and (2) membrane pore formation. Here, we use the toxin mutant LTX ...super(N4C) to selectively study the receptor-mediated actions of LTX. LTX super(N4C) binds to both LTX receptors (latrophilin and neurexin) and greatly enhances the frequency of spontaneous and miniature EPSCs recorded from CA3 pyramidal neurons in hippocampal slice cultures. The effect of LTX super(N4C) is reversible and is not attenuated by La super(3+) that is known to block LTX pores. On the other hand, LTX super(N4C) action, which requires extracellular Ca super(2+), is inhibited by thapsigargin, a drug depleting intracellular Ca super(2+) stores, by 2-aminoethoxydiphenyl borate, a blocker of inositol(1,4,5)-trisphosphate-induced Ca super(2+) release, and by U73122, a phospholipase C inhibitor. Furthermore, measurements using a fluorescent Ca super(2+) indicator directly demonstrate that LTX super(N4C) increases presynaptic, but not dendritic, free Ca super(2+) concentration; this Ca super(2+) rise is blocked by thapsigargin, suggesting, together with electrophysiological data, that the receptor-mediated action of LTX super(N4C) involves mobilization of Ca super(2+) from intracellular stores. Finally, in contrast to wild-type LTX, which inhibits evoked synaptic transmission probably attributable to pore formation, LTX super(N4C) actually potentiates synaptic currents elicited by electrical stimulation of afferent fibers. We suggest that the mutant LTX super(N4C), lacking the ionophore-like activity of wild-type LTX, activates a presynaptic receptor and stimulates Ca super(2+) release from intracellular stores, leading to the enhancement of synaptic vesicle exocytosis.
Zolpidem is a hypnotic benzodiazepine site agonist with some γ‐aminobutyric acid (GABA)A receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of γ2 subunit ...(γ2F77I) point mutant mice. Analysis of forebrain GABAA receptor expression with immunocytochemistry, quantitative 3Hmuscimol and 35S t‐butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with 3Hflunitrazepam and 3Hmuscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous γ2I77/I77 and γ2F77/F77 mice. However, quantitative immunoblot analysis of γ2I77/I77 hippocampi showed some increased levels of γ2, α1, α4 and δ subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 µm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, γ2F77/F77) mice by ∼ 60%, and peak amplitude by ∼ 20% at 33–34 °C in vitro. The actions of zolpidem (100 nm or 1 µm) were substantially reduced in γ2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 µm) on carbachol‐induced oscillations in the CA3 area of γ2I77/I77 mice was significantly different compared with controls. Thus, the γ2F77I point mutation virtually abolished the actions of zolpidem on GABAA receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the γ2 subunit.
Zolpidem is a hypnotic benzodiazepine site agonist with some gamma -aminobutyric acid (GABA) sub(A) receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of ...gamma 2 subunit ( gamma 2F77I) point mutant mice. Analysis of forebrain GABA sub(A) receptor expression with immunocytochemistry, quantitative super(3)Hmuscimol and super(35)S t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with super(3)Hflunitrazepam and super(3)Hmuscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous gamma 2I77-I77 and gamma 2F77-F77 mice. However, quantitative immunoblot analysis of gamma 2I77-I77 hippocampi showed some increased levels of gamma 2, alpha 1, alpha 4 and delta subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 mu m) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL-6J, gamma 2F77-F77) mice by similar to 60%, and peak amplitude by similar to 20% at 33-34 degree C in vitro. The actions of zolpidem (100 nm or 1 mu m) were substantially reduced in gamma 2I77-I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens-alveus interneurons. At network level, the effect of zolpidem (10 mu m) on carbachol-induced oscillations in the CA3 area of gamma 2I77-I77 mice was significantly different compared with controls. Thus, the gamma 2F77I point mutation virtually abolished the actions of zolpidem on GABA sub(A) receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the gamma 2 subunit.
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