Expression and cellular function of vSNARE proteins in brain astrocytes.

Gray matter protoplasmic astrocytes, a major type of glial cell in the mammalian brain, extend thin processes ensheathing neuronal synaptic terminals. Albeit electrically silent, astrocytes respond to neuronal activity with Ca(2+) signals that trigger the release of gliotransmitters, such as glutamate, d-serine, and ATP, which modulate synaptic transmission. It has been suggested that the astrocytic processes, together with neuronal pre- and post-synaptic elements, constitute a tripartite synapse, and that astrocytes actively regulate information processing. Astrocytic vesicles expressing VAMP2 and VAMP3 vesicular SNARE (vSNARE) proteins have been suggested to be a key feature of the tripartite synapse and mediate gliotransmitter release through Ca(2+)-regulated exocytosis. However, the concept of exocytotic release of gliotransmitters by astrocytes has been challenged. Here we review studies investigating the expression profile of VAMP2 and VAMP3 vSNARE proteins in rodent astrocytes, and the functional implication of VAMP2/VAMP3 vesicles in astrocyte signaling. We also discuss our recent data suggesting that astrocytic VAMP3 vesicles regulate the trafficking of glutamate transporters at the plasma membrane and glutamate uptake. A better understanding of the functional consequences of the astrocytic vSNARE vesicles on glutamate signaling, neuronal excitability and plasticity, will require the development of new strategies to selectively interrogate the astrocytic vesicles trafficking in vivo.

Intermediate zone cells express calcium-permeable AMPA receptors and establish close contact with growing axons.

Recent studies have shown that cells in the intermediate zone (IZ) of the embryonic neocortex originate in the basal telencephalon and migrate tangentially in the cortical wall (;; ). We had previously observed growing cortical axons closely apposed to calbindin-positive, tangentially oriented cells in the IZ (), and it has been shown that neurites in the IZ express a glutamate transporter (). To test if glutamate released by corticofugal growth cones could influence the tangential IZ cells, we characterized the glutamate receptors expressed by IZ cells using patch-clamp techniques, histochemical labeling, and immunostaining on slices of embryonic mice forebrain. We show that tangential IZ cells express inwardly rectifying kainate responses, but not NMDA responses, and accumulate cobalt after AMPA receptor activation. We conclude that IZ cells express calcium-permeable AMPA receptors. This property correlates with our observation that the GluR2 subunit is not expressed in the IZ. AMPA receptors are activated by a millimolar concentration of glutamate. To know whether this high level of glutamate could occur at the surface of IZ cells, we examined contacts made by corticofugal growth cones and calbindin-positive IZ cells using electron microscopy. We show vesicle-containing neurites tightly apposed to calbindin-positive IZ cells over remarkably long length. This suggests that glutamate released by growing corticofugal axons could reach high concentrations close to AMPA receptors of tangential IZ cells and efficiently activate them to control the intracellular calcium in embryonic IZ cells.

Inhibitory action of serotonin in CA1 hippocampal neurons in vitro.

The ionic mechanism of the inhibitory effect of serotonin was investigated in vitro in the CA1 region of the rat hippocampus by extra- and intracellular recordings. Local or bath applications of serotonin induced a long-lasting reduction of extracellularly recorded synaptic potentials and orthodromic population spikes without affecting the afferent volley or the antidromic population spike. Serotonin can also reduce the frequency of occurrence of spontaneous excitatory and inhibitory postsynaptic potentials without any reduction of input resistance of the pyramidal neuron. During the response to serotonin, the conductance increase evoked by GABA, the inhibitory neurotransmitter, was not changed. A direct postsynaptic effect of serotonin was demonstrated: local or bath applications of serotonin induced a tetrodotoxin-resistant hyperpolarization and conductance increase. The conductance change was not reduced by manual clamp of the neurons to the control resting membrane potential; therefore, a possible involvement of the sodium-potassium electrogenic pump is unlikely. When neurons were loaded with chloride, serotonin could still induce a hyperpolarization with an apparent reversal more negative than the resting membrane potential. When neurons were loaded with caesium, the hyperpolarization and the conductance increase evoked by serotonin were blocked. It is therefore concluded that serotonin increases potassium permeability. Similar effects were induced by a 5-HT1A ligand. The slow after hyperpolarization was reduced by serotonin; the calcium spike was reduced at the same time. In caesium loaded neurons, the spike duration was not modified by serotonin. In the presence of extracellular caesium (4-5 mM), the serotonin-induced hyperpolarization and the conductance change were blocked, but the effect of serotonin on calcium spikes persisted. Tetraethylammonium (5-10 mM) or 4-aminopyridine (0.5 mM) had no effect on the response to serotonin. These data indicate that serotonin has a postsynaptic inhibitory action by an activating potassium conductance. The possibility of a regulation of calcium currents is discussed. The possible role of serotonin on intrinsic synaptic transmission is also discussed.

Pharmacological characteristics of facilitation of hippocampal population spikes by cholinomimetics.

In rats under urethane anaesthesia, various cholinomimetics, acetylcholine-antagonists and other agents were released iontophoretically in the pyramidal layer of area CA1. Like acetylcholine, a variety of cholinomimetics readily enhanced population spikes evoked by fimbrial-commissural stimulation. Judging by the equipotent iontophoretic currents, the strongest muscarinic agonist was muscarine. Other potent agonists included carbachol, methacholine, propionylcholine, bethanechol and the much slower-acting arecoline, pilocarpine and oxotremorine. Choline was about 5 times weaker than acetylcholine. Though not as effective as acetylcholine, some nicotinic agonists also consistently enhanced population spikes, particularly dimethylphenylpiperazinium and acetylthiocholine. Other nicotinic agents, such as butyrylcholine, nicotine and tetramethylammonium were much less active. Both scopolamine and atropine, given systematically in high doses (10-80 mg/kg), strongly depressed or abolished the action of muscarinic agonists, but to a lesser and more variable extent the action of ACh. They did not antagonize dimethylphenylpiperazinium. When applied iontophoretically, alpha-bungarotoxin, tubocurarine or mecamylamine did not block the action of any of the cholinomimetics. Indeed, in higher doses they tended to promote population spikes (a comparable enhancement was also seen with larger iontophoretic doses of atropine or scopolamine). On the other hand, gallamine and dihydro-beta-erythroidine antagonized muscarine but not dimethylphenylpiperazinium; a less selective block of cholinomimetics was produced by suxamethonium. It was concluded that both muscarinic and nicotinic receptors (or receptors with mixed properties) appear to be involved in the facilitatory action of acetylcholine on population spikes evoked by fimbrial-commissural stimulation.

Electrophysiological and pharmacological characteristics of facilitation of hippocampal population spikes by stimulation of the medial septum.

In rats under urethane anesthesia, single shock or tetanic stimulation of the medial septum--which evoked only minimal field potentials--sharply enhanced population spikes evoked in area CA1 by commissural stimulation. An enhancement of population spikes was observed only (a) in areas CA1 and CA2 (adjacent to CA1 in the dorsal hippocampus), but not in the fascia dentata or the deep pyramidal layers CA3 or CA4; (b) in a narrow range of depth, close to the stratum pyramidale; (c) when the intensity of commissural stimulation was of adequate intensity. A comparable facilitation of population spikes was produced at the same sites by microiontophoretic release of acetylcholine. The septal facilitatory action increased in effectiveness with the number of tetanic pulses (up to 10-12) at a given frequency, and it had a maximum at frequencies of 50-100 Hz. It reached a maximum 20-50 ms after the end of septal stimulation, and then decayed slowly, the overall duration being up to 300 ms. The cholinergic nature of the facilitation induced by septal stimulation was confirmed by the parallel potentiation of septal action and that of acetylcholine by physostigmine and their depression by atropine and scopolamine.

Hemicholinium impairs septo-hippocampal facilitatory action.

Intraventricular injections of hemicholinium-3 led to a sharp reduction in hippocampal acetylcholine content (by 79% on the average). This was associated with the following changes in population spikes evoked in area CA1 by commissural stimulation: (1) a tendency to progressive increase, over 1-2 hours; (2) in a few cases (3 out of 12), a striking depression of the normal strong facilitation produced by brief tetanic stimulation of the medial septum (typically 10 pulses at 50-100 Hz); (3) a much more consistent tendency towards fading of the septal facilitatory effect during repeated applications of such brief septal tetani (in 10 cases out of 12); as well as, (4) diminished facilitation by sustained, lower frequency septal tetanic stimulation (20-50 Hz). The reduced efficiency of septal action--especially during repetitive stimulation--was not accompanied by a consistent reduction of the facilitation produced by local applications of acetylcholine; it is, therefore, best explained by the diminished availability of acetylcholine, and so provides further evidence that septo-hippocampal facilitation is mediated by a cholinergic mechanism.

Facilitation of hippocampal CA3 pyramidal cell firing by electrical fields generated antidromically.

In experiments on rats under urethane anaesthesia--in which the fimbria and hippocampal commissure had been cut previously to eliminate orthodromic inputs--the negative antidromic population spike evoked in CA3 by fimbrial stimulation was measured inside and outside 73 neurons in the stratum pyramidale. Subtraction of the extracellular from the intracellular records showed that on the average 39.2% (S.E. 1.93) of the extracellular population spikes appeared as a positive, depolarizing transmembrane potential. Similar measurements in the dendritic zone of CA3, where the extracellular antidromic population spike is positive, revealed a smaller and hyperpolarizing transmembrane potential, whereas presumed neuroglia showed no consistent transmembrane potential in either direction. Further tests demonstrated clear facilitation of individual pyramidal cell firing, synchronous with the antidromic population spike. These observations are consistent with the possibility that, owing to the unusually close packing and regular alignment of the pyramidal neurons, electrical field interactions in CA3 tend to promote synchronized mass discharges.

Chemical modulation of ephaptic activation of CA3 hippocampal pyramids.

In rats under urethane anaesthesia, antidromic population spikes were evoked in CA3 pyramidal layer by fimbrial/commissural stimulation at a very low frequency (approximately 0.5 Hz). Submaximal population spikes--between 20 and 90% of maximum--were enhanced by 8-38% by applications of acetylcholine and bicuculline, or by medial septal stimulation. Noradrenaline had a less pronounced and regular facilitatory action, whereas gamma-aminobutyrate and glutamate only depressed population spikes. Maximal enhancement by acetylcholine or bicuculline was observed when the antidromic population spike was initially at 38-53% of maximum amplitude. A simple explanation of these results is that acetylcholine and bicuculline, by raising their excitability, facilitate the excitation of non-invaded pyramidal cells by antidromic field potentials. They are fully in keeping with previous intracellular observations on ephaptic interactions between CA3 neurons, and provide a further illustration, in situ, of the importance of increased excitability and disinhibition--whether caused by drugs or synaptic action--in promoting synchronized excitation by ephaptic currents.

The protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) disinhibits CA1 pyramidal cells in rat hippocampal slices.

1. The effects of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) on evoked synaptic potentials were investigated in the CA1 region of rat hippocampal slices by use of extracellular and intracellular recording techniques. 2. Extracellular recordings showed that superfusion with H-7 (10-100 microM) increased the amplitude of the population spike and the initial slope of the dendritic field e.p.s.p. H-7 also produced the appearance of multiple population spikes in the somatic region and in the dendritic field e.p.s.p. 3. H-7 (30 microM) induced the disappearance of intracellularly recorded inhibitory potentials elicited by orthodromic stimulation of CA1 pyramidal cells. At this concentration H-7 had no effect on resting membrane potential, input membrane resistance, and spike threshold. In voltage-clamped neurones H-7 blocked the antidromically evoked inhibitory currents and the spontaneous miniature inhibitory currents. 4. The hyperpolarizing effect of bath applied gamma-aminobutyric acid (GABA, 500 microM) or isoguvacine (30 microM) was not affected by 30 microM H-7. 5. Neither the PKC activity regulator sphingosine (10-40 microM) nor the H-7 analogue N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004, 20-50 microM) which is devoid of activity on PKC at these concentrations, affected the extracellularly recorded dendritic field e.p.s.p. or population spike. 6. It is concluded that the disinhibitory effect produced by H-7 is due to the block of a H-7-sensitive PKC which is involved in the spontaneous and evoked release of GABA.

Diversity of glutamate receptors in neocortical neurons: implications for synaptic plasticity.

The biochemical and functional characteristics of the AMPA subtype of the glutamate receptors expressed by pyramidal and non-pyramidal neurons of the neocortex have been studied in acute slices by means of single-cell RT-PCR and fast applications of glutamate on outside-out patches. Our results suggest that the predominant expression of the flop splice variants of the GluR1-4 AMPA subunits contributes to the faster desensitization of these receptors in non-pyramidal neurons compared to pyramidal cells where flip variants of GluR1-4 are dominant. Alternative splicing of AMPA receptors may therefore play an important role in regulating synaptic function in a cell-type specific manner.