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In the present study, GABA was shown to induce a necrotic neuronal death in cultured striatal neurones from mouse embryos. This effect did not depend on the activation of GABA[sub A], GABA[sub B] or GABA[sub C] receptors as it was neither antagonized by bicuculline, saclofen or picrotoxin, respectively, nor reproduced by the GABA receptor agonists, muscimol and baclofen. Excluding the participation of glutamate, GABA neurotoxicity persisted in the presence of either the antagonists of ionotropic and metabotropic glutamate receptors or glutamate pyruvate transaminase, which induces an immediate catabolism of glutamate. A GABA transport-associated process is involved in GABA neurotoxicity as nipecotic acid and NO 711, two inhibitors of the high-affinity neuronal GABA transporters (GAT-1, in particular), completely prevented the neurotoxic effect of GABA. The activation of a subset of G proteins is also implicated in the GABA transport-mediated neuronal death as GABA neurotoxicity was completely suppressed when striatal neurones were pre-treated with pertussis toxin. Further demonstrating the specificity of this neurotoxic process, GABA-induced neurotoxicity was not observed in cortical neurones which, in contrast to striatal neurones, are largely represented by glutamatergic neurones. In conclusion, our study suggests that glutamate is not the sole neurotransmitter that can be responsible for brain damage and that GABA neurotoxicity involves both GABA transport and G protein transduction pathways. [ABSTRACT FROM AUTHOR]

Attempts were made to label tachykinin NK[sub 2] binding sites in the adult rat brain using [[sup 125]I]neurokinin A (NKA) as ligand in the presence of NK[sub 1] and NK[sub 3] agonist or antagonist to avoid labelling of NK[sub 1] and NK[sub 3] binding sites, respectively. A high-affinity, specifically NK[sub 2]-sensitive, [[sup 125]I]NKA-binding, temperature-dependent, reversible, sensitive to GTPγS and correspondence to a single population of binding sites (K[sub D] and B[sub max] values: 2.2 nm and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin-related compounds indicated that the pharmacological properties of these NK[sub 2]-sensitive [[sup 125]I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide γ, as well as the potent and selective NK[sub 2] antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK[sub 2]-sensitive [[sup 125]I]NKA binding sites differs markedly from those of NK[sub 1] and NK[sub 3] binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [[sup 125]I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei. [ABSTRACT FROM AUTHOR]

Sequential and opposite regulation of two outward K[sup +] currents by ET-1 in cultured striatal astrocytes. Am J Physiol Cell Physiol 281: C1373-C1384, 2001.—In the brain, astrocytes represent a major target for endothelins (ETs), a family of peptides that can be released by several cell types and that have potent and multiple effects on astrocytic functions. Four types of K+ currents (I[sub K]) were detected in various proportions by patchclamp recordings of cultured striatal astrocytes, including the A-type I[sub K], the inwardly rectifying I[sub K IR], the Ca[sup 2+] -dependent I[sub K] (I[sub K Ca]), and the delayed-rectified I[sub K] (I[sub K DR]). Variations in the shape of current-voltage relationships were related mainly to differences in the proportion of these currents. ET-1 was found to regulate with opposite effects the two more frequently recorded outward K[sup +] currents in striatal astrocytes. Indeed, this peptide induced an initial activation of I[sub K Ca] (composed of SK and BK channels) and a delayed long-lasting inhibition of I[sub K DR]. In current-clamp recordings, the activation of I[sub K Ca] correlated with a transient hyperpolarization, whereas the inhibition of I[sub K DR] correlated with a sustained depolarization. These ET-l-induced sequential changes in membrane potential in astrocytes may be important for the regulation of voltage gradients in astrocytic networks and the maintenance of K[sup +] homeostasis in the brain microenvironment. [ABSTRACT FROM AUTHOR]

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H[sub 2]O[sub 2] formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca[sup 2+] is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn[sup 2+] preferentially inhibits the initiation step. In this study, we provide evidence indicating that H[sub 2]O[sub 2] and thapsigargin mobilized a common intracellular Ca[sup 2+] pool. H[sub 2]O[sub 2] treatment stimulated a slow increase in intracellular Ca[sup 2+], and precluded the effect of thapsigargin on Ca[sup 2+] mobilization. H[sub 2]O[sub 2] stimulated the phosphorylation of both eIF-2α and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H[sub 2]O[sub 2]-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H[sub 2]O[sub 2] treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H[sub 2]O[sub 2] inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2α and eEF-2 and the relative contribution of these two events depends on the duration of H[sub 2]O[sub 2] treatment. [ABSTRACT FROM AUTHOR]

The release of H-DA was estimated in superfusates of a superfusion cannula introduced into the caudate of a superfusion cannula introduced into was continuously formed from l-3,5-H-tyrosine introduced into the superfusion medium. Injected at the periphery, picrotoxin (2.5 mg/kg, i.p.) markedly enhanced the release of H-DA the effect being particularly pronounced 90 min after the drug injection. The effects of the local introduction of picrotoxin into the substantia nigra and into the caudate nucleus on H-DA release were also examined to ensure the specificity of the result. Picrotoxin (10 M) added into the superfusing medium of a second superfusion cannula introduced into the substantia nigra enhanced by 2 to 5 times the release of H-DA during the drug application. No effect could be seen when the tip of the superfusion cannula was not exactly localized into the substantia nigra. Although less pronounced, a stimulation of H-DA release was also seen when picrotoxin (10 M) was added directly into the superfusion medium of the cannula introduced into the caudate nucleus. These results suggest that some gabaergic neurons are involved in the control of DA release by acting on dopaminergic cell bodies or dendrites into the substantia nigra. Other gabaergic neurons may directly or indirectly act on dopaminergic terminals within the caudate nucleus. [ABSTRACT FROM AUTHOR]

The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized H-5-HT were examined in rat hypothalamic slices. H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with l-H-tryptophan in the presence of the various durgs tested. ACh (10 M) in the presence of eserine (2×10 M), and carbachol (10 M) stimulated the release of H-5-HT. In contrast, oxotremorine (10 M) reduced the H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10 M) and d-tubocurarine (10 M). It was not reduced by the muscarinic antagonists, atropine (10 M) and scopolamine (10 M). In fact, each of two antagonists added alone to the incubating medium enhanced H-5-HT release. The scopolamine (10 M) stimulating effect on H-5-HT release was suppressed by d-tubocurarine (10 M). Finally, the inhibiting effect of oxotremorine on H-5-HT release was not prevented by d-tubocurarine (10 M) but was in the presence of atropine (10 M) or scopolamine (10 M). In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of H-5-HT and H-5-HIAA from l-H-tryptophan and on the accumulation of l-H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous H-5-HT in hypothalamic synaptosomes (P fraction). These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals. [ABSTRACT FROM AUTHOR]

Using crude synaptosomal fractions (P fractions) and I-Bolton and Hunter substance P (I-BHSP) as a ligand, the characteristics of specific binding sites were examined in various brain structures and in the spinal cord (dorsal and ventral parts) of the rat. Scatchard plots revealed the occurrence of a single class of binding sites in the various structures studied with comparable K values (from 0.46 to 1.10 nmol/l in the brain and 0.51, 0.56 nmol/l in the spinal cord dorsal and ventral parts respectively) and of marked differences in the number of binding sites (B) (septum > striatum > hippocampus, hypothalamus > mesencephalon > cerebral cortex and dorsal part of the spinal cord > ventral part). In the brain no correlation was found between the number of I-BHSP binding sites and the amount of substance P levels (substance P-like immunoreactivity) in synaptosomes, particularly in the hippocampus and the substantia nigra since the former structure was characterized by its low substance P content and its high number of binding sites and the reverse was observed in the substantia nigra. The ability of several C- and N-terminal fragments of substance P and of tachykinins to compete with I-BHSP binding to synaptosomes from the hippocampus, the hypothalamus and the dorsal part of the spinal cord was then determined. Results obtained were closely similar from one structure to another and comparable to those previously reported using whole brain synaptosomes. Although the presence of various types of central substance P receptors cannot be excluded, the present results indicate that only one class of sites can be demonstrated using I-BHSP as a ligand. [ABSTRACT FROM AUTHOR]

A single treatment with a large dose of methiothepin (20 mg/kg, i.p.) induced, as early as the 2nd day after injection, a significant increase (+20-35%) in the number of specific binding sites for H-5-HT in forebrain areas, particularly the hippocampus. Experiments with H-methiothepin indicated that the drug remained firmly bound to brain membranes thus maintaining a local concentration high enough to effectively block 5-HT receptors for 10-12 h after its peripheral administration. Accordingly, it can be concluded that the occupancy of central 5-HT receptor sites by methiothepin for several hours was sufficient to induce a supersensitivity phenomenon within the two following days. Although H-methiothepin was a useful marker for analyzing the disposition and the kinetics of the 5-HT antagonist in brain tissues, it could not be used as a specific ligand of 5-HT receptors in brain since under in vitro as well as in vivo conditions most of H-methiothepin bound to non-specific sites, especially to the lipid component of the membranes. [ABSTRACT FROM AUTHOR]

A perfusion technique has been used to study the mechanisms involved in the control of acetylcholine (ACh) release from rat brain cortical and striatal slices submitted to KCl depolarization. Tissues were superfused continuously with U-C-glucose, a very efficient precursor of the acetyl moiety of ACh, and with eserine to prevent the hydrolysis of the C-ACh released in the superfusion medium. This radioenzymatic approach was more flexible than those in which endogenous ACh outflows were estimated using a biological or a radioenzymatic method. Moreover, the modulation of the ester release process could be studied using the physiological ligand of the presynaptic muscarinic receptor, i.e. ACh itself. The continuous synthesis of C-ACh from U-C-glucose, throughout the superfusion experiment, allowed the rate of C-ACh release to reach a steady state, under resting conditions (5.6 mM KCl) as well as under conditions of stimulation by high concentrations of KCl. This method was very convenient for pharmacological studies as compared to other methods in which the efflux of H-ACh from performed intratissular stores of H-ACh was measured. Using this technique, it was shown that atropine (10 μM) enhanced the rate of C-ACh spontaneous release. Curiously, extraneously added ACh (0.6 mM) had a similar effect. When cortical prisms were submitted to a brief 31 mM KCl pulse stimulation (3 min), the negative feedback regulation process was not fully triggered. Indeed, atropine, which prevents the interaction of the released ACh with the regulatory muscarinic receptor, significantly stimulated the C-ACh outflow only when brain tissues were submitted to a sustained 31 mM KCl depolarisation, or when the ACh concentration in the superfusion fluid was raised (0.6 mM). These results suggest that the muscarinic negative control of ACh release from brain cholinergic terminals becomes fully operative only when the nerve endings are depolarized and when the ACh concentration within the synapse exceeds a threshold, i.e. during intense functioning of the cholinergic synapse. [ABSTRACT FROM AUTHOR]