Marie-Paule Roisin

Galanin reduces release of endogeneous excitatory amino acids in the rat hippocampus

Galanin is a 29-amino acid peptide widely distributed in the central nervous system where it modulates the release of several neurotransmitters and neurohormones. Because previous data had postulated that galanin could inhibit the release of excitatory amino acids and protect hippocampal neurons against anoxia, we have investigated the effect of galanin on the release of endogenous glutamate and aspartate evoked by potassium depolarization in rat hippocampal slices. Galanin, added to a concentration of 0.3 microM, produced a 50-60% reduction in the release of endogenous glutamate and aspartate as evoked by 40 mM K+. This effect was concentration-dependent with half-maximal effective galanin concentrations (EC50) of 1.7 and 5.9 nM for glutamate and aspartate, respectively. Such an effect was found to occur preferentially in the ventral rather than in the dorsal region of the hippocampus. The inhibitory effect of galanin on the K(+)-evoked release of excitatory amino acids was reversed by the specific galanin antagonist M-15 (0.3 microM), and by the ATP-sensitive potassium channel blocker glibenclamide (10 microM). Furthermore, M-15 alone increased the basal and the K(+)-evoked release of glutamate and aspartate from hippocampal slices. It is concluded that galanin exerts a tonic inhibition of excitatory glutamate/aspartate neurotransmission in the rat ventral hippocampus. The efficacy of glibenclamide in antagonizing the effect of galanin suggests the involvement of ATP-sensitive or -insensitive potassium channels in such a regulation.

Effect of potassium channel modulators on the release of glutamate induced by ischaemic-like conditions in rat hippocampal slices.

The effects of the potassium channel openers lemakalim, RP 52891 and galanin and the potassium channel blockers glibenclamide and gliquidone were evaluated by the release of endogenous glutamate from rat hippocampal slices subjected to a brief period of ischaemia (2-10 min). Ischaemia was mimicked by incubating slices in a glucose free medium equilibrated with 95% N2/5% CO2. These conditions evoked a release of glutamate which was insensitive to tetrodotoxin and Ca2+ indicating a non-vesicular origin. The release of glutamate evoked by a 6- or 8-min period of ischaemia was reduced by 25-40% in the presence of lemakalim (10 microM), RP 52891 (10 microM) or galanin (0.3 microM), whereas it was enhanced by 60 to 100% in the presence of glibenclamide (1 microM) and gliquidone (2 microM). These observations suggest that cellular damage resulting from ischaemia induced excessive release of glutamate in the hippocampus may be partly reduced by potassium channel openers, and conversely increased by sulfonylureas.

Long-term potentiation in the hippocampus of the anaesthetized rat is not associated with a sustained enhanced release of endogenous excitatory amino acids

The relationship between long-term potentiation of synaptic transmission and the release of endogenous glutamate and aspartate has been investigated in the CA1 region of the hippocampus and in the fascia dentata of the anaesthetized rat. A high-frequency train of electrical stimulation of afferent pathways produced a long lasting (greater than 2 h) enhancement of the field excitatory postsynaptic potential in CA1 and of the population spike in the fascia dentata. In both regions, this was not associated with a significant long lasting increase in the release of glutamate and aspartate. It is concluded that the maintenance of long-term potentiation is not associated with a sustained increase in the release of excitatory amino acids.

Basic Fibroblast Growth Factor-Induced Increase in zif/268 and c-fos mRNA Levels Is Ca2+ Dependent in Primary Cultures of Hippocampal Neurons

Abstract: Basic fibroblast growth factor (bFGF) is present in the developing rat brain and has been shown to provide critical trophic support for hippocampal neurons in culture. The influence of bFGF on the expression of mRNAs encoding the transcription factors zif/268 and c‐fos was studied in primary cultures of hippocampal neurons (derived from rat embryos) using reverse transcription‐coupled PCR. In these cultures grown for 3 days in the absence of serum, bFGF causes a dramatic and transient increase in the levels of zif/268 and c‐fos, within 15 and 30 min, respectively. A similar induction of these two early genes occurs following activation of protein kinase C (PKC). The bFGF‐induced activation persists after PKC desensitization but is inhibited by chelation of intracellular Ca2+. These results suggest that in primary cultures of hippocampal neurons, bFGF induces the expression of immediate early genes through a pathway that requires Ca2+ mobilization.

Two binding sites for [3H]glibenclamide in the rat brain

The binding of [3H]glibenclamide, a potent sulfonylurea which blocks ATP-sensitive potassium channels, was studied in the rat brain. A Scatchard plot of saturation isotherms suggests that [3H]glibenclamide binds in various brain regions to a high- and a low-affinity binding site (Kd values of 0.21 nM and 111 nM and Bmax values of 41 and 1060 fmol/mg protein, respectively). Competitive binding assays with various unlabelled sulfonylureas showed biphasic displacements of [3H]glibenclamide with pseudo-Hill coefficients significantly different from unity. These data indicate the existence of a heterogeneity of binding sites to [3H]glibenclamide in the rat brain; this may correlate with the variability of effects of sulfonylureas observed from physiological experiments.

Potassium Chloride Pulse Enhances Mitogen-Activated Protein Kinase Activity in Rat Hippocampal Slices

Abstract: Mitogen‐activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCI pulse. This activation correlates with an increased tyrosine phosphorylation of a 42‐kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti‐MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2‐amino‐5‐phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCI pulse used induces long‐term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.

Release of proteins during long-term potentiation in the hippocampus of the anaesthetized rat

Using a push-pull device, the release of endogenous proteins in the extracellular space was investigated in the CA1 region of the hippocampus of anaesthetized rats. With low-frequency stimulation of the Schaffer collaterals, there was a relatively stable release of 5 proteins (64, 54, 48, 45 and 16 kDa). A train of high-frequency stimulation produced a long-lasting enhancement of the negative field EPSP and a delayed (90-120 min) enhancement of the release of these proteins. An additional 19 kDa protein was present only 90 min after the train. These observations raise the possibility that release of proteins might be involved in the maintenance of LTP.

Subcellular Fractionation on Percoll Gradient of Mossy Fiber Synaptosomes: Morphological and Biochemical Characterization in Control and Degranulated Rat Hippocampus

Abstract: A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaption of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well‐preserved morphology (large size 3 μm, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1–8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.

Different GABAB‐Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1‐P60]. At P4, GABA (300 µM) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABAB receptors because (a) they were neither blocked by 10 µM bicuculline nor reproduced by 10 µM isoguvacine and (b) they were mimicked by the GABAB agonist baclofen (3–30 µM), an effect fully antagonized by the GABAB antagonist 2‐hydroxysaclofen (10 µM). A baclofen‐induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α‐, β‐, and ε‐PKCs). In contrast, after P21, PKC activity and α‐, β‐, ε‐, and γ‐PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABAB receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.

A new method for the measurement of endogenous transmitter release in localized regions of hippocampal slices

We describe here a method that allows measurement of the release of endogenous amino acids from localized regions of brain slices combined with conventional electrophysiological experiments. Hippocampal slices were placed in fully submerged chambers and a cannula was positioned just above the dendritic layers of CA1. The cannula was connected to a peristaltic pump and the content of amino acids in the perfusate was measured by HPLC. Extracellular field potentials were concomitantly recorded. Stable levels of aspartate and glutamate were found above the stratum radiatum of CA1. No detectable release was found when the cannula was located above the alveus, the fimbria or in the effluent of the slice. A pulse of K+ (50 mM) produced a brief 3-fold increase in glutamate, aspartate and a detectable release of GABA in CA1. Brief high frequency trains (10 Hz) also increased significantly the release. This method will be useful in determining alterations in transmitter release in the slice in relation to anoxia, epilepsy and long term potentiation.