Different patterns of induction of FGF-2, FGF-1 and BDNF mRNAs during kindling epileptogenesis in the rat.

Neurotrophic factors (NTF) play important roles in the developing and in the adult brain. NTF involvement in neuronal plasticity is suggested by the modulation of NTF expression patterns in different physiological and pathological situations and by the effects they produce in the adult brain (e.g. axonal sprouting induction and neuroprotection). We used the RNAase protection assay to investigate the expression patterns of some NTFs during amygdala kindling, an animal model of epilepsy in which 'pathological' neuronal plasticity appears to occur. After a single kindling stimulation, fibroblast growth factor-2 (FGF-2) mRNA levels were increased in the hippocampus, the cortex and the hypothalamus, whereas they were not significantly altered in the thalamus and the striatum. A single stimulation did not alter fibroblast growth factor-1 (FGF-1) and brain-derived neurotrophic factor (BDNF) gene expression. Fully kindled animals, left unstimulated for a week, did not exhibit any alteration in the mRNA levels for any of the NTFs examined. However, in contrast with the effect of a single stimulation, amygdala stimulation of kindled animals (evoking a generalized tonic-clonic seizure) produced a great increase in hippocampal and cortical BDNF mRNA levels, but FGF-1 mRNA levels were not altered, and FGF-2 mRNA levels were significantly increased only in the cortex. These results suggest that different NTFs can be recruited at different stages of kindling epileptogenesis and, accordingly, may play different parts in the adaptive changes taking place in this experimental paradigm.

Characterization of glutamate and [3H]D-aspartate outflow from various in vitro preparations of the rat hippocampus.

The characteristics of high-K+ and electrically evoked endogenous glutamate and [3H]D-aspartate release have been studied in multiple in vitro preparations of the rat hippocampus (transverse slices, granule cells cultures, synaptosomes and mossy fibre synaptosomes) under similar experimental conditions. High external K+ concentrations evoked [3H]D-aspartate and endogenous glutamate overflow in a concentration-dependent manner in all preparations (except it was not possible to measure endogenous glutamate outflow from granule cells). This effect was tetrodotoxin-insensitive but partially calcium-dependent. In slices, field electrical stimulation evoked an overflow of endogenous glutamate, but not of [3H]D-aspartate, in a frequency-dependent manner. This effect was concentration-dependently amplified by the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC). The electrically evoked glutamate overflow in the presence of t-PDC was tetrodotoxin-sensitive and calcium-dependent. In primary dentate gyrus cell cultures, electrical stimulation evoked an overflow of [3H]D-aspartate in a frequency-dependent manner, while endogenous glutamate outflow was not detectable. This effect could be inhibited by tetrodotoxin and by the N-type calcium channel blocker omega-conotoxin GVIA. Finally, the effect of adenosine has been studied in order to assess the pharmacological modulability of [3H]D-aspartate and endogenous glutamate stimulation-induced overflow. Adenosine was found to inhibit 35 mM K(+)- and 20 Hz electrical stimulation-induced [3H]D-aspartate and endogenous glutamate overflow. These effects were all prevented by the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). These data are in line with the hypothesis that reuptake plays a role in regulating glutamate release, and that [3H]D-aspartate represents a valid marker of endogenous glutamate under most (but not all) experimental conditions.

Biotin deficiency facilitates kindling hyperexcitability in rats.

Biotin-deficient conditions are frequently associated with epileptic disorders. Biotin deficiency may be caused by long-term treatment with anticonvulsants or excessive ingestion of avidin. Absence of biotinidase activity can also lead to biotin deficiency, and is characterized by developmental delay as well as neurological and dermatological abnormalities. Because seizures are one of the most frequent signs of the latter, biotin-deficient conditions could conceivably facilitate convulsive disorders. To test this hypothesis, we investigated the occurrence of a latent kindling hyperexcitability in biotin-deprived rats. In these animals, duration of after-discharge on the first stimulation was longer at threshold amplitude, kindling development through its early stages was accelerated and duration of the forelimb clonus of fully kindled seizures was increased. Biotin deprivation in mixed cerebellar granule cell-astrocyte cultures also produced a tetrodotoxin-sensitive delayed loss of the glutamatergic neuronal population. The data thus support a facilitatory role for biotin-deficient conditions in convulsive disorders.

Adenosine A1 receptors in the rat brain in the kindling model of epilepsy.

Adenosine and adenosine analogues have potent anticonvulsant effects on various seizure models, including kindling, an animal model of temporal lobe epilepsy. It is now reported that binding of a specific ligand (cyclohexyladenosine) to adenosine A1 receptors is not changed in the cerebral cortex of kindled rats. However, the affinity of cyclohexyladenosine to adenosine receptors is significantly increased in the hippocampus. In addition, cyclohexyladenosine is slightly more potent to inhibit [3H]D-aspartate outflow from hippocampal synaptosomes taken from kindled than from control rats. Taken together, these data suggest that an increased affinity of adenosine to A1 receptors may play a role in the anticonvulsant effect of adenosine A1 analogues in the kindling model.

Characterization of K(+)-evoked [3H]D-aspartate outflow in the rat hippocampus in vitro.

The characteristics of K(+)-evoked outflow of [3H]D-aspartate, a glutamate release marker, were systematically investigated in the rat hippocampus, using 35 mM K(+)-evoked [3H]noradrenaline outflow as a reference. Elevation of external K+ concentrations increased [3H]D-aspartate outflow in a concentration-dependent manner both in slices and synaptosomes. In the absence of external Ca2+, K(+)-evoked [3H]D-aspartate outflow was decreased by approx 60% in synaptosomes and 80% in slices. However, elimination of external Ca2+ in the presence of 2 mM EGTA significantly reduced only 100 mM K(+)-evoked outflow, both in slices and synaptosomes. In the absence of external Ca2+, 35 mM K(+)-evoked [3H]noradrenaline outflow was abolished even when EGTA was present in the solution. Furthermore, the Ca(2+)-channel blockers omega-conotoxin (10 nM) and nifedipine (0.5 microM) did not significantly reduce K(+)-evoked [3H]D-aspartate outflow; [3H]noradrenaline outflow, however, was reduced by more than one third by omega-conotoxin. Finally [3H]D-aspartate overflow was insensitive to tetrodotoxin (0.5 microM) both in synaptosomes and in slices, while that of [3H]noradrenaline was significantly reduced in slices. It is concluded that (1) [3H]D-aspartate outflow is partly Ca(2+)-dependent; (2) differences between K(+)-evoked [3H]D-aspartate and [3H]noradrenaline outflow include sensitivity to stimulation by EGTA, to Ca(2+)-channel blockers and to tetrodotoxin. Some of these discrepancies may be ascribed to the existence of a cytosolic, Ca(2+)-independent pool of releasable glutamate and [3H]D-aspartate. These observations pose some problems as to the experimental approach for the study of Ca(2+)-dependent [3H]D-aspartate release.

Temperature dependence of [3H]PAF binding to washed human platelets.

Our results indicate that the binding of [3H]PAF to a single class of high affinity sites on human platelets is endothermic and largely entropy-driven. The increase in entropy can be attributed to the disorganization of water molecules bound to both receptor and ligand during the binding process.