Cannabinoid type 1 (CB1) receptors play a central role in the protection against excitotoxicity induced by treatment of mice with kainic acid (KA). As inactivation of CB1 receptor function in mice blocks KA-induced increase of brain-derived neurotrophic factor (BDNF) mRNA levels in hippocampus, the notion was put forward that BDNF might be a mediator, at least in part, of CB1 receptor-dependent neuroprotection [Marsicano et al. (2003) Science, 302, 84-88]. To assess this signalling cascade in more detail, organotypic hippocampal slice cultures were used, as this in vitro system conserves morphological and functional properties of the hippocampus. Here, we show that both genetic ablation of CB1 receptors and pharmacological blockade with the specific CB1 receptor antagonist SR141716A increased the susceptibility of the in vitro cultures to KA-induced excitotoxicity, leading to extensive neuronal death. Next, we found that the application of SR141716A to hippocampal cultures from wild-type mice abolished the KA-induced increase in BDNF protein levels. Therefore, we tried to rescue these organotypic cultures from neuronal death by exogenously applied BDNF. Indeed, BDNF was sufficient to prevent KA-induced neuronal death after blockade of CB1 receptor signalling. In conclusion, our results strongly suggest that BDNF is a key mediator in CB1 receptor-dependent protection against excitotoxicity, and further underline the physiological importance of the endogenous cannabinoid system in neuroprotection.
Synucleins have attracted much attention because of their involvement in several neurodegenerative disorders. In a screening for genes differentially expressed after high‐dose cocaine exposure, we found γ‐synuclein as a major upregulated candidate in the tegmentum. Overexpression of both α‐ and γ‐synuclein after drug treatment was confirmed by means of microarrays, yielding an increase in the hippocampus, the striatum and the tegmentum (2.65 ×, 1.96 × and 3.5 ×, respectively, for α‐synuclein vs. 2.7 ×, 1.96 × and 7.16 × for γ‐synuclein), but no change in the nucleus accumbens. Investigation of the distribution of mRNA (by in situ hybridization) and of the proteins (by immunocytochemistry) shows in both cases a clearly distinct pattern of expression for α‐ and γ‐synuclein. α‐synuclein displays a very characteristic distribution, confined to specific nuclei, whereas γ‐synuclein is more widely expressed throughout the brain. mRNA of both α‐ and γ‐synucleins display a complementary pattern of expression all over the cortex. In contrast to γ‐synuclein, α‐synuclein is neuronal, being only found in NeuN‐expressing cells, and is expressed in the basal ganglia (faintly) and in the substantia nigra compacta where it is highly correlated with tyrosine hydroxylase. Immunocytochemistry shows that γ‐synuclein generally colocalizes with glial fibrillary acidic protein‐expressing cells and is abundant in the red nucleus, the substantia nigra reticulata and the anterior commissure, while γ‐synuclein mRNA labels the matrix compartments of the caudate‐putamen. The role of synucleins in relation to cocaine‐induced plasticity or neurotoxicity is discussed.
RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.
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