Figure 5

SNAP-25 isoform expression varies between anatomical regions of the adult brain. GFP-expressing cells from coronal sections of adult (P50) transgenic GAD67-GFP (ΔNeo) mouse brains were identified by epifluorescence and isolated by laser capture microdissection (LCM) from the reticular nucleus of the thalamus (Ret. Nuc.), caudate, cerebellum (Purkinje cells), and CA1 region of the hippocampus (Hp interneuron). In addition non-GFP expressing glutamatergic neurons collected from the pyramidal layer of the hippocampus (Hp Pyramidal) and granule layer of cerebellum (Cb Gran. Cell). The total RNA extracted from microdissected samples was assayed by real-time qRT-PCR analysis to assess transcript levels relative to β-actin as a control gene, as described in the Methods. Panel A is a representative micrograph of a GFP-expressing cell (arrowhead and hashed outline) before and after LCM, visualized with both phase contrast (left) and a FITC filter set (right). Note how neighboring cells (arrows) and tissue remain intact during the procedure. B, Histogram shows the relative ratio of SNAP-25a (solid bar) and 25b (white bar) RNA expression in selected brain regions (n = 6 pools taken from individual brains). Note that although, in all cases, SNAP-25b expression is significantly higher than SNAP-25a (***P < 0.001, Student's t-test), the ratio of isoform expression between anatomical regions does not change (n. s.; One-way ANOVA with Tukey post hoc comparison). Throughout the areas surveyed, however, the overall mRNA expression of SNAP-25a and SNAP-25b did vary widely (shown in Panels C and D, respectively). Statistical analysis with a one-way ANOVA and Tukey post hoc comparisons of total SNAP-25a and SNAP-25b transcript expression was carried out for each anatomical region (n = 3, except for cerebellar granule cells, n = 6). Asterisks denote significance of the difference between the level of SNAP-25 transcripts of each region compared to cerebellar Purkinje cells (* = p < 0.05; ** = p < 0.01; *** = p < 0.001); additional significant differences in isoform transcripts between GABAergic neurons of the reticular nucleus (Ret. Nuc.) and non-GABAergic neurons of the hippocampus (Hp Pyramidal) and cerebellum (Cb Gran. Cell) are also indicated.