We first examined whether total BK, wellhello free trial and splice variant, mRNA expression levels differed across distinct regions of the adult (postnatal day 35, P35) murine CNS. To compare the relative expression of total BK channel mRNA transcripts between different CNS regions of P35 mice all data were normalised to the expression of the housekeeping gene, ?-actin, in each region. Total BK channel mRNA expression was variable between different regions with the highest levels observed in frontal cortex and entorhinal cortex (Figure 1b). Regions with the lowest levels (< 50% of the level in entorhinal cortex) were cerebellum, medulla and posterior cortex. The spinal cord, midbrain, pons, thalamus, hypothalamus, hippocampus, olfactory bulb and striatum displayed intermediate total BK mRNA levels (Figure 1b).
Differential expression of the STREX and ZERO BK channel splice variant mRNA was also observed across these tissues at P35. The relative proportion of STREX transcripts (expressed as a percentage of total BK mRNA in each CNS region) was low in all regions tested, with levels typically < 10% of total BK mRNA transcripts. However, even across tissues, the relative proportion of STREX transcripts was significantly different (p < 0.05, Kruskal Wallis test with post-hoc Dunn's test compared to entorhinal cortex) with frontal cortex, thalamus and entorhinal cortex displaying the highest proportion of STREX transcripts and the pons and cerebellum displaying the lowest levels (Figure 1c). The proportion of total BK channel transcripts expressing the ZERO variant also varied significantly across tissues. The entorhinal cortex displayed a significantly (p < 0.01, Kruskal Wallis test with post-hoc Dunn's test) greater proportion of ZERO transcripts compared to any other tissue, with 85.6 ± 9.7 % of total BK transcripts encoded by the ZERO variant. (Figure 1c). In the majority of CNS regions, the proportion of ZERO transcripts was greater than 50% of total BK mRNA transcripts with the notable exception of cerebellum, in which 4.5 ± 2.3 % of transcripts encoded for the ZERO variant (Figure 1c). Taken together, these data suggest that the ZERO variant is the predominant transcript in most adult CNS tissues and that the STREX variant is expressed at a significantly lower level at postnatal day 35. A notable exception is the cerebellum, in which both ZERO and STREX transcripts represent < 10% of total transcripts at P35 suggesting that other site C2 splice variants are predominantly expressed in this region.
Developmental control from BK channel mRNA splicing on the CNS
In order to have a look at developmental changes in BK station mRNA expression in various areas of the new murine CNS, BK channel mRNA term is quantified in the embryonic weeks thirteen, 15 and you may 18, and you will postnatal weeks seven and you can thirty-five. For every single structure, the total BK channel mRNA phrase at every developmental date point is actually expressed due to the fact a percentage of these on P35. For every single splice version the term at each developmental time area was indicated just like the a portion of complete BK station transcripts into the each tissues.
Structures out of rhombencephalon, mesencephalon and you can spinal cord
From inside the spinal-cord a little, but significant, boost in total BK channel mRNA phrase are seen regarding E13 compared to P35 (Contour 2a). It was accompanied by a dramatic reduced total of STREX version term with an excellent concomitant increase in Zero variant mRNA expression (Shape 4a). Similar developmental up regulation out of full BK channel mRNA expression is as well as found in the newest midbrain, cerebellum, pons and medulla (Shape 2b–e). During these frameworks, significant developmental downregulation out of STREX variant term are once more noticed between embryonic and you may postnatal degrees (Figure 4b–e). Inside the midbrain it was paralleled from the a critical boost in Zero variant expression while in the pons and you may medulla zero extreme transform ranging from E13 and you may P35 was seen (Contour 4d 4e). Compared with most other countries, Zero variation term significantly ent from the cerebellum (Figure 4c).
