We first examined whether total BK, 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 regulation out of BK station mRNA splicing on the CNS
So you can investigate developmental changes in BK channel mRNA phrase in almost any regions of new murine CNS, BK channel mRNA phrase is quantified at the embryonic months thirteen, 15 and 18, and you will postnatal days 7 and you can thirty five. For every tissue, the entire BK station https://datingranking.net/marriagemindedpeoplemeet-review/ mRNA term at every developmental go out part are expressed due to the fact a share of this during the P35. Each splice variant the expression at every developmental time part is actually shown because the a portion of your own overall BK channel transcripts in for each and every muscle.
Tissues of rhombencephalon, mesencephalon and you may back
Into the spinal cord a small, however, significant, upsurge in total BK channel mRNA expression was seen from E13 than the P35 (Shape 2a). It was followed closely by a remarkable reduced total of STREX variation expression having a concomitant increase in Zero variant mRNA phrase (Contour 4a). Equivalent developmental up control off total BK channel mRNA expression was plus present in the fresh new midbrain, cerebellum, pons and you will medulla (Figure 2b–e). Within these structures, significant developmental downregulation from STREX variation expression is actually again noticed between embryonic and postnatal amounts (Figure 4b–e). Within the midbrain this was paralleled because of the a significant upsurge in No variant phrase whereas in the pons and you can medulla no significant changes ranging from E13 and you can P35 was basically seen (Shape 4d 4e). In contrast to most other nations, Zero variant term somewhat ent regarding cerebellum (Contour 4c).