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 control regarding BK station mRNA splicing about CNS
So you’re able to investigate developmental changes in BK route mRNA term in different areas of the latest murine CNS, BK station mRNA expression was quantified at the embryonic months 13, fifteen and you will 18, and you will postnatal weeks 7 and you will thirty-five. For every single tissues, the complete BK route mRNA expression at each developmental big date section is actually indicated due to the fact a percentage of these within P35. For each splice variant the phrase at every developmental go wellhello promo code out point are indicated because the a percentage of the overall BK route transcripts inside the for each and every muscle.
Structures away from rhombencephalon, mesencephalon and you will spinal cord
Within the back a little, however, high, upsurge in overall BK route mRNA expression is actually seen out of E13 versus P35 (Figure 2a). It was followed by a remarkable reduction of STREX variation expression that have a concomitant boost in Zero variation mRNA phrase (Contour 4a). Similar developmental upwards controls from overall BK route mRNA term are in addition to noticed in this new midbrain, cerebellum, pons and you may medulla (Figure 2b–e). Within these structures, extreme developmental downregulation out-of STREX variation expression are once more seen anywhere between embryonic and postnatal levels (Profile 4b–e). When you look at the midbrain this is paralleled by a serious upsurge in No variation phrase while when you look at the pons and medulla zero high alter ranging from E13 and P35 were seen (Contour 4d 4e). In contrast to most other regions, No variation term significantly ent regarding cerebellum (Profile 4c).