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 controls regarding BK station mRNA splicing throughout the CNS
To browse the developmental alterations in BK station mRNA phrase in numerous regions of the fresh new murine CNS, BK channel mRNA term try quantified from the embryonic days 13, fifteen and you may 18, and you can postnatal days seven and you can thirty five. For every single structure, the complete BK station mRNA phrase at each developmental date area is actually expressed since a percentage of this within P35. Per splice version the word at every developmental go out section try expressed just like the a share of complete BK station transcripts within the for each and every structure.
Structures off rhombencephalon, mesencephalon and you may back
During the spinal-cord a small, but extreme, escalation in overall BK channel mRNA phrase try noticed from E13 compared to P35 (Profile 2a). This was followed by a remarkable reduction of STREX version phrase which have good concomitant upsurge in Zero variation mRNA expression (Shape 4a). Equivalent developmental right up regulation from complete BK route mRNA term is actually and observed in the fresh new midbrain, cerebellum, pons and medulla (Contour 2b–e). On these buildings, high developmental downregulation away from STREX variation term try once again observed anywhere between embryonic and postnatal amount (Shape 4b–e). In midbrain this was paralleled of the a critical increase in Zero version expression whereas within the pons and yubo log in you may medulla no significant transform between E13 and you can P35 had been seen (Profile 4d 4e). Compared with other nations, No version expression significantly ent on the cerebellum (Contour 4c).