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 away from BK channel mRNA splicing throughout the CNS
So you can have a look at developmental changes in BK channel mRNA expression in numerous areas of the fresh murine CNS, BK channel mRNA expression is actually quantified on embryonic weeks 13, fifteen and you will 18, and you will postnatal days eight and thirty five. Each cells, the full BK route mRNA phrase at each and every developmental big date section are conveyed while the a portion of these at P35. Per splice variation the term at each and every developmental time area was indicated since a share of one’s total naughtydate BK station transcripts for the each muscle.
Buildings of rhombencephalon, mesencephalon and you will spinal-cord
Within the spinal cord a tiny, but high, upsurge in overall BK channel mRNA expression are noticed away from E13 compared to P35 (Figure 2a). This was accompanied by a dramatic decrease in STREX variant expression which have good concomitant escalation in No version mRNA phrase (Contour 4a). Comparable developmental up control of complete BK channel mRNA expression try along with noticed in the fresh midbrain, cerebellum, pons and you can medulla (Contour 2b–e). On these tissues, tall developmental downregulation regarding STREX variation term are once more seen between embryonic and you can postnatal level (Profile 4b–e). Into the midbrain this was paralleled from the a critical upsurge in No variant phrase whereas within the pons and you may medulla no significant change ranging from E13 and you will P35 was indeed observed (Contour 4d 4e). Compared with other places, Zero variation term notably ent regarding cerebellum (Profile 4c).