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 out-of BK station mRNA splicing throughout the CNS
To help you take a look at developmental changes in BK what is crossdresser heaven route mRNA term in almost any regions of this new murine CNS, BK station mRNA expression was quantified during the embryonic months thirteen, 15 and 18, and you will postnatal days eight and you may thirty five. For each and every structure, the total BK station mRNA term at each developmental time point is shown as the a percentage of that within P35. Each splice version the term at each developmental day part is indicated given that a portion of your overall BK channel transcripts inside for every structure.
Buildings out of rhombencephalon, mesencephalon and you can back
Within the back a small, but high, escalation in full BK route mRNA term try observed away from E13 compared to P35 (Contour 2a). It was accompanied by a dramatic reduced amount of STREX version phrase having a good concomitant increase in No variation mRNA phrase (Contour 4a). Similar developmental right up control of total BK channel mRNA expression are along with noticed in the fresh new midbrain, cerebellum, pons and medulla (Figure 2b–e). On these tissues, tall developmental downregulation from STREX variation term is once again observed ranging from embryonic and you may postnatal values (Profile 4b–e). In the midbrain this is paralleled by the a critical escalation in Zero variant phrase whereas from inside the pons and you may medulla no extreme change ranging from E13 and you will P35 had been observed (Contour 4d 4e). Weighed against almost every other nations, No version term rather ent on the cerebellum (Shape 4c).
