BK channel mRNA expression regarding the murine CNS
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 from BK channel mRNA splicing regarding CNS
So you can browse the developmental changes in BK channel mRNA expression in various aspects of the new murine CNS, BK route mRNA term are quantified within embryonic days thirteen, 15 and you may 18, and you may postnatal months seven and you may thirty-five. Per cells, the complete BK route mRNA phrase at every developmental time section is conveyed because the a share of that in the P35. For every single splice variant the term at each developmental big date part is shown as the a share of one’s full BK station transcripts for the for each tissues.
Tissues out-of rhombencephalon, mesencephalon and you will back
Inside spinal cord a little, however, significant, boost in total BK station mRNA expression is seen out of E13 as compared to P35 (Figure 2a). This is accompanied by a remarkable decrease in STREX variant term with an effective concomitant rise in Zero version mRNA expression (Shape 4a). Comparable developmental up control from total BK channel mRNA phrase was including noticed in this new midbrain, cerebellum, pons and you may medulla (Profile 2b–e). Within these structures, high developmental downregulation out-of STREX kostenlos nach Alter Erwachsenen Dating variant phrase was again observed ranging from embryonic and you will postnatal degrees (Figure 4b–e). For the midbrain this was paralleled by a life threatening boost in No variation phrase while into the pons and you will medulla zero significant transform between E13 and you may P35 was indeed seen (Contour 4d 4e). Weighed against almost every other countries, Zero version phrase significantly ent from the cerebellum (Figure 4c).