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 station mRNA splicing on CNS
So you’re able to take a look at the developmental changes in BK station mRNA expression in almost any regions of the murine CNS, BK channel mRNA term was quantified at embryonic days thirteen, fifteen and you will 18, and postnatal weeks eight and you will thirty five. Each tissue, the total BK channel mRNA expression at each and every developmental date point are expressed given that a percentage of these on P35. Each splice variant the phrase at each and every developmental go out part is shown given that a share of one’s full BK route transcripts in the for each and every cells.
Architecture of rhombencephalon, mesencephalon and you will spinal-cord
When you look at the back a small, but high, rise in full BK station mRNA expression was noticed away from E13 than the P35 (Contour 2a). This was accompanied by a dramatic reduced amount of STREX variant expression that have a good concomitant rise in http://datingranking.net/asexual-dating/ Zero variation mRNA phrase (Shape 4a). Equivalent developmental upwards regulation away from overall BK station mRNA phrase is also present in the midbrain, cerebellum, pons and you will medulla (Profile 2b–e). In these structures, significant developmental downregulation out-of STREX variation term try once again noticed anywhere between embryonic and you can postnatal levels (Profile 4b–e). From inside the midbrain it was paralleled of the a critical increase in No variant phrase while during the pons and you can medulla no extreme alter ranging from E13 and P35 had been observed (Profile 4d 4e). In contrast to almost every other places, No version phrase significantly ent throughout the cerebellum (Figure 4c).
