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 of BK route mRNA splicing on the CNS
So you can look at the developmental changes in BK station mRNA term in various areas of the fresh new murine CNS, BK channel mRNA term is actually quantified on embryonic months thirteen, fifteen and you will 18, and you may postnatal weeks eight and you may thirty-five. For every tissues, the full BK route mRNA expression at each and every developmental go out part try conveyed since the a portion of these within P35. For each splice version the phrase at each and every developmental go out section is conveyed given that a percentage of full BK route transcripts within the for every single tissues.
Tissues off rhombencephalon, mesencephalon and spinal cord
When you look at the spinal cord a tiny, however, significant, upsurge in full BK station mRNA term is actually observed away from E13 as compared to P35 (Contour 2a). It was followed closely by a dramatic reduced total of STREX version term with good concomitant increase in Zero version mRNA term (Contour 4a). Comparable developmental right up controls out of full BK station mRNA expression was as well as seen in the fresh midbrain, cerebellum, pons and you can medulla (Profile 2b–e). Within these buildings, extreme developmental downregulation away from STREX variant expression was once again seen ranging from embryonic and you may postnatal level (Shape 4b–e). Within the midbrain it was paralleled because of the a serious rise in Zero version term whereas from inside the pons and you can medulla no significant alter anywhere between E13 and P35 have been observed (Figure 4d 4e). Weighed against almost every other nations, No variation expression notably ent By ethnicity dating review about cerebellum (Contour 4c).