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 regarding BK route mRNA splicing regarding CNS
In order to investigate developmental changes in BK channel mRNA term in various aspects of brand new murine CNS, BK route mRNA term try quantified at the embryonic months 13, 15 and you may 18, and you may postnatal days seven and you will thirty-five skout tÃ¼rkiye. Per tissues, the complete BK station mRNA phrase at every developmental time part are expressed while the a portion of that during the P35. Each splice version the phrase at each developmental go out section are shown due to the fact a percentage of the complete BK route transcripts in the for every tissue.
Buildings away from rhombencephalon, mesencephalon and you can back
In the back a little, however, tall, boost in total BK channel mRNA term are seen off E13 compared to the P35 (Contour 2a). This was followed by a remarkable reduced amount of STREX variation phrase that have an effective concomitant increase in Zero variant mRNA phrase (Shape 4a). Similar developmental upwards regulation regarding complete BK channel mRNA phrase try and noticed in the fresh new midbrain, cerebellum, pons and you will medulla (Contour 2b–e). On these tissues, extreme developmental downregulation out-of STREX variation term is actually once again observed anywhere between embryonic and you can postnatal values (Figure 4b–e). From inside the midbrain this is paralleled because of the a life threatening increase in Zero version expression while when you look at the pons and you will medulla zero tall alter anywhere between E13 and you will P35 was indeed seen (Profile 4d 4e). In contrast to most other countries, Zero variation phrase significantly ent regarding the cerebellum (Contour 4c).