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 regulation out of BK channel mRNA splicing from the CNS
To check out the developmental alterations in BK route mRNA term in almost any areas of the newest murine CNS, BK route mRNA term is quantified from the embryonic weeks 13, fifteen and you may 18, and you may postnatal months seven and you will thirty-five. Each tissues, the BK route mRNA term at each developmental day section is shown once the a portion of this on P35. For every splice variant the term at each and every developmental big date point try indicated while the a percentage of one’s overall BK channel transcripts when you look at the per structure.
Structures regarding rhombencephalon, mesencephalon and spinal-cord
During the spinal-cord a small, however, extreme, boost in complete BK channel mRNA phrase was noticed out-of E13 as compared to P35 (Shape 2a). It was with a dramatic reduced total of STREX version expression which have good concomitant boost in Zero variant mRNA expression (Profile 4a). free Nudist dating websites Comparable developmental upwards control off total BK route mRNA term try and additionally seen in brand new midbrain, cerebellum, pons and medulla (Profile 2b–e). In these buildings, high developmental downregulation away from STREX variation phrase try once more seen ranging from embryonic and you will postnatal degrees (Profile 4b–e). For the midbrain this was paralleled of the a life threatening increase in No variant phrase while in pons and you can medulla no extreme alter ranging from E13 and P35 have been seen (Profile 4d 4e). Compared with most other countries, No version phrase significantly ent throughout the cerebellum (Shape 4c).