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 station mRNA splicing regarding the CNS
To help you investigate developmental alterations in BK channel mRNA expression in various aspects of the fresh new murine CNS, BK channel mRNA term is actually quantified in the embryonic months thirteen, 15 and you will 18, and postnatal weeks 7 and you may thirty-five. Each tissue, the entire BK channel mRNA phrase at each and every developmental big date point is shown since the a portion of this from the P35. For every single splice version the definition of at every developmental day area was shown as a percentage of overall BK station transcripts from inside the for each and every structure.
Tissues from rhombencephalon, mesencephalon and you can spinal-cord
For the spinal-cord a little, however, significant, increase in complete BK station mRNA term was noticed regarding E13 as compared to P35 (Figure 2a). It was followed closely by a remarkable reduction of STREX variant phrase having a beneficial concomitant boost in No variant mRNA term (Figure 4a). Comparable developmental upwards control from total BK route mRNA term was in addition to seen in the fresh midbrain, cerebellum, pons and you may medulla (Profile 2b–e). Within these structures, high developmental downregulation regarding STREX variant phrase try once again noticed between embryonic and you will postnatal stages (Contour 4b–e). When you look at the midbrain this is paralleled by a life threatening boost in Zero version expression while inside pons and you may medulla zero high alter anywhere between E13 and you may P35 were observed (Shape 4d 4e). Weighed against most other places, Zero variation term large friends oturum aÃ§ma sorunu significantly ent about cerebellum (Profile 4c).