Neuronal voltage-gated Na+ channels are composed of three glycoprotein subunits, a pore-forming α subunit and two auxiliary β subunits, β1 and β2 (Messner & Catterall, 1985). Recently, two additional β1-like subunits have been identified, β1A and β3 (Kazen-Gillespie et al. 2000; Morgan et al. 2000). In the present study we have compared the cellular distribution of β1A and β3 mRNAs in the developing rat brain by semi-quantitative in situ hybridisation histochemistry.
All animals (n = 5) used were killed by an approved Schedule I procedure. Adjacent 5µm thick sections of whole Wistar rat embryo at stages E10, E15, E17 and E19 and male Wistar rat brain at postnatal stages P1, P3, P9, P14 and adult (6-7 weeks) were cut using a cryostat onto slides. Hybridisation buffer containing 45 bp long radio-labelled oligoprobes was added and hybridised at 42 °C overnight. Slides were subjected to stringency washes and apposed to X-ray film. Slides were also coated with liquid emulsion, developed, and stained in Cresyl Violet. Quantification of autoradiographs was carried out using an MCID image analyser.
Expression of β3 mRNA was first observed at E10 whilst β1A expression was observed at E15. At E15 both subunits were expressed at similar levels in the telencephalon and myelencephalon. β1A mRNA was exclusively detected in the mesencephalon whilst β3 mRNA was exclusively observed in midbrain, thalamic and spinal cord regions. At E17 both subunits were expressed at similar levels in the cortex, superior and inferior colliculus and the cervical flexure. However, at E19 distinct differences in mRNA expression patterns were detectable. β1A was expressed in all cortical layers whereas β3 expression was absent from the intermediate cortical layer. In contrast, β3 was exclusively expressed in the differentiating hippocampus and the thalamic and hypothalamic nuclei.
In the postnatal CNS β1A and β3 mRNA distribution patterns were far more divergent. In the cortex, expression of β3 was maximal at P1, decreasing at P9 and P14 to a low level in the adult. By P1, observable β1A expression in the cortex was low, dropping after P3 to barely detectable levels in the adult. In the striatum expression levels of β3 were high at P1, peaked at P3, and decreased slightly thereafter to give high levels of expression in the adult. However, striatal β1A expression was just detectable above background at P1, and undetectable after P3. In the thalamus β1A expression was absent at P1 and did not change thereafter. Thalamic levels of β3 mRNA were particularly high at P1 and decreased at P3, 9 and 14 to very low expression levels in the adult. β1A expression was not detected in the hippocampus throughout postnatal development, whilst β3 expression remained high in all hippocampal regions analysed. However, the converse was true in the cerebellum. β3 expression was not detected in the cerebellum throughout postnatal development, whereas relatively high levels of β1A expression were observed in the postnatal cerebellum. β1A expression was detected in the molecular and granule cell layers of the cerebellum after stage P14, and at high levels in the Purkinje cell layer during all stages of postnatal development and in the adult.
In summary, we show that the two recently identified voltage-gated sodium channel auxiliary subunits β1A and β3 display markedly different expression profiles in the CNS throughout embryogenesis, which may reflect differences in their physiological function during this critical period of neuronal development.
- Kazen-Gillespie, K.A., Ragsdale, D.S., D’Andrea, M.R., Mattei, L.N., Rogers, K.E. & Isom, L.L. (2000). J. Biol. Chem. 275, 1079-1088.
Messner, D.J. & Catterall, W.A. (1985). J. Biol. Chem. 260, 10597-10604.
Morgan, K., Stevens, E.B., Shah, B., Cox, P.J., Dixon, A.K., Lee, K. et al. (2000). Proc. Natl Acad. Sci. USA 97, 2308-2313.