Proceedings of The Physiological Society
University of Cambridge (2004) J Physiol 555P, PC18
Connexin 45 in neurones of sensory, motor and autonomic pathways of murine brainstem and spinal cord
C.J. Milligan*, I.J. Edwards*, S. Maxeiner, O. Kruger, K. Willecke*, S.A. Deuchars* and J. Deuchars*
*School of Biomedical Sciences, University of Leeds, Leeds, UK and Institut fur Genetik, Rheinische Friedrich-Wilhelms Universitat Bonn, D-53117, Bonn, Germany
Electrical coupling typically mediated by gap junctions is evident between neurones such as sympathetic preganglionic neurones (e.g. Nolan et al. 1999) and motor neurones (Kiehn and Tresch, 2002). However, the gap junctional proteins involved remain to be identified. Here we investigate whether connexin45 (Cx45) is present in these neurones.
All animals were humanely killed by intraperitoneal injection of Sagatal (60 mg kg-1 I.P.) followed by transcardial perfusion with 0-4 % paraformaldehyde and 0-5 % glutaraldehyde in 0.1M phosphate buffer. Brainstem and spinal cord were sectioned on a vibrating microtome (50µm) and processed as indicated below. Expression of Cx45 was studied by localising expression of β-galactosidase in sections from 3 mice in which the entire coding region of the Cx45 gene was replaced by the lacZ gene encoding for β-galactosidase (Kruger et al. 2000). For Cx45 immunohistochemistry 8 male Wistar rats (150-200g) and 3 C57Bl/6 mice (6-8 weeks) were perfused transcardially as above. In rats, autonomic preganglionic and motor neurones were pre-labelled by intraperitoneal injection of 0.1ml of 0.1 % of Fluorogold (Fluorochrome Int.) 3-7 days prior to perfusion. Sections were incubated in mouse or rabbit anti-Cx45 (1:100, Chemicon, U.K.) and these localized with appropriate Cy3 conjugated secondary antibodies raised in donkey (1:800, Jackson Immunochemicals).
Localisation of Cx45 (Cx45/lacZ) expression and immunoreactivity of Cx45 labelled the same populations of cells in mice and rats. Cx45 was strongly expressed in autonomic and motor nuclei throughout the brainstem and spinal cord, including the following nuclei: facial, abducens, dorsal vagal, ambiguus, hypoglossal and in the spinal cord the intermediolateral cell column and ventral horn. In these nuclei, Cx45 positive neurones also contained Fluorogold confirming that they were motor or preganglionic neurones. Cx45 in neurones that are constituents of sensory pathways included the cochlear, spinal trigeminal, dorsal column, inferior olivary nuclei as well as the area postrema and the dorsal horn.
These data suggest widespread expression of Cx45 in neurones in murine brainstem and spinal cord and are the first identification of a particular connexin protein in many of these neuronal populations.
This work was funded by the Wellcome Trust (CJM), British Heart Foundation (SAD), the German Research Association (SM, KW) and a Physiological Society vacation studentship (IJE).
Where applicable, experiments conform with Society ethical requirements