Proceedings of The Physiological Society
University of Cambridge (2008) Proc Physiol Soc 11, PC105
Distribution of Cx45 expression throughout the murine spinal cord
R. J. Chapman1, A. E. King1, S. Maxeiner2, K. Willecke2, J. Deuchars1
1. Institute of Membrane and Systems Biology, University of Leeds, Leeds, United Kingdom. 2. Division of Molecular Genetics, University of Bonn, Bonn, Germany.
Gap junctions are clusters of connexin containing channels that are expressed in regions of apposing membranes in coupled cells (Evans & Martin, 2002). Characterisation of connexin expression within the CNS is vital to the ultimate functional understanding of gap junctions. To date, there are ~20 known isoforms of connexin proteins and about half of mammalian connexins are expressed in the central nervous system (Willecke et al., 2002). Additionally, the neuronal connexin subtype, Cx45, has been found in α-motorneurons of the spinal ventral horn using in situ hybridization (Chang et al., 1999). Using Cx45-reporter mice expressing GFP (Maxeiner et al., 2003), we aimed to characterise the distribution of GFP-positive cells throughout the adult mouse spinal cord.
Animals anaesthetised deeply with sagatal (60mg.kg ip) were perfused trans-cardially with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) and the spinal cords harvested. All procedures accord with the UK Animals (Scientific Procedures) Act 1986. 50μm transverse sections were cut of both thoracic and lumbar spinal cord. Sections were then incubated in either mouse anti-NeuN (1:1000, Chemicon) or mouse anti-GFAP (1:1000, Affinity) or biotinylated IB4 (1:100, Vector Labs), and chicken anti-GFP (1:1000, Invitrogen) at 4oC for 12-36hrs. Anti-GFP was then visualised using Alexa488-conjugated donkey anti-chicken (1:1000, Invitrogen). NeuN and GFAP were individually visualised using Alexa555-conjugated donkey anti-mouse (1:1000, Invitrogen), and IB4 visualised with Strepdavidin Alexa555 (1:100, Invitrogen).
The distribution of GFP-expressing cells was highly concentrated within laminae II/III in both thoracic and lumbar spinal dorsal horn (DH), with a few cells noted in lamina I. No cells expressing GFP were observed in the spinal ventral horn (cf. Chang et al., 1999). The total number of cells expressing GFP in the lumbar DH was significantly higher than in the thoracic spinal DH (22.6±0.7 cells lumbar and 17.8±0.7 cells thoracic; t-test, P<0.001, n=3), and the number of cells expressed per 10μm2 was also significantly higher (3.17±0.1 cells lumbar and 2.76±0.07 cells thoracic; t-test, P<0.05, n=3). Staining with isolectin B4 (IB4) revealed that GFP expressing cells within lamina III received close appositions from fibers originating from laminae I/II. The post-mitotic neuronal marker, NeuN, was found to co-localise with presumed Cx45-immunopositive neurons in the spinal DH laminae, but no co-localisation was observed with the glial fibrillary acidic protein, GFAP.
These data suggest a highly localised expression of this connexin protein which may potentially play a significant role in the processing of somatosensory and sensorimotor afferent information. Further studies are now required to determine the DH cell type expressing this Cx45 protein.
Where applicable, experiments conform with Society ethical requirements