Neurodegeneration, Neuroprotection, and Disease-Oriented NeuroscienceResearch PaperAtypical protein kinase C expression in phrenic motor neurons of the rat
Section snippets
Animals
All procedures were approved by the University of Wisconsin Animal Care and Use Committee. A total of 10 adult male Lewis rats were used in this study (3–6 months of age, Harlan, Colony 202A, Indianapolis, IN, USA).
Immunoblot analyses of ventral spinal homogenates
Four rats were anesthetized with isoflurane and euthanized with an overdose of Beuthanasia-D (Schering-Plough, Netherlands, pentobarbital, at least 120 mg/kg i.c.). C3–5 spinal segments were harvested and immediately placed on dry ice. The dorsal half of C3–5 was removed, and the
Immunoblot identification of atypical PKC isoforms
At least three atypical PKC isoforms including PKCζ, PKMζ, and PKCι, were detected in ventral spinal segments C3–5 in each of four un-operated rats (Fig. 1A). A control immunoblot, obtained by omission of primary antibody, confirmed that label in Fig. 1A was not due to non-specific label from the secondary antibody (control data not shown). Using a different primary antibody (PKCι; Table 1), a separate immunoblot confirmed the presence of PKCι (Fig. 1B). Background staining from the secondary
Discussion
Here we demonstrate the presence of atypical PKC isoforms in identified phrenic motor neurons. This is one of the first demonstrations of atypical PKC expression in any type of vertebrate motor neuron. Previous studies examined the distribution of atypical PKCs in the brain, including mRNA analysis in mice (Oster et al., 2004) and protein analysis in rats (Naik et al., 2000). However, only a few studies have investigated spinal atypical PKC expression (Hu et al., 2003, Wolf et al., 2008, Narita
Acknowledgments
Supported by National Institutes of Health (HL80209, HL69064 and HL07654). Dr. Stéphane Vinit is supported by a Craig H. Neilsen Foundation Fellowship.
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2012, Respiratory Physiology and NeurobiologyCitation Excerpt :Microglia and astrocyte activation were also estimated caudal to injury in the C4–C5 ventral horn, segments that contain most of the phrenic motor nucleus. Image fields were chosen based on the presence of presumptive phrenic motor neurons (large Neurotrace-positive cells often localized in a centralized cluster of the ventral horn; Guenther et al., 2010). The fractional area occupied by OX42 fluorescence in the ipsilateral C4 ventral horn significantly increased in injured vs. sham rats at 1 (17.6 ± 1.8% vs. 5.0 ± 0.7%, p < 0.001), 3 (38.6 ± 1.9% vs. 9.7 ± 0.5%, p < 0.001), 14 (41.7 ± 3.1% vs. 15.6 ± 2.9%, p < 0.001) and 28 (32.9 ± 2.0% vs. 13.7 ± 1.5%, p < 0.001) days post-C2LI (Fig. 4A, black bars, and C, red).