Elsevier

Neuroscience

Volume 169, Issue 2, 25 August 2010, Pages 787-793
Neuroscience

Neurodegeneration, Neuroprotection, and Disease-Oriented Neuroscience
Research Paper
Atypical protein kinase C expression in phrenic motor neurons of the rat

https://doi.org/10.1016/j.neuroscience.2010.05.018Get rights and content

Abstract

Atypical protein kinase C (PKC) isoforms play important roles in many neural processes, including synaptic plasticity and neurodegenerative diseases. Although atypical PKCs are expressed throughout the brain, there are no reports concerning their expression in central neural regions associated with respiratory motor control. Therefore, we explored the neuroanatomical distribution of atypical PKCs in identified phrenic motor neurons, a motor pool that plays a key role in breathing. Diaphragm injections of cholera toxin B were used to retrogradely label and identify phrenic motor neurons; immunohistochemistry was used to localize atypical PKCs in and near labeled motor neurons (i.e. the phrenic motor nucleus). Atypical PKC expression in the phrenic motor nucleus appears specific to neurons; aPKC expression could not be detected in adjacent astrocytes or microglia. Strong atypical PKC labeling was observed within cholera toxin B labeled phrenic motor neurons. Documenting the expression of atypical PKCs in phrenic motor neurons provides a framework within which to assess their role in respiratory motor control, including novel forms of respiratory plasticity known to occur in this region.

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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