Increased atypical PKC expression and activity in the phrenic motor nucleus following cervical spinal injury.

Atypical protein kinase C (aPKC) isoforms are expressed in phrenic motor neurons, a group of motor neurons critical for breathing. Following C2 cervical hemisection (C2HS), spontaneous plasticity occurs in crossed-spinal synaptic pathways to phrenic motor neurons, at least partially restoring inspiratory phrenic activity below the injury. Since aPKCs are necessary for synaptic plasticity in other systems, we tested the hypothesis that C2HS increases aPKC expression and activity in spinal regions associated with the phrenic motor nucleus. C2 laminectomy (sham) or C2HS was performed on adult, male Lewis rats. Ventral spinal segments C3-5 were harvested 1, 3 or 28 days post-surgery, and prepared for aPKC enzyme activity assays and immunoblots. Ventral cervical aPKC activity was elevated 1 and 28, but not 3, days post-C2HS (1 day: 63% vs sham ipsilateral to injury; p<0.05; 28 day: 426% vs sham; p<0.05; no difference in ipsilateral vs contralateral response). Total PKCζ/ι protein expression was unchanged by C2HS, but total and phosphorylated PKMζ (constitutively active PKCζ isoform) increased ipsilateral to injury 28 days post-C2HS (p<0.05). Ipsilateral aPKC activity and expression were strongly correlated (r(2)=0.675, p<0.001). In a distinct group of rats, immunohistochemistry confirmed that aPKCs are expressed in neurons 28 days post-C2HS, including large, presumptive phrenic motor neurons; aPKCs were not detected in adjacent microglia (OX-42 positive cells) or astrocytes (GFAP positive cells). Changes in aPKC expression in the phrenic motor nucleus following C2HS suggests that aPKCs may contribute to functional recovery following cervical spinal injury.

Systemic inflammation impairs respiratory chemoreflexes and plasticity.

Many lung and central nervous system disorders require robust and appropriate physiological responses to assure adequate breathing. Factors undermining the efficacy of ventilatory control will diminish the ability to compensate for pathology, threatening life itself. Although most of these same disorders are associated with systemic and/or neuroinflammation, and inflammation affects neural function, we are only beginning to understand interactions between inflammation and any aspect of ventilatory control (e.g. sensory receptors, rhythm generation, chemoreflexes, plasticity). Here we review available evidence, and present limited new data suggesting that systemic (or neural) inflammation impairs two key elements of ventilatory control: chemoreflexes and respiratory motor (versus sensory) plasticity. Achieving an understanding of mechanisms whereby inflammation undermines ventilatory control is fundamental since inflammation may diminish the capacity for natural, compensatory responses during pathological states, and the ability to harness respiratory plasticity as a therapeutic strategy in the treatment of devastating breathing disorders, such as during cervical spinal injury or motor neuron disease.

Atypical protein kinase C expression in phrenic motor neurons of the rat.

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.

NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation.

Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O(2), 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (approximately C(4)) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C(4) spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91(phox), whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47(phox) and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C(4) ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.