Spinal Atypical Protein Kinase C Activity Is Necessary to Stabilize Inactivity-Induced Phrenic Motor Facilitation

Abstract

The neural network controlling breathing must establish rhythmic motor output at a level adequate to sustain life. Reduced respiratory neural activity elicits a novel form of plasticity in circuits driving the diaphragm known as inactivity-induced phrenic motor facilitation (iPMF), a rebound increase in phrenic inspiratory output observed once respiratory neural drive is restored. The mechanisms underlying iPMF are unknown. Here, we demonstrate in anesthetized rats that spinal mechanisms give rise to iPMF, and that iPMF consists of at least two mechanistically distinct phases: 1) an early, labile phase that requires atypical PKC (PKCζ and/or PKCΙ/λ) activity to transition to a 2) late, stable phase. Early (but not late) iPMF is associated with increased interactions between PKCζ/Ι and the scaffolding protein ZIP/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/Ι activity is necessary for iPMF, spinal aPKC activity is not necessary for phrenic long-term facilitation (pLTF) following acute intermittent hypoxia, an activity-independent form of spinal respiratory plasticity. Thus, while iPMF and pLTF both manifest as prolonged increases in phrenic burst amplitude, they arise from distinct spinal cellular pathways. Our data are consistent with the hypotheses that: 1) local mechanisms sense and respond to reduced respiratory-related activity in the phrenic motor pool, and 2) inactivity-induced increases in phrenic inspiratory output require local PKCζ/Ι activity to stabilize into a long-lasting iPMF. Although the physiological role of iPMF is unknown, we suspect that iPMF represents a compensatory mechanism, assuring adequate motor output in a physiological system where prolonged inactivity ends life.

Decreased spinal synaptic inputs to phrenic motor neurons elicit localized inactivity-induced phrenic motor facilitation

“…This differential response is demonstrated by the profound reduction in ipsilateral phrenic motor output without any significant reduction in contralateral phrenic activity during procaine injections. As hypothesized, a unilateral reduction in phrenic synaptic inputs elicited ipsilateral iPMF that was mechanistically similar to those following central neural apnea (Broytman et al, 2013; Strey et al, 2012). Thus, iPMF is induced by inactivity-dependent mechanisms operating in the spinal cord near the phrenic motor nucleus, indicating local, spinal mechanisms are capable of shaping phrenic neural responses to inactivity.…”

“…Since iPMF following central neural apnea requires spinal atypical protein kinase C (aPKC) activity (Strey et al, 2012), we hypothesized that spinal aPKC activity is necessary for iPMF following disruption of spinal synaptic inputs to phrenic motor neurons. To test this hypothesis, a cell-permeable pseudosubstrate inhibitory peptide (PKCζ-PS) which binds to and inhibits all aPKC isoforms (i.e., PKCζ, PKCι/λ and PKMζ; Reyland, 2009) or the inactive scrambled version of the peptide (scrPKCζ-PS) was delivered intrathecally prior to intraspinal procaine or aCSF.…”

“…Unilateral axon conduction blockade reduced synaptic inputs to a subset of phrenic motor neurons; upon axon conduction blockade reversal, a robust increase in phrenic burst amplitude was immediately apparent in the ipsilateral phrenic nerve (iPMF). Similar to iPMF following central neural apnea (Broytman et al, 2013; Strey et al, 2012), spinal TNFα and aPKC activity were required for late, but not early iPMF. Surprisingly, a small, crossed spinal facilitation (csPMF) was also apparent in the contralateral phrenic motor pool, even though synaptic inputs were preserved.…”

“…However, it remains possible that iPMF is induced by factors other than respiratory neural inactivity per se . For example, the most common method to elicit iPMF is hyperventilation (Baertsch and Baker-Herman, 2013; Broytman et al, 2013; Mahamed et al, 2011; Strey et al, 2012), which creates a central neural apnea by lowering arterial CO 2 below the threshold for breathing. However, apart from stopping respiratory neural drive, the attendant hypocapnia and/or alkalosis may decrease cerebral blood flow and reduce oxygen unloading in the CNS (Brian, 1998; Vogel et al, 1996), both of which could lead to brain hypoxia (Nwaigwe et al, 2000; Schneider et al, 1998), a stimulus known to elicit prolonged increases in respiratory motor output (Bavis and Mitchell, 2003; Blitz and Ramirez, 2002).…”

Neither serotonin nor adenosine-dependent mechanisms preserve ventilatory capacity in ALS rats

“…Lastly, phrenic inactivity due to hypocapnia, vagal stimulation and/or anesthetic depression (Zhang et al, 2004; Mahamed et al, 2011) induces pMF by a mechanism that requires spinal PKCζ activity (Strey et al, 2012). One or all of these pathways to pMF may contribute to compensatory plasticity.…”