Phrenic Long-Term Facilitation Requires PKCθ Activity within Phrenic Motor Neurons

Abstract

Acute intermittent hypoxia (AIH) induces a form of spinal motor plasticity known as phrenic long-term facilitation (pLTF); pLTF is a prolonged increase in phrenic motor output after AIH has ended. In anesthetized rats, we demonstrate that pLTF requires activity of the novel PKC isoform, PKC, and that the relevant PKC is within phrenic motor neurons. Whereas spinal PKC inhibitors block pLTF, inhibitors targeting other PKC isoforms do not. PKC is highly expressed in phrenic motor neurons, and PKC knockdown with intrapleural siRNAs abolishes pLTF. Intrapleural siRNAs targeting PKC, an atypical PKC isoform expressed in phrenic motor neurons that underlies a distinct form of phrenic motor plasticity, does not affect pLTF. Thus, PKC plays a critical role in spinal AIH-induced respiratory motor plasticity, and the relevant PKC is localized within phrenic motor neurons. Intrapleural siRNA delivery has considerable potential as a therapeutic tool to selectively manipulate plasticity in vital respiratory motor neurons.

Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

“…We now show that TrkB activation within phrenic motor neurons is necessary for a well-studied form of respiratory neuroplasticity, BDNF dependent AIH-induced pLTF. We postulate that AIH triggers serotonin release and 5HT 2 receptor activation on phrenic motor neurons (Fuller et al 2001; Baker-Herman and Mitchell, 2002), activating PKC-θ (Devinney et al, 2015) and ERK/MAP kinases (Hoffman et al, 2012), initating new BDNF synthesis (Baker-Herman et al, 2004) and release followed by autocrine TrkB activation (this study). The ultimate outcome of these signaling evnts is a long-term increase in phrenic motor output, possibly by enhancing glutamate receptor funtion (Figure 4; Fuller et al, 2000; McGuire et al, 2008).…”

“…In a previous study from our laboratory, we used intrapleural siRNAs targeting PKC-Θ (Devinney et al, 2015). In this study, ventral spinal PKC-Θ was knocked down >50% and AIH-induced pLTF was abolished, yet this treatment had no effect on XII LTF (Devinney et al, 2015). These results provide additional evidence supporting the idea that intrapleural siRNA effects are localized to spinal motor neurons (or at least motor nuclei).…”

“…Intrapleural siRNAs were delivered daily as 30μl injections bilaterally (2×30μL of 5μM siRNA) from a 50μl Hamilton syringe 3, 2 and 1 day prior to tissue harvest or neurophysiology experiments (outlined in Figure 1A). This technique was adapted from previous reports using intrapleural siRNA injections to knockdown gene expression within phrenic motor neurons (Mantilla et al, 2013; Devinney et al, 2015). After injections, isoflurane was discontinued and chest movements were monitored for signs of distress and/or pneumothorax (none observed).…”

“…Considerable effort has been focused on understanding cellular and synaptic mechanisms of AIH-induced phrenic long-term facilitation (pLTF), and we know that it: 1) is initiated by spinal, serotonin type 2 receptor activation (Baker-Herman and Mitchell, 2002; Fuller et al, 2001; Kinkead et al, 1998; MacFarlane et al, 2011); 2) requires protein kinas C-θ activity within phrenic motor neurons (Devinney et al, 2015); 3) requires new synthesis of spinal brain derived neurotrophic factor (BDNF; Baker-Herman et al, 2004); and 4) requires ERK/MAP kinase signaling (Hoffman et al, 2012). Although new BDNF synthesis is necessary and sufficient for AIH-induced pLTF (Baker-Herman et al, 2004), the involvement of its high affinity receptor, tropomyosin receptor kinase B (TrkB), has not been conclusively demonstrated.…”

Intrathecal Intermittent Orexin-A Causes Sympathetic Long-Term Facilitation and Sensitizes the Peripheral Chemoreceptor Response to Hypoxia in Rats

“…Phrenic facilitation is evoked in the cervical phrenic motor nucleus by intracellular pathways activated by serotonergic G protein-coupled receptors coupled to protein kinase Cu (PKCu) and protein kinase A (PKA) effector proteins. PKCu activates extracellular signal-regulated kinase/mitogenactivated protein kinase pathways (Devinney et al, 2015), whereas PKA phosphorylates phosphatidylinositol-3-kinase/ protein kinase B pathways . Both mechanisms strengthen excitatory responses mediated by postsynaptic glutamatergic signals (Hoffman et al, 2012).…”