Spinal 5-HT₇ receptors induce phrenic motor facilitation via EPAC-mTORC1 signaling

Abstract

Fields DP, Springborn SR, Mitchell GS. Spinal 5-HT 7 receptors induce phrenic motor facilitation via EPAC-mTORC1 signaling. J Neurophysiol 114: -2022. First published August 12, 2015 doi:10.1152/jn.00374.2015.-Spinal serotonin type 7 (5-HT 7 ) receptors elicit complex effects on motor activity. Whereas 5-HT 7 receptor activation gives rise to long-lasting phrenic motor facilitation (pMF), it also constrains 5-HT 2 receptor-induced pMF via "cross-talk inhibition." We hypothesized that divergent cAMP-dependent signaling pathways give rise to these distinct 5-HT 7 receptor actions. Specifically, we hypothesized that protein kinase A (PKA) mediates cross-talk inhibition of 5-HT 2 receptor-induced pMF whereas 5-HT 7 receptor-induced pMF results from exchange protein activated by cAMP (EPAC) signaling. Anesthetized, paralyzed, and ventilated rats receiving intrathecal (C 4 ) 5-HT 7 receptor agonist (AS-19) injections expressed pMF for Ͼ90 min, an effect abolished by pretreatment with a selective EPAC inhibitor (ESI-05) but not a selective PKA inhibitor (KT-5720). Furthermore, intrathecal injections of a selective EPAC activator (8-pCPT-2=-Me-cAMP) were sufficient to elicit pMF. Finally, spinal mammalian target of rapamycin complex-1 (mTORC1) inhibition via intrathecal rapamycin abolished 5-HT 7 receptor-and EPAC-induced pMF, demonstrating that spinal 5-HT 7 receptors elicit pMF by an EPAC-mTORC1 signaling pathway. Thus 5-HT 7 receptors elicit and constrain spinal phrenic motor plasticity via distinct signaling mechanisms that diverge at cAMP (EPAC vs. PKA). Selective manipulation of these molecules may enable refined regulation of serotonin-dependent spinal motor plasticity for therapeutic advantage. motor neuron; phrenic nerve; spinal cord; respiratory plasticity; neuroplasticity; 5-HT 7 ; receptor; exchange protein activated by cAMP; protein kinase A; rapamycin; mTOR SEROTONIN PLAYS A KEY ROLE in important forms of sensorymotor plasticity, including sensitization of the gill withdrawal reflex in Aplysia (reviewed in Kandel 2012). For example, episodic serotonin presentations enhance sensory motor synaptic transmission, giving rise to the gill withdrawal reflex (Brunelli et al. 1976). This well-studied form of plasticity in an invertebrate model system relies on multiple serotonin receptor subtypes, each activating unique kinase signaling cascades (Barbas et al. 2003).In ways similar to sensory motor facilitation in Aplysia, episodic serotonin receptor activation is necessary and sufficient for important forms of spinal respiratory motor plasticity, such as long-lasting (Ͼ90 min) phrenic motor facilitation (pMF) following acute intermittent hypoxia (AIH; reviewed by Mahamed and Mitchell 2007;Dale-Nagle et al. 2010;Devinney et al. 2013) or direct injections of serotonin or serotonin receptor agonists into the cervical spinal cord of rats (Hoffman

Divergent cAMP signaling differentially regulates serotonin-induced spinal motor plasticity

“…Intrathecal EPACa (10μL, 100μM) elicited pMF (Fig. 2, B; 58.9 ± 8.2% 90 min post-injection; n = 6; p < 0.001) similar to our previous report (Fields et al, 2015). EPACa induced pMF was attenuated by EPACi (Fig.…”

“…Aliquots of stock solutions remained viable for up to one week if stored frozen (−20° C) in 100% DMSO; after this time unused drug solutions were discarded. Prior studies using a similar protocol confirmed that EPACa is a selective EPAC activator (Fields et al 2015) and DOI is a selective 5-HT 2A receptor agonist (MacFarlane et al 2011). Separate studies using cell culture assays have shown that ESI-05 and Rp-8-Br-cAMP are selective inhibitors of EPAC (Tsalkova et al 2012; Rehmann 2013) and PKA (Poppe et al 2008; Harmati et al 2011) respectively (Table 1).…”

“…However, the functional implications of serotonin receptor co-activation have seldom been explored. In spinal pMF, Gq-coupled 5-HT 2A and Gs-coupled (cAMP-linked) 5-HT 7 receptors give rise to pMF through mechanistically distinct signaling cascades (MacFarlane et al, 2011; Hoffman and Mitchell, 2011; Fields et al, 2015). Although each receptor is sufficient to elicit pMF when stimulated alone, inter-receptor, cross-talk inhibition within the spinal respiratory control network limits pMF when they are co-activated (MacFarlane et al, 2009).…”

“…Previous studies have shown that intermittent 5-HT 2A agonist injections are required to elicit this form of pMF (MacFarlane et al 2011), whereas single injections (not intermittent) are necessary for PKA and EPAC induced pMF (Fields et al 2015). To confirm individual molecules are sufficient to elicit pMF, intrathecal 5-HT 2A receptor agonist (3×6μL, 100μM), PKAa (10μL, 100μM) or EPACa (10μL, 100μM) injections were given intrathecally.…”

“…To maintain volume consistency vehicle injections were given in each of these groups via a second intrathecal catheter. Dosing for 5-HT 2A (MacFarlane et al, 2011) and EPACa (Fields et al, 2015) were determined from previous studies; a limited dose response curve was completed for PKAa (data not shown). Intrathecal injections of the 5-HT 2A agonist, PKAa, or EPACa, gave rise to pMF without affecting hypoglossal (XII) nerve activity.…”