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

Abstract

Spinal serotonin type 7 (5-HT₇) receptors elicit complex effects on motor activity. Whereas 5-HT₇ receptor activation gives rise to long-lasting phrenic motor facilitation (pMF), it also constrains 5-HT₂ receptor-induced pMF via “cross-talk inhibition.” We hypothesized that divergent cAMP-dependent signaling pathways give rise to these distinct 5-HT₇ receptor actions. Specifically, we hypothesized that protein kinase A (PKA) mediates cross-talk inhibition of 5-HT₂ receptor-induced pMF whereas 5-HT₇ receptor-induced pMF results from exchange protein activated by cAMP (EPAC) signaling. Anesthetized, paralyzed, and ventilated rats receiving intrathecal (C₄) 5-HT₇ receptor agonist (AS-19) injections expressed pMF for >90 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-HT₇ receptor- and EPAC-induced pMF, demonstrating that spinal 5-HT₇ receptors elicit pMF by an EPAC-mTORC1 signaling pathway. Thus 5-HT₇ 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.

serotonin plays a key role in important forms of sensory-motor 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 (>90 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 and Mitchell 2011; MacFarlane et al. 2009, 2011). Indeed, multiple serotonin receptor subtypes elicit pMF via mechanistically distinct signaling cascades named for the G proteins most often coupled with the initiating metabotropic serotonin receptor (Dale-Nagle et al. 2010). Specifically, G_s protein-coupled serotonin type 7 (5-HT₇) receptors give rise to pMF through the “S pathway” (Dale-Nagle et al. 2010; Hoffman and Mitchell 2011), whereas G_q protein-coupled 5-HT₂ receptors induce the “Q pathway” to pMF (Dale-Nagle et al. 2010; MacFarlane et al. 2011).

While the S and Q pathways elicit pMF through distinct signaling cascades, they interact via mutual “cross-talk inhibition” (Dale-Nagle et al. 2010; Devinney et al. 2013; Hoffman and Mitchell 2013). One manifestation of cross-talk inhibition is the bell-shaped dose-response curve of pMF in response to intermittent intrathecal serotonin injections (MacFarlane and Mitchell 2009). Although low serotonin doses elicit pMF by activating spinal 5-HT₂ receptors (Fuller et al. 2001; MacFarlane et al. 2011; MacFarlane and Mitchell 2009), high doses do not unless spinal 5-HT₇ receptors are inhibited. Thus, when activated alone, 5-HT₂ (MacFarlane et al. 2011) and 5-HT₇ (Hoffman and Mitchell 2011) receptors each give rise to distinct forms of pMF. When activated concurrently, they effectively cancel each other because of balanced cross-talk inhibition (Devinney et al. 2013; Hoffman and Mitchell 2013; MacFarlane and Mitchell 2009). Cross-talk inhibition from 5-HT₇ receptors to Q pathway-induced pMF is mediated by protein kinase A (PKA) activity (Hoffman and Mitchell 2013); however, it is not known how 5-HT₇ receptor activation initiates pMF when acting alone (Hoffman and Mitchell 2011).

An alternative, but only recently appreciated, cAMP-dependent signaling cascade operating through exchange protein activated by cAMP (EPAC; de Rooij et al. 1998; Kawasaki et al. 1998) contributes to at least some forms of cAMP-dependent synaptic plasticity (Fernandes et al. 2015; Ster et al. 2007; Woolfrey et al. 2009). A key downstream molecule differentiating the PKA vs. EPAC signaling pathway is protein kinase B or Akt, a molecule necessary for 5-HT₇-induced pMF (Hoffman and Mitchell 2011). Whereas PKA inhibits neuronal Akt activity, EPAC stabilizes Akt's association with scaffolding proteins to enhance its kinase function (Nijholt et al. 2008). Thus we hypothesized that EPAC (not PKA) is both necessary and sufficient for 5-HT₇ receptor-induced pMF, whereas PKA mediates cross-talk inhibition of the Q pathway to pMF. Furthermore, Akt may elicit plasticity by increasing activity of mammalian target of rapamycin complex-1 (mTORC1), a major regulator of new protein synthesis (Ma and Blenis 2009). Thus we tested the hypothesis that spinal 5-HT₇ receptors give rise to pMF through an EPAC-mTORC1 signaling pathway.

We found that spinal EPAC activation is necessary and sufficient for 5-HT₇ receptor-induced pMF and that these effects require mTORC1 signaling. We confirm that PKA is not necessary for 5-HT₇ receptor-induced pMF and conclude that divergent cAMP signaling pathways underlie the distinct functions of 5-HT₇ receptors as they elicit (EPAC) and constrain (PKA) spinal serotonin-dependent respiratory motor plasticity.

METHODS

Experiments were performed on anesthetized 300- to 400-g male Lewis rats (Colony 202c, Harlan, Indianapolis, IN). The Animal Care and Use Committee at the University of Wisconsin, Madison approved all procedures.

Immunohistochemistry.

Although PKA constrains 5-HT₂ receptor-dependent pMF through cross-talk inhibition (Hoffman and Mitchell 2013), several lines of evidence suggest that EPAC (not PKA) mediates 5-HT₇ receptor-induced pMF. However, no study has attempted to localize these proteins in spinal respiratory neurons in general, or phrenic motor neurons (PMNs) in specific. Thus we investigated 5-HT₇ receptor, PKA, and EPAC protein expression within PMNs.

To identify PMNs, four untreated rats received intrapleural injections of the retrograde tracer cholera toxin B fragment (CtB; List Biological Laboratories, Campbell, CA; Mantilla et al. 2009). Briefly, each rat was anesthetized with isoflurane (1–1.5% in 100% O₂), and a 50-μl Hamilton syringe with a sterile, slightly beveled 27-gauge needle was used to inject 12.5 μl of 0.2% CtB in sterile saline. Bilateral injections were made at the fifth intercostal space (25 μl of 0.2% CtB total). After injections rats were carefully monitored for signs of respiratory distress associated with potential pneumothorax as isoflurane was discontinued (15 min). Seven days after injection rats were again anesthetized with isoflurane, euthanized, and transcardially perfused with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 4.0% paraformaldehyde (PFA; freshly made in 0.1 M phosphate buffer, pH 7.4). The cervical spinal cord was removed and placed in 4.0% PFA overnight at 4°C and then transferred to 20% and 30% sucrose in PBS buffer until sinking. Serial coronal sections (40 μm) of cervical C₃–C₅ were prepared with a microtome (Leica SM2000R) before storage in a cryoprotectant solution (30% ethylene glycol, 30% glycerol in 0.1 M PBS).

Free-floating sections were washed with 0.1 M PBS and incubated in a blocking solution (2% normal donkey serum, 0.1% Triton in PBS) for 60 min. Afterwards, tissues were costained for CtB:EPAC, CtB:PKA, or CtB:5-HT₇ with the following antibodies: goat anti-CtB (1:5,000; Calbiochem), rabbit anti-EPAC (1:100; Santa Cruz), rabbit anti-PKA (1:100; Abcam), and rabbit anti 5-HT₇ (1:100; Abcam). Unbound primary antibodies were removed with several PBS (0.1 M, pH 7.4) washes prior to incubation with secondary antibodies (Alexa 594 anti-goat 1:1,000 for CtB, Alexa 488 anti-rabbit 1:500 for EPAC and 5-HT₇, Alexa 488 anti-rabbit 1:1,000 for PKA; Invitrogen) for 2 h at room temperature. Afterwards, sections were washed in PBS and mounted on charged slides with an antifade solution (Prolong Gold antifade reagent; Invitrogen). Control staining with each primary antibody (without secondary antibodies) and all secondary antibodies (without primary antibodies) ruled out nonspecific, off-target staining (see Fig. 1).

Fig. 1.

Identified phrenic motor neurons (PMNs) express exchange protein activated by cAMP (EPAC), protein kinase A (PKA), and serotonin type 7 (5-HT₇) receptors: immunofluorescence images (×100) from 3 rats (left to right) for EPAC (A–C), PKA (D–F), and 5-HT₇ receptor (G–I) protein (red) with retrograde cholera toxin B fragment (CtB; green). A–C: EPAC + CtB staining of PMNs reveals perinuclear EPAC distribution (*). D–F: PKA + CtB staining of PMNs exhibits diffuse cytoplasmic PKA distribution (*). G–I: 5-HT₇ receptor + CtB staining shows abundant neuropil (*) but limited cytoplasmic/cell body 5-HT₇ receptor distribution. CtB staining was consistent in all groups, with prominent cytoplasmic staining and limited immunofluorescence in the nucleus or neuropil.

Slides were examined with a confocal microscope (Eclipse TE 2000-U, Nikon) using EZ-C1 software (Nikon). Z stacks were taken (26 μm thick, 2-μm steps) with a ×100 magnification objective. Images were rendered and finished with EZ-C1 software.

Experimental preparation.

Rat anesthesia was induced with isoflurane (3.5%) before placement on a heated stainless steel surgical table. The inspired oxygen concentration was continuously monitored (TED 60T; Teledyne Analytical Instruments), with adjustments made as necessary by changing nitrogen and/or oxygen flow rates. A tail vein catheter (24 gauge; Surflo, Elkton, MD) was inserted, and an infusion pump (Cole-Palmer, Vernon Hills, IL) was used to deliver intravenous fluids throughout the experiment; the solution used was 9:1 lactated Ringer solution (Baxter)-sodium bicarbonate solution (8.4%; Hospira; 1.5–2 ml/h). Rats were tracheotomized and bilaterally vagotomized through a ventral midline incision. A polyethylene catheter (PE50, ID/OD 0.58 mm/0.965 mm; Intramedic) was inserted into the femoral artery to monitor blood pressure (Gould Pressure Transducer, P23) and allow for arterial blood sampling (ABL 500; Radiometer, Copenhagen, Denmark). Dorsal laminectomy and durotomy (C₁/C₂) were performed to enable intrathecal drug delivery. Muscles overlying the cervical spinal cord were separated to expose the C₁-C₂ cervical vertebrae; after C₂ laminectomy, a silicone catheter (OD 0.6 mm; Access Technologies; primed with drug/vehicle) was inserted through a small hole in the dura and advanced caudally (3 mm), resting just over spinal regions C₃-C₄. This catheter was not placed in position until the end of surgical preparations to minimize unintended drug diffusion from the catheter. The left phrenic and hypoglossal (XII) nerves were dissected via a dorsal approach, desheathed, and covered with saline-soaked cotton to prevent desiccation. The rat was then slowly converted to urethane anesthesia (1.8 g/kg) while simultaneously being weaned from isoflurane (over 15–20 min). During anesthetic conversion, a rectal thermometer (Fisher Scientific) was inserted and body temperature was maintained at 37.5 ± 1.0°C throughout the experiment by adjusting the temperature of the surgical table. After conversion to urethane anesthesia, the rat was allowed at least 1 h for stabilization before experimental protocols started.

During the stabilization period the intrathecal catheter was inserted while both nerves were placed on bipolar silver recording electrodes and submerged in mineral oil. Once adequacy of anesthesia was confirmed (absent blood pressure spike in response to toe pinch) and respiratory nerve activity was detected, the rat was paralyzed with pancuronium bromide (3 mg/kg; Sicor Pharmaceuticals); 20 min was allowed for further stabilization. Nerve activity was amplified (gain, 10,000; A-M Systems, Everett, WA), band-pass filtered (100 Hz to 10 kHz), and integrated (CWE 821 filter; Paynter, Ardmore, PA; time constant 50 ms). The signal was digitized and recorded with a WINDAQ data acquisition system (DATAQ Instruments, Akron, OH). The resulting signal was analyzed with custom-designed software on a LabVIEW platform.

Throughout surgery and experimental protocols rats were ventilated with 60% inspired oxygen. End-tidal CO₂ was monitored with a flow-through capnoguard with sufficient response time to detect end-tidal CO₂ levels in a rat (Novametrix, Wallingford, CT). Rats were ventilated at a frequency of 70 breaths/min and a tidal volume of 2.5 ml or less (rodent ventilator model 683; Harvard Apparatus, South Natick, MA). This ventilation level caused hypocapnia (reduced end-tidal CO₂); thus inspired CO₂ was increased until the desired end-tidal value was attained. The CO₂ apneic threshold was determined by progressively lowering inspired CO₂ until respiratory nerve activity ceased; the recruitment threshold was subsequently determined by progressively raising inspired CO₂ until nerve activity resumed. To establish baseline levels, end-tidal CO₂ was set 2–3 mmHg above the CO₂ recruitment threshold as done previously (Bach and Mitchell 1996). Arterial partial pressure of CO₂ (Pa_CO2) was maintained within 1 mmHg of baseline values throughout the experiment by manipulating inspired CO₂ and monitoring arterial blood gases. Base excess values more negative than −3.0 meq/l were corrected with intravenous sodium bicarbonate (8.4%; Hospira) prior to baseline measurements. Blood was sampled (0.3 ml using a heparinized capillary) once baseline nerve recordings had stabilized and then again at 30, 60 and 90 min after injection of intrathecal drugs (see below). Measurements of nerve activity (burst amplitude and frequency) were monitored continuously and evaluated immediately prior to blood samples in 1-min bins. At the end of experiments rats were euthanized by urethane overdose.

Physiological variables for respective groups are summarized in Table 1. While several groups showed a significant increase in firing frequency, this was mild relative to phrenic nerve amplitude changes. Because of the inconsistency and faintness of this difference we did not pursue this further.

Table 1.

Physiological variables at baseline and 30, 60, and 90 min after final injection

	Time, min	Control	5-HT7 Agonist	EPACi + 5-HT7 Agonist	Rapamycin + 5-HT7 Agonist	PKAi + 5-HT7 Agonist	EPACa	EPACi + EPACa	Rapamycin + EPACa
PaCO2, mmHg	Baseline	49.8 ± 1.5	47.2 ± 1.5	52.3 ± 2.0	52.6 ± 0.9	50.5 ± 1.7	52.7 ± 0.9	55.5 ± 0.6	52.6 ± 0.9
	30	48.7 ± 2.4	48.2 ± 1.7	52.5 ± 1.9	52.2 ± 1.2	50.6 ± 1.7	53.4 ± 1.1	55.5 ± 0.9	52.2 ± 1.2
	60	50.7 ± 1.5	47.5 ± 1.6	54.0 ± 2.1	52.9 ± 1.5	50.5 ± 1.5	53.0 ± 0.8	55.4 ± 0.7	52.9 ± 1.5
	90	50.6 ± 1.9	47.0 ± 1.5	51.9 ± 1.9	52.5 ± 1.4	50.7 ± 1.8	52.8 ± 1.0	55.7 ± 0.8	52.5 ± 1.4
PaO2, mmHg	Baseline	310 ± 27	301 ± 11	306 ± 17	341 ± 25	310 ± 8	322 ± 12	369 ± 18	309 ± 23
	30	291 ± 29	303 ± 18	297 ± 22	337 ± 26	296 ± 6.0	302 ± 11	347 ± 22	299 ± 16
	60	289 ± 26	289 ± 9.9	295 ± 25	359 ± 26	291 ± 7.5	304 ± 14	344 ± 25	302 ± 15
	90	291 ± 29	283 ± 11	289 ± 30	364 ± 27	305 ± 15	288 ± 7.7	344 ± 27	305 ± 15
MAP, mmHg	Baseline	50.7 ± 3.0	49.8 ± 4.6	73.4 ± 14	58.5 ± 3.6	54.1 ± 4.8	44.5 ± 2.6	49.3 ± 3.3	39.8 ± 3.5
	30	50.0 ± 3.4	48.5 ± 3.8	69.5 ± 12	47.9 ± 3.9	50.5 ± 4.8	40.4 ± 2.3	41.1 ± 2.9	31.7 ± 3.4
	60	42.5 ± 3.4	44.6 ± 3.6	67.4 ± 11	45.9 ± 7.3	45.3 ± 3.0	37.5 ± 2.5	41.1 ± 2.5	28.5 ± 3.0
	90	41.9 ± 2.5	44.9 ± 2.4	66.0 ± 12	43.7 ± 6.5	42.32.8	31.1 ± 3.4	40.9 ± 3.8	24.5 ± 3.4
Phrenic burst frequency	Baseline	55.8 ± 8.4	44.8 ± 1.39	49.3 ± 0.5	47.9 ± 1.1	50.0 ± 0.8	44.4 ± 0.8	41.0 ± 0.9	45.1 ± 1.1
	30	54.9 ± 8.5	47.4 ± 1.3	48.3 ± 1.8	48.3 ± 1.1	49.4 ± 0.9	47.1 ± 1.0	41.9 ± 1.2	44.5 ± 1.5
	60	55.6 ± 9.5	48.6 ± 1.4*	48.8 ± 1.4	49.3 ± 2.1	51.0 ± 1.1	47.5 ± 1.1*	43.3 ± 0.8	43.4 ± 1.5
	90	56.3 ± 10.2	48.8 ± 1.5*	47.1 ± 0.5	50.0 ± 1.8	49.6 ± 1.9	46.3 ± 1.2	44.6 ± 0.8*	43.0 ± 1.5

Values are expressed as means ± SE. EPAC, exchange protein activated by cAMP; PKA, protein kinase A; EPACi, EPAC-selective inhibitor; PKAi, PKA-selective inhibitor; EPACa, EPAC-selective activator; Pa_CO2, arterial partial pressure of CO₂; Pa_O2, arterial partial pressure of O₂; MAP, mean arterial pressure.

^*Significantly different from baseline within same group (P ≤ 0.05).

Drugs and vehicles.

The following drugs were obtained from Santa Cruz (Dallas, TX): AS-19 (5-HT₇ receptor agonist), 8-pCPT-2′-Me-cAMP [8-pCPT; EPAC-selective activator (EPACa)], and KT-5720 [PKA-selective inhibitor (PKAi)]. Rapamycin (mTORC1 inhibitor) was obtained from Thermo-Fisher (Waltham, MA), while ESI-05 [EPAC-selective inhibitor (EPACi)] was obtained from BioLog Life Science Institute (Germany). All drugs were initially dissolved in dimethyl sulfoxide (DMSO) before dilution with vehicle (20% DMSO in sterile saline) before use. Aliquots of stock solutions remained viable for up to 1 wk if stored frozen (−20°C) in 100% DMSO; after this time, unused drug solutions were discarded. Prior studies confirm that EPACa is a selective EPAC activator (Christensen et al. 2003; Poppe et al. 2008); conversely, EPACi, PKAi, and rapamycin are regarded as selective inhibitors of EPAC (Rehmann 2013; Tsalkova et al. 2012), PKA (Davies et al. 2000), and mTORC1 (Davies et al. 2000), respectively.

Experimental protocols.

After stabilization of nerve signals a baseline blood sample was drawn, followed by a control intrathecal injection of vehicle (12 μl), a 15-min gap, and three consecutive intrathecal injections (C₄) of 5-HT₇ receptor agonist (3 × 5 μl, 100 μM; 5-min intervals) or a single injection of EPACa (10 μl, 100 μM). The 5-HT₇ receptor agonist dose was determined from a previous study using the same experimental protocol (Hoffman and Mitchell 2011), while a limited dose-response curve was completed for EPACa (data not shown).

Whereas the 5-HT₇ receptor agonist gave rise to pMF when injected intermittently (not as a single bolus), intrathecal EPACa gave rise to pMF when given as a single bolus (not intermittently). This requirement for intermittent 5-HT₇ receptor activation is consistent with previous studies demonstrating pattern sensitivity of both serotonin-induced and serotonin-dependent pMF (Baker and Mitchell 2000; MacFarlane and Mitchell 2009). In contrast, single (vs. intermittent) injection requirements for 8-pCPT (EPACa) are consistent with previous studies of EPAC-induced plasticity (Ster et al. 2007).

To identify molecules necessary for 5-HT₇ receptor- and EPAC-induced pMF, additional groups received intrathecal injections of selective inhibitors prior to the 5-HT₇ receptor agonist or EPAC activator. All inhibitors were given intrathecally via a second catheter (over a period of 2 min) 15 min prior to 5-HT₇ receptor agonist or EPACa injections. To determine whether PKA is necessary for 5-HT₇ receptor-induced pMF KT-5720, a PKAi (n = 6; 12 μl, 100 μM), was given prior to 5-HT₇ receptor agonist injections. We previously demonstrated that KT-5720 at this dose prevents PKA-mediated constraint of 5-HT_2A receptor-dependent, AIH-induced pMF (Hoffman and Mitchell 2013). In addition, this dose prevents PKA- but not EPAC-dependent signaling within cell cultures (Davies et al. 2000). Using an EPACi (12 μl, 2 mM), ESI-05, we tested whether EPAC is necessary for 5-HT₇ receptor (n = 7)- and EPACa (n = 6)-induced pMF. Finally, by pretreating with the highly selective inhibitor rapamycin (12 μl, 100 μM) we determined whether mTORC1 was necessary for 5-HT₇ receptor (n = 6)- or EPACa (n = 6)-induced pMF.

Additional control groups were completed for vehicle (n = 6), PKAi (n = 5), EPACi (n = 5), and rapamycin (n = 4) in which the drug was given followed by 3 × 5-μl injections of vehicle 15 min later. None of the control groups affected phrenic nerve activity, and they were not significantly different from each other; thus these groups were assembled into a single control group (n = 20; P ≥ 0.26).

Statistical analyses.

Peak integrated amplitude and frequency of phrenic and XII nerves were averaged in 1-min bins at baseline (preinjection) and then 30, 60, and 90 min after intrathecal injections. Amplitude values are expressed as percent change from baseline, while frequency is expressed as change from baseline in bursts per minute. Statistical comparisons were made for control and experimental groups with a two-way repeated-measures ANOVA; a Tukey post hoc test was used to identify pairwise differences. Significance was accepted as P < 0.05. All values are expressed as means ± SE.

RESULTS

Phrenic motor neuron expression of EPAC, PKA, and 5-HT₇ receptors.

PMNs were identified via intrapleural CtB injections. Antibodies targeting EPAC₂, PKA, and 5-HT₇ receptors all revealed immunofluorescence within/near CtB-identified PMNs. EPAC₂ immunoreactivity was cytoplasmic, with the densest staining in the perinuclear region (Fig. 1, A–C). PKA immunoreactivity was observed within the cytoplasm of CtB-colabeled PMNs (Fig. 1, D–F). Immunofluorescence for 5-HT₇ receptors was localized to the neuropil, with relatively limited cytoplasmic or nuclear staining (Fig. 1, G–I). Thus PMNs can directly respond to 5-HT₇ receptor agonists via EPAC and/or PKA signaling, although we cannot rule out additional contributions to pMF from surrounding cells: glia, presynaptic neurons, or interneurons.

EPAC, but not PKA, is necessary for 5-HT₇-induced pMF.

Intermittent intrathecal injections of 5-HT₇ receptor agonist (3 × 5 μl, 100 μM) AS-19 elicited long-lasting pMF expressed as a progressive enhancement in phrenic nerve amplitude (>90 min: 88.8 ± 7.2%; Fig. 2, A and Bii). The dose/selectivity of this same 5-HT₇ receptor agonist was determined in a previous study using the same experimental protocol (Hoffman and Mitchell 2011).

Fig. 2.

5-HT₇ receptor-induced phrenic motor facilitation (pMF) requires EPAC but not PKA activity. A: average change in phrenic burst amplitude from baseline to 90 min after injections. Lines are from rats with intrathecal injections of the 5-HT₇ receptor agonist (AS-19; 3 × 5 μl, 100 μM) with saline (◇, n = 7; 10 μl), EPAC-selective inhibitor (EPACi, △, n = 7; 10 μl, 2 mM), or PKA-selective inhibitor (PKAi, ×, n = 5; 10 μl, 100 μM) pretreatment or control rats that did not receive the 5-HT₇ receptor agonist (□, n = 20; 10 μl). Data represent mean ± SE values. Significant differences from ^#baseline, *control, or ^&PKAi + 5-HT₇ agonist: all P ≤ 0.05. B: representative traces of phrenic neurograms are shown before, during, and after intrathecal injections of vehicle + 5-HT₇ receptor agonist, EPACi + 5-HT₇ receptor agonist, or PKAi + 5-HT₇ receptor agonist. First arrow (below trace iv) represents the pretreatment injection; 2nd arrow represents the first of 3 5-HT₇ receptor agonist injections. Data from control and 5-HT₇ agonist groups are repeated within multiple figures.

Pretreatment with an intrathecal EPACi (10 μl, 2 mM), ESI-05, abolished 5-HT₇ receptor-induced pMF (90 min: 15.1 ± 10.5%, P = 0.0002; Fig. 2, A and Biii) demonstrating EPAC as necessary for 5-HT₇ receptor-induced pMF. In contrast, pretreating with a PKAi (10 μl, 100 μM), KT-5720, did not significantly reduce pMF relative to the vehicle-pretreated 5-HT₇ receptor agonist group (81.8 ± 10.0%, P = 0.61; Fig. 2, A and Biv). The KT-5720 dose is based on previous work using the same in vivo protocol (Hoffman and Mitchell 2013) and PKA cell culture activity assays (Davies et al. 2000). Cervical spinal injections of the 5-HT₇ receptor agonist did not affect hypoglossal nerve (XII) activity (n = 4; 90 min: −0.1 ± 13.1%, P = 0.95), suggesting that 5-HT₇ receptor agonist-induced pMF requires activation of spinal (vs. brain stem) receptors (Baker-Herman and Mitchell 2002; MacFarlane and Mitchell 2009).

Spinal EPAC activation elicits pMF.

Intrathecal injection of an EPACa (10 μl, 100 μM), 8-pCPT, elicited robust pMF lasting at least 90 min (104.4 ± 7.8%, P = 0.0000040; Fig. 3, A and Bii). Pretreatment with an intrathecal EPACi (10 μl, 2 mM), ESI-05, significantly attenuated EPACa-induced pMF (90 min: 24.5 ± 7.8%, P = 0.00064; Fig. 3, A and Biii), confirming the selectivity of EPACa. Although cervical spinal 5-HT₇ receptor agonist injections did not cause XII facilitation, similar EPACa injections did elicit significant XII burst amplitude facilitation (data not shown) at 10 μM (n = 2; 90 min: 59.5 ± 8.2%) and 100 μM (n = 4; 90 min: 77.2 ± 23.1%); this effect was blocked by intrathecal EPACi pretreatment (n = 3; 90 min: 7.9 ± 11.6%, P = 0.00064). These differential 5-HT₇ vs. EPAC effects on XII activity are difficult to explain, and we did not pursue this issue further in the present study.

Fig. 3.

Spinal EPAC activation is sufficient to elicit pMF. A: average change in phrenic burst amplitude from baseline. Individual curves represent EPACa-treated rats (10 μl, 100 μM) with saline (◇, n = 7; 10 μl) or with EPACi pretreatment (△, n = 6; 10 μl, 2 mM) or control injections alone (□; n = 20; 10 μl). Data represent mean ± SE values. Significant differences from ^#baseline, *control, or ^&EPACi + 5-HT₇ receptor agonist: all P ≤ 0.05. B: representative phrenic neurograms: time control (i), vehicle + EPACa (ii), or EPACi + EPACa (iii). First arrow represents pretreatment injection; 2nd arrow represents single EPACa injection. Data from control and EPACa groups are shown within multiple figures.

mTORC1 is necessary for 5-HT₇- and EPAC-induced pMF.

Pretreatment with the mTORC1 inhibitor rapamycin (10 μl; 100 μM) blocked both 5-HT₇ receptor (11.2 ± 9.2%, P = 0.00007)- and EPACa (9.9 ± 10.0%, P = 0.000039)-induced pMF (Fig. 4, A and B, respectively), demonstrating that mTORC1 is necessary for both processes.

Fig. 4.

Mammalian target of rapamycin complex-1 (mTORC1) activity is necessary for 5-HT₇ receptor- and EPAC-induced pMF. A: average change in phrenic burst amplitude from baseline for 5-HT₇ receptor agonist (10 μl, 100 μM) with saline (◇, n = 7; 10 μl) or rapamycin (△, n = 6; 10 μl, 100 μM) pretreatment or control injection alone (□, n = 20; 10 μl). Data represent mean ± SE values. Significant differences from ^#baseline, *controls, and ^&groups with rapamycin pretreatment: all P ≤ 0.05. Data from control, 5-HT₇ agonist, and EPACa groups are shown in multiple figures.

DISCUSSION

Although cervical spinal 5-HT₇ receptor activation elicits pMF, it also constrains 5-HT₂ receptor-induced pMF via cross-talk inhibition (Hoffman and Mitchell 2013; MacFarlane and Mitchell 2009). Here we show that these diverse actions result from divergent cAMP signaling. Specifically, 5-HT₇ receptors constrain 5-HT₂ receptor-induced pMF through cAMP/PKA signaling (Hoffman and Mitchell 2013) but elicit pMF via a mechanistically distinct cAMP/EPAC pathway. Since 5-HT₇ receptor- and EPAC-induced pMF are rapamycin sensitive, this pathway requires mTORC1 signaling. We conclude that serotonin elicits spinal plasticity via diverse mechanisms. First, serotonin elicits phrenic motor plasticity via unique mechanisms associated with distinct serotonin receptor subtypes (e.g., 5-HT₇ vs. 5-HT₂ receptors). Second, 5-HT₇ receptors elicit heterogeneous effects (presumably) within the same cell via divergent cAMP signaling (PKA and EPAC). Understanding distinct serotonin and cAMP functions may be critical as we attempt to harness serotonin-dependent plasticity as a treatment for severe neuro-motor disorders that compromise breathing, such as spinal injury and amyotrophic lateral sclerosis (Dale et al. 2014; Mitchell 2007).

While EPAC activation is necessary and sufficient for some forms of cAMP-dependent plasticity, selective PKA activation (EPAC independent) also gives rise to plasticity in some model systems (Castellucci et al. 1980). Thus EPAC and PKA may give rise to alternate, parallel signaling cascades for cAMP-dependent plasticity. Although PKA-dependent plasticity has been well studied, considerably less is known concerning how EPAC activation elicits plasticity. Here we demonstrate that EPAC-dependent, 5-HT₇ receptor-induced pMF is mechanistically distinct from the serotonin-induced PKA-ERK-brain-derived neurotrophic factor (BDNF) signaling pathway described in invertebrate model systems (for review see Kandel 2012). As shown by the available evidence concerning 5-HT₇ receptor-induced pMF, 5-HT₇ receptors elicit pMF via EPAC-Akt-mTORC1 signaling, resulting in new synthesis of an immature TrkB isoform (Golder et al. 2008; Hoffman and Mitchell 2011). This mechanism is independent of PKA, ERK signaling, or new BDNF synthesis (Hoffman and Mitchell 2011).

We are not aware of any previous studies connecting EPAC or mTORC1 signaling with serotonin-induced plasticity. By showing that the canonical pathway of PKA-ERK-BDNF does not play a role in 5-HT₇ receptor-induced spinal respiratory motor plasticity we have uncovered an interesting question: what purpose do parallel plasticity pathways operating downstream from a single receptor serve? We suggest that mechanistic heterogeneity in spinal motor plasticity signaling is important to maintain appropriate network activity throughout maturation, during changing environmental conditions, and during severe disease states (Dale et al. 2014). Although PKA signaling does not contribute to 5-HT₇-induced pMF, it may serve as a “reserve” pathway when EPAC-dependent signaling is impaired. For example, while wild-type mice express EPAC-dependent (PKA-independent) mossy fiber plasticity, EPAC₂-knockout mice retain certain forms of mossy fiber plasticity by shifting to PKA-dependent signaling (Fernandes et al. 2015). Thus in some conditions PKA activity is sufficient to induce plasticity during compromised EPAC signaling.

Finally, since PKA does not normally contribute to 5-HT₇ receptor-induced pMF our findings suggest that reduced PKA signaling may prevent cross-talk inhibition without compromising 5-HT₇ receptor-induced pMF. By minimizing cross-talk inhibition with PKA-selective inhibitors it may be possible for both 5-HT₂ and 5-HT₇ receptors to independently contribute to an enhanced form of pMF (i.e., metaplasticity; Fields and Mitchell 2015). In agreement, whereas PKA mediates cross-talk inhibition (Hoffman and Mitchell 2013), EPAC enables concurrent activation of signaling pathways operating downstream from 5-HT₂ and 5-HT₇ receptors (Johnson-Farley et al. 2005). The respective contributions of reduced PKA and/or enhanced EPAC activity to enhanced AIH-induced pMF following intermittent hypoxia preconditioning (Fields and Mitchell 2015) remain to be explored.

Although we are only beginning to understand interpathway interactions in serotonin receptor-induced spinal motor plasticity, the ability of nonessential accessory signaling pathways to regulate plasticity has considerable significance, from both a biological and a therapeutic perspective.

GRANTS

This work was funded by National Institutes of Health (NIH) Grants HL-80209 and HL-69064. D. P. Fields was supported by an Advanced Opportunity Fellowship at the University of Wisconsin and the Medical Scientist Training Program (NIH T32 GM-008692).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: D.P.F. and G.S.M. conception and design of research; D.P.F. and G.S.M. interpreted results of experiments; D.P.F. and G.S.M. edited and revised manuscript; D.P.F. and G.S.M. approved final version of manuscript; S.R.S. and G.S.M. performed experiments; S.R.S. and G.S.M. prepared figures; G.S.M. analyzed data; G.S.M. drafted manuscript.