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. Author manuscript; available in PMC: 2024 Jan 29.
Published in final edited form as: Exp Neurol. 2020 Sep 9;334:113460. doi: 10.1016/j.expneurol.2020.113460

Differential mechanisms are required for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections

Lauren F Borkowski 1, Nicole L Nichols 1,*
PMCID: PMC10823911  NIHMSID: NIHMS1957020  PMID: 32916172

Abstract

Selective elimination of respiratory motor neurons using intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. This CTB-SAP model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons. After 7(d) days of CTB-SAP, phrenic long-term facilitation (pLTF, a form of respiratory plasticity) is enhanced, but returns towards control levels at 28d. However, the mechanism responsible for this difference in magnitude of pLTF is unknown. In naïve rats, pLTF predominately requires 5-HT2 receptors, the new synthesis of BDNF, and MEK/ERK signaling; however, pLTF can alternatively be induced via A2A receptors, the new synthesis of TrkB, and PI3K/Akt signaling. Since A2A receptor-dependent pLTF is enhanced in naïve rats, we suggest that 7d CTB-SAP treated rats utilize the alternative mechanism for pLTF. Here, we tested the hypothesis that pLTF following CTB-SAP is: 1) TrkB and PI3K/Akt, not BDNF and MEK/ERK, dependent at 7d; and 2) BDNF and MEK/ERK, not TrkB and PI3K/Akt, dependent at 28d. Adult Sprague Dawley male rats were anesthetized, paralyzed, ventilated, and were exposed to acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) following bilateral, intrapleural injections at 7d and 28d of: 1) CTB-SAP (25 μg), or 2) un-conjugated CTB and SAP (control). Intrathecal C4 delivery included either: 1) small interfering RNA that targeted BDNF or TrkB mRNA; 2) UO126 (MEK/ERK inhibitor); or 3) PI828 (PI3K/Akt inhibitor). Our data suggest that pLTF in 7d CTB-SAP treated rats is elicited primarily through TrkB and PI3K/Akt-dependent mechanisms, whereas BDNF and MEK/ERK-dependent mechanisms induce pLTF in 28d CTB-SAP treated rats. This project increases our understanding of respiratory plasticity and its implications for breathing following motor neuron death.

Keywords: Respiratory plasticity, Phrenic motor neuron death, Spinal cord, Breathing, Respiratory motor output

1. Introduction

Neuromuscular/neurodegenerative diseases (e.g., Pompe disease, spinal muscular atrophy (SMA), progressive muscular atrophy, and amyotrophic lateral sclerosis (ALS)) and injuries (e.g., spinal cord injury) result in loss of respiratory motor neurons, which subsequently leads to respiratory muscle weakness and/or paralysis, ventilator dependence, and ultimately death (Boentert et al., 2017; Bourke et al., 2001; Lechtzin et al., 2002; Lyall et al., 2001; Nichols et al., 2013b; Nogués et al., 2002; Wong et al., 2002). However, breathing is somehow maintained before ventilatory failure occurs. For example, patients with ALS and a genetic rat model of ALS (SOD1G93A) maintain breathing until disease end-stage (Lyall et al., 2001; Nichols et al., 2013a). Although genetic models have been used to study the effects of motor neuron loss in the aforementioned diseases, these models also develop global symptoms that make it difficult to understand solely the effects of respiratory motor neuron loss. To specifically study the effects caused by respiratory motor neuron loss, we utilize intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) to induce targeted respiratory motor neuron death (Nichols et al., 2018).

When CTB-SAP is injected intrapleurally, the CTB component binds to and enters the axolemma of neurons inhabiting the intrapleural space, allowing for the entire CTB-SAP construct to enter the axons. The construct is then retrogradely transported to the neuron cell bodies of phrenic and intercostal motor neurons in the cervical (C3–5) and thoracic (T2–7) spinal cord, respectively (Lencer and Tsai, 2003; Mantilla et al., 2009; Nichols et al., 2015b). Once inside the cell bodies, CTB and SAP will then dissociate (Llewellyn-Smith et al., 2000) allowing SAP to bind to ribosomes. This binding disables protein synthesis resulting in motor neuron cell death over a matter of hours to days via mechanisms of apoptosis (Llewellyn-Smith et al., 1999; Lujan et al., 2010). Significant respiratory motor neuron death is observed in 7 day (d) and 28d CTB-SAP treated rats compared to control rats (un-conjugated CTB + SAP injection) (Nichols et al., 2015b). Despite respiratory motor neuron loss and a decrease in phrenic output, eupneic breathing is maintained while breathing in response to maximum chemoreceptor activation is decreased in CTB-SAP treated rats (Nichols et al., 2015b).

One way that eupneic breathing is potentially maintained over the course of motor neuron loss is via compensatory mechanisms such as respiratory plasticity. We speculate these are mechanisms that enable surviving motor neurons to increase their output in order to compensate for the loss of neighboring motor neurons. This long-lasting increase in phrenic motor output is a form of phrenic plasticity known as phrenic motor facilitation (pMF) (Mitchell et al., 2001). pMF can be elicited pharmacologically (e.g., via activation of Gq- or Gs-coupled receptors) or physiologically (e.g., via acute intermittent hypoxia (AIH)). AIH-induced phrenic plasticity is specifically termed phrenic long-term facilitation (pLTF). pLTF occurs through the activation of Gq (5-HT2A/B) (Bach and Mitchell, 1996; Fuller et al., 2001; MacFarlane and Mitchell, 2007; MacFarlane et al., 2011) or Gs-coupled receptor-dependent pathways (A2A or 5-HT7) (Hoffman et al., 2010; Hoffman and Mitchell, 2013). Moderate exposure to AIH (35–55 mmHg PaO2) in naïve adult rats leads to the induction of pLTF through serotonin (5-HT) release in the phrenic motor nucleus. 5-HT2 receptors on or near phrenic motor neurons in the phrenic motor nucleus of the C3–6 spinal cord then become activated (Bach and Mitchell, 1996). Protein kinase C theta (PKCΦ) is subsequently activated,(Devinney et al., 2015; Devinney et al., 2013) and leads to the new synthesis of brain-derived neurotrophic factor (BDNF) (Baker-Herman et al., 2004; McGuire and Ling, 2004). BDNF then binds to the mature TrkB receptor (Baker-Herman et al., 2004), which then activates MEK and the phosphorylation of ERK (Hoffman et al., 2012), ultimately resulting in pLTF. pLTF also requires reactive oxygen species (ROS) formation via NADPH oxidase activity since ROS disinhibits phosphatase action on PKC (i.e., ROS formation allows pLTF to be evoked) (MacFarlane and Mitchell, 2008; MacFarlane et al., 2011).

As mentioned above, activation of spinal metabotropic receptors coupled to Gs proteins, such as adenosine 2A (A2A) (Golder et al., 2008) or 5-HT7 receptors (Hoffman and Mitchell, 2011), can also result in pMF. Pharmacological A2A receptor agonism-induced pMF requires the new synthesis of an immature TrkB isoform that is thought to signal from within phrenic motor neurons through auto-activation (Golder et al., 2008). pMF induced via the activation of A2A receptor agonism occurs independently from 5-HT receptor activation (Golder et al., 2008). Physiological A2A receptor activation occurs when naïve rats are exposed to more severe AIH (i.e., 25–35 mmHg PaO2). This leads to the release of adenosine into the extracellular space and activates adenosinergic mechanisms of pLTF, which includes the activation of protein kinase B (pAkt) via phosphatidylinositol 3-kinases (PI3K) (Agosto-Marlin et al., 2016; Devinney et al., 2013; Golder et al., 2008; Hoffman and Mitchell, 2011; Nichols et al., 2012). This severe protocol of AIH elicits pLTF that is enhanced from those rats exposed to the moderate AIH protocol (Nichols et al., 2012). Interestingly, when exposed to moderate AIH (35–55 mmHg PaO2), 7d CTB-SAP rats exhibit enhanced pLTF that is similar to the amount of A2A receptor-induced pLTF in naïve rats, while 28d CTB-SAP rats exhibit a more moderate amount of pLTF that is similar to that of 5-HT2 receptor-induced pLTF in naïve rats (Baker-Herman et al., 2004; Fuller et al., 2000). However, the underlying mechanisms required for producing varying amounts of AIH-induced pLTF following CTB-SAP-induced phrenic motor neuron death have yet to be understood.

Using the CTB-SAP model, we aim to understand and delineate the underlying mechanisms of AIH-induced pLTF that are activated following phrenic motor neuron loss at 7d and 28d. Therefore, we hypothesized that 7d CTB-SAP rats elicit enhanced AIH-induced pLTF through TrkB and PI3K/Akt mechanisms, while 28d CTB-SAP rats elicit moderate pLTF through BDNF and MEK/ERK signaling mechanisms. Using intrathecal delivery of siRNAs to target BDNF and TrkB mRNA or inhibitors to target MEK/ERK or PI3K/Akt activity, we found that pLTF is elicited predominately through TrkB and PI3K/Akt-dependent mechanisms in 7d CTB-SAP treated rats, whereas BDNF and MEK/ERK-dependent mechanisms induce pLTF in 28d CTB-SAP treated rats.

2. Methods

2.1. Animals

Experiments were conducted on adult (3–4 months old) male Sprague Dawley rats (Envigo Colony 208; Indianapolis, IN). Rats were housed in pairs and maintained under a 12:12 light:dark cycle. Animals had access to a standard commercial pelleted diet and water ad libitum. All procedures in this manuscript were approved by the Institutional Animal Care and Use Committee at the University of Missouri in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals. The University of Missouri is an AAALAC-accredited institution that operates under Animal Welfare Assurance ID A3394–01.

2.2. Intrapleural injections

Rats received bilateral intrapleural injections (6 mm deep, fifth intercostal space) using a 50 μl Hamilton syringe and a custom needle (6 mm, 23 gauge, semi-blunt to prevent lung puncture) while under isoflurane anesthesia (1.5% in 100% oxygen as previously described by Mantilla et al. (2009). Control rats were intrapleurally injected with cholera toxin B subunit (CTB; 25 μg dissolved in doubly distilled H2O; Calbiochem; Billerica, MA) unconjugated to saporin (SAP; 25 μg dissolved in phosphate buffered saline (PBS); Advanced Targeting Systems; San Diego, CA) to enable comparisons for respiratory plasticity. CTB conjugated to saporin (CTB-SAP; 25 μg dissolved in PBS; Advanced Targeting Systems; San Diego, CA) was intrapleurally injected to target respiratory motor neurons as described previously (Nichols et al., 2015b, 2017). CTB-SAP treated rats also received an additional 20 μg of CTB dissolved in double distilled H2O (Calbiochem; Billerica, MA) in their administered intrapleural injections. Rats were then monitored to ensure respiration was not compromised following intrapleural injections and were housed for 7 or 28 days before the below surgical preparation and neurophysiological experiments were performed.

2.3. Surgical preparation

Experimental procedures were performed as described previously (e.g., Hoffman et al., 2012; Nichols et al., 2018). Briefly, rats were isoflurane anesthetized, tracheotomized, and ventilated (Small Animal SAR-1000 Ventilator; CWE, Ardmore, PA, USA; tidal volume ~ 2.5 mL, frequency ~ 70–80 breaths per minute). Rats remained under isoflurane anesthesia (3.5% in 50% O2, balance N2) throughout surgical procedures before being converted to urethane anesthesia over 15–20 min (1.85 g kg− 1, i.v.) while isoflurane was slowly withdrawn. Once converted to urethane anesthesia, rats were then paralyzed using pancuronium bromide for neuromuscular blockade (2.5 mg kg− 1 i.v.). To maintain body fluid and acid-base balance, rats were given a 1:2:0.13 mixture of 6% Hetastarch (in 0.9% sodium chloride), lactated Ringer's solution, and 8.4% sodium bicarbonate via continuous intravenous infusion (1.5–6 mL kg− 1 h− 1). Lack of the pressor responses or obvious respiratory neural responses to a toe pinch with a hemostat was used to confirm the adequacy of anesthesia before and immediately after surgical and neurophysiological protocols were complete. Body temperature was maintained (37 ± 1 °C) with a custom-made heated surgical table, and was accessed with a rectal thermometer (Physitemp, Clifton, NJ, USA). Throughout the surgical preparation, end-tidal PCO2 (PETCO2) was maintained at ~45 mmHg and monitored with a flow-through carbon dioxide analyzer designed to sufficiently measure response time for PETCO2 measurements in rats (CapStar-100, CWE, Ardmore, PA).

Rats were bilaterally vagotomized and blood pressure was monitored in the right femoral artery by the insertion of a polyethylene catheter (PE50 ID: 0.58 mm, OD: 0.965 mm; Intramedic, MD, USA) connected to a pressure transducer (APT300 Pressure Transducer, Harvard Apparatus, Holliston, MA, USA). Arterial blood samples were taken during baseline, the first hypoxic episode, and at 15, 30 and at 60 min post-AIH and analyzed for partial pressures of O2 (PO2) and CO2 (PCO2), pH, and base excess (BE) using a blood gas analyzer (ABL80 Flex, Radiometer, Brea, CA).

Through a dorsal approach, the left phrenic nerve was isolated, distally cut, desheathed, and covered with a saline soaked cotton ball until it was placed on a bipolar silver electrode (described in the following section). For intrathecal delivery of drugs (see below), a laminectomy was performed at cervical level 2 (C2), and a small incision was made in the dura. A soft silicone intrathecal catheter (2 Fr, Access Technologies, Skokie, IL) was caudally inserted subdurally (3–4 mm) until the tip of the catheter rested over the phrenic motor nucleus in the C4 section of the spinal cord. The catheter was attached to a 50 μl Hamilton syringe filled with drug or vehicle solutions as described below. A minimum of 1 h was allowed following the conversion to urethane anesthesia before neurophysiological recordings began to eliminate the dampening effect of isoflurane on phrenic nerve output.

2.4. Neurophysiological recordings

The left phrenic nerve was submerged in mineral oil and placed on bipolar silver electrodes to record nerve activity. Neural signals were amplified (10,000 ×), band-pass filtered (300–10,000 Hz, Model 1800, A-M Systems, Carlsborg, WA, USA), full-wave rectified, and integrated (50 ms time constant, MA-821, CWE Inc., Ardmore, PA, USA). Integrated nerve bursts were digitized (8 kHz) and analyzed using a WINDAQ data acquisition system (DATAQ Instruments, Akron, OH, USA).

2.5. Experimental protocol

Apneic and recruitment thresholds were determined at least 1-h post urethane anesthesia conversion. First, ventilation was increased and PETCO2 was reduced until rhythmic nerve bursts had ceased (i.e., apneic threshold). After 1 min of nerve cessation, the ventilator rate was decreased and PETCO2 was slowly increased until the resumption of rhythmic nerve bursts occurred (i.e., recruitment threshold). To establish baseline conditions, PETCO2 was held approximately 2 mmHg above the recruitment threshold until stabilization of neural activity had occurred (≥15 min). A baseline arterial blood sample was taken to document baseline blood gas levels. Following baseline measurements, rats were exposed to three 5-min episodes of isocapnic (± 1.5 mmHg) acute intermittent hypoxia [10% inspired O2, arterial PO2 (PaO2): 35–45 mmHg] separated by 5-min intervals of baseline O2 conditions (50% inspired O2, PaO2: ≥ 150 mmHg). Rats were returned to baseline inspired O2 levels after the third bout of hypoxia, and inspired O2 levels were maintained for the duration of the experiments. Manipulation of inspired CO2 and/or the ventilation rate was done to maintain isocapnic arterial PCO2 (PaCO2) within ± 1.5 mmHg of the respective baseline value.

2.6. Intrathecal drug delivery protocol

To test the hypothesis that new spinal TrkB and/or BDNF protein synthesis are required for AIH-induced pLTF, small interfering RNAs (siRNAs) targeting BDNF and TrkB mRNA were injected via an intrathecal catheter over the cervical spinal cord (~C4) before AIH to prevent the translation and new BDNF or TrkB protein synthesis. TrkB siRNAs were obtained as a pool of four 21-nucleotide duplexes (siTrkB; ON-TARGET plus, Dharmacon; gene, Rat NTRK2; GenBank Accession No. NM 012731, Lafayette, CO); this same pool has been shown previously to effectively block new TrkB synthesis using the same experimental preparation.(Golder et al., 2008) BDNF siRNAs were also obtained as a pool of four 21-nucleotide duplexes (siBDNF; ON-TARGET plus, Dharmacon; gene, Rat BDNF; GenBank accession number, NM012513); these same pool has also previously been shown to prevent new synthesis of spinal BDNF in the same experimental preparation.(Baker-Herman et al., 2004) We used a nontargeting siRNA sequence (siNT; ON-TARGETplus Nontargeting siRNA #1; Dharmacon) as a negative control for potential non-specific effects of siRNAs. siRNAs were reconstituted with siRNA universal buffer (Dharmacon), and stored at −20 °C. Stock siTrkB or siBDNF (4 μl of 5 μM solution) was combined with: siNT (4 μl of 5 μM solution) so that the same amount of total siRNA was used for all groups; the transfection reagent, oligofectamine (32 μl, Invitrogen, Carlsbad, CA); and RNase-free water (160 μl, final concentration: 100 nM), and incubated at room temperature for 20 min. siNT experiments were performed by taking the stock siNT solution (8 μl of 5 μM solution), oligofectamine (32 μl), and RNase-free water (160 μl; final concentration, 100 nM), and then incubated as described above. siRNAs were slowly injected 2 h before baseline measurements and AIH exposures and were performed over the C4 spinal segment via an intrathecal catheter (two, 10 μl injections separated by 10 min). 7d control treated control groups included three groups that received AIH treatment: 1) siBDNF (n = 6), 2) siTrkB (n = 6), and 3) siNT (n = 8); and one group that received no AIH treatment: time controls (TCs; 32 μl oligofectamine, 168 μl of RNase-free water; n = 4). 28d control treated control groups included three groups that received AIH treatment: 1) siBDNF (n = 7), 2) siTrkB (n = 6), and 3) siNT (n = 8); and one group that received no AIH treatment: TCs (n = 4). 7d CTB-SAP treated groups included three groups that received AIH treatment: 1) siBDNF (n = 6), 2) siTrkB (n = 6), and 3) siNT (n = 6), and one group that received no AIH treatment: TCs (n = 3). 28d CTB-SAP treated groups included three groups that received AIH treatment: 1) siBDNF (n = 6), 2) siTrkB (n = 8), and 3) siNT (n = 8); and one group that received no AIH treatment: TCs (n = 4). All TC treated rats were grouped together within control and CTB-SAP rats. Approximately 2 h after siRNA delivery, animals were exposed to either AIH or no AIH (TC), and phrenic nerve activity was recorded for 60 min after AIH or sham AIH.

Additionally, selective inhibitors were intrathecally injected for MEK/ERK (UO126, [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene; 12 μl, 100 μM; MEK/ERK inhibitor; Promega]), and PI3K/Akt (PI828 [2-(4-Morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one; 12 μl, 100 μM; PI3K/Akt inhibitor; Tocris Bioscience]) to evaluate their involvement in AIH-induced pLTF. 7d control treated groups included the following: 1) UO126 + AIH (n = 7), 2) UO126 TC (n = 4), 3) PI828 + AIH (n = 7), 4) PI828 TC (n = 3), and 5) vehicle + AIH (12 μl, 20% DMSO; n = 6). 28d control treated groups included the following: 1) UO126 + AIH (n = 6), 2) UO126 TC (n = 4), 3) PI828 + AIH (n = 7), 4) PI828 TC (n = 4), and 5) vehicle + AIH (n = 8). 7d CTB-SAP treated groups included the following: 1) UO126 + AIH (n = 6), 2) UO126 TC (n = 4), 3) PI828 + AIH (n = 7), 4) PI828 TC (n = 3), and 5) vehicle + AIH (n = 6). 28d CTB-SAP treated groups included the following: 1) UO126 + AIH (n = 7), 2) UO126 TC (n = 5), 3) PI828 + AIH (n = 7), 4) PI828 TC (n = 3), and 5) vehicle + AIH (n = 8). All TC treated rats were grouped together within control and CTB-SAP rats. Approximately 20 min after drug or vehicle delivery, animals were exposed to either AIH or no AIH (TC), and phrenic nerve activity was recorded for 60 min after AIH or sham AIH.

2.7. Data and statistical analysis

Integrated phrenic nerve burst amplitudes were averaged over 1 min during baseline and 15, 30, and 60 min after AIH. Peak integrated inspiratory phrenic nerve bursts at baseline were similar within treatment groups; thus nerve bursts were normalized to baseline measurements to appropriately quantify the magnitude of pLTF (expressed as a percentage change from baseline). Statistical comparisons between treatment groups for AIH studies (amplitude, PaCO2, PaO2, pH, BE, and mean arterial pressure) were done using a two-way ANOVA with a repeated-measures design. Comparisons of the short-term hypoxic phrenic response were made using a one-way ANOVA, in which phrenic burst amplitude during the fifth minute of the first hypoxic episode was compared. For TC rats receiving no AIH exposure, a two-way ANOVA with repeated-measures design was performed; since there were no differences among them, they were grouped into a single TC group within control and CTB-SAP rats (siTC control, n = 8; siTC CTB-SAP, n = 7; UO126 TC control, n = 8; UO126 TC CTB-SAP, n = 9; PI828 TC control, n = 7; and PI828 TC CTB-SAP, n = 6). A one-way ANOVA was used when comparing phrenic nerve burst amplitudes across groups at 60 min post-hypoxia. A one-way ANOVA with ranks was used when comparing phrenic nerve burst frequency (expressed as an absolute change from baseline (bursts/min)) across groups at 60 min post-hypoxia. When significant ANOVA differences were detected, individual comparisons were made with Fisher's least significant difference post hoc test (Sigma Plot version 13.0; Systat Software Inc., San Jose, CA, USA). Differences between the groups were considered significant if p < 0.05; all values are expressed as means ± S.E.M.

3. Results

3.1. Blood gases and mean arterial pressures

Despite small but significant differences within and across groups for PaCO2, it was successfully maintained within 1.5 mmHg from its baseline value in all groups (Tables 1 & 2). Therefore, changes in integrated phrenic nerve burst amplitude following AIH are not attributed to differences in chemoreceptor feedback (changes > 1.5 mmHg of baseline in PaCO2 can influence pLTF; Bach and Mitchell, 1996). While apneic and recruitment thresholds were significantly different in some instances (AT: control siTC vs. 7d control siBDNF (p = 0.036), control siTC vs. 28d control siBDNF (p = 0.017), 7d CTB-SAP siNT vs. 7d CTB-SAP siTrkB (p = 0.026), 28d CTB-SAP siNT vs. 7d CTB-SAP siTrkB (p = 0.037), control UO126 TC vs. 28d control vehicle (p = 0.022), 7d control vehicle vs. 28d control vehicle (p = 0.040), 28d control vehicle vs. 28d control UO126 (p = 0.049); RT: control siTC vs. 7d control siBDNF (p = 0.037), CTB-SAP PI828 TC vs. 7d CTB-SAP PI828 (p = 0.024), 7d control vehicle vs. 7d CTB-SAP PI828 (p = 0.037), 7d CTB-SAP PI828 vs. 28d control UO126 (p = 0.045), 7d CTB-SAP PI828 vs. 28d control PI828 (p = 0.045), and 7d control vehicle vs. 28d control vehicle (p = 0.042); data not shown), drugs were not given prior to apneic and recruitment threshold acquisition and cannot contribute to differences in PaCO2. Since PaO2 was successfully regulated above 150 mmHg at baseline and remained above 150 mmHg post-hypoxia, and was held within the target range for AIH (35-45 mmHg) (Tables 1 & 2), we speculate that significant differences for PaO2 across groups for baseline and 60 mins post-hypoxia were likely not biologically relevant. For mean arterial blood pressure, slight but significant differences within groups were < 20 mmHg at 60 mins post-hypoxia, and this was consistent among groups (Tables 1 & 2; changes in mean arterial pressure of < 20 mmHg from baseline values have minimal effect on respiratory activity in rats (Walker and Jennings, 1995; Bach and Mitchell, 1996)). As expected, PaO2 and mean arterial pressure differed among groups when AIH vs. TCs were compared during hypoxic episodes. There were no differences detected for pH in rats that received intrathecal siRNA pre-treatment, whereas there were small but significant differences for pH in rats that received pre-treatment of intrathecal vehicle or inhibitors for MEK/ERK (UO126) or PI3K/Akt (PI828) (Supplementary Tables 1 & 2). When comparing BE values, we did detect small but significant differences for rats pre-treated with intrathecal siRNA or vehicle or drug inhibitors, but BE was successfully maintained within 2.5 mEq*L−1 from its baseline value in all groups (Supplementary Tables 1 & 2). Thus, the differential pLTF expression observed in this study was not affected by differences in blood gases or blood pressure regulation.

Table 1:

Arterial PCO2, PO2 and mean arterial pressure (MAP) during baseline, hypoxia (HX) and 60 minutes post-hypoxia for control (CON) and CTB-SAP treated rats with acute intermittent hypoxia (AIH) or without AIH (time-control or TC). Rats received intrathecal delivery of siRNAs targeting BDNF (siBDNF), TrkB (siTrkB) or non-target (siNT) mRNA. Significant differences within groups included versus hypoxia (a), and 60 min (b), and across groups included versus: respective TC (c), 28d CON siTrkB (d), and 28d CON siBDNF (e). Values are expressed as means ± S.E.M. Differences were considered significant if p<0.05.

PaCO2 (mmHg) PaO2 (mmHg) MAP (mmHg)
Experimental
Groups		 baseline HX 60 min baseline HX 60 min baseline HX 60 min
With AIH
siNT
  7d CON 45.1±1.5d 44.1±1.8d 46.5±1.3ad 310±4ab 38.0±1c 269±7a 121±4a 84±7c 110±5a
  7d CTB-SAP 49.5±1.5 49.6±1.6 51.0±2.1 296±8ab 39.1±1c 267±4a 114±4ab 72±6c 98±7a
  28d CON 48.2±1.0a 46.7±1.1d 48.1±1.2d 303±19ab 40±3c 267±19a 115±7ab 87±7c 98±7
  28d CTB-SAP 46.3±2.2bd 45.4±2.2d 47.8±1.9ad 305±11ab 41.3±1.3c 276±20a 110±6a 73±4c 103±5a
siBDNF
  7d CON 45.3±1.0d 45.1±1.2d 46.0±0.9d 301±7ab 38.5±1.4c 246±22acde 114±5ab 74±6c 95±5a
  7d CTB-SAP 44.9±2.3d 45.2±3.1d 46.4±2.5d 290±11a 40.4±1.8c 276±7a 116±5ab 70±8c 102±7a
  28d CON 48.5±3.1 47.8±3.2 47.9±3.3d 299±10a 40.0±1.5c 285±5a 127±6ab 85±11c 101±9a
  28d CTB-SAP 46.1±1.3d 45.3±1.4d 46.1±1.5d 307±6ab 41.7±1.0c 273±11a 107±7a 69±12c 103±7a
siTrkB
  7d CON 44.3±1.2d 43.5±1.4d 45.8±0.8ad 299±10ab 39.0±1.3c 271±12a 118±9a 72±5c 109±9a
  7d CTB-SAP 47.9±1.3 48.8±1.4 47.8±1.2d 281±32ad 37.3±1.1c 257±26ad 118±6a 71±11c 111±6a
  28d CON 53.2±1.4 52.8±1.3 54.6±1.5a 316±10a 38.6±0.9c 293±6a 119±14ab 92±13c 99±15
  28d CTB-SAP 49.1±0.7 48.6±1.0 49.6±1.0 308±17ab 42.3±0.7c 265±19a 118±6ab 75±10c 93±7a
Without AIH (TCs)
  CON siTC 47.3±1.4d 47.8±1.5 47.7±1.3d 306±7 307±6 285±9 115±5 114±5 105±5
  CTB-SAP siTC 50.1±2.4 49.8±2.2 50.5±3.4 302±10 303±8 283±12 112±6 122±12 104±11a
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Table 2:

Arterial PCO2, PO2 and mean arterial pressure (MAP) during baseline, hypoxia (HX) and 60 minutes post-hypoxia for control (CON) and CTB-SAP treated rats with acute intermittent hypoxia (AIH) or without AIH (time-control or TC). Rats received intrathecal delivery of either: 1) vehicle (20% DMSO); or 2) MEK/ERK inhibitor (UO126); or 3) PI3K/Akt inhibitor (PI828). Significant differences within groups included versus hypoxia (a), and 60 min (b), and across groups included versus: respective TC (c), 7d CTB-SAP PI828 (d), 28d CON vehicle (e), 28d CTB-SAP vehicle (f), 28d CTB-SAP PI828 (g), 7d CTB-SAP vehicle (h), 28d CON UO126 (i), 7d CON UO126 (j), and 28d CTB-SAP UO126 (k). Values are expressed as means ± S.E.M. Differences were considered significant if p<0.05.

PaCO2 (mmHg) PaO2 (mmHg) MAP (mmHg)
Experimental
Groups		 baseline HX 60 min baseline HX 60 min baseline HX 60 min
With AIH
Vehicle
  7d CON 49.0±1.3 49.3±1.2 49.4±1.3 294±15a 39.7±2.3c 264±9a 108±9a 77±12c 106±4a
  7d CTB-SAP 50.3±1.2 49.8±1.0 49.6±1.5 317±12ab 45.0±1.2c 273±9ac 108±8a 79±8c 110±9a
  28d CON 51.2±1.0 50.9±0.9 51.1±1.2 263±21ae 42.1±2.5c 252±12a 107±4a 70±6ce 109±7a
  28d CTB-SAP 51.1±0.6 50.8±0.7 51.5±0.7 249±17a 40.1±1.7c 246±20a 117±4a 80±12c 107±6a
UO126
  7d CON 48.0±1.1 48.1±1.2 47.9±1.3 285±10a 37.4±0.8c 271±7a 123±8ac 91±11 110±7a
  7d CTB-SAP 45.5±1.9bcdef 46.4±1.9cd 47.0±1.6c 275±18a 38.0±1.7c 270±10ac 101±5ai 71±7c 97±8a
  28d CON 45.8±2.0def 46.1±2.2d 46.6±1.1g 294±9a 37.5±1.7c 267±14a 127±9acg 91±9 107±10a
  28d CTB-SAP 48.0±1.3 48.3±1.5c 47.4±1.5c 307±4af 39.3±1.5c 283±6ac 120±6abg 81±5 101±9a
PI828
  7d CON 45.6±1.5def 46.4±1.8d 46.1±1.5defg 302±10af 37.6±0.9c 278±12a 107±5a 80±7 100±6a
  7d CTB-SAP 51.4±1.5 52.1±1.9 51.2±1.7 288±16ab 38.2±0.8c 260±15a 118±6a 87±10 106±9a
  28d CON 45.0±1.9defgh 45.4±1.8def 45.5±1.4defg 303±8af 39.9±1.4c 282±5a 94±6dfijk 78±9 85±9efhij
  28d CTB-SAP 50.5±1.0b 49.8±1.0 52.0±1.1a 300±9af 41.8±1.1c 275±9a 98±6a 79±13c 84±6efhij
Without AIH (TCs)
  CON UO126 48.2±2.0 48.5±1.8 47.8±1.5 284±27 287±26 289±10 100±6 110±11 91±7a
  CTB-SAP UO126 52.6±3.0a 53.8±3.3 53.0±3.0 278±28b 280±27 222±35a 95±8df 93±11 96±5
  CON PI828 46.0±2.4def 45.2±2.7def 45.9±2.1defg 298±8f 295±9 277±12 103±4 91±6 93±6
  CTB-SAP PI828 52.7±1.5 52.9±1.8 52.8±1.6 280±30b 279±31 243±24a 102±7 106±7 85±9aefhj
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3.2. Short-term hypoxic phrenic responses

Since time control (TC) groups were not exposed to AIH, these groups should not have a hypoxic response. Thus, all AIH-treated groups had hypoxic responses that were significantly increased vs. corresponding TC groups (Figs. 1A & 1B; respective TC vs. 7d control siNT (p < 0.001), 7d control siBDNF (p < 0.001), 7d control siTrkB (p < 0.001), 28d control siNT (p < 0.001), 28d control siBDNF (p = 0.030),28 control siTrkB (p < 0.001), 7d CTB-SAP siNT (p = 0.002), 7d CTB-SAP siBDNF (p = 0.004), 7d CTB-SAP siTrkB (p = 0.004), 28d CTB-SAP siNT (p < 0.001), 28d CTB-SAP siBDNF (p = 0.049), 28 CTB-SAP siTrkB (p = 0.010), 7d control vehicle (p < 0.001), 7d control UO126 (p = 0.001), 7d control PI828 (p < 0.001), 28d control vehicle (p < 0.001), 28d control UO126 (p = 0.005), 28d control PI828 (p = 0.006), 7d CTB-SAP vehicle (p < 0.001), 7d CTB-SAP UO126 (p = 0.001), 7d CTB-SAP PI828 (p < 0.001), 28d CTB-SAP vehicle (p < 0.001), 28d CTB-SAP UO126 (p = 0.005), and 28d CTB-SAP PI828 (p = 0.009)). 7d CTB-SAP treated rats had an increased hypoxic response compared to 7d control rats pre-treated with the same siRNA (Fig. 1A; siNT (p < 0.001), siBDNF (p = 0.004), and siTrkB (p = 0.004)). However, the short-term hypoxic phrenic response was decreased in 28d CTB-SAP rats pre-treated with siBDNF or siTrkB vs. all 7d CTB-SAP groups regardless of siRNA pre-treatment (Fig. 1A; 28d CTB-SAP siBDNF vs. 7d CTB-SAP siNT (p = 0.002), 7d CTB-SAP siBDNF (p = 0.010), and 7d CTB-SAP siTrkB (p = 0.012); 28d CTB-SAP siTrkB vs. 7d CTB-SAP siNT (p = 0.007), 7d CTB-SAP siBDNF (p = 0.028), and 7d CTB-SAP siTrkB (p = 0.032)), and it was decreased in 28d CTB-SAP rats pre-treated with siBDNF vs. 28d CTB-SAP rats pre-treated with siNT (Fig. 1A; p = 0.048). The short-term hypoxic phrenic response was enhanced in vehicle treated 7d CTB-SAP rats vs. vehicle treated 7d control rats and 28d CTB-SAP rats pre-treated with vehicle (Fig. 1B; 7d CTB-SAP vehicle vs. 7d control vehicle (p = 0.008), and 28d CTB-SAP vehicle (p = 0.003)). 7d CTB-SAP rats pre-treated with PI828 had a hypoxic response that was significantly greater than 7d control rats pre-treated with PI828 and 28d CTB-SAP vehicle pre-treated rats (Fig. 1B; 7d CTB-SAP PI828 vs. 7d control PI828 (p = 0.001) and 28d CTB-SAP vehicle (p = 0.004)), whereas 7d controls pre-treated with PI828 had a hypoxic response that was significantly decreased vs. 28d controls pre-treated with vehicle (Fig. 1B; p = 0.042). Lastly, vehicle treated 28d control rats and 7d CTB-SAP rats pre-treated with UO126 had hypoxic responses that were significantly greater than 7d control rats pre-treated with UO126 (Fig. 1B; 7d control UO126 vs. 28d control vehicle (p = 0.025), and 7d CTB-SAP UO126 (p = 0.013)).

Fig. 1.

Fig. 1.

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Short-term phrenic nerve hypoxic response in 7d & 28d control and CTB SAP treated rats. A. Short-term phrenic nerve hypoxic responses were compared in 7d and 28d control and CTB-SAP treated rats pre-treated with siNT, siBDNF, siTrkB, and siTC. As expected, all treatment groups exposed to AIH had a significantly greater hypoxic response vs. the corresponding siTC group (†). All 7d CTB-SAP rats pre-treated with siRNA had a significantly greater hypoxic response vs. respective pre-treated 7d control rats (+). 28d CTB-SAP rats pre-treated with siBDNF and siTrkB had hypoxic reponses that were significantly less than 7d CTB-SAP rats pre-treated with siNT (#), 7d CTB-SAP rats pre-treated with siBDNF ($), and 7d CTB-SAP rats pre-treated with siTrkB (@). 28d CTB-SAP rats pre-treated with siNT had a significantly higher hypoxic response vs. 28d CTB-SAP rats pre-treated with siBDNF (€). B. Short-term phrenic nerve hypoxic responses were compared in 7 and 28d control and CTB-SAP treated rats pre-treated with vehicle (DMSO), UO126, PI828, and TC for both inhibitors (UO126 TC and PI828 TC). As expected, all treatment groups exposed to AIH had a significantly greater hypoxic response vs. the corresponding TC group (†). All 7d CTB-SAP rats pre-treated with vehicle or inhibitors had a significantly greater hypoxic response vs. respective pre-treated 7d control rats (+). 7d CTB-SAP rats pre-treated with vehicle had a significantly greater hypoxic response compared to 7d controls pre-treated with vehicle, and 28d CTB-SAP rats with vehicle (‡). 7d CTB-SAP rats pre-treated with PI828 had a hypoxic response that was significantly greater than 7d control rats pre-treated with PI828, and 28d CTB-SAP rats pre-treated with vehicle (*). The hypoxic response in 7d control rats pre-treated with UO126 was significantly less than 7d CTB-SAP rats pre-treated with UO126, and 28d control rats pre-treated with vehicle (>). Lastly, 28d controls pre-treated with vehicle had a significantly greater hypoxic response compared to 7d controls pre-treated with PI828 (§). Values are means ± SEM, and all significant differences are p < 0.05.

3.3. AIH-induced pLTF in 7d, not 28d, CTB-SAP treated rats is dependent on new TrkB synthesis

Previous studies have shown that AIH-induced pLTF is enhanced in 7d CTB-SAP treated rats compared to control rats and 28d CTB-SAP treated rats (Nichols et al., 2018). Thus, the purpose of the current study is to understand if underlying mechanisms contribute to the differential pLTF observed in CTB-SAP treated rats. One way we targeted the underlying mechanisms was to deliver intrathecal siBDNF or siTrkB. Representative phrenic neurograms are shown for AIH-exposed 7d CTB-SAP rats approximately 2 h following siNT, siBDNF or siTrkB pre-treatment (Figs 2A-C). As expected, pLTF in 7d control treated rats was abolished by siBDNF pre-treatment (significantly less than controls pre-treated with siNT (p < 0.001) and siTrkB (p = 0.001) at 60 min post-hypoxia; not different from controls pre-treated with siTC, p > 0.05; Fig. 2D), which is consistent with new BDNF synthesis being required for pLTF in control treated rats. siTrkB and siNT pre-treatment in 7d controls had no effect on pLTF (p > 0.05 for siTrkB vs. siNT; both significantly greater than rats pre-treated with siTC, both p < 0.001; Fig. 2D). In contrast, siBDNF and siNT pre-treatment had no effect on pLTF in 7d CTB-SAP treated rats (p > 0.05 for siBDNF vs. siNT; both significantly greater than rats pre-treated with siTC (30 min post-hypoxia: siNT, p < 0.001; 60 min post-hypoxia: both p < 0.001); Fig. 2E). Pre-treatment with siTrkB nearly abolished AIH-induced pLTF in 7d CTB-SAP rats (significantly less than pLTF exhibited by 7d CTB-SAP treated rats pre-treated with siNT (15, 30, and 60 min post-hypoxia, all p < 0.001) and siBDNF (15 min post-hypoxia, p = 0.038; 60 min post-hypoxia, p = 0.001); not different from CTB-SAP rats pre-treated with siTC, p > 0.05, Fig. 2E). These data indicate that new TrkB synthesis contributes to AIH-induced pLTF in 7d CTB-SAP rats.

Fig. 2.

Fig. 2.

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pLTF in 7d siRNA treated rats. A–C, Representative traces of compressed, integrated phrenic nerve activity before and after AIH in 7d CTB-SAP rats pre-treated with siNT (A), siBDNF (B), or siTrkB (C). Baseline is indicated in each trace by a white, dashed line. AIH elicits pLTF in 7d CTB-SAP rats pre-treated with siNT and siBDNF, but appears to be reduced in rats pre-treated with siTrkB. D, E. Phrenic burst amplitude (expressed as a percent change from baseline) in 7d control (D) and 7d CTB-SAP (E) rats pre-treated with siNT, siBDNF, siTrkB, or siTC. pLTF was significantly increased from baseline (*) and siTC (†) at 60 min in 7d control rats pre-treated with siNT or siTrkB. In contrast, pLTF was significantly increased from baseline (*) at all time points in 7d CTB-SAP rats pre-treated with siNT or siBDNF, and from siTC at 30 (siBDNF) and 60 min post-hypoxia (both siBDNF and siNT (†)). pLTF in 7d CTB-SAP rats pre-treated with siTrkB was significantly reduced from 7d CTB-SAP rats pre-treated with siNT at all time points, and 7d CTB-SAP pre-treated with siBDNF at 15 min and 60 min post-hypoxia (#). Values are means ± SEM, and all significant differences are p < 0.05.

In contrast, we have observed that the amount of pLTF exhibited by 28d CTB-SAP treated rats is similar to that of 28d control treated rats, suggesting the underlying pathways used to elicit pLTF may be similar (Nichols et al., 2018). Representative phrenic neurograms are shown for AIH-exposed 28d CTB-SAP rats approximately 2 h following siNT, siBDNF or siTrkB pre-treatment (Fig 3A-C). As expected, siBDNF pre-treatment nearly abolished pLTF in 28d control and CTB-SAP treated rats (significantly less than 28d control and CTB-SAP treated rats pre-treated with siNT (control: 30 min post-hypoxia, p = 0.016; 60 min post-hypoxia, p = 0.003; CTB-SAP: 15 min post-hypoxia, p = 0.038; 30 min post-hypoxia, p < 0.001; 60 min post-hypoxia, p = 0.002) and siTrkB (control: 30 min post-hypoxia, p = 0.041; 60 min post-hypoxia, p = 0.034; CTB-SAP: 30 min post-hypoxia, p = 0.003; 60 min post-hypoxia, p = 0.026); not different from rats pre-treated with siTC, p > 0.05; Figs. 3D & 3E). However, siTrkB and siNT pre-treatment had no effect on pLTF in both groups (p > 0.05 for siTrkB vs. siNT; both significantly greater than rats pre-treated with siTC (control siTrkB: 60 min post-hypoxia, p = 0.012; control siNT: 60 min post-hypoxia, p < 0.001; CTB-SAP siTrkB: 60 min post-hypoxia, p = 0.031; CTB-SAP siNT: 60 min post-hypoxia, p = 0.003; Figs. 3D & 3E). These data indicate that new BDNF synthesis is required for AIH-induced pLTF in 28d control and CTB-SAP treated rats.

Fig. 3.

Fig. 3.

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pLTF in 28d siRNA treated rats. A–C, Representative traces of compressed, integrated phrenic nerve activity before and after AIH in 28d CTB-SAP rats pre-treated with siNT (A), siBDNF (B), or siTrkB (C). Baseline is indicated in each trace by a white, dashed line. AIH elicits pLTF in 28d CTB-SAP rats pre-treated with siNT and siTrkB, but appears to be reduced in rats pre-treated with siBDNF. D, E. Phrenic burst amplitude (expressed as a percent change from baseline) in 28d control (D) and 28d CTB-SAP (E) rats pre-treated with siNT, siBDNF, siTrkB, or siTC. pLTF was significantly increased from baseline (*) and siTC (†) in 28d control and CTB-SAP rats pre-treated with siNT at all time points post-hypoxia exposure vs. baseline and at 30 and 60 mins vs. siTC, and at 30 and 60 min in both groups pre-treated with siTrkB. In contrast, pLTF in 28d controls pre-treated with siBDNF was significantly reduced from 28d controls pre-treated with siNT and siTrkB at 30 and 60 min post-hypoxia (+). Similarly, pLTF in 28d CTB-SAP treated rats pre-treated with siBDNF was significantly reduced from 28d CTB-SAP treated rats pre-treated with siTC at 15 min post-hypoxia (†), siNT at all time points, and siTrkB at 30 and 60 min post-hypoxia (+). Values are means ± SEM, and all significant differences are p < 0.05.

3.4. Role of MEK/ERK and PI3K/Akt activity for pLTF in CTB-SAP treated rats

To further determine if underlying mechanisms contribute to the differential pLTF observed in CTB-SAP treated rats, we targeted the downstream signaling targets of BDNF and TrkB which include MEK/ERK and PI3K/Akt. Specifically, we intrathecally delivered UO126 or PI828 to inhibit MEK/ERK and PI3K/Akt signaling, respectively. Representative phrenic neurograms are shown for AIH-exposed 7d CTB-SAP rats approximately 20 mins following vehicle, UO126, or PI828 pre-treatment (Figs 4A-C). UO126 pre-treatment abolished pLTF in 7d control rats (significantly less than controls pre-treated with vehicle (60 min post-hypoxia, p < 0.001) and PI828 (60 min post-hypoxia, p = 0.017); not different from controls pre-treated with UO126 TC, p > 0.05; Fig. 4D), which is consistent with the requirement of MEK/ERK for pLTF in control rats. Pre-treatment with PI828 significantly attenuated AIH-induced pLTF in 7d CTB-SAP rats (significantly less than pLTF exhibited by 7d CTB-SAP treated rats pre-treated with vehicle (15 min post-hypoxia, p = 0.011; 30 min post-hypoxia, p = 0.001; 60 min post-hypoxia, p < 0.001) and UO126 (15 min post-hypoxia, p = 0.049; 30 min post-hypoxia, p = 0.027; 60 min post-hypoxia, p = 0.022); significantly greater than CTB-SAP rats pre-treated with PI828 TC (60 min post-hypoxia, p = 0.009); Fig. 4E). Surprisingly, MEK/ERK inhibition also significantly attenuated pLTF in 7d CTB-SAP rats (significantly less than pLTF exhibited by 7d CTB-SAP treated rats pre-treated with vehicle (60 min post-hypoxia, p < 0.001); significantly greater than CTB-SAP rats pre-treated with UO126 TC (15 min post-hypoxia, p = 0.030; 30 min post-hypoxia, p = 0.001; 60 min post-hypoxia, p < 0.001); Fig. 4E). These data indicate that both MEK/ERK and PI3K/Akt activity are required for pLTF in 7d CTB-SAP rats, where PI3K/Akt has a greater contribution than MEK/ERK activity (Fig. 4E). Overall, these data suggest that pLTF observed in 7d CTB-SAP treated rats is elicited through an alternative mechanism that utilizes new TrkB synthesis, and downstream signaling via PI3K/Akt and MEK/ERK.

Fig. 4.

Fig. 4.

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pLTF in 7d UO126 and PI828 treated rats. A–C, Representative traces of compressed, integrated phrenic nerve activity before and after AIH in 7d CTB-SAP rats pre-treated with vehicle (A; DMSO), a MEK/ERK inhibitor (B; UO126), or a PI3K/Akt inhibitor (C; PI828). Baseline is indicated in each trace by a white, dashed line. AIH elicits pLTF in 7d CTB-SAP rats pre-treated with the vehicle or UO126, but appears to be reduced in rats pre-treated with PI828. D, E, Phrenic burst amplitude (expressed as a percent change from baseline) in 7d control (D) and 7d CTB-SAP (E) rats pre-treated with vehicle, UO126, PI828, or drug inhibitor time controls (UO126 TC and PI828 TC). pLTF was significantly increased from baseline (*) in 7d control rats pre-treated with vehicle at 30 and 60 min, and at 60 min in rats pre-treated with PI828. Vehicle and PI828 pre-treated controls also exhibited pLTF that was significantly increased vs. TCs at 60 min post-hypoxia (vehicle vs. UO126 TC, +; vehicle and PI828 vs. PI828 TC, $). 7d CTB-SAP rats pre-treated with vehicle or UO126 exhibited significantly greater pLTF from baseline and TCs at all time points (vehicle vs. UO126 TC (+) and PI828 TC ($); UO126 vs. UO126 TC, +), and 60 min post-hypoxia in rats pre-treated with PI828 vs. baseline and PI828 TC ($). pLTF was significantly reduced at 60 min post-hypoxia in 7d controls and 7d CTB-SAP treated rats pre-treated with UO126 vs. 7d controls and 7d CTB-SAP rats pre-treated with vehicle, as well as 7d controls pre-treated with PI828 (†). 7d CTB-SAP rats pre-treated with PI828 also exhibited significantly reduced pLTF at all time points vs. 7d CTB-SAP rats pre-treated with vehicle or UO126 (#). Values are means ± SEM, and all significant differences are p < 0.05.

As mentioned above, we hypothesized that 28d control treated rats and 28d CTB-SAP treated rats utilize the same underlying pathways to elicit pLTF since they exhibit a similar magnitude of pLTF (Nichols et al., 2018). Representative phrenic neurograms are shown for AIH-exposed 28d CTB-SAP rats approximately 20 mins following vehicle, UO126, or PI828 pre-treatment (Fig. 5A-C). As expected, UO126 pre-treatment abolished pLTF in 28d control and CTB-SAP treated rats (significantly less than 28d control and CTB-SAP treated rats pre-treated with vehicle (control: 60 min post-hypoxia, p = 0.008; CTB-SAP: 60 min post-hypoxia, p = 0.004) or PI828 (CTB-SAP: 60 min post-hypoxia, p = 0.021); not different from rats pre-treated with UO126 TC, p > 0.05; Fig. 5D & E). PI828 pre-treatment exhibited pLTF that was not different from vehicle pre-treatment for both 28d control and CTB-SAP groups (p > 0.05; Fig. 5D & E). Thus, these data indicate that MEK/ERK activity is required for AIH-induced pLTF in 28d control and CTB-SAP treated rats.

Fig. 5.

Fig. 5.

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pLTF in 28d UO126 and PI828 treated rats. A–C, Representative traces of compressed, integrated phrenic nerve activity before and after AIH in 28d CTB-SAP rats pre-treated with vehicle (A; DMSO), a MEK/ERK inhibitor (B; UO126), or a PI3K/Akt inhibitor (C; PI828). Baseline is indicated in each trace by a white, dashed line. AIH elicits pLTF in 28d CTB-SAP rats pre-treated with the vehicle or PI828, but appears to be reduced in rats pre-treated with UO126. D, E, Phrenic burst amplitude (expressed as a percent change from baseline) in 28d control (D) and 28d CTB-SAP (E) rats pre-treated with vehicle, UO126, PI828 or drug inhibitor time controls (UO126 TC and PI828 TC). pLTF was significantly increased from baseline (*) in 28d control rats pre-treated with vehicle at all time points, and at 60 min post-hypoxia in rats pre-treated with PI828. Vehicle pre-treated 28d controls also exhibited pLTF that was significantly increased vs. TCs at 60 min post-hypoxia (vehicle vs. UO126 TC, +; vehicle vs. PI828 TC, $). Similarly, 28d CTB-SAP rats pre-treated with vehicle or PI828 exhibited significantly greater pLTF from baseline at 30 and 60 min post-hypoxia, and from TCs at 60 min post-hypoxia (vehicle vs. UO126 TC (+) and PI828 TC ($); PI828 vs. PI828 TC, +). pLTF was significantly reduced at 60 min post-hypoxia in 28d controls and 28d CTB-SAP treated rats pre-treated with UO126 vs. 28d controls and 28d CTB-SAP rats pre-treated with vehicle, and vs. 28d CTB-SAP treated rats pre-treated with PI828 (†). Values are means ± SEM, and all significant differences are p < 0.05.

An overall comparison of all groups is depicted in Fig. 6 for pLTF at 60 mins post-hypoxia. When comparing the two time points (7d vs. 28d) following siRNA pre-treatment, 7d CTB-SAP treated rats pre-treated with siNT exhibited significantly greater pLTF compared to 28d CTB-SAP treated rats with the same pre-treatment (p = 0.019; Fig. 6A). 7d and 28d control rats pre-treated with siBDNF had pLTF that mirrored that of siTC pre-treatment, while pre-treatment with siTrkB had no effect on pLTF when compared to siNT pre-treated groups, regardless of time-point (p > 0.05; Fig. 6A). Similarly, pLTF in 28d CTB-SAP treated rats pre-treated with siBDNF mirrored that of siTC pre-treatment, and was reduced compared to 7d CTB-SAP treated rats pre-treated with siBDNF (p = 0.001; Fig. 6A), while pLTF in 7d CTB-SAP rats pre-treated with siTrkB was not different vs. 28d CTB-SAP rats following the same pre-treatment (p > 0.05; Fig. 6A). This confirms that new synthesis of BDNF is required for pLTF in controls and 28d CTB-SAP treated rats, but not 7d CTB-SAP treated rats. When comparing the two time points (7d vs. 28d) following pre-treatment with UO126 (MEK/ERK inhibitor) or PI828 (PI3K/Akt inhibitor), all drug treatments within the CTB-SAP group attenuated pLTF from 7d CTB-SAP rats pre-treated with the vehicle (all p < 0.001; Fig. 6B). Con-current with previous findings, 7d CTB-SAP rats pre-treated with the vehicle had an enhanced pLTF from 28d CTB-SAP rats of the same treatment (p < 0.001; Fig. 6B) (Nichols et al., 2018). In control rats, MEK/ERK inhibition attenuated pLTF to that of TCs regardless of time point, suggesting MEK/ERK signaling is required for AIH-induced pLTF in control rats (7d control: p = 0.006 vs. vehicle pre-treated rats; 28d control: p = 0.030 vs. vehicle pre-treated rats; Fig. 6B). MEK/ERK inhibition also attenuated pLTF in 28d CTB-SAP rats when compared to 7d CTB-SAP rats of the same treatment (p = 0.001; Fig. 6B). pLTF following PI3K/Akt pre-treatment was not different when comparing 7d to 28d controls or 7d to 28d CTB-SAP treated rats (p > 0.05; Fig. 6B). These data confirm that pLTF in 7d CTB-SAP rats requires both MEK/ERK and PI3K/Akt activity, whereas 28d CTB-SAP rats require only MEK/ERK activity for pLTF. Lastly, we also compared phrenic nerve burst frequency at 60 min post-hypoxia (expressed as a percent change from baseline) for all groups (data not shown), in which we detected one significant difference: 28d CTB-SAP treated rats with vehicle pre-treatment exhibited significantly decreased phrenic nerve burst frequency when compared to 7d control rats pre-treated with vehicle (p = 0.020).

Fig. 6.

Fig. 6.

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Direct comparisons of the change in phrenic amplitude (percent baseline) following AIH at 60 min post-hypoxia in 7d & 28d control and CTB-SAP treated rats. A. pLTF was compared in 7d and 28d control and CTB-SAP treated rats pre-treated with siNT, siBDNF, siTrkB, and siTC. 7d and 28d control rats pre-treated with siNT and siTrkB had greater pLTF vs. the corresponding siTC group (†). 7d CTB-SAP rats pre-treated with siNT and siBDNF and 28d rats pre-treated with siNT and siTrkB had greater pLTF vs. the corresponding siTC group (†). 7d CTB-SAP rats pre-treated with siNT and sBDNF elicited greater pLTF than that of respective 7d control rats (+). 7d and 28d control rats pre-treated with siBDNF had an attenuated pLTF from 28d control rats pre-treated with siNT (&) and siTrkB (¥). 28d CTB-SAP rats pre-treated with siBDNF had pLTF that was significantly attenuated from that of 7d CTB-SAP rats with the same pre-treatment (*), and from that of 28d CTB-SAP rats pre-treated with siTrkB (€). pLTF was reduced in 7d controls as well as 7d and 28d CTB-SAP treated rats pre-treated with siTrkB vs. 7d CTB-SAP rats pre-treated with siBDNF ($). 7d CTB-SAP rats pre-treated with siNT exhibited greater pLTF vs. 28d CTB-SAP rats pre-treated with siBDNF and 7d CTB-SAP rats as well as 28d CTB-SAP treated rats pre-treated with siTrkB (#). pLTF was significantly different when comparing 28d CTB-SAP treated rats pre-treated with siNT vs. 28d CTB-SAP rats pre-treated with siBDNF, and 7d CTB-SAP rats pre-treated with siNT or siTrkB (@). B. pLTF was compared in 7d and 28d control and CTB-SAP treated rats pre-treated with vehicle, UO126, PI828, and TC for both drug inhibitors (UO126 TC and PI828 TC). All CTB-SAP treatment groups and 7d control rats pre-treated with vehicle had significantly reduced pLTF vs. 7d CTB-SAP rats pre-treated with vehicle (‡). 7d and 28d control rats pre-treated with UO126 and control rats pre-treated with UO126 TC and PI828 TC had pLTF that was attenuated from 7d and 28d control rats pre-treated with vehicle (£ and *, respectively). 7d control rats and 28d CTB-SAP rats pre-treated with UO126, 7d CTB-SAP rats pre-treated with PI828, and control and CTB-SAP rats pre-treated with UO126 TC had pLTF that was signifirantly reduced from 7d CTB-SAP rats pre-treated with UO126 (+). 7d control rats pre-treated with UO126 and control rats pre-treated with PI828 TC had pLTF that was reduced from 7d control rats pre-treated with PI828 (¶). 7d CTB-SAP rats pre-treated with PI828 had pLTF that was greater than PI828 TC (Φ). 28d CTB-SAP rats pre-treated with UO126 and CTB-SAP rats pre-treated with PI828 TC had pLTF that was attenuated from 28d CTB-SAP rats pre-treated with PI828 (§). 28d CTB-SAP rats pre-treated with UO126 and CTB-SAP rats pre-treated with UO126 TC and PI828 TC had pLTF that was reduced from 28d CTB-SAP rats pre-treated with vehicle (>). Values are mean for each group ± SEM, and all significant differences are p < 0.05.

4. Discussion

Here, we demonstrate that diverse underlying mechanisms may be responsible for the difference in pLTF seen at 7d and 28d following CTB-SAP-induced respiratory motor neuron loss. When bilateral intrapleural injections of CTB-SAP (25 μg) are given, respiratory motor neurons die, but breathing is maintained (Nichols et al., 2015b). Previous studies have shown that not only do 7d CTB-SAP rats elicit respiratory plasticity, but it is enhanced from that of control, and 28d CTB-SAP rats (Nichols et al., 2018). The major findings of this study include: 1) new TrkB synthesis and both MEK/ERK and PI3K/Akt activity are required for pLTF observed in 7d CTB-SAP treated rats; and 2) pLTF is elicited through BDNF and MEK/ERK signaling in 28d CTB-SAP rats

4.1. Requirement for TrkB, and both PI3K/Akt and MEK/ERK signaling for pLTF in 7d CTB-SAP rats

Our findings in the current study suggest that initiation of the enhanced pLTF observed in 7d CTB-SAP rats is mediated predominately through new synthesis of TrkB (Figs. 2 & 6). As shown in previous studies, activation of the adenosine 2A (A2A) receptor via pharmacological activation (Golder et al., 2008) or severe AIH (Nichols et al., 2012) requires the new synthesis of an immature TrkB isoform, activation of PI3K and Akt, and leads to enhanced pLTF. This A2A induced phrenic plasticity occurs independently from the predominant mechanism which requires 5-HT2 activation (Hoffman et al., 2012; Nichols et al., 2012). Therefore, we hypothesized that the enhancement observed in 7d CTB-SAP rats was occurring through the same signaling pathway. Because siTrkB pre-treatment attenuated but did not completely abolish pLTF in 7d CTB-SAP rats (p < 0.05; Figs. 2 & 6), we cannot eliminate the potential contributions of other pLTF signaling mechanisms, such as through BDNF and MEK/ERK signaling (Baker-Herman et al., 2004; Hoffman et al., 2012). Other signaling pathways that may contribute to the enhanced pLTF observed in 7d CTB-rats, but were not investigated in the current study, include signaling through α1-adrenoreceptors (Huxtable et al., 2014; Neverova et al., 2007), 5-HT7 receptors (Hoffman and Mitchell, 2013) which may require mammalian target of rampamycin (mTOR) (Fields et al., 2015), vascular endothelial growth factor (VEGF) (Dale-Nagle et al., 2011), or erythropoietin (EPO) (Dale and Mitchell, 2013). In addition, pre-treatment with inhibitors of either MEK/ERK or PI3K/Akt did not result in complete abolishment of pLTF in 7d CTB-SAP treated rats (Figs. 4 & 6); however, we suggest that PI3K/Akt has a greater contribution vs. MEK/ERK to pLTF observed in 7d CTB-SAP rats (pLTF following PI3K/Akt pre-treatment was attenuated when compared to pLTF following MEK/ERK pre-treatment; p < 0.05; Figs. 4 & 6). Interestingly, blocking new synthesis of TrkB did not affect the phrenic response to hypoxia, as amplitude was not different from 7d CTB-SAP rats pre-treated with siNT (p > 0.05; Fig. 1). Conversely, both MEK/ERK and PI3K/Akt inhibition attenuated the phrenic hypoxic response in 7d CTB-SAP rats vs. vehicle pre-treated 7d CTB-SAP rats (p < 0.05; Fig. 1). We speculate that activation of both MEK/ERK and PI3K/Akt signaling contribute to pLTF in 7d CTB-SAP treated rats similar to what has been observed for VEGF-induced pMF (Dale-Nagle et al., 2011) either through: 1) separate mechanisms elicited by AIH exposure; or 2) that these signaling pathways converge on each other in a synergistic fashion to elicit pLTF.

Since we observe that TrkB synthesis and primarily PI3K/Akt signaling are required for pLTF in 7d CTB-SAP treated rats, we suggest that pLTF is dependent on A2A receptor activation in these rats. Following respiratory motor neuron loss induced by bilateral, intrapleural CTB-SAP (25 μg), we speculate phrenic plasticity elicited by activation of the A2A receptor and its downstream TrkB and PI3K/Akt pathway is an effective way to increase phrenic output, and maintain eupneic breathing. However, recent studies using a higher concentration of bilateral, intrapleural CTB-SAP (50 μg) that induced more severe respiratory motor neuron loss suggest otherwise (Seven et al., 2020). In studies further investigating the role of A2A receptor in neuroprotection using intrapleurally injected CTB-SAP (50 μg per bilateral injection), it was found that A2A receptors are significantly upregulated before peak phrenic motor neuron death occurs (Seven et al., 2020). This upregulation and activation directly contribute to the acceleration of phrenic motor neuron death. Blockade of the A2A receptor not only significantly increased phrenic motor neuron survival, but also improved diaphragmatic function (Seven et al., 2020). Therefore, the plasticity that we observe in 7d CTB-SAP rats (25 μg) may be in response to the activation of the A2A receptor through the accumulation of adenosine released from neighboring phrenic motor neuron cells experiencing neurotoxic apoptosis. Future studies will need to investigate the role of the A2A receptor in pLTF and neuroprotection following less severe respiratory motor neuron death induced by CTB-SAP (25 μg per bilateral injection) by: 1) determining if A2A receptor antagonism elicits an additive neuroprotective response to further enhance pLTF; and 2) determining if A2A receptor agonism itself results in further enhanced pLTF, and if its activation is beneficial long-term to overcome respiratory deficits observed with maximal chemoreceptor activation. Furthermore, we suggest that different treatment options are required over the course of respiratory motor neuron loss, and that A2A receptor activation is potentially beneficial when respiratory motor neuron loss is less severe.

4.2. BDNF and MEK/ERK signaling are utilized for pLTF in 28d CTB-SAP treated rats

Conversely, the current study has shown that pLTF in 28d CTB-SAP rats is similar in magnitude to control rats and naïve rats exposed to moderate AIH, as reported previously (Baker-Herman et al., 2004; Fuller et al., 2000; Hoffman et al., 2012; Nichols et al., 2018). This suggests that 28d CTB-SAP rats may also be eliciting plasticity through the same mechanisms as those documented in naïve rats (Baker-Herman et al., 2004; Hoffman et al., 2012). In short, AIH-induced pLTF requires the new synthesis of BDNF in 28d CTB-SAP treated rats (Figs 3 & 6), similar to previous reports in naïve rats (Agosto-Marlin and Mitchell, 2017; Baker-Herman et al., 2004). Additionally, pre-treatment with the MEK/ERK inhibitor (UO126) abolished pLTF in 28d CTB-SAP rats (Figs. 5 & 6), consistent with previous studies in naïve and SOD1G93A rats (Hoffman et al., 2012; Nichols et al., 2017). However, siTrkB and inhibition of PI3K/Akt had no effect on pLTF in 28d CTB-SAP rats (Figs. 3, 5, & 6). Additionally, it should be noted that no pre-treatment in 28d CTB-SAP treated rats resulted in a restored or greater magnitude of pLTF like what we observe in 7d CTB-SAP treated rats. Thus, the constraint on pLTF in 28d CTB-SAP treated rats remains unknown.

Since pLTF in 28d CTB-SAP treated rats was not enhanced following any pre-treatment in our study, we suggest that neither the Gq pathway (i.e., BDNF and MEK/ERK) nor the Gs pathway (i.e., TrkB and PI3K/Akt) are responsible for constraining pLTF. We speculate that this lack of enhancement may be due to the A2A receptors no longer being activated to contribute to the pLTF exhibited in 28d treated rats. Additionally, phrenic plasticity has previously been shown to be constrained by peripheral and/or local influences such as inflammation. Specifically, inflammation abolishes phrenic plasticity induced by moderate AIH (i.e., 5-HT2 induced pLTF) or via 5-HT2A/B receptor pharmacological activation in naïve rats (Agosto-Marlin et al., 2018; Huxtable et al., 2011; Huxtable et al., 2015; Huxtable et al., 2013; Vinit et al., 2011). We have previously shown that microglial number is increased in the phrenic motor nucleus of CTB-SAP rats (Nichols et al., 2015b), and we have preliminary data that suggest systemically inhibiting inflammation (presumably caused by motor neuron death) using a nonsteroidal anti-inflammatory drug (ketoprofen) reveals enhanced pLTF in 28d CTB-SAP rats (Nichols and Tanner, 2018). Together, this suggests that inflammation may hinder pLTF in 28d CTB-SAP rats, and future studies will be focused on understanding what is responsible for this constraint (e.g., cytokines such as TNF-α).

Lastly, the requirement for 5-HT2A/B receptor activation as well as their downstream signaling mechanisms in the initiation and maintenance of pMF and pLTF have been previously studied, and may be required in 28d CTB-SAP treated rats. For example, 5-HT2 receptor activation and MEK/ERK signaling is required for the induction, but not the maintenance, of AIH-induced pLTF in naïve rats (Fuller et al., 2001; Hoffman et al., 2012). Furthermore, inhibition of MEK/ERK blocks 5-HT2A, but not 5-HT2B pMF (Fuller et al., 2001; Hoffman et al., 2012; Tadjalli and Mitchell, 2019). It is also known that pMF via 5-HT2A activation is NADPH oxidase-independent, while 5-HT2B-induced pMF is NADPH oxidase-dependent (MacFarlane et al., 2011). Following respiratory motor neuron loss in an ALS rodent model, it has been shown that inhibition of 5-HT receptors or downstream signaling modulators results in compromised breathing or pLTF, respectively (Nichols et al., 2014; Nichols et al., 2011; Nichols et al., 2017). In this same model, pre-symptomatic SOD1G93A rats exhibit NADPH oxidase-dependent AIH-induced pLTF, but pLTF becomes NADPH oxidase-independent at end-stage (Nichols et al., 2015a). Together, this suggests a differential requirement for 5-HT2A/B receptor activation over the course of disease. Here, we show that 28d CTB-SAP rats require new BDNF synthesis and MEK/ERK activity for pLTF (Figs. 3, 5 & 6). However, it remains unknown if 5-HT2A/B receptors are also required for pLTF, and if further activating these receptors would increase breathing in 28d CTB-SAP treated rats. Once the complete underlying mechanism for pLTF is elucidated in 28d CTB-SAP rats, pharmacological interventions at this time point can be tested to either maintain or elicit enhanced pLTF. Since the pathways evaluated in the current study are intracellular, future studies will be focused on the requirement of 5-HT2A/B and A2A receptors for pLTF in 7d and 28d CTB-SAP treated rats.

4.3. Significance

The CTB-SAP model effectively mimics the respiratory motor neuron loss and deficits observed in a variety of neuromuscular diseases. Studying AIH-induced pLTF in this model not only assists us in teasing out the underlying mechanisms of pLTF over the course of motor neuron death, but also provides insight for potential targets for pharmacological or gene-therapy treatments to prevent respiratory deficits, ventilator dependence, and mortality. Additionally, because our data show that the mechanisms by which pLTF is elicited differs by time point, we must understand these mechanisms in order to pharmacologically target them in patients at varying stages of disease. This may provide insight as to why patients do not respond to therapy and pharmacological intervention in the same way. Finally, because symptoms vary vastly in motor neuron and neuromuscular diseases, it is important to investigate the multiple signaling pathways to provide more treatment options when one therapy does not work.

The following are the supplementary data related to this article.

Supplementary Material

Supplementary Table 1
Supplementatry Table 2

Acknowledgements

The authors thank Safraaz Mahammed for the custom-designed computer program used for data analysis.

Abbreviations:

CTB

cholera toxin B

CTB-SAP

cholera toxin B conjugated to saporin

BDNF

brain-derived neurotrophic factor

PI3K

phosphatidylinositol 3-kinases

Akt

protein kinase B (pAkt)

Footnotes

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.expneurol.2020.113460.

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Associated Data

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Supplementary Materials

Supplementary Table 1
NIHMS1957020-supplement-Supplementary_Table_1.docx (17.7KB, docx)
Supplementatry Table 2
NIHMS1957020-supplement-Supplementatry_Table_2.docx (19.1KB, docx)

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