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. 2016 Jun 15;116(3):1232–1238. doi: 10.1152/jn.00210.2016

Ampakine CX717 potentiates intermittent hypoxia-induced hypoglossal long-term facilitation

S M Turner 1,4,5, M K ElMallah 2, A K Hoyt 1, J J Greer 3, D D Fuller 1,4,5,✉
PMCID: PMC5018053  PMID: 27306673

The emerging interest in intermittent hypoxia as an adjunct to conventional neurorehabilitation strategies led us to examine if pharmacologic modulation of AMPA currents could enhance the respiratory motor response to intermittent hypoxia. In particular, this work focused on a class of drugs known as ampakines. Using a mouse model, we found that under appropriate conditions ampakine pretreatment can potentiate intermittent hypoxia-induced long-term facilitation of inspiratory burst amplitude recorded in the hypoglossal nerve.

Keywords: respiratory, plasticity, AMPA, mouse

Abstract

Glutamatergic currents play a fundamental role in regulating respiratory motor output and are partially mediated by α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors throughout the premotor and motor respiratory circuitry. Ampakines are pharmacological compounds that enhance glutamatergic transmission by altering AMPA receptor channel kinetics. Here, we examined if ampakines alter the expression of respiratory long-term facilitation (LTF), a form of neuroplasticity manifested as a persistent increase in inspiratory activity following brief periods of reduced O2 [intermittent hypoxia (IH)]. Current synaptic models indicate enhanced effectiveness of glutamatergic synapses after IH, and we hypothesized that ampakine pretreatment would potentiate IH-induced LTF of respiratory activity. Inspiratory bursting was recorded from the hypoglossal nerve of anesthetized and mechanically ventilated mice. During baseline (BL) recording conditions, burst amplitude was stable for at least 90 min (98 ± 5% BL). Exposure to IH (3 × 1 min, 15% O2) resulted in a sustained increase in burst amplitude (218 ± 44% BL at 90 min following final bout of hypoxia). Mice given an intraperitoneal injection of ampakine CX717 (15 mg/kg) 10 min before IH showed enhanced LTF (500 ± 110% BL at 90 min). Post hoc analyses indicated that CX717 potentiated LTF only when initial baseline burst amplitude was low. We conclude that under appropriate conditions ampakine pretreatment can potentiate IH-induced respiratory LTF. These data suggest that ampakines may have therapeutic value in the context of hypoxia-based neurorehabilitation strategies, particularly in disorders with blunted respiratory motor output such as spinal cord injury.

NEW & NOTEWORTHY

The emerging interest in intermittent hypoxia as an adjunct to conventional neurorehabilitation strategies led us to examine if pharmacologic modulation of AMPA currents could enhance the respiratory motor response to intermittent hypoxia. In particular, this work focused on a class of drugs known as ampakines. Using a mouse model, we found that under appropriate conditions ampakine pretreatment can potentiate intermittent hypoxia-induced long-term facilitation of inspiratory burst amplitude recorded in the hypoglossal nerve.

glutamatergic currents play a fundamental role in regulating respiratory motor output. These currents are mediated in part by the α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors that are expressed throughout the premotor (medullary) and motor respiratory circuitry. Glutamate, acting via AMPA receptors, is a critical component of the excitatory drive for respiratory rhythmogenesis and also the synaptic input to respiratory motoneurons (Funk and Feldman 1995). Thus, on a breath-by-breath basis, AMPA-mediated currents are essential to the control of breathing. Neuroplastic changes in respiratory motor output, as can occur after neurologic injury or altered environmental conditions, are also likely to involve increases (or decreases) in AMPA-mediated glutamatergic currents (Alilain and Goshgarian 2008). One of the most well characterized examples of respiratory neuroplasticity is “long-term facilitation” (LTF) following intermittent exposure to brief periods of reduced oxygen [i.e., intermittent hypoxia (IH)]. This response manifests as an increase in inspiratory motor activity that persists well beyond the period of IH (Mitchell et al. 2001a), and there is a body of evidence suggesting that IH can enhance glutamatergic synaptic transmission to respiratory motoneurons (Gonzalez-Rothi et al. 2015). Potentiation of neuromotor output following IH can occur in both respiratory and nonrespiratory motor systems (Gonzalez-Rothi et al. 2015) and has been documented in species ranging from mouse to human (Gonzalez-Rothi et al. 2015; Mitchell et al. 2001a). The ability for a few short episodes of mild to moderate hypoxia to trigger functional neuroplasticity has led several research groups to explore the use of IH as a therapeutic tool in neurologic rehabilitation settings (Gonzalez-Rothi et al. 2015). For example, IH paradigms have recently been shown to increase walking ability (Trumbower et al. 2012) and respiratory motor activity in persons with chronic spinal cord injury (SCI) (Tester et al. 2014).

The emerging interest in IH as an adjunct to conventional neurorehabilitation strategies (Gonzalez-Rothi et al. 2015) led us to examine if pharmacologic modulation of AMPA currents could enhance the respiratory motor response to IH. Treatments that enhance the impact of IH or reduce the number of hypoxic exposures required to evoke neuroplastic changes could have therapeutic value. In particular, this work focused on a class of drugs known as “ampakines.” These drugs enhance excitatory glutamatergic neural transmission by altering AMPA receptor channel kinetics (Ren et al. 2012) and can facilitate respiratory motor output in conditions of blunted respiratory drive. For example, ampakines are an effective treatment in rodent models of opiate or GABAergic drug-induced respiratory depression (Lorier et al. 2010; Ren et al. 2012, 2013), neuromuscular disease (ElMallah et al. 2015a), and apnea of prematurity (Ren et al. 2015). Ampakines can also enhance the expression of hippocampal long-term potentiation (Chang et al. 2014), which is one of the premier models of neuroplasticity over the last 45 yr (Lee and Kirkwood 2011). Given that IH exposure appears to enhance the effectiveness of glutamatergic synapses on respiratory motoneurons (Mitchell et al. 2001a) and that ampakines enhance glutamatergic AMPA channel function (Suppiramaniam et al. 2001), we hypothesized that ampakine pretreatment would potentiate the magnitude of IH-induced respiratory LTF. To test this hypothesis, inspiratory bursts were recorded from the hypoglossal nerves (XII) of anesthetized mice, and ampakine CX717 was systemically administered before a brief IH exposure (3 × 1 min, 15% O2). Aspects of this work were previously published in abstract form (Turner et al. 2014, 2016).

METHODS

All procedures were approved by the University of Florida Institutional Animal Care and Use Committee; experimental groups are summarized in Table 1. Adult male 129SVE mice (n = 48; Taconic, Hudson, NY) were anesthetized with urethane (1.0–1.6 mg/kg ip; Sigma, St. Louis, MO) and supplements were given if indicated (0.3 g/kg ip). Body temperature was maintained at 37.5°C using a servo-controlled heating pad (TC-1000; CWE, Ardmore, PA) and hemoglobin saturation (SaO2) was measured with pulse oximetry (MouseOx; STARR Life Science, Oakmont, PA). The trachea was cannulated for mechanical ventilation (MicroVent; Harvard Apparatus, Holliston, MA) with hyperoxic gas (FiO2 = 0.60). End tidal carbon dioxide (ETCO2) (MicroCapStar; CWE, Ardmore, PA) was set and maintained at 28 ± 3mmHg by adjusting ventilation frequency (140–160 breaths/min). Mice were bilaterally vagotomized and the carotid artery was catheterized (FunnelCath; Instech Laboratories, Plymouth Meeting, PA) for blood pressure measurements (Statham P-10EZ pressure transducer and TA-100 Transducer Amplifier; CWE) or sampling of arterial blood. Pancuronium bromide (2.5 mg/kg, Hospira, Lake Forest, IL) administration eliminated respiratory muscle contraction. The XII nerve was cut and efferent activity was recorded using monopolar tungsten electrodes (1,000× amplification, Model 1700; A-M Systems, Carlsborg, WA); band-pass filtering was 0.01–10 KHz. Raw neurograms were integrated using a 100-ms time constant (MA-1000; CWE). Data were digitized (CED Power 1401; Cambridge Electronic Design, Cambridge, England) and recorded (Spike2 software; Cambridge Electronic Design). In n = 22 mice, arterial partial pressure of O2 (PaO2) and CO2 (PaCO2), and pH were measured from 0.2-ml arterial blood samples (i-Stat; Heska, Fort Collins, CO). A 50:50 sodium bicarbonate:saline solution was administered intraperitoneally (0.1 ml) at 15-min intervals.

Table 1.

Summary of baseline recording conditions

Group (n) Burst Amplitude, V Burst Frequency, breaths/min ETCO2, mmHg SaO2, % MAP, mmHg Body Weight, g Rectal Temperature, °C
TC (n = 9) 0.64 ± 0.3 98 ± 9.0 28.7 ± 5.8 96.1 ± 2.1 64.0 ± 2.6 30.6 ± 1.2 37.5 ± 0
CX717 TC (n = 6) 0.91 ± 0.2 69 ± 14.9 27.7 ± 1.5 98.2 ± 0.1 76.8 ± 7.1 29.9 ± 1.3 37.5 ± 0
IH (n = 18) 0.49 ± 0.1 99 ± 7.7 27.7 ± 2.7 97.1 ± 0.6 83.6 ± 5.1 29.1 ± 0.7 37.5 ± 0
CX717 + IH (n = 15) 0.49 ± 0.1 102 ± 10.4 30.8 ± 1.9 97.8 ± 0.4 76.2 ± 2.6 28.3 ± 0.7 37.5 ± 0
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Mice were randomly distributed across 4 experimental groups. The table depicts the average values for each experimental group during baseline conditions for the following parameters: raw hypoglossal nerve (XII) burst amplitude (e.g., the value of the amplified, rectified signal from the extracellular recording electrode), XII burst frequency, ETCO2, SaO2, mean arterial pressure (MAP), body weight, and rectal temperature.

TC, body core remperature; IH, intermittent hypoxia.

Experimental protocols.

Baseline recordings were made for 5–10-min followed by either 1) no injection, 2) vehicle injection [10% 2-hydroxypropyl-β-cyclodextrin (HPCD) in 0.45% saline solution; Sigma], or 3) ampakine CX717 injection (15 mg/kg dissolved in vehicle; RespireRx, New Jersey). After 10 min, mice were treated with three 1-min episodes of hypoxia (FiO2 = 0.15) separated by 3 min (FiO2 = 0.60) or sustained baseline conditions (i.e., no IH). Nerve recordings were maintained for 60–90 min following the baseline period.

Data analysis.

Neurograms were analyzed as previously described (ElMallah et al. 2015a,b). The moving time average or “integrated” XII signal was calculated as the peak to peak raw amplitude obtained from the extracellular recording [i.e., volts or arbitrary units (AU)] and normalized relative to baseline output (i.e., %BL). Inspiratory burst frequency (i.e., bursts per min) was also normalized relative to baseline output. Data were averaged during BL (5 min), hypoxic episodes (last 10 s), and following hypoxia (5 min) at 15, 30, 45, 60, and 90 min.

Statistics were performed using SigmaStat v12.0 software. Two-way repeated-measures ANOVA with Student-Newman-Keuls post hoc analyses compared changes between groups over time. Baseline conditions were compared across the experimental groups using one-way ANOVA. If normality and equal variance assumptions failed, the data were log10 transformed. The statistical significance threshold was P < 0.05.

RESULTS

There were no significant differences in baseline recording parameters across groups (Table 1). The rectal temperature was maintained at 37.5 ± 0.1°C, and SaO2 was maintained above 96% throughout each experiment (except during hypoxia when it reached 45–55%). The ETCO2 was maintained at 28 ± 3mmHg by adjusting ventilation frequency (140–160 breaths/min). The baseline mean arterial pressure across groups (n = 30; Table 1) averaged 77 ± 3.0 mmHg and decreased by ∼15% over the course of the experiments. The ability to take arterial blood samples is limited in the mouse; thus, a single sample was drawn at the end of the experiment (pH 7.31 ± 0.03, PaO2 = 94 ± 3.7 mmHg, PaCO2 = 44 ± 3.7 mmHg).

Initial control experiments.

To confirm that inspiratory XII nerve bursting remained stable in absence of IH, recordings were made for 90 min while SaO2 was maintained ≥96%. Data were collected from mice that received an intraperitoneal vehicle injection at the onset of the recording period (10% HPCD, n = 4) or no injection (n = 5). These results were pooled into a single “control” group since there were no differences (or trends) between the two groups for either burst frequency (P = 0.693) or amplitude (P = 0.890) over the recording duration. Fig. 1, A and B, demonstrates that burst amplitude (%BL) was stable over the 90-min time control protocol, with no evidence for “facilitation-like” increases in burst amplitude. We also examined the impact of a single ampakine injection (without exposure to IH) on XII motor output over a 90-min recording period. In two of six experiments, XII burst amplitude increased immediately following the ampakine injection (15 mg/kg ip), but this increase was not sustained. Thus, on average, XII burst amplitude was 107 ± 8% baseline at 10 min following the ampakine injection (P = 0.39 vs. preinjection value). None of the mice showed evidence for XII burst amplitude facilitation following a single ampakine injection (e.g., see the scatter plot in Fig. 1B) over the 90-min recording period, and the mean XII burst amplitude was similar to the control group (Fig. 1A, P = 0.808) at 90 min.

Fig. 1.

Fig. 1.

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In the absence of intermittent hypoxia (IH), hypoglossal nerves (XII) bursting is stable for 90 min. A: mean burst amplitude [% baseline (BL)] was stable over the 90-min time control protocol in both control (□) and CX717 (○) groups. B: scatter plot of the individual data, along with the mean data, for burst amplitude at 60 min. C: burst frequency (%BL) was unchanged over the 90 min post-BL recording period in control (□) and CX717 (○) groups. D: scatter plot of the burst frequency response at 60 min.

In the control experiments, inspiratory burst frequency (%BL, Fig. 1, C and D) was stable over the 90-min recording. The ampakine injection caused burst frequency to transiently increase in two of six mice, but the frequency rapidly returned to baseline values and after 10 min was 104 ± 7% BL (P = 1.00 vs. preinjection baseline). Over the 90-min recording period following ampakine injection, the mean burst frequency (P = 0.309) was not different than the control response. However, two of the six ampakine-treated mice showed a gradual increase in inspiratory burst frequency (e.g., Fig. 1D).

XII output during and following IH.

The mean XII motor response during the IH exposure is presented in Fig. 2. There was a significant interaction (P = 0.010) between hypoxic episode number (i.e., 1–3) and treatment (control vs. ampakine). In the control group, the initial bout of hypoxia evoked a 247 ± 33% increase in burst amplitude (Fig. 2A), and similar responses were observed during episodes 2–3 (P ≥ 0.90 vs. episode 1 within the IH group; Fig. 2A). Ampakine-treated mice had more robust hypoxia-induced increases in burst amplitude, with values reaching 386 ± 86% BL during the first hypoxic episode (Fig. 2A). Acute hypoxic responses in the ampakine-treated mice also showed progressive augmentation (Mitchell et al. 2001b) with burst amplitude reaching 475 ± 105 and 474 ± 107% during the second and third hypoxic episodes, respectively (both P ≤ 0.001 vs. episode 1 within the CX717 + IH group; Fig. 2A). The hypoxic responses during episodes 2 and 3 were significantly greater in CX717 pretreated mice compared with the IH-only group (P = 0.024 and P = 0.030, respectively; Fig. 2A). There were no differences in the inspiratory burst frequency response to IH between IH and CX717 + IH groups (P = 0.598, Fig. 2B).

Fig. 2.

Fig. 2.

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The acute response to IH is increased following CX717. A: increase in XII inspiratory burst amplitude (%BL) during each of 3 successive hypoxic episodes. B: there were no differences in the inspiratory burst frequency response (%BL) between IH (■) and CX717 + IH (●)-treated mice. *P < 0.05 vs. IH (treatment effect). #P < 0.05 for time effect. T̂ime × treatment interaction.

An example of inspiratory XII activity over the 90-min period following IH is presented in Fig. 3A. Consistent with prior reports in mice (ElMallah et al. 2015b; Toyama et al. 2009), the IH group showed LTF of inspiratory burst amplitude with values reaching ∼200% baseline at 90-min post-IH (Fig. 3B). Statistical comparison to the time control group that was not exposed to IH also confirmed that burst amplitude LTF was present (treatment effect, P = 0.006; data not shown). The scatter plot in Fig. 3C is provided to illustrate the variability burst amplitude LTF in the IH group. Note that some of the IH-treated mice did not express LTF (i.e., burst amplitudes were maintained near BL values after IH), but on average, LTF was present.

Fig. 3.

Fig. 3.

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CX717 pretreatment increase the magnitude of long-term facilitation (LTF) of XII burst amplitude. A: an example of inspiratory XII activity over the 90-min period following IH for IH (□) and CX717 + IH (○)-treated mice. B: IH alone was sufficient to elicit LTF values reaching ∼200% baseline at 90 min post-IH and that CX717 enhanced LTF. C: scatter plot illustrates the variability in XII burst amplitude LTF (data are from 60 min post-IH), and that CX717 altered the distribution of LTF values. D: the average inspiratory burst frequency response following IH. E: burst frequency scatter plot showing that most mice had inspiratory burst frequencies below the baseline value. *P < 0.05 vs. IH (treatment effect). #P < 0.05 for time effect compared with baseline within the group. $P < 0.001, overall time effect.

Ampakine-treated mice had considerably more robust LTF of inspiratory burst amplitude, and this is illustrated by the mean data shown in Fig. 3B. Statistically, there was an interaction (P < 0.001) between time (i.e., duration following hypoxia) and treatment (control vs. ampakine). A scatter plot to illustrate the variability of the individual data points at a single time point (60 min) following CX717 + IH is provided in Fig. 3C. Note that 9 of 15 CX717-treated mice had LTF which exceed the mean of the IH group, and direct statistical comparison of burst amplitudes at this time point indicates a significant impact of ampakine treatment on the LTF response (P = 0.010). The change in raw amplitude of the inspiratory hypoglossal burst (i.e., absolute voltage, referred to as AU) from baseline to LTF was also significantly greater in the CX717 + IH group (0.64 ± 0.13 AU) vs. the IH group (0.27 ± 0.09 AU, P = 0.019; data not shown).

Inspiratory burst frequency declined following IH in both IH and CX717 + IH groups (Fig. 3D; P < 0.001 for time effect; P = 0.903 for treatment effect). The scatter plots shown in Fig. 3E illustrate that this was a robust effect, with nearly all of the IH-treated mice having inspiratory burst frequencies below the baseline value at 60 min post-IH. We did not detect any differences in the inspiratory burst frequency following IH between IH and CX717 + IH-treated mice (P = 0.820).

The scatter in the data revealed variability in the relative magnitude of burst amplitude LTF (e.g., see the scatter plot in Fig. 3C), especially in the CX717 + IH group. This led us to conduct a post hoc evaluation of the relationship between initial (i.e., baseline) XII motor output and subsequent expression of LTF (Fig. 4). We first noted that across the entire data set there was a considerable range in the raw amplitude of the inspiratory hypoglossal burst [i.e., arbitrary units (AU)] following IH or CX717 + IH. When data were grouped according to the magnitude of the baseline inspiratory burst (AU), it became apparent that there was a relationship between LTF and the initial baseline output. Figure 4, A and B, shows the LTF data grouped according to the initial baseline output (AU), which was defined as “low” (i.e., <0.6 AU) or “high” (i.e., >0.6 AU) based on clustering of the data points (see Fig. 4C). Regardless of whether the LTF burst amplitude is expressed in AU (Fig. 4A) or %BL (Fig. 4B), the CX717-treated group with initially low baseline output has greater LTF (P < 0.05 vs. other groups). However, there are a number of caveats that must be considered when evaluating the raw amplitude of extracellularly recorded biopotentials (Fuller et al. 2009; Nichols and Mitchell 2015). To provide a comprehensive and transparent analysis, Fig. 4, C–F, provides individual data points from each mouse at 90 min post-IH. The approach we used was to consider the baseline output as the independent variable and to determine if BL output was predictive of LTF magnitude (dependent variable). Figure 4C demonstrates the overall scatter of the raw burst amplitude (AU) at baseline and during LTF and shows that there is no statistically significant relationship between these two variables. However, regression analyses reveals a highly significant relationship between baseline burst amplitude and the relative magnitude of LTF (%BL) in CX717 + IH-treated mice (P < 0.001, Fig. 4D). Thus CX717-treated mice with low initial baseline bursting were much more likely to express robust XII LTF. In contrast, no such relationship was present in the IH group (P = 0.852, Fig. 4D). No statistically significant relationships were detected between baseline burst frequency (burst/min) and burst amplitude LTF (Fig. 4E). However, a strong tendency was noted in the IH group for LTF baseline burst frequency to be predictive of LTF (P = 0.062); a similar trend was not observed in CX717 + IH-treated mice (P = 0.248). In contrast, a highly significant relationship was observed in both the IH and CX717 + IH mice (both P < 0.001) between baseline burst frequency (burst/min) and burst frequency following IH. Thus, in both experimental groups, a high baseline burst frequency predicted a depression in burst frequency following IH (Fig. 4F). These data suggest the variable LTF response to CX717 before IH may be dependent on the initial baseline amplitude.

Fig. 4.

Fig. 4.

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The initial burst amplitude impacts expression of LTF When data are grouped according to the magnitude of the initial (baseline) inspiratory burst [arbitrary units (a.u.)], it is apparent that this measure is predictive of LTF in the ampakine-treated group. A and B: regardless of whether LTF expression (dependent variable) is presented as burst amplitude (a.u.) or normalized to baseline (%BL). Note that CX717-treated mice with initially low baseline output were more likely to show enhanced LTF. C: overall scatter of the raw burst amplitude (a.u.) data with no significant relationship between baseline and LTF values. D: in contrast, a highly significant relationship between baseline burst amplitude and the relative magnitude of LTF (%BL) in CX717 + IH-treated mice was found. E: there was a tendency for baseline burst frequency to be predictive of LTF in the IH group, but a similar trend was not observed in CX717 + IH-treated mice. F: a highly significant relationship between baseline burst frequency (burst/min) and the subsequent impact of IH on burst frequency. *P < 0.05 compared with IH alone (control).

DISCUSSION

Our primary finding is that ampakine CX717 can substantially potentiate LTF of XII motor output when given before a brief period of IH. This result is consistent with reports from other experimental models suggesting that ampakines can enhance neural plasticity (Baudry et al. 2012; Chang et al. 2014). The current data suggest an interaction between positive allosteric modulation of AMPA receptors and exposure to IH that manifests only when the initial respiratory neuromotor output is (relatively) low. We suggest that further studies are warranted to elucidate the underlying mechanisms and to refine dosing strategies.

Ampakines: mechanism of action and potential impact on neuroplasticity.

Within the central nervous system, AMPA receptors mediate fast synaptic excitation and are dynamically regulated in an activity-dependent manner (Widagdo et al. 2015). Changes in synaptic strength can be mediated through altering AMPA receptor copy number and subunit composition in the postsynaptic membrane (Malenka 2003) and also through posttranslational modification (Anggono and Huganir 2012). When glutamate exposure is brief, AMPA receptor channels close, and the agonist may dissociate (deactivation), leaving receptors in an activatable state (Timm et al. 2011). Positive allosteric modulators of AMPA receptors are hypothesized to bind in a modulator binding pocket within the GluR2 subunit (Ptak et al. 2009) and lead to increased open-channel time (Arai et al. 1996a,b; Partin et al. 1996; Staubli et al. 1994a,b; Suppiramaniam et al. 2001). In turn, this prolongs EPSP duration and increases ion flux through AMPA receptor channels; thereby increasing likelihood of synaptic plasticity (Lynch et al. 2011; Staubli et al. 1994a,b). Indeed, several previous reports suggest that ampakine treatment can enhance long-term potentiation (Arai et al. 1996a; Baudry et al. 2012; Lynch et al. 2011; Staubli et al. 1994a) and a recent report provides evidence for dendritic growth and facilitated learning following chronic ampakine treatment in middle aged rats (Lauterborn et al. 2016). Based on these and other previous publications (e.g., Lorier et al. 2010), we speculate that ampakine-mediated potentiation of AMPA currents in XII motoneurons enhance the subsequent ability of IH to trigger mechanisms to elicit neuroplasticity and therefore LTF (Devinney et al. 2013).

IH, respiratory LTF, and ampakines.

Intermittent hypoxia-induced LTF is a powerful and well-studied model of respiratory motor plasticity (reviewed in Fields and Mitchell 2015) with implications for neurorehabilitation (Gonzalez-Rothi et al. 2015). LTF typically manifests as a sustained increase in inspiratory burst amplitude (vs. burst frequency) after IH and has been demonstrated in multiple motor outputs including phrenic, XII, and intercostal (Baker-Herman and Strey 2011; Navarrete-Opazo and Mitchell 2014). The optimal paradigm of IH exposure for triggering LTF is not known, but, the need for repeated (intermittent) exposures to hypoxia is a consistent finding across published studies (Devinney et al. 2013). Mitchell and colleagues have established there is not a unique signaling pathway that gives rise to LTF but rather there are multiple pathways that can be triggered depending on the particular IH paradigm (Devinney et al. 2013). Studies of cellular mechanisms giving rise to ampakine-enhancement of IH-induced respiratory neuroplasticity is an important direction for future work.

Post hoc evaluation of the current data suggested that the initial (baseline) XII motor output had a substantial impact on the ability of CX717 to impact IH-induced LTF. Thus there appears to be a “ceiling effect” in which CX717 is not effective at modulating neuroplasticity when respiratory output is initially high. To our knowledge, ours is the first study of respiratory neuroplasticity and ampakines, but prior data support the notion that ampakines are only effective at modulating breathing when initial output is low (ElMallah et al. 2015a; Lorier et al. 2010; Ren et al. 2006, 2009, 2012, 2013, 2015, 2012). First, ampakines cause no significant shifts in respiratory parameters in awake, spontaneously breathing mice (ElMallah et al. 2015a) or in perinatal (Ren et al. 2012, 2013, 2015, 2012), juvenile (Ren et al. 2009, 2012, 2013), and adult rats (Ren et al. 2009, 2012, 2013) or in humans (Oertel et al. 2010). Likewise, ampakines have little effect on respiratory motor output recorded from hypoglossal (XII) nerves in vivo from anesthetized and ventilated mice (present study and ElMallah et al. 2015a) and from nerve rootlets in vitro (Lorier et al. 2010, 2012, 2013) or in situ (Ren et al. 2009, 2013) under baseline conditions with strong, rhythmic respiratory output. This phenomenon was clearly demonstrated in a study of breathing in perinatal rats: CX1739 robustly stimulated breathing in animals with unstable, low breath frequencies but had no impact on animals with stable breathing (Ren et al. 2015).

Conclusion.

The function and regulation of AMPA receptors have been established as critical components of many models of neuroplasticity, including respiratory LTF after IH. In addition, enhancing AMPA-mediated signaling through positive allosteric modulation has become a pharmacological target in neurodegenerative diseases, ADHD, schizophrenia, autism and stroke (Black 2005; Menniti et al. 2013; Morrow et al. 2006; Ward and Harries 2010). In regards to breathing, ampakine CX717 is an effective stimulant in rat models of severe drug-induced respiratory depression (i.e., >50% reduction in breath frequency) in vivo (Ren et al. 2012, 2013), in situ (Ren et al. 2013), and in vitro (Ren et al. 2012, 2013). The present data indicate that systemic ampakine administration can create preconditions within the respiratory neural control system that promote expression of neuroplasticity. We speculate that enhanced LTF in the current study reflects an interaction between IH-triggered signaling pathways in XII motoneurons, and positive allosteric modulation of XII motoneuron AMPA receptors.

The current results are intriguing in light of recent evidence that IH can be a highly effective neurorehabilitation in clinical settings (Gonzalez-Rothi et al. 2015; Trumbower et al. 2012). The ampakine used here, CX717, is metabolically stable and has been deemed safe in primate studies and clinical trials for cognitive disorders (Oertel et al. 2010; Ren et al. 2009). Importantly, CX717 also readily crosses the blood-brain barrier and there is no evidence for significant side effects (Ren et al. 2009). Thus, we suggest that pairing IH with ampakines may have value in the context of neurorehabilitation. Recent work from Trumbower et al. (2012) establishes that persons with SCI can experience significant improvements in locomotor function after IH “training.” In fact, even a single session of IH therapy can trigger an increase in the force that can be generated by the leg muscles in patients with chronic SCI (Trumbower et al. 2012). IH can also effectively evoke a modest LTF of breathing in individuals with chronic cervical and thoracic SCI (Tester et al. 2014), and this has recently been confirmed by an independent laboratory (Sankari et al. 2015). Importantly, SCI represents a condition in which activation of many motoneuron pools is typically reduced. Thus, SCI may create preconditions that enable ampakines to potentiate respiratory and somatic motor output, and the current data suggest that this may be even more effective when paired with an appropriate IH paradigm.

GRANTS

This work was supported by the following grants: National Institute of Neurological Disorders and Stroke Grant 1-R01-NS-080180-01A1 (to D. D. Fuller), State of Florida Brain and Spinal Cord Injury Research Program (to D. D. Fuller), Canadian Institutes for Health Research (to J. J. Greer), and Neilsen Foundation 313369 (to S. M. Turner).

DISCLOSURES

J. J. Greer is a consultant for RespireRx Pharmaceuticals, which is the company that supplied the ampakine drug used in these experiments.

AUTHOR CONTRIBUTIONS

S.M.T., M.K.E., J.J.G., and D.D.F. conception and design of research; S.M.T. and M.K.E. performed experiments; S.M.T., M.K.E., A.K.H., and D.D.F. analyzed data; S.M.T., M.K.E., A.K.H., J.J.G., and D.D.F. interpreted results of experiments; S.M.T., A.K.H., and D.D.F. prepared figures; S.M.T. drafted manuscript; S.M.T., M.K.E., A.K.H., J.J.G., and D.D.F. edited and revised manuscript; S.M.T., M.K.E., A.K.H., J.J.G., and D.D.F. approved final version of manuscript.

ACKNOWLEDGMENTS

We gratefully acknowledge Arnold Lippa and RespireRx Pharmaceuticals for kindly providing ampakine CX717.

Present address for M. K. ElMallah: Dept. of Pediatrics and Gene Therapy, Division of Pulmonary Medicine, Univ. of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655.

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