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Published in final edited form as: Respir Physiol Neurobiol. 2021 Nov 11;296:103814. doi: 10.1016/j.resp.2021.103814

Spinally delivered ampakine CX717 increases phrenic motor output in adult rats

Prajwal P Thakre 1,2,3, Michael D Sunshine 1,2,3, David D Fuller 1,2,3,*
PMCID: PMC9235873  NIHMSID: NIHMS1816754  PMID: 34775071

Abstract

Ampakines are synthetic molecules that allosterically modulate AMPA-type glutamate receptors. We tested the hypothesis that delivery of ampakines to the intrathecal space could stimulate neural drive to the diaphragm. Ampakine CX717 (20mM, dissolved in 10% HPCD) or an HPCD vehicle solution were delivered via a catheter placed in the intrathecal space at the fourth cervical segment in urethane-anesthetized, mechanically ventilated adult male Sprague-Dawley rats. The electrical activity of the phrenic nerve was recorded for 60-minutes following drug application. Intrathecal application of CX717 produced a gradual and sustained increase in phrenic inspiratory burst amplitude (n=10). In contrast, application of HPCD (n=10) caused no sustained change in phrenic motor output. Phrenic burst rate, heart rate, and mean arterial pressure were similar between CX717 and HPCD treated rats. We conclude that intrathecally delivered ampakines can modulate phrenic motor output. This approach may have value for targeted induction of spinal neuroplasticity in the context of neurorehabiliation.

Keywords: phrenic nerve, ampakine CX717, intrathecal delivery

Introduction

Ampakines are small molecules that are positive allosteric modulators of α-amino-3-hydro-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By modulating AMPA receptor kinetics, ampakines can enhance excitatory glutamatergic transmission (Hampson et al. 1998). In vitro studies indicate that ampakines are not direct agonists of AMPA receptors (Arai et al. 1996; Arai et al. 2002; Arai et al. 2004) and also have no direct impact on others such as NMDA or kainite glutamate receptors (Arai and Kessler 2007). Ampakines can enhance synaptic transmission and promote plasticity without altering the fundamental aspects of AMPA receptor activation (Ward et al. 2010).

Ampakines can stimulate breathing through actions on premotor “rhythm generating” neurons (Ren et al. 2006) as well as respiratory motoneurons (Lorier et al. 2010). Ampakines are most effective at stimulating breathing during conditions associated with reduced or impaired respiratory motor activity such as opioid overdose (Oertel et al. 2010; Ren et al. 2009), spinal cord injury (Wollman et al. 2020b) or neuromuscular disease (ElMallah et al. 2015; Ogier et al. 2007). Ampakines can also enhance the expression of respiratory neuroplasticity following hypoxia (Turner et al. 2016; Wollman et al. 2020a).

Drug delivery to the intrathecal space, targeting the spinal cord, has been used extensively in basic science studies of respiratory neural control and plasticity (Devinney et al. 2016; Gill et al. 2016; Nichols et al. 2015). Intrathecal drug delivery is also used to treat chronic pain (Deer et al. 2017) and has promise in restoring motor function after spinal cord injury when used in conjunction with electrical stimulation (Capogrosso et al. 2018; Minev et al. 2015). Intrathecal delivery of ampakines, if effective at stimulating respiratory motor output, could be useful in the context of pre-clinical mechanistic studies (Baker-Herman et al. 2004), or could possibly have application to neurorehabilitation in conditions with impaired motor activity (Capogrosso et al. 2018). AMPA receptors are densely distributed on phrenic motoneurons (Rana et al. 2019; Rana et al. 2020) and contribute to depolarization during inspiration (Chitravanshi and Sapru 1996). Accordingly, AMPA receptors provide a potential substrate for increasing phrenic motor output following spinal ampakine delivery. Accordingly, these proof-of-concept studies tested the hypothesis that small volume (15μl) intrathecal delivery of ampakine CX717 (20mM) could produce a sustained increase in inspiratory phrenic nerve burst amplitude in adult rats.

Methods

All experiments were performed upon approval from the University of Florida Institutional Animal Care and Use Committee (Protocol ID: 201807438). Adult male Sprague Dawley rats (208A Colony, Envigo) housed in pairs were maintained on a 12:12-hr light-dark cycle with free access to food and water. Initially, anesthesia was induced with 3% isoflurane in 100% oxygen in an enclosed Plexiglas chamber. Rats were then transferred to a heated pad on a surgical table where anesthesia was maintained using a nose cone. After ensuring adequate anesthetic depth rats were tracheotomized, ventilated and a bilateral vagotomy was performed after which they were maintained within a normal physiological range (end-tidal CO2=40). Animals were slowly converted from isoflurane to urethane (2.1 g/kg at 6 ml/hr) anesthesia delivered via tail vein catheter. Femoral artery catheterization was carried out for blood pressure measurements and withdrawal of arterial blood samples.

A midline incision was made and muscles overlying the dorsal aspect of the cervical spinal cord were exposed. A C2 laminectomy was performed and a silicone catheter attached to a 50μl Hamilton syringe was inserted after giving a small cut to the dura. The catheter was advanced caudally to reach C4 segment of the spinal cord and held in position with saline soaked cotton plugs. Unilateral phrenic nerve (left) was isolated via dorsal approach and was cut distally. The cut end was de-sheathed and placed in a glass suction electrode filled with 0.9% saline solution. Phrenic nerve activity was amplified (x10k Hz) using a differential AC amplifier (Model 1700, A-M systems, Everett, WA), band-pass filtered (100Hz-3kHz), digitized (16 bit, 25k samples/channel, Power 1401, CED) and integrated with (0.05s time constant) using Spike2 software. All analysis was carried out offline using a custom written MATLAB code.

An apneic threshold for phrenic inspiratory bursting was determined by slowly reducing inspired fraction of CO2 until the animal went into apnea (measured as absence of phrenic activity for 60 sec) and then slowly increasing the CO2 to establish recruitment threshold. Animals were maintained between 2–3 mmHg above the CO2 recruitment threshold. After establishing a stable phrenic activity for at least 15 min with stable blood gas values, either CX717 (20 mM, provided by Dr. Arnold Lippa of RespireRx) or 10% HPCD were slowly delivered intrathecally (15 μl volume, over 60 sec). Phrenic nerve output was measured for 60 min and blood gas evaluations were made every 15 min. Preparations were included in final analysis if the 60 min PaCO2 were within 1.5 mmHg from the baseline values. Intrathecal delivery of several doses of ampakine were attempted in preliminary experiments including 500μM, 1-, 5- and 20-milliM. However, only 20milliM dose resulted in a clear change in phrenic activity and hence was selected for a formal study, the results of which are reported here.

Data collection and analyses

All neurophysiology data was collected using Spike2 software (version 8.01, CED). A custom MATLAB code (MathWorks, R2019a) was generated to analyze phrenic nerve recordings. Responses were averaged over a duration of 3 min just prior to arterial blood sampling. Integrated phrenic burst amplitude was normalized to the baseline output and calculated as percent change from baseline (%BL). Inspiratory phrenic burst frequency was expressed as breaths per minute. All other parameters including blood pressure, blood gas values are reported as absolute values.

Statistical analysis was performed using SigmaPlot 14 (Systat Software) and GraphPad Prism (Version 9). Comparisons at multiple time points for phrenic burst amplitude, frequency, heart rate and mean arterial pressure between CX717 and vehicle (HPCD) was done by two-way repeated measures analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc analysis for further multiple comparisons. Changes in blood gas values within the same group were analyzed using ordinary one-way ANOVA to with Dunnett’s multiple comparison to compare changes from baseline. In separate analysis, for comparison of individual data points at 60 min between the two groups, an unpaired two-tailed t-test was applied to determine significance.

Results

Representative examples of phrenic nerve activity, respiratory rate, heart rate and blood pressure at baseline and after intrathecal application of CX717 or the HPCD vehicle are shown in Fig. 1A–B. The group mean data are shown in Fig. 1C–F. The scatter plots at the right of each panel depict the individual data points at 60 min. As evident from Fig. 1C, intrathecal application of CX717 produced a gradual and sustained increase in phrenic inspiratory burst amplitude. In contrast, application of HPCD caused no sustained change in phrenic motor output. Phrenic burst amplitude showed an interaction between treatment (CX717 or HPCD) and time [F(4,179)=11.7, P <0.001]. Multiple comparison tests indicated that burst amplitude in CX717 treated rats was greater at 30- (P=0.001), 45- (P=0.03) and 60-min (P<0.001) min as compared to HPCD treatment. Fig. 1D shows group mean data for the effect of CX717 and HPCD on phrenic burst frequency. There was a change in burst frequency over time [F(4,179)=2.9, P=0.02] but no effect of treatment was observed [F(1,179)=0.02, P=0.9]. Analysis of heart rate is shown in Fig. 1E. The heart rate was similar between groups [treatment effect: F(1,95)=0.005, P=0.9], however there was a slight be statistically significant decline in heart rate over the course of the experiment [time effect: F(4,95)=4.8, P=0.002]. Mean arterial pressure (Fig. 1F) was stable throughout the experimental protocol with no difference between groups (treatment: F(1,95)=2.77, P=0.11; time: F(4,95)=0.82, P=0.51).

Figure 1. Intrathecally directed low-impact ampakine CX717 to the cervical spinal cord increases phrenic motor output.

Figure 1.

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A and B showing 60 sec of representative phrenic neurograms along with respiratory rate, heart rate and blood pressure before and after intrathecal delivery of HPCD or CX717 recorded till 60 min post-application. Expanded time scale traces for baseline and end of 60th min time points are also shown for both conditions. C-F: Quantified data showing changes in mean phrenic burst amplitude, burst frequency, heart rate and mean arterial pressure at baseline, 15-, 30-, 45-, and 60-min along with scatter plot depicting the individual data points at 60 min alone. Mean phrenic amplitude showed a gradual increase in phrenic burst amplitude after CX717. Scatter plot shows a significant increase in the CX717 group (vs HPCD) at 60 min (unpaired t-test, P=0.004). Group mean changes over time and scatter plots at 60 min for burst frequency, heart rate and mean arterial pressure show no significant changes. Data represented as mean ± SD, n=10.

Discussion

Our data show that intrathecal delivery of ampakine CX717 to the cerebrospinal fluid above the dorsal mid-cervical spinal cord results in a gradual and sustained increase in phrenic motor output. The increased phrenic motor output was not associated with changes in heart rate or mean arterial pressure. Thus, delivery of ampakines with this approach can impact neurons which regulate phrenic motor control, most likely phrenic motoneurons in the ventral spinal cord.

Ampakines and respiratory control.

Ampakines are family of compounds divided into two general classes: type I (“high impact”) and type II (“low impact”) (reviewed in Arai and Kessler 2007). The drugs first described by Lynch and Rogers (Lynch 2004) are considered high impact, whereas the ampakine used in the present study, CX717, can be classified as low impact. Low impact ampakines retain the ability to act as positive allosteric AMPA receptor modulators, but the receptor kinetics differ by a shorter decay time constant (Arai and Kessler 2007). High impact ampakines are typically more potent, but also may have greater concerns regarding safety and off-target effects. Both the high and low impact ampakines can impact synaptic plasticity. For example, enhanced expression of hippocampal long-term potentiation has been described after treatment with high or low impact ampakines (Arai and Kessler 2007).

Initial work which showed that ampakines can stimulate respiratory-related neural activity was done by Greer and colleagues (Greer and Ren 2009; Lorier et al. 2010; Ren et al. 2009; Ren et al. 2006). One of the first studies showed that a high impact ampakine CX546 could act on medullary respiratory networks to increase the frequency of inspiratory bursting (Ren et al. 2006). The CX546 treatment was particularly effective at increasing inspiratory burst rate after opioid-induced respiratory depression, suggesting a possible therapeutic application of ampakines. Subsequent work confirmed that ampakines can act directly on respiratory motoneurons (Lorier et al. 2010). Specifically, a medullary slice preparation was used to enable whole-cell patch recordings from the inspiratory hypoglossal motoneurons. Application of the high impact ampakine CX614 caused an increase in AMPA-evoked current amplitude, area and duration in XII motoneurons. In addition, the low impact ampakine CX717, also used in the current study, also produced an increase in AMPA current area and duration in XII motoneurons (Lorier et al. 2010). Thus, both low- and high-impact ampakines can enhance AMPA currents in motoneurons.

Ampakines are also potent stimulators of respiratory motor output in neuromuscular disease (ElMallah et al. 2015) and after cervical spinal cord injury (Wollman et al. 2020b). For example, when ampakine CX717 is administered intravenously (15 mg/kg) to anesthetized adult rats with a high cervical spinal cord injury, there is a rapid and bilateral increase in phrenic nerve inspiratory burst amplitude (Wollman et al. 2020b). The increase in burst amplitude occurred with very little impact on the overall respiratory rate, which suggests a direct impact on phrenic motoneuron AMPA currents (Wollman et al. 2020b). A study by Elmallah and colleagues explored the impact of ampakine CX717 on breathing patterns in a mouse model of Pompe disease (ElMallah et al. 2015). Pompe disease is a lysosomal storage disorder, and is associated with pathology and degeneration of respiratory muscles and motoneurons (Fuller et al. 2013). In awake Pompe mice, studied using whole body plethysmography, an intraperitoneal injection of ampakine CX717 (15 mg/kg) resulted in an increase in inspiratory tidal volume during quiet breathing (ElMallah et al. 2015).

In addition to being a respiratory stimulant, ampakines can also enhance the expression of respiratory neuroplasticity (Turner et al. 2018; Turner et al. 2016; Wollman et al. 2020a). In specific, several published reports indicate that ampakines can enhance the ability of hypoxia to induce neuroplasticity changes in respiratory motor output. One of most well studied examples of plasticity in respiratory control is intermittent hypoxia-induced long-term facilitation or LTF (Fuller et al. 2000). This response is manifested as a sustained increase in respiratory motor output lasting well beyond the hypoxia exposures. Phrenic LTF can occur via serotonin-dependent mechanisms, or adenosinergic mechanisms, depending on the relative severity of the hypoxia stimulus (Devinney et al. 2013). The first report of ampakine and LTF was a study of hypoglossal inspiratory motor output in anesthetized mice (Turner et al. 2016). When, ampakine CX717 was administered prior to three brief (1-min) bouts of hypoxia, the magnitude of hypoglossal LTF was considerable enhanced. A subsequent study in a rat model showed that pretreatment with ampakine CX717 enabled a single 5-min episode of hypoxia to evoke phrenic LTF. This is noteworthy because induction of respiratory LTF is generally agreed to require repeated episodes of hypoxia (Baker et al. 2001). This finding was reproduced in a recent study of phrenic motor output in spinal intact rats that also reported no added benefit of 3 episodes vs. 1 episode of hypoxia when given in conjunction with CX717 (Thakre et al. 2021).

One of the overarching goals of studying ampakines and phrenic motor plasticity is to determine if there may be therapeutic benefit in conditions associated with impaired phrenic activation, such as spinal cord injury. The first such study was by Wollman et al., who evaluated the impact of intravenously delivered CX717 on phrenic inspiratory bursting after high cervical spinal cord injury in rats (Wollman et al. 2020b). The CX717 (15 mg/kg) caused an immediate increase in phrenic bursting in anesthetized rats at 2 or 8 weeks following spinal injury. However, pairing CX717 with a single bout of hypoxia produced inconsistent effects. At 2-wks following spinal injury, there was an apparently enhanced phrenic motor response when CX717 was paired with a hypoxia. In contrast, at 8-wks post-injury, there was no evidence that pairing CX717 with hypoxia enhanced the phrenic response. This differential response could reflect dynamic remodeling that occurs over time in the injured spinal cord, including alterations in glutamate receptor expression (Rana et al. 2019). Collectively, the published work firmly establishes that pretreatment with low-impact ampakines can create conditions which enable the respiratory neural control system to exhibit enhanced plasticity (Thakre et al. 2021; Turner et al. 2016; Wollman et al. 2020a; Wollman et al. 2020b). However, much work remains to be done to understand how to optimize hypoxia dosing in conjunction with ampakines, particularly after neurologic injury.

Intrathecal drug delivery.

To our knowledge the current study is the first to deliver ampakines using an intrathecal approach. Clinically, intrathecal drug delivery is a viable and a relatively safe option for management of chronic pain (Deer et al. 2017). Further, this approach has potential to improve motor function after spinal cord injury (Courtine et al. 2009; Hayakawa et al. 2015; Ichiyama et al. 2008; Wenger et al. 2014). For example, several therapeutic compounds including antibodies and growth factors have been safely delivered intrathecally in human subjects with spinal cord injury (Kucher et al. 2018) (Nagoshi et al. 2020). Recently, a novel approach to intrathecal drug delivery has been developed (Minev et al. 2015), and this may have potential as a method for ampakine delivery. Specifically, a novel silicone-based neural interface, termed “e-dura”, can be used for both electrical and chemical stimulation of the spinal cord (Minev et al. 2015). When this soft neural interface was sub-durally implanted, it was safe and allowed for electrical stimulation simultaneously with drug delivery. When agonists for serotonin receptors 5-HT1, 5-HT2 and 5-HT7 were given intrathecally, and coupled with an electrical stimulation paradigm, it resulted in restoration of locomotion in spinal cord injured rats (Capogrosso et al. 2018). Our results confirm that intrathecal application of ampakines can modulate phrenic motor output. This could represent a method that could be used to increase spinal motoneuron excitability, and pairing this with electrical stimulation (Courtine et al. 2009; Ichiyama et al. 2008; Wenger et al. 2014) could have value in the context of neurorehabilitation.

Potential mechanism.

While ampakine CX717 likely produced the observed results via allosteric modulation of AMPA receptors, our studies cannot pinpoint the location of the neurons directly affected by CX717. The most obvious possibility is that the intrathecal application of CX717 acted directly on spinal neurons, and in particular phrenic motoneurons. Phrenic motoneurons express AMPA receptors (Rana et al. 2019; Rana et al. 2020), and focal application of AMPA agonists to the phrenic motor nucleus produces an increase in phrenic nerve burst activity (Chitravanshi and Sapru 1996). Prior studies using a similar intrathecal mid-cervical drug delivery method have confirmed that this approach can target phrenic motoneurons (Baker-Herman et al. 2004; Baker-Herman and Mitchell 2002). Accordingly, a direct effect of ampakine CX717 on AMPA currents in phrenic motoneurons may at least partially explain the increase in phrenic bursting which occurred in the current study. However, phrenic motoneurons are not the only possible target of the intrathecally delivered CX717. The cervical spinal cord contains a population of “pre-phrenic” cervical interneurons that have synaptic projections to phrenic motor neurons (Lane et al. 2008). These pre-phrenic interneurons can excite phrenic motoneurons (Streeter et al. 2017), but the distribution of AMPA receptors on these cells is not known. The CX717 could also have modulated the activity of spinal rhythm generating neurons (Dubayle and Viala 1996) or spino-bulbar neurons (Lane et al. 2008) that project to brainstem respiratory neurons.

Another possibility is that the intrathecally delivered CX717 may have moved rostrally via the cerebrospinal fluid to directly impact brainstem or high cervical neurons and networks. Several studies have identified important respiratory related neuronal populations not only at the high cervical level e.g. C1–2 (Oku et al. 2008) but also rostral to this area e.g. nucleus retroambiguus (Jones et al. 2012). It remains a possibility that ampakine in the present study spread beyond the intended C3-C6 region of cervical spinal cord and was unintentionally distributed to the rostral rhythm generating circuits following intrathecal injections impacting breathing.

For the current study, we speculate that the primary location of the neurons impacted by the CX717 was in the spinal cord. The volume of solution delivered intrathecally was low (15 μl), consistent with prior work that used the approach to deliver drugs to the mid-cervical spinal cord (Baker-Herman et al. 2004; Baker-Herman and Mitchell 2002). Care was also taken to precisely place the catheter tip at the C3-C4 border, and fluid was very gradually ejected to minimize caudal flow down the spinal column. To minimize the possibility of rostral flow upon delivery, the animals head was slightly tilted upwards throughout the experiments.

The current work was conducted using a urethane anesthetized rat preparation so that factors such as arterial blood gases and lung volume could be rigorously controlled. However, anesthesia can certainly impact the neural control of breathing, and therefore the phrenic motor responses to amapkine CX717. However, prior work establishes that ampakines can stimulate breathing in urethane anesthetized animals (Thakre et al. 2021; Wollman et al. 2020a), awake animals without anesthesia (ElMallah et al. 2015), and also in vitro using brainstem slice preparations (Lorier et al. 2010). Thus, the impact of ampakines on breathing is preserved across a range of conditions, and the impact of anesthesia is likely to be modest.

Conclusion.

Our proof-of-concept study establish that intrathecal delivery of ampakines can modulate phrenic motor output. Prior work shows that ampakine treatment can enhance neuroplasticity in the respiratory system (Wollman et al. 2020a) and other circuits (Lynch 1998), and can stimulate phrenic motor output after cervical spinal cord injury (Wollman et al. 2020b). Intrathecal drug delivery shows great promise for neurorehabilitation, in particular when performed in conjunction with electrical stimulation of the spinal cord (Capogrosso et al. 2018; Minev et al. 2015). Thus, intrathecal delivery of low dose, low-impact ampakine may have value the context of neurorehabilitation.

Table 1. Physiological values for different groups.

Blood gas values at baseline, 15-, 30-, 45- and 60-min post-application of either CX717 or HPCD in rats under different conditions. Data represented as mean ± SD. HPCD, 2-hydroxypropyl-ß-cyclodextrin; PaCO2, arterial CO2 pressure; PaO2, arterial O2 pressure and SBEc, standard excess base.

Experimental Group PaCO2 (mmHg) PaO2 (mmHg) SBEc (mmol/L) Weight (g) Age (weeks)
CX717 alone n=10 357.2 ± 28 11.8 ± 1.6
Baseline 43.1 ± 2.7 263.2 ± 23.3 −0.5 ± 2.2
15 min 43.4 ± 3 256.9 ± 26.7 −0.6 ± 1.8
30 min 44.5 ± 2.3 253.3 ± 23 −0.1 ± 1.9
45 min 44.4 ± 3 247 ± 30.3 0.1 ± 1.7
60 min 44.1 ± 2.5 238.5 ± 30.9 −0.5 ± 2
HPCD alone n=10 362.6 ± 23.7 12.8 ± 1.8
Baseline 42.8 ± 2.3 263.5 ± 22.1 −0.5 ± 1.4
15 min 43 ± 2.4 253.7 ± 23.6 −0.2 ± 1.8
30 min 42.6 ± 2.8 253.2 ±23.4 −0.1 ±1.7
45 min 43.9 ± 3.2 243.8 ± 29 0.3 ± 2
60 min 43 ± 2.5 240.3 ± 32 −0.1 ± 2.1
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Acknowledgements.

We thank Dr Arash Tadjalli for his help with preliminary studies. Funding support for this work was received from the National Institute of Health: 5 R01 HL139708 02 (DDF). MDS was supported by F31 HL145831-01. Ampakine used in this study was kindly provided by Dr. Arnold Lippa of RespireRx.

Footnotes

Conflicts of interest:

None.

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