AI
Wednesday, January 23, 2008 9:40:55 PM
Neuro, Yes, but as the old saying goes, sometimes it's better to be lucky than good :o) I've been neither lately unfortunately..
BTW, here's the full text of Greer's RD study using CX-546. I couldn't copy the graphs, but the entire paper is available free at Pubmed. It looks like Greer did a thorough job, with in vitro and in vivo studies (scroll down for the in vivo). Cortex's presentation slides of similar rat studies using CX-614, CX-717, and CX-701 also looked very compelling. These studies all appear to use a rescue type design however, and I haven't seen any data from a premedication type rat study. Perhaps in the next conf call someone can ask Dr. Stoll if any premed type animal studies have been done. If they have and the results were as good as these rescue type, that would be most reassuring -
http://ajrccm.atsjournals.org/cgi/content/full/174/12/1384
>>> Ampakines Alleviate Respiratory Depression in Rats
Jun Ren, Betty Y. Poon, Yun Tang, Gregory D. Funk and John J. Greer. Division of Neuroscience, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada
Correspondence and requests for reprints should be addressed to John J. Greer, Ph.D., Department of Physiology, University of Alberta, 513 HMRC, Edmonton, AB, T6G 2S2 Canada. E-mail: john.greer@ualberta.ca
ABSTRACT
TOP
ABSTRACT
AT A GLANCE COMMENTARY
METHODS
RESULTS
DISCUSSION
REFERENCES
Rationale: There is a need for improved therapeutic interventions to treat both drug- and sleep-induced respiratory depression. Increased understanding of the neurochemical control of respiration will help identify a basis for advances. Activation of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type glutamate receptors positively modulates respiratory drive and rhythmogenesis in several brain regions including the pre-Bötzinger complex. Ampakines are a diverse group of small molecules that activate subsets of these receptors.
Objective: We determined whether the ampakine CX546 would enhance respiratory drive and rhythmogenesis across various stages of development and whether this ampakine could counter opioid- and barbiturate-induced respiratory depression.
Methods: Respiratory frequency and amplitude were measured in the following rat models: (1) perinatal in vitro brainstem–spinal cord, (2) neonatal in vitro medullary slice, (3) juvenile in situ perfused, working heart–brainstem preparation, and (4) newborn and adult in vivo.
Results: Administration of CX546 stimulated baseline respiratory frequency in perinatal in vitro preparations but not in older animals (greater than Postnatal Day 0). Furthermore, pharmacologic depression of respiratory frequency and amplitude was countered at all ages studied by the administration of CX546 in vitro, in situ, and in vivo. Significantly, CX546 countered opioid-induced breathing depression in all preparations, without altering analgesia as assessed by measuring the time to foot withdrawal in response to a thermal stimulus.
Conclusions: CX546 effectively reverses opioid- and barbiturate-induced respiratory depression without reversing the analgesic response. These studies suggest that ampakines may be useful in preventing or reversing opioid-induced respiratory depression and identify the potential of ampakines for alleviating other forms of respiratory depression including sedative use and sleep apnea.
Scientific Knowledge on the Subject
There is a need for improved therapeutic interventions to treat respiratory depression and apnea. An increased understanding of the neurochemical control of respiration is providing a basis for advances.
What This Study Adds to the Field
We demonstrate that ampakines, which have been administered clinically without significant side effects, may prove useful in the realm of pulmonary medicine to alleviate respiratory depressions.
There is a concerted effort to understand the neurochemical control of respiratory rhythm–generating networks as well as the premotor and motoneuron circuits that determine activity patterns of respiratory muscles. Insights derived from experimental work will be important for developing pharmacologic interventions to alleviate respiratory depression and apneas. A specific region within the ventrolateral medulla, the pre-Bötzinger complex (pre-BötC), plays a critical role in generating rhythmic inspiratory drive, and thus is a major region of investigative focus (reviewed in Reference 1). Within the pre-BötC, glutamatergic synaptic signaling mediated by non–N-methyl-D-aspartate receptors (primarily -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] receptors) is particularly important for maintaining respiratory rhythm (2, 3). Blockage of non–N-methyl-D-aspartate receptors with the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione causes a dose-dependent decline, and eventual cessation, of respiratory frequency and inspiratory drive to cranial and spinal motoneurons. Elevation of endogenously released glutamate levels with glutamatergic uptake inhibitors (2) or reduction of AMPA receptor desensitization (4) leads to increases in respiratory frequency in vitro. In this study, we test the hypothesis that the ampakine CX546 provides a pharmacologic means to counteract respiratory depression. This drug is a member of the ampakine family of compounds that modulate the AMPA receptor complex by increasing the duration of glutamate-induced AMPA receptor–gated inward currents (5). Specifically, CX546 binds to the receptor in its agonist-bound state, not its desensitized or agonist-unbound state, and modulates the kinetics of deactivation (channel closing and transmitter dissociation) and desensitization (6). The combined use of in vitro, in situ, and in vivo rat models allowed analyses of the effect of CX546 on pre-BötC activity and respiratory motor pools in reduced and intact preparations across multiple developmental stages.
In Vitro Perinatal Rat Preparations
Brainstem–spinal cord perinatal preparations.
Sprague-Dawley rat fetuses were delivered from timed-pregnant dams anesthetized with halothane (2% delivered in 95% O2 and 5% CO2) and maintained at 37°C by radiant heat, in accordance with procedures approved by the Animal Welfare Committee of the University of Alberta (Edmonton, AB, Canada). The timing of pregnancies was determined by vaginal lavage of the dam. Newborn rat pups were anesthetized with metofane. Newborn pups and embryos were then decerebrated and the brainstem–spinal cord was dissected according to procedures similar to those established for perinatal rats (7, 8). The neuraxis was continuously perfused at 27 ± 1°C (perfusion rate, 5 ml/min; chamber volume, 1.5 ml) with modified Kreb's solution that contained (mM): 128 NaCl, 3.0 KCl, 1.5 CaCl2, 1.0 MgSO4, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose equilibrated with 95% O2–5% CO2 (pH 7.4).
Medullary slice preparations.
Brainstem–spinal cords isolated from newborn rats were pinned down, ventral surface upward, on a paraffin-coated block (3, 9). The block was mounted in the vise of a vibratory microtome (Leica VT1000 S; Leica Microsystems, Wetzlar, Germany). The brainstem was sectioned serially in the transverse plane, starting from the rostral medulla to within approximately 150 µm of the rostral boundary of the pre-BötC, as judged by the appearance of the inferior olive. A single transverse slice containing the pre-BötC and more caudal reticular formation regions was then cut (500–600-µm thick), transferred to a recording chamber, and pinned down onto Sylgard elastomer (Dow Corning, Midland, MI). The medullary slice was continuously perfused with a bathing solution identical to that used for the brainstem–spinal cord preparation with the exception that in some cases the K+ concentration was increased to 9 mM, which facilitates long-term generation of stable rhythm (greater than 5 h) by these preparations (3, 9).
Perfused heart in situ preparations.
Methods are described in detail elsewhere (10, 11). In brief, juvenile Sprague-Dawley rats (between 3 and 4 wk of age, 80–120 g) were anesthetized with isoflurane, submerged in ice-cold oxygenated perfusate, decerebrated and transected caudal to the diaphragm, and the descending aorta was cannulated (less than 8 min). The torso and brainstem were then transferred to a recording chamber, where the descending aorta was cannulated with a double-lumen cannula (one line to deliver perfusate, the second to monitor blood pressure) and perfused with saline (bubbled with 95% O2–5% CO2) at a flow rate sufficient to generate and maintain arterial pressure at 60 mm Hg. Once perfusion was initiated and arterial pressure was stabilized, the animal was gradually warmed to 32°C by heating the perfusate. The preparation was then allowed to stabilize for 1 h (from the start of the dissection), during which time the left phrenic nerve was dissected to monitor inspiratory activity (frequency and burst amplitude). After the 1-h stabilization period, baseline respiratory output was recorded and the various drugs (fentanyl and CX546) were added directly to the perfusate.
Recording.
For in vitro perinatal rat preparations, recordings of hypoglossal (XII) cranial nerve roots, cervical (C4) ventral roots, and neuronal population discharge within the ventrolateral medulla in vitro were made with suction electrodes placed over the nerve roots or on the surface of the rhythmic slice preparations. Respiratory rhythm in perfused, in situ preparations was monitored from the phrenic nerve, which was placed over two platinum hook electrodes. Signals were amplified, rectified, low-pass filtered, and recorded to a computer, using an analog–digital converter (Axon Instruments Digidata 1200; Molecular Devices, Sunnyvale, CA) and data acquisition software (Axon Instruments AxoScope; Molecular Devices).
Plethysmographic Measurements
Whole-body plethysmographic measurements of the frequency and depth of breathing were made from unrestrained Sprague-Dawley rats of either sex. Pressure changes associated with breathing were measured with either a 27-ml chamber for postnatal (P)0–P1 rats or a 2,200-ml chamber for adult rats (150–300 g), a pressure transducer (model DP103; Validyne Engineering, Northridge, CA), and a signal conditioner (CD-15; Validyne Engineering). For newborns, the plethysmograph was contained within an infant incubator (Isolette, model C-86; Air-Shields/Dräger Medical, Hatboro, PA) to maintain the ambient temperature at the approximate nest temperature of 32°C (12).
Nociceptive Testing
Thermal nociception was measured by a modification of a previously reported method (13). Briefly, unrestrained rats were placed in a plastic chamber (14 x 16 x 22 cm) and allowed to acclimate for 10 min before testing. The plantar test apparatus (Ugo Basile, Comerio, Italy) consisted of a movable infrared heat source (8-V, 50-W halogen lamp; OSRAM, Munich, Germany) which was positioned directly beneath the hind paw, 20 mm below the chamber floor. Heat settings were 30 and 50 (manufacturer settings) for newborn and adult rats, respectively. The instrument detected paw withdrawal latency from the onset of heat exposure, with accuracy to 0.1 s. When the rat perceived pain and withdrew its paw, the instrument automatically detected the withdrawal latency to the nearest 0.1 s. Withdrawal latencies were recorded before, during, and after each drug administration. The heat stimulus was automatically terminated if a withdrawal response was not observed within 20 s of its onset.
Pharmacologic Agents
All drugs were purchased from Sigma (St. Louis, MO). The ampakine CX546 was dissolved in dimethyl sulfoxide to make a 50–200 mM stock solution. CX546 (50–400 µM) and the µ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO, 800 nM) were used in vitro and added directly to the bathing medium. Fentanyl (4 nM) and CX546 (50 µM) were added to the perfusate of the working heart in situ preparation. For the in vivo plethysmographic studies, CX546 was administered intraperitoneally at a dose of 16 mg/kg (dose based on Lauterborn and coworkers [14]). Fentanyl HCl (60 µg/kg for newborn rats and 130 µg/kg for adult rats) and phenobarbital (28 mg/kg for newborn rats and 100 mg/kg for adult rats) were dissolved in physiologic saline and administered intraperitoneally (total volume, 5–10 µl for newborn rats and 150–300 µl for adult rats) to reduce baseline respiratory frequency by about 50%. Administration of vehicle did not affect the respiratory parameters studied in any preparation.
Analyses
For in vitro and in situ experiments, values of respiratory period (or frequency) and peak inspiratory burst amplitude were measured from the integrated nerve recording and reported as means relative to control values. For in vivo plethysmography, interbreath intervals and the relative amplitude of volume excursions associated with each breath were calculated before and after (5 min) intraperitoneal drug administration. In all cases, values are given as means and standard deviations. Statistical significance was tested by Student t test for paired or unpaired data (two groups) or by one-way repeated measures analysis of variance (multiple groups) followed by the Holm-Sidak test for multiple comparisons. Significance was accepted at p values lower than 0.05.
In Vitro Perinatal Preparations
Fetal and neonatal brainstem–spinal cord preparations have been well characterized and shown to generate complex, coordinated patterns of respiratory-related activity (7, 8). Recordings from cervical ventral and hypoglossal cranial nerve roots provide information regarding the pharmacology of respiratory rhythm–generating networks and the pathways transmitting that respiratory drive to key output components of the respiratory motor system, without the confounding influence of peripheral chemoreceptors and supramedullary structures. Figure 1A shows a representative example of the respiratory discharge produced by an Embryonic Day 20 (E20) brainstem–spinal cord preparation. The frequency of rhythmic respiratory discharge was markedly enhanced by the addition of CX546 (50–100 µM) to the bathing medium. Population data showing the increase in respiratory frequency compared with control for ages E18–P3 are provided in Figure 1B. In perinatal preparations (E18–P0), which often have a slower baseline rhythm compared with older neonates (8), CX546 caused a significant increase in frequency. By P3, however, CX546 had no effect on the baseline frequency of the more robust respiratory output generated by these older brainstem–spinal cord preparations.
Figure 1. CX546 stimulates frequency of rhythmic respiratory activity generated by brainstem–spinal cord preparations. (A) Rectified and integrated suction electrode recordings of C4 ventral root discharge from an Embryonic Day 20 (E20) brainstem–spinal cord preparation in response to bath application of CX546. (B) Population data showing changes in respiratory frequency of brainstem–spinal cord preparations in response to bath application of CX546 at various perinatal ages (n = 4 or 5 for each age; # significant difference between control and CX546; p < 0.05).
The medullary slice preparation is a derivative of the brainstem–spinal cord preparation (9). It contains the minimum component of neuronal populations within the ventrolateral medulla necessary for generating a respiratory rhythm, the pre-BötC. The medullary slice also contains a significant portion of the rostral ventral respiratory group, hypoglossal nucleus, and XII cranial nerve rootlets from which inspiratory motor discharge is recorded. Figure 2 illustrates the effects of CX546 on the respiratory rhythm generated by medullary slice preparations. The respiratory-related output generated by these slices typically shows a gradual decrease in frequency and burst amplitude and stops within approximately 60 min when medullary slices are prepared and bathed in solution containing 3 mM [K+]o. Bath application of CX546 (400 µM) to the slices after rhythmic activity ceased in 3 mM [K+]o caused a rapid and potent stimulation of respiratory networks. Within 2 min of CX546 application, frequency and amplitude were restored to levels as great as, and in some cases greater than, observed at any point in bathing medium containing 3 mM [K+]o.
Figure 2. CX546 stimulates frequency of rhythmic respiratory activity generated by medullary slice preparations. (A) Long-term recording (more than 3 h) from a Postnatal Day 2 (P2) medullary slice preparation perfused with bathing medium containing 3 mM [K+]o, showing rectified and integrated hypoglossal nerve root (XII) activity. The bottom traces in A, a–d, show expanded records of respiratory discharge at specific periods during the recording. Note that when perfused with 3 mM [K+]o bathing solution, rhythmic activity was present shortly after slice production (trace a), but burst amplitude and frequency gradually diminished and stopped in less than 60 min (trace b). Trace c, after cessation of rhythmic activity (in 3 mM [K+]o), bath application of CX546 restored rhythmic activity of XII motoneurons. Moreover, the respiratory frequency and amplitude in CX546 were greater than under initial control conditions. In trace d, respiratory rhythm persisted for more than 3 h in the presence of CX546 (longest period tested). (B) Population data showing time course of changes in respiratory frequency (relative to control levels averaged over the first 2 min of recording) of P2 medullary slice preparations in 3 mM [K+]o solution or 3 mM [K+]o followed by addition of CX546 at t = 60 min (n = 3 for each data point after 45 min, n = 6 for data points from 0 to 45 min). * Significant difference relative to control.
The next series of experiments examined the ability of CX546 to counter the depression of respiratory frequency and amplitude caused by the µ-opioid receptor agonist DAGO. Figures 3A and 3B show recordings of rhythmic respiratory discharge generated by P1 brainstem–spinal cord and medullary slice (bathed in 9 mM [K+]o) preparations. DAGO (800 nM) markedly suppressed respiratory frequency and amplitude in both preparations. The DAGO-induced depression was alleviated by the subsequent administration of CX546 (200 µM). Population data are provided in Figures 3C and 3D. The administration of CX546 on its own did not significantly alter the control values of respiratory frequency or amplitude of motor nerve discharge generated by medullary slice preparations bathed in 9 mM [K+]o.
Figure 3. CX546 counters opioid-induced respiratory depression in vitro. Rectified and integrated recordings of (A) C4 ventral roots (brainstem–spinal cord of a P1 rat) and (B) XII nerve roots (medullary slice of a P1 rat perfused with 9 mM [K+]o bathing medium) in response to bath application of the µ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO). DAGO-induced suppression of respiratory frequency and amplitude in these preparations was partially reversed by the subsequent bath application of CX546. (C and D) Population data for brainstem–spinal cord and medullary slice preparations (P1–P2 rats; n = 7 for each preparation), showing the effects of bath-applied DAGO alone and then CX546 (in the continued presence of DAGO) on inspiratory frequency and burst amplitude relative to control values (of 1.0). * Significant difference relative to control; # significant difference between value in DAGO alone and after subsequent application of CX546. (Values of frequency and amplitude are reported relative to control values standardized to 1.0.)
Perfused Heart In Situ Data
Rhythmically active brainstem–spinal cord, and medullary slice preparations in rat are not viable beyond the newborn period. However, it is possible to examine central respiratory control and its pharmacology in reduced preparations of older rodents, using the in situ working heart–brainstem preparation (10). These preparations generate a normal (in vivo–like) breathing motor pattern that includes augmenting phrenic bursts. They are also oxygenated throughout and have uniform brain tissue pH (15). Figure 4A shows a representative example of phrenic nerve discharge recorded from a P24 rat in situ preparation. Administration of fentanyl (4 nM) to the perfusate produced a significant decrease in respiratory frequency and phrenic burst amplitude that was countered by subsequent administration of CX546 (50 µM). Population data are presented in Figure 4B.
Figure 4. CX546 counters opioid-induced depression of respiratory frequency and amplitude generated by perfused heart in situ preparations. (A) Long-term recording of raw and integrated phrenic nerve discharge in a P24 rat. Time points at which fentanyl and CX546 were added to the reservoir containing oxygenated perfusion medium are indicated by arrows. Note that it took approximately 90 s for solution in the reservoir to reach the preparation. Bottom panels show expanded sections of phrenic nerve respiratory discharge taken from the regions labeled a–e in the long-term recording. (B) Population data (n = 8) showing the relative effects (compared with control period) of fentanyl and then CX546 (in the continued presence of fentanyl) on amplitude and frequency of integrated phrenic nerve discharge. Open bars, amplitude; solid bars, frequency. *p < 0.001; #p < 0.05; +p < 0.01.
In Vivo Plethysmography
The final stage of the study was to examine the actions of CX546 in vivo. Whole body plethysmography was used to examine the breathing patterns generated by unanesthetized newborn rats (P0–P2) and adult rats. Respiration was depressed by either intraperitoneal administration of the µ-opioid receptor agonist fentanyl or the barbiturate phenobarbital. As shown in representative examples in Figures 5A–5C and in the population data of Figures 5D–5F, both agents significantly decreased respiratory frequency and burst amplitude. Subsequent intraperitoneal injection of CX546 (16 mg/kg) reversed the opiate- and phenobarbital-mediated respiratory depression. The baseline frequency and amplitude of newborn rats (P0–P2, n = 5) and adult rats (n = 3, data not shown), however, were not significantly altered by the same dose of CX546 on its own. CX546 was also without any obvious effect on the behavior or arousal state (i.e., increase/decrease in spontaneous movement, agitation, indication of sedation)
Figure 5. CX546 counters opioid- and phenobarbital-induced respiratory depression in vivo. Plethysmographic recordings show that the inhibitory effect of fentanyl (A) and phenobarbital (B) on the breathing frequency and amplitude in unanesthetized P0 pups is partially countered by CX546. (C) Plethysmographic recording from an adult rat showing that, as seen in P0 pups, administration of fentanyl inhibits breathing frequency and amplitude and that this is partially reversed by CX546. (D and E) Population data for P0 rat pups, showing changes in frequency and amplitude relative to control evoked by fentanyl or phenobarbital both before and after the administration of CX546 (n = 4). (F) Population data for adult rats, showing relative changes in frequency and amplitude evoked by fentanyl alone and after the administration of CX546 (n = 4). * Significant difference relative to control; # significant difference between values in the presence of the respiratory depressant alone and after subsequent application of CX546. All drugs were delivered intraperitoneally.
Nociceptive Testing
The preceding experiments demonstrated that CX546 counteracts the respiratory depression induced by DAGO or fentanyl. We then investigated whether the intraperitoneal administration of CX546 affects fentanyl-induced analgesia in vivo. Using a thermal nociception test, the latency of hind paw withdrawal was 4.9 ± 3.8 and 5.6 ± 1.7 s for newborns and adult rats, respectively (see Table 1). Administration of fentanyl at doses that suppressed respiratory rhythm (60 µg/kg for newborn rats and 130 µg/kg for adult rats) extended the latency to paw withdrawal to the 20-s cutoff limit. This fentanyl-induced analgesia, which manifests itself as the loss of foot withdrawal, persisted despite the subsequent intraperitoneal administration of CX546 (16 mg/kg). The fentanyl-induced analgesic effect was, however, blocked by the subsequent administration of naloxone (1 mg/kg) in both newborn and adult rats. When CX546 (16 mg/kg) was administrated on its own, it did not change the sensitivity of thermal nociception tests in the control condition. These results demonstrate that CX546 reduces the deleterious respiratory-depressant effects of opioids without inhibiting their desirable, analgesic actions.
TABLE 1. DRUG EFFECTS ON THERMAL NOCICEPTION DETERMINED IN HIND PAW WITHDRAWAL TESTS
DISCUSSION
Respiratory depression has serious clinical implications and manifests itself in a number of situations including pharmacologically induced and endogenous bradypnea and apneas (central and obstructive). Advancements in drug treatments based on an understanding of the neurochemical control of breathing are needed. Here, we demonstrate that an ampakine, which potentiates AMPA receptor–mediated conductances (6) critical for inspiratory rhythmogenesis and synaptic drive to respiratory motor nuclei, is an effective means of countering respiratory depression.
Ampakine-mediated Modulation of Respiratory Activity
Sites of action.
Administration of the ampakine CX546 provided a powerful and robust means of alleviating depressed central respiratory drive; respiratory activity was enhanced when the network was examined in its most reduced (medullary slice) and most intact (in vivo) states. Potentiation of output in medullary slices and brainstem–spinal cord preparations indicates that CX546 acts on medullary respiratory networks. Direct action within the pre-BötC rhythm-generating networks is likely because frequency is enhanced. However, it is also possible that the increased frequency reflects secondary actions through a population of neurons that provides tonic excitatory glutamatergic drive to the rhythm generator. Amplitude effects in vitro and in vivo also suggest that premotor and motoneuron pools are sites of ampakine action. Certainly, inhibition of desensitization, which is one effect of ampakines, within the XII motor nucleus potentiates inspiratory burst amplitude (4).
The observation that the potentiating actions of ampakines are also present in situ and in vivo, and therefore not limited to in vitro preparations, indicates that potentiation is not dependent on some unique condition established in vitro. Ampakines counteract respiratory depression in the presence of excitatory drive to the central respiratory rhythm generation from peripheral chemoreceptors, mechanoreceptors, and supramedullary centers. In fact, it is possible that in more intact preparations, ampakines act not only within the pre-BötC and motor pools, but also enhance activity in excitatory afferent feedback pathways.
Development.
Ampakine-mediated enhancement of respiratory activity was observed in animals ranging in age from E18 to adult. In animals P0 or younger, ampakines enhanced baseline activity. In older preparations, ampakines enhanced activity only if respiration was initially depressed. Whether this reflects a developmental change in sensitivity to ampakines, or whether the potentiating actions of ampakines become manifest only if respiratory activity is first suppressed, is unclear. Paradoxically, if ampakines potentiate respiratory output by blocking desensitization, one would predict greater efficacy in older animals with conditions of increased network activity due to increased glutamate receptor activation and the greater potential for desensitization. Further, the inhibition of desensitization kinetics by CX546 should become more significant with maturation. AMPA receptor subunits, GluR1–4, exist as either flip or flop splice variants. In embryonic animals, the flip variants predominate (16). With maturation there is a shift in expression such that the flop isoforms, which have more rapid desensitization kinetics, become increasingly dominant. Thus, these data suggest that the most significant action of ampakines in relation to controlling activity of respiratory networks may not be modulation of glutamate receptor desensitization properties. Rather, enhancement of binding affinity and prolongation of channel open time may be the important parameters.
Defining the molecular mechanism(s) underlying the ampakine-mediated enhancement of depressed respiratory output will require analysis at the single-cell and most likely single-channel levels, and is beyond the scope of this study. Despite an incomplete mechanistic understanding, potentiation of depressed but not baseline activity is a desirable feature of ampakine action from a clinical perspective. Enhancing baseline ventilation or increasing depressed respiratory output to levels greater than baseline (i.e., hyperventilation) would have independent deleterious consequences including the disruption of blood gases, and predisposition to respiratory instability.
Specificity of action on respiratory networks.
The finding that CX546 stimulated breathing without widespread activation of neuronal circuits may not be expected given the ubiquitous distribution of AMPA receptors within the central nervous system. However, behavioral selectivity of ampakines has been reported in other studies and might be explained by regional and network selectivity of ampakines (17). AMPA subunits (GluR1–4 subunits, each with flip and flop splice variants and RNA-editing R/G and Q/R site; see Reference 18) are differentially expressed throughout the brain. Ampakines have subunit-specific effects and could act with regional selectivity. For example, ampakines have a severalfold greater effect on excitatory postsynaptic currents in hippocampus (high concentrations of GluR1 and GluR2) than in thalamus (GluR3 and GluR4). Even within the hippocampus, ampakines have different effects across subgroups of cells. It is therefore possible that ampakines have particularly strong effects on the neurons regulating respiration.
Additional ampakine actions.
In addition to their direct effects on glutamatergic signaling, ampakines, including CX546, appear to chronically elevate the production of brain-derived neurotrophic factor (BDNF) in hippocampal and cortical neurons via the positive modulation of AMPA receptors (14). However, the rapid kinetics of ampakine CX546 treatment on respiratory drive is inconsistent with an upregulation of BDNF synthesis. Moreover, BDNF decreases, rather than increases, the frequency of respiratory rhythm in medullary slice preparations (19). A further mechanism of CX546 action is the enhancement of astrocyte metabolism, which increases glucose utilization and lactate production (20). The possibility of such astrocyte-mediated actions influencing respiratory drive, in conjunction with direct modulation of neuronal AMPA receptor conductances, should be considered given the proposed metabolic coupling between glia and medullary respiratory neurons (21). However, the acute actions of CX546 on respiratory frequency are unlikely to reflect solely glia-derived increases in extracellular lactic acid because bath application of lactic acid (5 mM) to brainstem–spinal cord or medullary slice preparations did not mimic the actions of CX546 (J. Ren and J. J. Greer, unpublished observation).
Implications for Analysis of Respiratory Rhythm Generation
The first series of experiments was performed with in vitro brainstem–spinal cord and medullary slice preparations. These in vitro preparations isolated from embryonic or P0 rats typically generate a respiratory rhythm of low frequency compared with older neonatal animals (reviewed in Reference 22). This is in part due to lack of sufficient excitatory drive from glutamatergic and neuromodulatory systems. Data from this study demonstrate that CX546, presumably via the modulation/potentiation of the AMPA receptor conductance, markedly enhances the frequency of respiratory rhythm in vitro in E18–P0 rats, and restores depressed rhythm in older neonates in both brainstem–spinal cord and medullary slice preparations.
It is significant that the medullary slice preparation produces robust respiratory-related rhythm when perfused with bathing medium containing 3 mM [K+]o and CX546. The rhythmic slice preparation has become a standard model in respiratory control studies and its use has significantly advanced understanding of the sites and mechanisms of mammalian rhythm generation (9). A limitation of these slice preparations, however, has been the necessity of bathing the tissue in elevated [K+]o (typically 9 mM) for generation of long-lasting rhythmic activity (although see Reference 23). The perceived need for elevated [K+]o underlies the argument of some investigators that mechanisms of rhythm generation identified in vitro are not relevant to breathing in vivo. The opposing view, presented with the original description of the slice (3, 9), is that elevated [K+]o is required in the slice to compensate for the loss of tonic and phasic excitatory glutamatergic synaptic drive to rhythm-generating networks that is present in more intact in vitro preparations, such as the brainstem–spinal cord preparation, and under in vivo conditions. Accumulating evidence emphasizes the potential importance of [K+]o in determining the effects of neuromodulatory cascades on rhythm, but also suggests that many of the basic mechanisms of rhythm generation described in vitro apply in vivo. For example, the importance of the pre-BötC in rhythm generation, and the pacemaker behavior of pre-BötC neurons, and so on, are not dependent on elevated [K+]o (1, 24). The demonstration in this study that ampakines evoke a robust, long-lasting respiratory rhythm at physiological [K+]o adds further support to the thesis. It also provides an alternative to the use of elevated [K+]o or addition of neurotransmitter receptor agonists for producing long-lasting rhythmic respiratory activity in medullary slices.
Functional/Clinical Significance
The in vivo data from newborn and adult rats demonstrate that the actions of ampakines observed in vitro and in situ occur in the intact animal. Ampakines act as a powerful stimulant of respiratory frequency and tidal volume in perinates and rats of all ages in which respiration was depressed by the µ-opioid receptor agonist fentanyl or the barbiturate phenobarbital. Opiates are widely used analgesics and phenobarbital is the longest acting of the barbiturates, which are used primarily for sedation or as an anticonvulsant. However, both have serious adverse effects, perhaps the most significant being respiratory depression. Richter's group has demonstrated that a serotonin (5-hydroxytryptamine) receptor subtype 4a (5-HT4a) agonist counters opioid-induced breathing depression without loss of analgesia in rats (25, 26). The mechanisms underlying 5-HT4a receptor functions as an antidote to fentanyl have not been clearly delineated although they may interact via convergent cAMP intracellular signaling. This is supported by the finding that nociception-induced respiratory depression in vitro is reversed by activation of adenylyl cyclase or blockage of phosphodiesterase activity (27). It is important to note that administration of CX546 countered drug-induced respiratory depression without significantly altering the baseline frequency or amplitude of breathing in control animals, inhibiting the opioid-induced analgesia, or noticeably affecting the behavior of the animals. Besides the effectiveness of ampakines to counter drug-induced respiratory depression, it will be of interest to determine whether ampakines are effective in treating central and obstructive apneas in newborns and adults, areas in which advancements in treatment are certainly needed (28, 29). Genetic mouse models with clear central hypoventilation during the postnatal period (reviewed in Reference 30) should provide experimental opportunities to explore this area. Future studies will be necessary to determine the relative efficacy of various ampakines and to perform a more thorough examination for potentially harmful side effects. Further, this study was limited to an examination of the acute actions of ampakines over minutes. A drug delivery system that provides ampakine-mediated actions over longer periods will likely be necessary for uses beyond countering drug-induced respiratory depression.
Conclusions
Data from the current study indicate that ampakines may provide a novel pharmacologic means of countering respiratory depression. Importantly, ampakines readily cross the blood–brain barrier and appear to be well tolerated (17, 31, 32). Further work evaluating the efficacy of the various members of currently available and developing members of the ampakine family as respiratory stimulants toward applicability in clinical settings is warranted. <<<
BTW, here's the full text of Greer's RD study using CX-546. I couldn't copy the graphs, but the entire paper is available free at Pubmed. It looks like Greer did a thorough job, with in vitro and in vivo studies (scroll down for the in vivo). Cortex's presentation slides of similar rat studies using CX-614, CX-717, and CX-701 also looked very compelling. These studies all appear to use a rescue type design however, and I haven't seen any data from a premedication type rat study. Perhaps in the next conf call someone can ask Dr. Stoll if any premed type animal studies have been done. If they have and the results were as good as these rescue type, that would be most reassuring -
http://ajrccm.atsjournals.org/cgi/content/full/174/12/1384
>>> Ampakines Alleviate Respiratory Depression in Rats
Jun Ren, Betty Y. Poon, Yun Tang, Gregory D. Funk and John J. Greer. Division of Neuroscience, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada
Correspondence and requests for reprints should be addressed to John J. Greer, Ph.D., Department of Physiology, University of Alberta, 513 HMRC, Edmonton, AB, T6G 2S2 Canada. E-mail: john.greer@ualberta.ca
ABSTRACT
TOP
ABSTRACT
AT A GLANCE COMMENTARY
METHODS
RESULTS
DISCUSSION
REFERENCES
Rationale: There is a need for improved therapeutic interventions to treat both drug- and sleep-induced respiratory depression. Increased understanding of the neurochemical control of respiration will help identify a basis for advances. Activation of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type glutamate receptors positively modulates respiratory drive and rhythmogenesis in several brain regions including the pre-Bötzinger complex. Ampakines are a diverse group of small molecules that activate subsets of these receptors.
Objective: We determined whether the ampakine CX546 would enhance respiratory drive and rhythmogenesis across various stages of development and whether this ampakine could counter opioid- and barbiturate-induced respiratory depression.
Methods: Respiratory frequency and amplitude were measured in the following rat models: (1) perinatal in vitro brainstem–spinal cord, (2) neonatal in vitro medullary slice, (3) juvenile in situ perfused, working heart–brainstem preparation, and (4) newborn and adult in vivo.
Results: Administration of CX546 stimulated baseline respiratory frequency in perinatal in vitro preparations but not in older animals (greater than Postnatal Day 0). Furthermore, pharmacologic depression of respiratory frequency and amplitude was countered at all ages studied by the administration of CX546 in vitro, in situ, and in vivo. Significantly, CX546 countered opioid-induced breathing depression in all preparations, without altering analgesia as assessed by measuring the time to foot withdrawal in response to a thermal stimulus.
Conclusions: CX546 effectively reverses opioid- and barbiturate-induced respiratory depression without reversing the analgesic response. These studies suggest that ampakines may be useful in preventing or reversing opioid-induced respiratory depression and identify the potential of ampakines for alleviating other forms of respiratory depression including sedative use and sleep apnea.
Scientific Knowledge on the Subject
There is a need for improved therapeutic interventions to treat respiratory depression and apnea. An increased understanding of the neurochemical control of respiration is providing a basis for advances.
What This Study Adds to the Field
We demonstrate that ampakines, which have been administered clinically without significant side effects, may prove useful in the realm of pulmonary medicine to alleviate respiratory depressions.
There is a concerted effort to understand the neurochemical control of respiratory rhythm–generating networks as well as the premotor and motoneuron circuits that determine activity patterns of respiratory muscles. Insights derived from experimental work will be important for developing pharmacologic interventions to alleviate respiratory depression and apneas. A specific region within the ventrolateral medulla, the pre-Bötzinger complex (pre-BötC), plays a critical role in generating rhythmic inspiratory drive, and thus is a major region of investigative focus (reviewed in Reference 1). Within the pre-BötC, glutamatergic synaptic signaling mediated by non–N-methyl-D-aspartate receptors (primarily -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] receptors) is particularly important for maintaining respiratory rhythm (2, 3). Blockage of non–N-methyl-D-aspartate receptors with the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione causes a dose-dependent decline, and eventual cessation, of respiratory frequency and inspiratory drive to cranial and spinal motoneurons. Elevation of endogenously released glutamate levels with glutamatergic uptake inhibitors (2) or reduction of AMPA receptor desensitization (4) leads to increases in respiratory frequency in vitro. In this study, we test the hypothesis that the ampakine CX546 provides a pharmacologic means to counteract respiratory depression. This drug is a member of the ampakine family of compounds that modulate the AMPA receptor complex by increasing the duration of glutamate-induced AMPA receptor–gated inward currents (5). Specifically, CX546 binds to the receptor in its agonist-bound state, not its desensitized or agonist-unbound state, and modulates the kinetics of deactivation (channel closing and transmitter dissociation) and desensitization (6). The combined use of in vitro, in situ, and in vivo rat models allowed analyses of the effect of CX546 on pre-BötC activity and respiratory motor pools in reduced and intact preparations across multiple developmental stages.
In Vitro Perinatal Rat Preparations
Brainstem–spinal cord perinatal preparations.
Sprague-Dawley rat fetuses were delivered from timed-pregnant dams anesthetized with halothane (2% delivered in 95% O2 and 5% CO2) and maintained at 37°C by radiant heat, in accordance with procedures approved by the Animal Welfare Committee of the University of Alberta (Edmonton, AB, Canada). The timing of pregnancies was determined by vaginal lavage of the dam. Newborn rat pups were anesthetized with metofane. Newborn pups and embryos were then decerebrated and the brainstem–spinal cord was dissected according to procedures similar to those established for perinatal rats (7, 8). The neuraxis was continuously perfused at 27 ± 1°C (perfusion rate, 5 ml/min; chamber volume, 1.5 ml) with modified Kreb's solution that contained (mM): 128 NaCl, 3.0 KCl, 1.5 CaCl2, 1.0 MgSO4, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose equilibrated with 95% O2–5% CO2 (pH 7.4).
Medullary slice preparations.
Brainstem–spinal cords isolated from newborn rats were pinned down, ventral surface upward, on a paraffin-coated block (3, 9). The block was mounted in the vise of a vibratory microtome (Leica VT1000 S; Leica Microsystems, Wetzlar, Germany). The brainstem was sectioned serially in the transverse plane, starting from the rostral medulla to within approximately 150 µm of the rostral boundary of the pre-BötC, as judged by the appearance of the inferior olive. A single transverse slice containing the pre-BötC and more caudal reticular formation regions was then cut (500–600-µm thick), transferred to a recording chamber, and pinned down onto Sylgard elastomer (Dow Corning, Midland, MI). The medullary slice was continuously perfused with a bathing solution identical to that used for the brainstem–spinal cord preparation with the exception that in some cases the K+ concentration was increased to 9 mM, which facilitates long-term generation of stable rhythm (greater than 5 h) by these preparations (3, 9).
Perfused heart in situ preparations.
Methods are described in detail elsewhere (10, 11). In brief, juvenile Sprague-Dawley rats (between 3 and 4 wk of age, 80–120 g) were anesthetized with isoflurane, submerged in ice-cold oxygenated perfusate, decerebrated and transected caudal to the diaphragm, and the descending aorta was cannulated (less than 8 min). The torso and brainstem were then transferred to a recording chamber, where the descending aorta was cannulated with a double-lumen cannula (one line to deliver perfusate, the second to monitor blood pressure) and perfused with saline (bubbled with 95% O2–5% CO2) at a flow rate sufficient to generate and maintain arterial pressure at 60 mm Hg. Once perfusion was initiated and arterial pressure was stabilized, the animal was gradually warmed to 32°C by heating the perfusate. The preparation was then allowed to stabilize for 1 h (from the start of the dissection), during which time the left phrenic nerve was dissected to monitor inspiratory activity (frequency and burst amplitude). After the 1-h stabilization period, baseline respiratory output was recorded and the various drugs (fentanyl and CX546) were added directly to the perfusate.
Recording.
For in vitro perinatal rat preparations, recordings of hypoglossal (XII) cranial nerve roots, cervical (C4) ventral roots, and neuronal population discharge within the ventrolateral medulla in vitro were made with suction electrodes placed over the nerve roots or on the surface of the rhythmic slice preparations. Respiratory rhythm in perfused, in situ preparations was monitored from the phrenic nerve, which was placed over two platinum hook electrodes. Signals were amplified, rectified, low-pass filtered, and recorded to a computer, using an analog–digital converter (Axon Instruments Digidata 1200; Molecular Devices, Sunnyvale, CA) and data acquisition software (Axon Instruments AxoScope; Molecular Devices).
Plethysmographic Measurements
Whole-body plethysmographic measurements of the frequency and depth of breathing were made from unrestrained Sprague-Dawley rats of either sex. Pressure changes associated with breathing were measured with either a 27-ml chamber for postnatal (P)0–P1 rats or a 2,200-ml chamber for adult rats (150–300 g), a pressure transducer (model DP103; Validyne Engineering, Northridge, CA), and a signal conditioner (CD-15; Validyne Engineering). For newborns, the plethysmograph was contained within an infant incubator (Isolette, model C-86; Air-Shields/Dräger Medical, Hatboro, PA) to maintain the ambient temperature at the approximate nest temperature of 32°C (12).
Nociceptive Testing
Thermal nociception was measured by a modification of a previously reported method (13). Briefly, unrestrained rats were placed in a plastic chamber (14 x 16 x 22 cm) and allowed to acclimate for 10 min before testing. The plantar test apparatus (Ugo Basile, Comerio, Italy) consisted of a movable infrared heat source (8-V, 50-W halogen lamp; OSRAM, Munich, Germany) which was positioned directly beneath the hind paw, 20 mm below the chamber floor. Heat settings were 30 and 50 (manufacturer settings) for newborn and adult rats, respectively. The instrument detected paw withdrawal latency from the onset of heat exposure, with accuracy to 0.1 s. When the rat perceived pain and withdrew its paw, the instrument automatically detected the withdrawal latency to the nearest 0.1 s. Withdrawal latencies were recorded before, during, and after each drug administration. The heat stimulus was automatically terminated if a withdrawal response was not observed within 20 s of its onset.
Pharmacologic Agents
All drugs were purchased from Sigma (St. Louis, MO). The ampakine CX546 was dissolved in dimethyl sulfoxide to make a 50–200 mM stock solution. CX546 (50–400 µM) and the µ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO, 800 nM) were used in vitro and added directly to the bathing medium. Fentanyl (4 nM) and CX546 (50 µM) were added to the perfusate of the working heart in situ preparation. For the in vivo plethysmographic studies, CX546 was administered intraperitoneally at a dose of 16 mg/kg (dose based on Lauterborn and coworkers [14]). Fentanyl HCl (60 µg/kg for newborn rats and 130 µg/kg for adult rats) and phenobarbital (28 mg/kg for newborn rats and 100 mg/kg for adult rats) were dissolved in physiologic saline and administered intraperitoneally (total volume, 5–10 µl for newborn rats and 150–300 µl for adult rats) to reduce baseline respiratory frequency by about 50%. Administration of vehicle did not affect the respiratory parameters studied in any preparation.
Analyses
For in vitro and in situ experiments, values of respiratory period (or frequency) and peak inspiratory burst amplitude were measured from the integrated nerve recording and reported as means relative to control values. For in vivo plethysmography, interbreath intervals and the relative amplitude of volume excursions associated with each breath were calculated before and after (5 min) intraperitoneal drug administration. In all cases, values are given as means and standard deviations. Statistical significance was tested by Student t test for paired or unpaired data (two groups) or by one-way repeated measures analysis of variance (multiple groups) followed by the Holm-Sidak test for multiple comparisons. Significance was accepted at p values lower than 0.05.
In Vitro Perinatal Preparations
Fetal and neonatal brainstem–spinal cord preparations have been well characterized and shown to generate complex, coordinated patterns of respiratory-related activity (7, 8). Recordings from cervical ventral and hypoglossal cranial nerve roots provide information regarding the pharmacology of respiratory rhythm–generating networks and the pathways transmitting that respiratory drive to key output components of the respiratory motor system, without the confounding influence of peripheral chemoreceptors and supramedullary structures. Figure 1A shows a representative example of the respiratory discharge produced by an Embryonic Day 20 (E20) brainstem–spinal cord preparation. The frequency of rhythmic respiratory discharge was markedly enhanced by the addition of CX546 (50–100 µM) to the bathing medium. Population data showing the increase in respiratory frequency compared with control for ages E18–P3 are provided in Figure 1B. In perinatal preparations (E18–P0), which often have a slower baseline rhythm compared with older neonates (8), CX546 caused a significant increase in frequency. By P3, however, CX546 had no effect on the baseline frequency of the more robust respiratory output generated by these older brainstem–spinal cord preparations.
Figure 1. CX546 stimulates frequency of rhythmic respiratory activity generated by brainstem–spinal cord preparations. (A) Rectified and integrated suction electrode recordings of C4 ventral root discharge from an Embryonic Day 20 (E20) brainstem–spinal cord preparation in response to bath application of CX546. (B) Population data showing changes in respiratory frequency of brainstem–spinal cord preparations in response to bath application of CX546 at various perinatal ages (n = 4 or 5 for each age; # significant difference between control and CX546; p < 0.05).
The medullary slice preparation is a derivative of the brainstem–spinal cord preparation (9). It contains the minimum component of neuronal populations within the ventrolateral medulla necessary for generating a respiratory rhythm, the pre-BötC. The medullary slice also contains a significant portion of the rostral ventral respiratory group, hypoglossal nucleus, and XII cranial nerve rootlets from which inspiratory motor discharge is recorded. Figure 2 illustrates the effects of CX546 on the respiratory rhythm generated by medullary slice preparations. The respiratory-related output generated by these slices typically shows a gradual decrease in frequency and burst amplitude and stops within approximately 60 min when medullary slices are prepared and bathed in solution containing 3 mM [K+]o. Bath application of CX546 (400 µM) to the slices after rhythmic activity ceased in 3 mM [K+]o caused a rapid and potent stimulation of respiratory networks. Within 2 min of CX546 application, frequency and amplitude were restored to levels as great as, and in some cases greater than, observed at any point in bathing medium containing 3 mM [K+]o.
Figure 2. CX546 stimulates frequency of rhythmic respiratory activity generated by medullary slice preparations. (A) Long-term recording (more than 3 h) from a Postnatal Day 2 (P2) medullary slice preparation perfused with bathing medium containing 3 mM [K+]o, showing rectified and integrated hypoglossal nerve root (XII) activity. The bottom traces in A, a–d, show expanded records of respiratory discharge at specific periods during the recording. Note that when perfused with 3 mM [K+]o bathing solution, rhythmic activity was present shortly after slice production (trace a), but burst amplitude and frequency gradually diminished and stopped in less than 60 min (trace b). Trace c, after cessation of rhythmic activity (in 3 mM [K+]o), bath application of CX546 restored rhythmic activity of XII motoneurons. Moreover, the respiratory frequency and amplitude in CX546 were greater than under initial control conditions. In trace d, respiratory rhythm persisted for more than 3 h in the presence of CX546 (longest period tested). (B) Population data showing time course of changes in respiratory frequency (relative to control levels averaged over the first 2 min of recording) of P2 medullary slice preparations in 3 mM [K+]o solution or 3 mM [K+]o followed by addition of CX546 at t = 60 min (n = 3 for each data point after 45 min, n = 6 for data points from 0 to 45 min). * Significant difference relative to control.
The next series of experiments examined the ability of CX546 to counter the depression of respiratory frequency and amplitude caused by the µ-opioid receptor agonist DAGO. Figures 3A and 3B show recordings of rhythmic respiratory discharge generated by P1 brainstem–spinal cord and medullary slice (bathed in 9 mM [K+]o) preparations. DAGO (800 nM) markedly suppressed respiratory frequency and amplitude in both preparations. The DAGO-induced depression was alleviated by the subsequent administration of CX546 (200 µM). Population data are provided in Figures 3C and 3D. The administration of CX546 on its own did not significantly alter the control values of respiratory frequency or amplitude of motor nerve discharge generated by medullary slice preparations bathed in 9 mM [K+]o.
Figure 3. CX546 counters opioid-induced respiratory depression in vitro. Rectified and integrated recordings of (A) C4 ventral roots (brainstem–spinal cord of a P1 rat) and (B) XII nerve roots (medullary slice of a P1 rat perfused with 9 mM [K+]o bathing medium) in response to bath application of the µ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO). DAGO-induced suppression of respiratory frequency and amplitude in these preparations was partially reversed by the subsequent bath application of CX546. (C and D) Population data for brainstem–spinal cord and medullary slice preparations (P1–P2 rats; n = 7 for each preparation), showing the effects of bath-applied DAGO alone and then CX546 (in the continued presence of DAGO) on inspiratory frequency and burst amplitude relative to control values (of 1.0). * Significant difference relative to control; # significant difference between value in DAGO alone and after subsequent application of CX546. (Values of frequency and amplitude are reported relative to control values standardized to 1.0.)
Perfused Heart In Situ Data
Rhythmically active brainstem–spinal cord, and medullary slice preparations in rat are not viable beyond the newborn period. However, it is possible to examine central respiratory control and its pharmacology in reduced preparations of older rodents, using the in situ working heart–brainstem preparation (10). These preparations generate a normal (in vivo–like) breathing motor pattern that includes augmenting phrenic bursts. They are also oxygenated throughout and have uniform brain tissue pH (15). Figure 4A shows a representative example of phrenic nerve discharge recorded from a P24 rat in situ preparation. Administration of fentanyl (4 nM) to the perfusate produced a significant decrease in respiratory frequency and phrenic burst amplitude that was countered by subsequent administration of CX546 (50 µM). Population data are presented in Figure 4B.
Figure 4. CX546 counters opioid-induced depression of respiratory frequency and amplitude generated by perfused heart in situ preparations. (A) Long-term recording of raw and integrated phrenic nerve discharge in a P24 rat. Time points at which fentanyl and CX546 were added to the reservoir containing oxygenated perfusion medium are indicated by arrows. Note that it took approximately 90 s for solution in the reservoir to reach the preparation. Bottom panels show expanded sections of phrenic nerve respiratory discharge taken from the regions labeled a–e in the long-term recording. (B) Population data (n = 8) showing the relative effects (compared with control period) of fentanyl and then CX546 (in the continued presence of fentanyl) on amplitude and frequency of integrated phrenic nerve discharge. Open bars, amplitude; solid bars, frequency. *p < 0.001; #p < 0.05; +p < 0.01.
In Vivo Plethysmography
The final stage of the study was to examine the actions of CX546 in vivo. Whole body plethysmography was used to examine the breathing patterns generated by unanesthetized newborn rats (P0–P2) and adult rats. Respiration was depressed by either intraperitoneal administration of the µ-opioid receptor agonist fentanyl or the barbiturate phenobarbital. As shown in representative examples in Figures 5A–5C and in the population data of Figures 5D–5F, both agents significantly decreased respiratory frequency and burst amplitude. Subsequent intraperitoneal injection of CX546 (16 mg/kg) reversed the opiate- and phenobarbital-mediated respiratory depression. The baseline frequency and amplitude of newborn rats (P0–P2, n = 5) and adult rats (n = 3, data not shown), however, were not significantly altered by the same dose of CX546 on its own. CX546 was also without any obvious effect on the behavior or arousal state (i.e., increase/decrease in spontaneous movement, agitation, indication of sedation)
Figure 5. CX546 counters opioid- and phenobarbital-induced respiratory depression in vivo. Plethysmographic recordings show that the inhibitory effect of fentanyl (A) and phenobarbital (B) on the breathing frequency and amplitude in unanesthetized P0 pups is partially countered by CX546. (C) Plethysmographic recording from an adult rat showing that, as seen in P0 pups, administration of fentanyl inhibits breathing frequency and amplitude and that this is partially reversed by CX546. (D and E) Population data for P0 rat pups, showing changes in frequency and amplitude relative to control evoked by fentanyl or phenobarbital both before and after the administration of CX546 (n = 4). (F) Population data for adult rats, showing relative changes in frequency and amplitude evoked by fentanyl alone and after the administration of CX546 (n = 4). * Significant difference relative to control; # significant difference between values in the presence of the respiratory depressant alone and after subsequent application of CX546. All drugs were delivered intraperitoneally.
Nociceptive Testing
The preceding experiments demonstrated that CX546 counteracts the respiratory depression induced by DAGO or fentanyl. We then investigated whether the intraperitoneal administration of CX546 affects fentanyl-induced analgesia in vivo. Using a thermal nociception test, the latency of hind paw withdrawal was 4.9 ± 3.8 and 5.6 ± 1.7 s for newborns and adult rats, respectively (see Table 1). Administration of fentanyl at doses that suppressed respiratory rhythm (60 µg/kg for newborn rats and 130 µg/kg for adult rats) extended the latency to paw withdrawal to the 20-s cutoff limit. This fentanyl-induced analgesia, which manifests itself as the loss of foot withdrawal, persisted despite the subsequent intraperitoneal administration of CX546 (16 mg/kg). The fentanyl-induced analgesic effect was, however, blocked by the subsequent administration of naloxone (1 mg/kg) in both newborn and adult rats. When CX546 (16 mg/kg) was administrated on its own, it did not change the sensitivity of thermal nociception tests in the control condition. These results demonstrate that CX546 reduces the deleterious respiratory-depressant effects of opioids without inhibiting their desirable, analgesic actions.
TABLE 1. DRUG EFFECTS ON THERMAL NOCICEPTION DETERMINED IN HIND PAW WITHDRAWAL TESTS
DISCUSSION
Respiratory depression has serious clinical implications and manifests itself in a number of situations including pharmacologically induced and endogenous bradypnea and apneas (central and obstructive). Advancements in drug treatments based on an understanding of the neurochemical control of breathing are needed. Here, we demonstrate that an ampakine, which potentiates AMPA receptor–mediated conductances (6) critical for inspiratory rhythmogenesis and synaptic drive to respiratory motor nuclei, is an effective means of countering respiratory depression.
Ampakine-mediated Modulation of Respiratory Activity
Sites of action.
Administration of the ampakine CX546 provided a powerful and robust means of alleviating depressed central respiratory drive; respiratory activity was enhanced when the network was examined in its most reduced (medullary slice) and most intact (in vivo) states. Potentiation of output in medullary slices and brainstem–spinal cord preparations indicates that CX546 acts on medullary respiratory networks. Direct action within the pre-BötC rhythm-generating networks is likely because frequency is enhanced. However, it is also possible that the increased frequency reflects secondary actions through a population of neurons that provides tonic excitatory glutamatergic drive to the rhythm generator. Amplitude effects in vitro and in vivo also suggest that premotor and motoneuron pools are sites of ampakine action. Certainly, inhibition of desensitization, which is one effect of ampakines, within the XII motor nucleus potentiates inspiratory burst amplitude (4).
The observation that the potentiating actions of ampakines are also present in situ and in vivo, and therefore not limited to in vitro preparations, indicates that potentiation is not dependent on some unique condition established in vitro. Ampakines counteract respiratory depression in the presence of excitatory drive to the central respiratory rhythm generation from peripheral chemoreceptors, mechanoreceptors, and supramedullary centers. In fact, it is possible that in more intact preparations, ampakines act not only within the pre-BötC and motor pools, but also enhance activity in excitatory afferent feedback pathways.
Development.
Ampakine-mediated enhancement of respiratory activity was observed in animals ranging in age from E18 to adult. In animals P0 or younger, ampakines enhanced baseline activity. In older preparations, ampakines enhanced activity only if respiration was initially depressed. Whether this reflects a developmental change in sensitivity to ampakines, or whether the potentiating actions of ampakines become manifest only if respiratory activity is first suppressed, is unclear. Paradoxically, if ampakines potentiate respiratory output by blocking desensitization, one would predict greater efficacy in older animals with conditions of increased network activity due to increased glutamate receptor activation and the greater potential for desensitization. Further, the inhibition of desensitization kinetics by CX546 should become more significant with maturation. AMPA receptor subunits, GluR1–4, exist as either flip or flop splice variants. In embryonic animals, the flip variants predominate (16). With maturation there is a shift in expression such that the flop isoforms, which have more rapid desensitization kinetics, become increasingly dominant. Thus, these data suggest that the most significant action of ampakines in relation to controlling activity of respiratory networks may not be modulation of glutamate receptor desensitization properties. Rather, enhancement of binding affinity and prolongation of channel open time may be the important parameters.
Defining the molecular mechanism(s) underlying the ampakine-mediated enhancement of depressed respiratory output will require analysis at the single-cell and most likely single-channel levels, and is beyond the scope of this study. Despite an incomplete mechanistic understanding, potentiation of depressed but not baseline activity is a desirable feature of ampakine action from a clinical perspective. Enhancing baseline ventilation or increasing depressed respiratory output to levels greater than baseline (i.e., hyperventilation) would have independent deleterious consequences including the disruption of blood gases, and predisposition to respiratory instability.
Specificity of action on respiratory networks.
The finding that CX546 stimulated breathing without widespread activation of neuronal circuits may not be expected given the ubiquitous distribution of AMPA receptors within the central nervous system. However, behavioral selectivity of ampakines has been reported in other studies and might be explained by regional and network selectivity of ampakines (17). AMPA subunits (GluR1–4 subunits, each with flip and flop splice variants and RNA-editing R/G and Q/R site; see Reference 18) are differentially expressed throughout the brain. Ampakines have subunit-specific effects and could act with regional selectivity. For example, ampakines have a severalfold greater effect on excitatory postsynaptic currents in hippocampus (high concentrations of GluR1 and GluR2) than in thalamus (GluR3 and GluR4). Even within the hippocampus, ampakines have different effects across subgroups of cells. It is therefore possible that ampakines have particularly strong effects on the neurons regulating respiration.
Additional ampakine actions.
In addition to their direct effects on glutamatergic signaling, ampakines, including CX546, appear to chronically elevate the production of brain-derived neurotrophic factor (BDNF) in hippocampal and cortical neurons via the positive modulation of AMPA receptors (14). However, the rapid kinetics of ampakine CX546 treatment on respiratory drive is inconsistent with an upregulation of BDNF synthesis. Moreover, BDNF decreases, rather than increases, the frequency of respiratory rhythm in medullary slice preparations (19). A further mechanism of CX546 action is the enhancement of astrocyte metabolism, which increases glucose utilization and lactate production (20). The possibility of such astrocyte-mediated actions influencing respiratory drive, in conjunction with direct modulation of neuronal AMPA receptor conductances, should be considered given the proposed metabolic coupling between glia and medullary respiratory neurons (21). However, the acute actions of CX546 on respiratory frequency are unlikely to reflect solely glia-derived increases in extracellular lactic acid because bath application of lactic acid (5 mM) to brainstem–spinal cord or medullary slice preparations did not mimic the actions of CX546 (J. Ren and J. J. Greer, unpublished observation).
Implications for Analysis of Respiratory Rhythm Generation
The first series of experiments was performed with in vitro brainstem–spinal cord and medullary slice preparations. These in vitro preparations isolated from embryonic or P0 rats typically generate a respiratory rhythm of low frequency compared with older neonatal animals (reviewed in Reference 22). This is in part due to lack of sufficient excitatory drive from glutamatergic and neuromodulatory systems. Data from this study demonstrate that CX546, presumably via the modulation/potentiation of the AMPA receptor conductance, markedly enhances the frequency of respiratory rhythm in vitro in E18–P0 rats, and restores depressed rhythm in older neonates in both brainstem–spinal cord and medullary slice preparations.
It is significant that the medullary slice preparation produces robust respiratory-related rhythm when perfused with bathing medium containing 3 mM [K+]o and CX546. The rhythmic slice preparation has become a standard model in respiratory control studies and its use has significantly advanced understanding of the sites and mechanisms of mammalian rhythm generation (9). A limitation of these slice preparations, however, has been the necessity of bathing the tissue in elevated [K+]o (typically 9 mM) for generation of long-lasting rhythmic activity (although see Reference 23). The perceived need for elevated [K+]o underlies the argument of some investigators that mechanisms of rhythm generation identified in vitro are not relevant to breathing in vivo. The opposing view, presented with the original description of the slice (3, 9), is that elevated [K+]o is required in the slice to compensate for the loss of tonic and phasic excitatory glutamatergic synaptic drive to rhythm-generating networks that is present in more intact in vitro preparations, such as the brainstem–spinal cord preparation, and under in vivo conditions. Accumulating evidence emphasizes the potential importance of [K+]o in determining the effects of neuromodulatory cascades on rhythm, but also suggests that many of the basic mechanisms of rhythm generation described in vitro apply in vivo. For example, the importance of the pre-BötC in rhythm generation, and the pacemaker behavior of pre-BötC neurons, and so on, are not dependent on elevated [K+]o (1, 24). The demonstration in this study that ampakines evoke a robust, long-lasting respiratory rhythm at physiological [K+]o adds further support to the thesis. It also provides an alternative to the use of elevated [K+]o or addition of neurotransmitter receptor agonists for producing long-lasting rhythmic respiratory activity in medullary slices.
Functional/Clinical Significance
The in vivo data from newborn and adult rats demonstrate that the actions of ampakines observed in vitro and in situ occur in the intact animal. Ampakines act as a powerful stimulant of respiratory frequency and tidal volume in perinates and rats of all ages in which respiration was depressed by the µ-opioid receptor agonist fentanyl or the barbiturate phenobarbital. Opiates are widely used analgesics and phenobarbital is the longest acting of the barbiturates, which are used primarily for sedation or as an anticonvulsant. However, both have serious adverse effects, perhaps the most significant being respiratory depression. Richter's group has demonstrated that a serotonin (5-hydroxytryptamine) receptor subtype 4a (5-HT4a) agonist counters opioid-induced breathing depression without loss of analgesia in rats (25, 26). The mechanisms underlying 5-HT4a receptor functions as an antidote to fentanyl have not been clearly delineated although they may interact via convergent cAMP intracellular signaling. This is supported by the finding that nociception-induced respiratory depression in vitro is reversed by activation of adenylyl cyclase or blockage of phosphodiesterase activity (27). It is important to note that administration of CX546 countered drug-induced respiratory depression without significantly altering the baseline frequency or amplitude of breathing in control animals, inhibiting the opioid-induced analgesia, or noticeably affecting the behavior of the animals. Besides the effectiveness of ampakines to counter drug-induced respiratory depression, it will be of interest to determine whether ampakines are effective in treating central and obstructive apneas in newborns and adults, areas in which advancements in treatment are certainly needed (28, 29). Genetic mouse models with clear central hypoventilation during the postnatal period (reviewed in Reference 30) should provide experimental opportunities to explore this area. Future studies will be necessary to determine the relative efficacy of various ampakines and to perform a more thorough examination for potentially harmful side effects. Further, this study was limited to an examination of the acute actions of ampakines over minutes. A drug delivery system that provides ampakine-mediated actions over longer periods will likely be necessary for uses beyond countering drug-induced respiratory depression.
Conclusions
Data from the current study indicate that ampakines may provide a novel pharmacologic means of countering respiratory depression. Importantly, ampakines readily cross the blood–brain barrier and appear to be well tolerated (17, 31, 32). Further work evaluating the efficacy of the various members of currently available and developing members of the ampakine family as respiratory stimulants toward applicability in clinical settings is warranted. <<<
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