Endogenous Glutamatergic Control of Rhythmically Active Mammalian Respiratory Motoneurons In Vivo

Instrumentation for studies in freely behaving rats.

Sterile surgery was performed with the rats anesthetized with intraperitoneal ketamine (85 mg/kg) and xylazine (15 mg/kg). In addition, the rats were intraperitoneally injected with saline (3 ml, 0.9%) for fluid loading, atropine sulfate (1 mg/kg) to minimize airway secretions, and buprenorphine (0.03 mg/kg) to reduce potential postoperative pain. An anesthesia mask was placed over the snout, and the rats spontaneously breathed a 50:50 mixture of room air and oxygen. Any additional anesthesia during surgery was administered, as necessary, by inhalation (isoflurane, typically 0.2–2%). Effective anesthesia was judged by abolition of the pedal withdrawal and corneal blink reflexes. The rats were then implanted with electroencephalogram (EEG) and neck electromyogram (EMG) electrodes for determination of sleep–wake states and with genioglossus and diaphragm electrodes for respiratory muscle recordings (Morrison et al., 2003; Sood et al., 2005; Chan et al., 2006). Tests for the accurate placement of the genioglossus electrodes and their function throughout the experiments included EMG recordings during surgery and observing tongue movements in response to electrical stimulation of the genioglossal wires at both the beginning and end of the experiments (Morrison et al., 2003; Sood et al., 2005; Chan et al., 2006). Removal of the tongue, by dissection, at the completion of all experiments confirmed the physical location of the electrodes to the genioglossus muscle. The size, configuration, and placement of the genioglossus and diaphragm electrodes were consistent across experiments, and each animal served as its own control.

Using a stereotaxic frame (model 962; David Kopf Instruments), microdialysis guides (CMA/11; Chromatography Sciences Company) were targeted 3 mm above the HMN during surgery (Morrison et al., 2003; Sood et al., 2005; Chan et al., 2006). The guides were placed 13.89 ± 0.11 mm posterior to bregma, 0.12 ± 0.012 mm lateral to the midline, and 6.99 ± 0.071 mm ventral to bregma. A dummy cannula was placed inside the microdialysis guide to keep it patent until the day of the experiment. Electrodes were connected to pins and inserted into a miniature plug (model STC-89PI-220ABS; Carleton University). The plug and microdialysis guides were affixed to the skull with dental acrylic and anchor screws.

On completion of surgery, the rats were transferred to a clean cage and kept warm under a heating lamp until full recovery as judged by normal locomotor activity, grooming, drinking, and eating. The rats were given soft food for the first day after surgery. The rats recovered for an average of 8.4 d (range, 6–14 d) before the experiments were performed.

Follow-up studies under anesthesia.

After completion of the protocol in the freely behaving rats (see below), the same animals were then acutely anesthetized with intraperitoneal injection of urethane (1 g/kg). The microdialysis probe was left in the hypoglossal motor nucleus, but the inlet and outlet tubing were connected together to prevent loss of the internal solution. After the onset of surgical anesthesia, the rats were tracheotomized and spontaneously breathed a 50:50 mixture of room air and oxygen; the rats were also given atropine (1 mg/kg) to minimize airway secretions. Subsequent anesthesia was administered as necessary by inhalation (halothane, typically 0.2–0.9%). When halothane was initiated within an animal, typically no, or only minor, adjustments were necessary across the experiment. We routinely use this anesthetic regimen because it provides highly reliable preparations with stable respiratory and EEG activities over the course of the experiments (Liu et al., 2005; Steenland et al., 2006) that is not possible, for example, with periodic bolus doses of supplemental urethane, which can transiently suppress genioglossus activity. The femoral artery and vein were cannulated for blood pressure measurement and intravenous infusions, respectively. The rats received continuous intravenous fluid (0.4 ml/h) containing 7.6 ml of saline, 2 ml of 5% dextrose, and 0.4 ml of 1 m NaHCO₃. Core body temperature was monitored with a rectal probe and maintained between 36 and 38°C with a water pump and heating pad (T/Pump-Heat Therapy System; Gaymar).

Freely behaving rats.

For recordings, a lightweight shielded cable was connected to the plug on the rat's head. The cable was attached to a counterbalanced swivel that permitted free movement. All rats were studied in a noise-attenuated, electrically shielded cubicle (EPC-010; BRS/LVE) and supplied with fresh bedding, food, and water. A video camera inside the cubicle allowed for continuous monitoring without disturbing the animal. For habituation, the rats were connected to the cable and swivel apparatus the day before the experiments.

Experiments started between 7:00 A.M. and 8:00 A.M. and were performed during the day, i.e., the normal sleep period of rats. At the start of the experiment, the internal cannula was removed from the guide, and the microdialysis probe (240 μm diameter, 1 mm membrane; CMA/11 14/01; Chromatography Sciences Company) was inserted. The probes projected 3 mm from the tip of the guide and were therefore targeted to the HMN. The ease of insertion of the probe ensured that the rat was minimally disturbed by the procedure. The probes were perfused with artificial CSF (ACSF) at a flow rate of 2.1 μl/min, with the ACSF being made fresh on the day of each experiment. The composition of the ACSF was as follows (in mm): 125 NaCl, 3 KCl, 1 KH₂PO₄, 2 CaCl₂, 1 MgSO₄, 25 NaHCO₃, and 30 glucose. The ACSF was warmed to 37°C and bubbled with CO₂ to a pH of 7.40 ± 0.003 (SEM). The CaCl₂ was added after the ACSF was warmed to 37°C.

After insertion of the microdialysis probe, at least 45 min were allowed to elapse before sleep–wake states and respiratory muscle activities were analyzed (Morrison et al., 2003; Sood et al., 2005; Chan et al., 2006). At least two full cycles containing periods of active and quiet wakefulness, and non-rapid-eye-movement (NREM) and REM sleep, were recorded in each animal for each applied drug (see below). At the end of the experiment, the microdialysis probes were washed out with ACSF for at least 1 h.

Follow-up studies under anesthesia.

After completion of the protocol in the freely behaving rats, the same animals were then prepared for acute, i.e., nonrecovery, studies under anesthesia (see above). To ensure consistent positioning between rats, they were placed in a stereotaxic apparatus (model 962; David Kopf Instruments), and the head was secured with blunt ear bars. The same EEG, genioglossus, and diaphragm EMG signals were recorded by reconnecting the amplifiers to the appropriate electrical contacts on the head plug. The microdialysis tubing was also reconnected to the perfusion pump, and ACSF was perfused through the microdialysis probe for at least 1 h before any interventions were performed. After perfusion with ACSF, the same drugs that were applied in the same rat during wakefulness and sleep were then reapplied under anesthesia. The time for drugs to be applied under anesthesia was at least 3 h after the last applied drugs in the conscious experiments. The studies under anesthesia were performed as positive controls to confirm our previous observations in the same anesthetized preparation (Steenland et al., 2006).

Study 1: NMDA receptor antagonism at the HMN.

Experiments were performed in seven rats to determine whether NMDA receptors at the HMN modulate genioglossus activity (1) across natural sleep–wake states and (2) during anesthesia. In the same rats studied under both conditions, genioglossus activity was recorded during control perfusion of ACSF into the HMN, followed by d-2-amino-5-phosphonovaleric acid (d-APV) dissolved in ACSF (1 mm, pH 7.21 ± 0.034; Sigma). d-APV is a competitive NMDA receptor antagonist (Olverman et al., 1984; Rekling, 1992; O'Brien et al., 1997; Wang et al., 2002). In a previous study, we showed that this dose of d-APV delivered by microdialysis perfusion into the HMN produced appropriate antagonism of the NMDA receptor in response to application of exogenous agonist but did not affect responses to non-NMDA receptor stimulation (Steenland et al., 2006). The protocol for delivery of drugs in the freely behaving rats was described above. For the experiments under anesthesia, d-APV was applied for 15 min and washed out for at least 30 min. At the end of the anesthesia experiments, serotonin (10 mm, pH 6.36 ± 0.026; Sigma) dissolved in ACSF was perfused as a positive control to activate the genioglossus and confirm that the HMN was intact and able to respond to manipulation of neurotransmission (Jelev et al., 2001; Sood et al., 2005).

Study 2: non-NMDA receptor antagonism at the HMN.

Experiments were performed in seven separate rats to determine whether non-NMDA receptors at the HMN modulate genioglossus activity (1) across natural sleep–wake states and (2) during anesthesia. In the same rats studied under both conditions, genioglossus activity was recorded during control perfusion of ACSF into the HMN, followed by 6-cyano-7-nitroquinoxaline-2, 3-dione disodium salt (CNQX) dissolved in ACSF (0.1 mm, pH 7.39 ± 0.006; Sigma). CNQX is a competitive non-NMDA receptor antagonist (Honore et al., 1988; Funk et al., 1993; Wang et al., 2002). In a previous study, we showed that microdialysis perfusion of this dose of CNQX into the HMN produced appropriate antagonism of the non-NMDA receptor in response to application of exogenous agonist but did not affect responses to NMDA receptor stimulation (Steenland et al., 2006). The protocol for delivery of drugs in the freely behaving rats was described above. For the anesthetized experiments, CNQX was applied for 15 min and washed out for at least 30 min. At the end of all the experiments, serotonin (10 mm, pH 6.35 ± 0.041) was also applied as a positive control to activate genioglossus muscle (Jelev et al., 2001; Sood et al., 2005). In a separate group of six rats, additional experiments were also performed across sleep–wake states during microdialysis perfusion of ACSF and 1 and 5 mm CNQX into the HMN using the identical protocol for the 0.1 mm CNQX and the 1 mm d-APV experiments. In anesthetized rats, 1 mm CNQX is sufficient to fully abolish genioglossus activity, even in vagotomized animals with high respiratory-related genioglossus activity (Steenland et al., 2006).

Study 3: endogenous stimulation of glutamate receptors at the HMN.

Experiments were performed in seven rats to determine the effect on genioglossus activity of elevating endogenous glutamate across sleep-wake states. Genioglossus activity was recorded during perfusion of ACSF into the HMN, followed by the glutamate uptake inhibitor dihydrokainate (DHK) dissolved in ACSF (2 mm, pH 7.32 ± 0.060; Sigma) (Funk et al., 1993; Fallgren and Paulsen, 1996). At the end of the experiments in three rats, DHK was also coadministered with 0.1 mm of the non-NMDA receptor antagonist CNQX. At the end of the experiments in another three rats, DHK was coadministered with 1 mm of the NMDA receptor antagonist d-APV. At the conclusion of each study, drugs were washed out for at least 1 h, and then serotonin (10 mm, pH 6.36 ± 0.026) was also applied as a positive control as in studies 1 and 2 to activate genioglossus muscle.

Analysis of EMG and EEG signals.

The EMGs were analyzed every 5 s from the respective moving-time average signals (above electrical zero). The genioglossus and diaphragm signals were analyzed breath-by-breath, which corresponded to ∼7–10 breaths for each 5 s epoch. For each breath in each 5 s epoch, the analysis of the genioglossus EMG was time locked to breathing as defined by the peak and trough of the diaphragm signal. Visual inspection of the computer-identified peaks and troughs of the diaphragm and genioglossus signals was performed during the automated analysis, and any artifacts were discarded and not used in the analysis. Peak inspiratory genioglossus activity was defined as the peak moving-time average of the genioglossus EMG in the 100 ms spanning the end of diaphragm inspiration, and tonic activity was defined as the minimal activity in expiration. For each 5 s epoch, genioglossus activity was then quantified as mean tonic activity (i.e., the mean minimal activity in expiration from the number of breaths in that epoch) and the mean amplitude of respiratory-related activity (i.e., mean peak inspiratory activity − tonic activity). Tonic genioglossus activity will include both basal tone and any background spontaneous activity that may occur during certain behaviors, e.g., twitches in REM sleep or behaviors in active wakefulness. Likewise, although clear phasic variations in genioglossus activity can occasionally occur in behaviors such as REM sleep or active wakefulness, whether the activity that occurs in synchrony with the diaphragm is truly “respiratory” or random activation attributable to behavioral or REM sleep processes cannot be determined with this or other methods, but such activations do not affect measurements in quiet wakefulness or NREM sleep. Mean neck muscle activity, diaphragm amplitude, and respiratory rate were also calculated in consecutive 5 s bins for all the periods of sleep and wakefulness in each rat. The EEG was sampled by computer at 500 Hz and then analyzed on overlapping segments of 1024 samples, windowed using a raised cosine (Hamming) function, and subjected to a fast Fourier transform to yield the power spectrum. The window was advanced in steps of 512 samples, and the mean power spectrum of the EEG signal over each 5 s epoch was calculated. The power contained within six frequency bands was recorded as absolute power and also as a percentage of the total power of the signal. The band limits were δ₂ (0.5–2 Hz), δ₁ (2–4 Hz), θ (4–7.5 Hz), α (7.5–13.5 Hz), β₁ (13.5–20 Hz), and β₂ (20–30 Hz); the ratio of high (20–30 Hz) to low (0.5–2 Hz) frequency activity was also calculated as an index of relative EEG activation (Hamrahi et al., 2001).

Measurements.

The moving-time averages of the genioglossus, neck, and diaphragm EMGs were averaged for each 5 s period across sleep–wake states in each rat and subsequently sorted into periods of active and quiet wakefulness, NREM sleep, and REM sleep as identified by standard EEG and EMG criteria (Morrison et al., 2003; Sood et al., 2005; Chan et al., 2006). The EEG and neck EMG signals were scored visually for identification of sleep–wake states but without reference to the genioglossus or diaphragm signals to minimize bias when selecting periods for analysis. Active wakefulness was identified as low-voltage EEG activity with high levels of neck muscle activity associated with behaviors such as grooming, eating, and drinking. Quiet wakefulness was associated with an absence of such overt behaviors. For the studies in the freely behaving rats, data were analyzed at least 30 min after a switch between drugs to allow for the lag time of the tubing connected to the microdialysis probe. For the anesthetized rats, measurements were taken over 1 min periods at the end of each period of drug perfusion as described previously (Steenland et al., 2006).