GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep

Abstract

The pharyngeal muscles, such as the genioglossus (GG) muscle of the tongue, are important for effective lung ventilation since they maintain an open airspace. Rapid-eye-movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity, even during strong reflex respiratory stimulation by elevated CO2. In vitro studies have demonstrated the presence of GABAA receptors on hypoglossal motoneurons, and these and other data have led to the speculation that GABAA mechanisms may contribute to the suppression of hypoglossal motor outflow to the GG muscle in REM sleep. We have developed an animal model that allows us to chronically manipulate neurotransmission at the hypoglossal motor nucleus using microdialysis across natural sleep-wake states in rats. The present study tests the hypothesis that microdialysis perfusion of the GABAA receptor antagonist bicuculline into the hypoglossal motor nucleus will prevent the suppression of GG muscle activity in REM sleep during both room-air and CO2-stimulated breathing. Ten rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) or 100 microM bicuculline during room-air and CO2-stimulated breathing (7 % inspired CO2). GABAA receptor antagonism at the hypoglossal motor nucleus increased respiratory-related GG activity during both room-air (P = 0.01) and CO2-stimulated breathing (P = 0.007), indicating a background inhibitory GABA tone. However, the effects of bicuculline on GG activity depended on the prevailing sleep-wake state (P < 0.005), with bicuculline increasing GG activity in non-REM (NREM) sleep and wakefulness both in room air and hypercapnia (P < 0.01), but GG activity was effectively abolished in those REM periods without phasic twitches in the GG muscle. This abolition of GG activity in REM sleep occurred regardless of ACSF or bicuculline at the hypoglossal motor nucleus, or room-air or CO2-stimulated breathing (P > 0.63). We conclude that these data in freely behaving rats confirm previous in vitro studies that GABAA receptor mechanisms are present at the hypoglossal motor nucleus and are tonically active, but the data also show that GABAA receptor antagonism at the hypoglossal motor nucleus does not increase GG muscle activity in natural REM sleep.

Figure 4. Group data showing GG responses to bicuculline at the hypoglossal motor nucleus

Group data showing changes in respiratory-related GG activity across all sleep-wake states during microdialysis perfusion of ACSF and bicuculline into the hypoglossal motor nucleus during both room-air and CO₂-stimulated breathing. Note that GG activity was effectively abolished in periods of REM_{TONIC GG} across all conditions. All values are means ±s.e.m.

Figure 2. Distribution of fluorescein dye dialysed into the hypoglossal motor nucleus

The top two photographs show examples in two rats of the spread of fluorescein dialysed for 1 h and 6 h into the hypoglossal motor nucleus. Neutral Red-stained sections containing the lesion sites left by the microdialysis probe are shown. The arrow indicates the sites of microdialysis. The distribution of fluorescence around the probe site is also shown from the adjacent histological section. The distribution of fluorescence in the hypoglossal motor nuclei for each individual rat is shown in the lower panels. Each rat is shown in a different colour and the bars indicate the sites of microdialysis. Note that the spread of fluorescence is largely within the medullary regions containing the hypoglossal motor nuclei. The traces on the left show the anatomical structures for orientation. Abbreviations as for Fig. 1.

Figure 1. Location of the microdialysis probes

Distribution of individual microdialysis sites from all rats. Probes were located in the hypoglossal motor nucleus in seven rats and in the midline immediately adjacent to both nuclei in three rats. The size of the bar represents the apparent size of the lesion from the histological sections. Abbreviations: Cer, cerebellum; 4V, fourth ventricle; Sol, nucleus of the tractus solitarius; XII, hypoglossal motor nucleus; ROb, raphe obscurus; Py, pyramidal tract; AP, area postrema.

Figure 3. Genioglossus (GG) responses to bicuculline at the hypoglossal motor nucleus in natural sleep

Sleep patterns and respiratory muscle activities with microdialysis perfusion of artificial cerebrospinal fluid (ACSF) and bicuculline into the hypoglossal motor nucleus during room-air and CO₂-stimulated breathing. In non-rapid-eye-movement (NREM) sleep bicuculline produced clear increases in GG activity compared to ACSF both in room air (C vs. A) and with CO₂ (D vs. B). However, major suppression of GG activity occurred in periods of rapid eye movement (REM) sleep without phasic GG twitches (i.e. REM_{TONIC GG}) across all conditions. The GG and diaphragm (DIA) signals are displayed as their moving-time averages (MTA) in arbitrary (Arb.) units. The integrator baseline (i.e. electrical zero) is shown for the GG MTA. The arrow on the DIA and GG calibration bars denotes the direction of inspiration. REM_{PHASIC GG}, REM associated with phasic GG muscle twitches; NREM_PRE-REM, periods of NREM that were analysed occurring immediately prior to REM; EEG, electroencephalogram; EMG, electromyogram.

Figure 5. The percentage of REM sleep associated with periods of tonic and phasic GG activity

Group data showing the changes in tonic and phasic GG activities in REM sleep with bicuculline (BIC) and ACSF at the hypoglossal motor nucleus compared to neck muscle activity during room-air and CO₂-stimulated breathing. The percentage of REM sleep accompanied by phasic bursts of GG activity was decreased by bicuculline compared to ACSF. No other changes were statistically significant. See text for further details. Abbreviations are as for Fig. 3.

Figure 6. GG activity analysed across the entire REM period

Group data showing GG activity measured throughout the entire REM episodes. Although there was a tendency for GG activity to increase in overall REM sleep in the presence of combined CO₂ stimulation and bicuculline (BIC) at the hypoglossal motor nucleus there was no statistically significant effect of bicuculline on GG activity in total REM sleep.

Figure 7. GG activity upon transition from NREM to REM sleep with bicuculline at the hypoglossal motor nucleus

Example showing the abolition of GG activity at the onset of REM sleep despite the presence of bicuculline at the hypoglossal motor nucleus. Abbreviations are as for Fig. 3.

Figure 8. Group data showing changes in GG activity from NREM to REM sleep

Group data showing changes in respiratory-related GG activity from NREM to REM sleep during microdialysis perfusion of ACSF and bicuculline into the hypoglossal motor nucleus during both room-air and CO₂-stimulated breathing. GG activity in REM sleep was similar across all conditions. See text for further details.

Figure 9. Group data showing specificity of responses to bicuculline at the hypoglossal motor nucleus

Responses of respiratory rate (A), phasic diaphragm (DIA) activity (B), the ratio of high to low frequencies in the EEG (C) and neck EMG (D) are shown across all sleep-wake states for microdialysis perfusion of ACSF and bicuculline into the hypoglossal motor nucleus during both room-air and CO₂-stimulated breathing. There are no effects of bicuculline on any parameter in hypercapnia but bicuculline decreased respiratory rate and increased diaphragm activity in room air. See text for further details.

Figure 10. GG activation by 5-hydroxytryptamine (5-HT) at the hypoglossal motor nucleus

Example showing an increase in GG activity with microdialysis perfusion of serotonin (5-HT) into the hypoglossal motor nucleus. These traces from one rat were both obtained in NREM sleep. Abbreviations are as for Fig. 3.