Deletion of guanine nucleotide binding protein alpha z subunit in mice induces a gene dose dependent tolerance to morphine.

The mechanism underlying the development of tolerance to morphine is still incompletely understood. Morphine binds to opioid receptors, which in turn activates downstream second messenger cascades through heterotrimeric guanine nucleotide binding proteins (G proteins). In this paper, we show that G(z), a member of the inhibitory G protein family, plays an important role in mediating the analgesic and lethality effects of morphine after tolerance development. We blocked signaling through the G(z) second messenger cascade by genetic ablation of the alpha subunit of the G protein in mice. The Galpha(z) knockout mouse develops significantly increased tolerance to morphine, which depends on Galpha(z) gene dosage. Further experiments demonstrate that the enhanced morphine tolerance is not caused by pharmacokinetic and behavioural learning mechanisms. The results suggest that G(z) signaling pathways are involved in transducing the analgesic and lethality effects of morphine following chronic morphine treatment.

Hypertolerance to morphine in G(z alpha)-deficient mice.

Our laboratory has generated a mouse deficient in the alpha (alpha) subunit of the G protein, G(z), (G(z alpha)) gene and we have examined the involvement of G(z alpha) in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by the response times to heat or cold tail flick times in a water bath at 50 degrees C or -5 degrees C and supraspinal analgesia was tested by the times for paw licking and jumping from a plate at 52 degrees C or 0.5 degrees C. Tolerance to morphine was induced in wild type and G(z alpha)-deficient mice over a 5 day period and the behavioral tests were performed daily. The tail flick reaction times to both hot and cold stimuli did not differ between the wild type and G(z alpha)-deficient mice. Analysis of the reaction times from the hot and cold plate tests showed the G(z alpha)-deficient mice developed tolerance to morphine to a greater degree and at a faster rate than wild type mice. Opioid binding assays were performed on synaptic membranes prepared from naive and morphine tolerant wild type and G(z alpha)-deficient brains. No changes in the affinity of morphine for its receptor or in the density of mu and delta opioid receptors were found between the two groups of mice in the naive or morphine tolerant state. This indicates that the absence of G(z alpha) does not affect opioid receptor affinity or receptor up or down regulation. Our results suggest that the presence of G(z alpha) delays the development of morphine tolerance and represents a possible therapeutic target for improving the clinical use of morphine.

Effect of changing body temperature on the ventilatory and metabolic responses of lean and obese Zucker rats.

We measured body temperature (Tb) and ventilatory and metabolic variables in lean (n = 8) and obese (n = 8) Zucker rats. Measurements were made while rats breathed air, 4% CO2, and 10% O2. Under control conditions, Tb in obese rats was always less than that of their lean counterparts. Obese rats adopted a more rapid, shallow breathing pattern than lean rats in air and had a lower ventilation rate in 4% CO2. Respiration in 10% O2 was similar for the two groups. Metabolic variables did not differ between lean and obese rats whatever the gas breathed. When lean rats were cooled to match Tb in control obese rats with an implanted abdominal heat exchanger, they increased ventilation and metabolism in air; there was no effect of cooling on responses to 4% CO2; and ventilation increased while metabolism decreased in 10% O2. When obese rats were warmed to match Tb in control lean rats, trends in ventilation and metabolism resulted in a tendency toward hyperventilation in air and 4% CO2, but not in 10% O2. Taken overall, matching Tb in lean and obese rats accentuated differences in respiratory and metabolic variables between the two groups. We conclude that differences in respiration between lean and obese Zucker rats are not due to the difference in Tb.

Mechanism controlling sleep organization of the obese Zucker rats.

We tested the hypothesis that the obese (fa/fa) Zucker rat has a sleep organization that differs from that of lean Zucker rats. We used the polygraphic technique to identify and to quantify the distribution of the three main states of the rat: wakefulness (W), non-rapid-eye-movement (NREM), and rapid-eye movement (REM) sleep states. Assessment of states was made with light present (1000-1600), at the rats thermoneutral temperature of 29 degrees C. Obese rats, compared with lean ones, did not show significant differences in the total time spent in the three main states. Whereas the mean durations of W and REM states did not differ statistically, that of NREM did (P = 0.046). However, in the obese rats, the frequencies of switching from NREM sleep to W, which increased, and from NREM to REM sleep, which decreased, were statistically significantly different (P = 0.019). Frequency of switching from either REM or W state was not significantly different. We conclude that sleep organization differs between lean and obese Zucker rats and that it is due to a disparity in switching from NREM sleep to either W or REM sleep and the mean duration of NREM sleep.

Respiratory muscle reserve in rats during heavy exercise.

The extent to which the respiratory pump muscles limit maximal aerobic capacity in quadrupeds is not entirely clear. To examine the effect of reduced respiratory muscle reserve on aerobic capacity, whole body peak oxygen consumption (VO2 peak) was measured in healthy Sprague-Dawley rats before and after Sham, unilateral, or bilateral hemidiaphragm denervation (Dnv) surgery. VO2 peak was determined by using a graded treadmill running test. Hemidiaphragm paralysis was verified after testing by recording the absence of electromyographic activity during inspiration. Before surgery, VO2 peak averaged 86, 87, and 92 ml . kg-1 . min-1 for the Sham, unilateral, and bilateral Dnv groups, respectively. Two weeks after surgery, there was no significant change in VO2 peak for either the Sham or unilateral Dnv group. However, VO2 peak decreased approximately 19% in the bilateral Dnv group 2 wk after surgery. These findings strongly suggest that the pulmonary system in rats is designed such that during heavy exercise, the remaining respiratory pump muscles are able to compensate for the loss of one hemidiaphragm, but not of both.

Influence of carotid denervation on the body-core temperature and metabolic responses to changes in ambient temperature during normoxemia and acute hypoxemia in guinea pigs during postnatal maturation.

Acute hypoxemia produces a decrease in body-core temperature (Tbc) in guinea pigs during postnatal maturation, although the factors mediating the response remain unknown. Experiments were therefore carried out to test the hypothesis that the carotid chemoreceptors and (or) baroreceptors mediate the decrease in Tbc during acute hypoxemia. Twelve guinea pigs, six carotid intact and six carotid denervated, were studied in a metabolic chamber to determine the influence of carotid denervation on the Tbc and metabolic (i.e., oxygen consumption) responses to changes in ambient temperature during normoxemia and during acute hypoxemia at 2, 3, and 4 weeks of age. Carotid denervation accentuated the decrease in Tbc in response to a decrease in ambient temperature during normoxemia at 2 and 3 weeks of age but not at 4 weeks of age without altering the metabolic response. This suggests that carotid denervation disrupted heat conservation mechanisms rather than heat production mechanisms in an age-specific manner. Furthermore, carotid denervation accentuated the decrease in Tbc and oxygen consumption in response to acute hypoxemia at all ages studied. This provides evidence that the carotid chemoreceptors and (or) baroreceptors do not mediate the decrease in Tbc that occurs in response to acute hypoxemia in guinea pigs during postnatal maturation.

Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies.

Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick-synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG.

Hypoxia similarly impairs metabolic responses to cutaneous and core cold stimuli in conscious rats.

Cold exposure elicits several thermoregulatory responses, including an increased metabolic heat production from shivering and nonshivering thermogenesis. The increased metabolism can be in response to body core and/or body cutaneous cooling. Hypoxic hypoxia has been shown to attenuate the metabolic response to cutaneous cooling. We measured metabolic heat production in adult conscious rats during independent cutaneous and core cooling, during normoxia and hypoxia, to 1) test the hypothesis that hypoxia suppresses the metabolic response to independent core cooling and 2) determine whether hypoxia acts preferentially on the response to cutaneous or core cooling. The animals were studied in a temperature-controlled metabolic chamber, and body core temperature was controlled by an abdominal heat exchange coil. Ambient temperature was varied (10, 19, and 28 degrees C) while core temperature was clamped at 37 degrees C or core temperature was varied (33, 35, and 37 degrees C) at a stable ambient temperature of 28 degrees C. Our data indicate that although the sensitivity of the metabolic response to core cooling is about five to six times that to cutaneous cooling. Hypoxia similarly attenuates thermoregulatory responses to both stimuli.

Contribution of the parasternal intercostals to inspiratory rib elevation in dogs.

To estimate the contribution of the parasternal intercostals to rib elevation during quiet breathing, parasternal intramuscular pressure, Pim, in the fourth interspace and displacement of the rib just below were measured in eight supine anesthetized dogs during: (1) bilateral stimulation of the parasternals, (2) quiet breathing before, after phrenicotomy, and subsequent vagotomy. During quiet breathing, the parasternal contribution averaged 66 +/- 12% of the rib elevation caused by inspiratory rib cage muscles. This contribution decreased in relative terms after phrenicotomy (37 +/- 14%) and subsequent vagotomy (26 +/- 14%) while it tended to increase in absolute terms (from 1.9 +/- 2.4 to 2.1 +/- 2.5 NS, and 2.4 +/- 2.4 mm P < 0.01, respectively). Rib elevation caused by inspiratory rib cage muscles increased after phrenicotomy (116 +/- 63%, P < 0.001) and subsequent vagotomy (279 +/- 60%, P < 0.001) as did Pim (19 +/- 10% NS and 41 +/- 36% P < 0.01, respectively). Moreover, the mechanical interaction of the parasternals among different interspaces measured in three other dogs, was likely to be limited during quiet breathing. We conclude that after diaphragm paralysis, the parasternals played a progressively smaller role while other rib cage muscles were increasingly recruited.

Circadian rhythms of body temperature and drinking and responses to thermal challenge in rats after PCPA.

Body temperature (Tb) and drinking were measured for five days in male and female rats. On day 6 (S1) the rats were injected with saline. On day 7 (P1) they were injected with PCPA (300 mg/kg IP). Measurements continued for 12 days. Immediately after PCPA Tb dropped. After that, the amplitude of the daily Tb rhythm was significantly decreased from days P2-P5. Females were more affected than males. Nocturnality of drinking was decreased on days P2-P4. Because the peak of the Tb rhythm advanced after PCPA, while the peak of the drinking rhythm was delayed, we conclude that the attenuation of the Tb rhythm was a direct result of PCPA treatment rather than a masking effect due to the attenuation of other rhythms. Other rats were thermally challenged during the first week post-PCPA. There were no differences in ability to regulate Tb in the cold, and the small variations in the heat were overshadowed by gender differences.

After phrenicotomy the rat alters the output of the remaining respiratory muscles without changing its sleep-waking pattern.

The respiratory activity of selected rib cage and abdominal wall muscles was studied in intact and bilaterally phrenicotomized (PNX) rats during non-rapid eye movement sleep (nREMS) and REMS. The polygraphic method was used to identify the animal's sleep-waking states before and after PNX. Electromyographic (EMG) recordings were made from the following muscles: the parasternals of the 1st, 3rd, and 5th interspaces; the external and internal intercostals of the 1st, 6th and 10th interspaces; the levator costae attaching to the 10th rib; the scalenus medius; and, the abdominal wall muscles, the external and internal obliques and rectus abdominis. After PNX, all rib cage muscles contracted exclusively during inspiration and all but one increased their activity. The exception was the internal intercostal muscle of the 10th interspace; its activity decreased. The external and internal oblique muscles, both of which were active during expiration in nREMS, also increased their output after PNX: rectus abdominis became an inspiratory muscle. The persistence of phasic activity of respiratory muscles during REMS varied not only from muscle to muscle but from one REMS epoch to another. The sleep-waking pattern of the PNX rat differed in only a minor way from that of the intact rat. Therefore, we conclude that the rat with total paralysis of its diaphragm uses mainly its neurometabolic mechanisms to achieve an adequate level of alveolar ventilation and not neurobehavioral mechanisms.

Alteration in breathing of the awake rat after laryngeal and diaphragmatic muscle paralysis.

The respiratory rate (f), tidal volume (VT) and ventilation (V) were measured in 3 groups of rats: 10 rats before and after cutting both recurrent laryngeal nerves (RLNX), 10 rats before and after bilateral phrenicotomy (PNX) and 5 sham transected (SHAMX) rats. All rats were exposed to air and gas mixtures, deficient in O2 and/or enriched with CO2. The barometric method was used to measure ventilatory parameters. The sham operation did not affect breathing pattern or ventilation. In RLNX rats, breathing the various gas mixtures exhibited no changes in V because f uniformly increased as VT declined. Therefore, loss of the neural control of the respiratory functions of the larynx in awake rats exposed to selected gas mixtures has no untoward effects on alveolar ventilation. Changes in ventilation of PNX rats, compared with SHAMX rats, depends on the gas composition breathed. With increasing severity of hypoxia and/or hypercapnia, PNX rats show a marked reduction in alveolar ventilation over that of the SHAMX rats. Thus, when the diaphragm is no longer able to participate in ventilatory responses, gas exchange is likely to become deficient.

Proprioceptive, chemoreceptive and sleep state modulation of expiratory muscle activity in the rat.

The purpose of this study was to assess the respiratory and tonic activity of the abdominal muscles and the postinspiratory activity of the diaphragm (stage 1 expiration) in rats during sleep while they breathed air, hypercapnic, and hypoxic gas mixtures. ECoG and neck EMG recordings enabled the differentiation to be made between nonrapid eye movement sleep (nREMS) and rapid eye movement sleep (REMS). EMGs of the rectus abdominis, internal and external oblique, and diaphragm muscles were displayed on a CRT and polygraph. During nREMS the rectus abdominis showed no respiratory activity, whereas the oblique muscles showed activity confined to stage 2 expiration. This activity was modulated by proprioceptive (sleep postures) and chemoreceptive activation (5% CO2 in air and 10-12% O2 in nitrogen): tonic activity was not consistently affected by such inputs. During REMS tonic activity disappeared, whereas phasic activity either remained unchanged or was abolished. If phasic activity ceased it could reappear periodically during the same REMS epoch. While breathing air, rats in nREMS showed postinspiratory diaphragmatic activity which was sustained or slightly increased while breathing a hypoxic gas mixture but was virtually abolished during hypercapnia. In REMS postinspiratory discharges almost disappeared. The data support the conclusion that the diaphragm provides expiratory braking and that the external and internal oblique muscles contribute to active exhalation during nREMS as well as priming the diaphragm for the next inspiration by improving its length-tension relationship. A three-phase neural respiratory pattern generator operates in nREMS: it changes temporarily to a two-phase system while breathing CO2 and during REMS due to the inhibition of the postinspiratory phase.

Differential effects of hypoxia on sleep of warm- and cold-acclimated rats.

We studied the effect of different levels of hypoxia (10, 12 or 13, 15, and 18% O2) on the sleep-waking pattern (SWP) and the maximum-minimum core temperature of warm-acclimated (WA) and cold-acclimated (CA) rats at their neutral temperature, 29 degrees C. Whereas the SWP of WA rats showed a trend toward increasing disruption as the degree of hypoxia increased, CA rats exhibited no such trend. The effect was chiefly on the frequency of state changes and less on epoch durations. The SWP of WA rats was more vulnerable to hypoxia than that of CA rats. Maximum and minimum body temperatures of WA and CA rats were not significantly affected by O2 lack down to 10% inspired O2. We conclude that in the rat 1) hypoxia primarily affects the neural mechanism that governs the frequency of changes in sleep-waking states; 2) the extent of alterations in SWP's depends on the ambient temperature to which the rats are acclimated; and 3) hypoxia does not significantly affect deep body temperature at the animal's neutral temperature.

The labile respiratory activity of ribcage muscles of the rat during sleep.

1. Sleep-waking states of chronically implanted rats were identified polygraphically while recording the integrated electromyogram (e.m.g.) of extrinsic (scalenus medius and levator costae) and intrinsic (external and internal interosseous intercostal and parasternal) muscles of the thoracic cage. Rats breathed air, air enriched in CO2 (5%) or air deficient in O2 (10% O2 in N2) and were free to adopt any desired posture. 2. In non-rapid eye movement (non-r.e.m.) sleep, the scalenus medius and intercostal muscles of the cephalic spaces were always inspiratory; intercostal muscles of the mid-thoracic spaces were commonly expiratory while the more caudal ones were only occasionally expiratory. Expiratory activity, when present in quiet wakefulness, extended for a variable period of time into non-r.e.m. sleep and always disappeared in r.e.m. sleep regardless of the ribcage muscle under study. 3. Inspiratory activity, when present in non-r.e.m. sleep, was unaffected, partially attenuated or abolished at entry into r.e.m. sleep. The peak integrated e.m.g. activity of ribcage muscles was measured as a function of posture, gas mixture breathed and ribcage site: (a) the greater the degree of curled-up posture, the greater the respiratory activity of scalenus medius, an effect augmented by CO2 but depressed by hypoxia, and (b) the more caudally placed ribcage muscles exhibited respiratory activity which was essentially unaffected by posture and gas mixture inspired. 4. The presence or absence of tonic activity in ribcage respiratory muscles during non-r.e.m. sleep was unrelated to posture. When tonic activity was present, it always disappeared in r.e.m. sleep. When expiratory activity was present in non-r.e.m. sleep, it too always disappeared in r.e.m. sleep. Inspiratory activity present in non-r.e.m. sleep was variably affected at entry into r.e.m. sleep; it was unchanged, partially attenuated or abolished. 5. It is concluded that thoracic cage muscles exhibit marked variability in their respiratory activity depending on posture, sleep-waking states and gas mixture breathed. It is postulated that the presence of tonic and/or expiratory activity in ribcage muscles during non-r.e.m. sleep reflects an increase in functional residual capacity (F.R.C.).

Respiratory functions of the inferior pharyngeal constrictor and sternohyoid muscles during sleep.

We studied the respiratory activity of the inferior pharyngeal constrictor and sternohyoid muscles of the rat during non-rapid eye movement (non-REM) and REM sleep. Each animal carried chronically implanted electrodes for recording the integrated EMG activity of respiratory muscles as well as the electrocorticogram (ECoG) and postural tone (dorsal neck EMG). The latter permitted polygraphic identification of sleep states. Curled up postures enhanced inspiratory activity of both upper airway muscles during non-REM sleep, an effect which CO2 breathing failed to augment except in the well curled up position. Hypoxia reduced their activity. During REM sleep, the inferior pharyngeal constrictor and sternohyoid muscles retained their inspiratory activity. No tonic activity could be detected in either muscle. We conclude that the inferior pharyngeal constrictor and sternohyoid muscles safeguard upper airway patency in the two main sleep states.

Unity of costal and crural diaphragmatic activity in respiration.

In chronically implanted rats, we examined the respiratory EMG activity of the two parts of the diaphragm, costal and crural, during sleep and wakefulness. Their activity was compared and contrasted with that of the EMG activity of the cricothyroid muscle. Whether in wakefulness, while grooming and drinking, or in nonrapid eye movement (non-REM) sleep, and independent of the gas mixture breathed (4 to 5% CO2 or 10% O2 in nitrogen), the two parts of the diaphragm paused during REM apnea episodes whereas the cricothyroid muscle ceased its activity or exhibited sustained activity. We conclude that the diaphragm, mainly an inspiratory muscle, acts as a single functional unit when under the respiratory control system. The cricothyroid muscle functions as an inspiratory and/or expiratory muscle, also under the respiratory control systems. Both muscles in the rat come under other neural control mechanisms governing nonrespiratory functions, e.g., swallowing, defecation, and coughing, but not vomiting.

Respiratory roles of genioglossus, sternothyroid, and sternohyoid muscles during sleep.

We examined the respiratory activity of the genioglossus, sternothyroid, and sternohyoid muscles of the rat during nonrapid eye movement (non-REM) and REM sleep. Each animal carried implanted electrodes for recording the integrated EMG activity of respiratory muscles, the postural tone (EMG), and electrocortical activity (polygraphic identification of sleep-waking states). The three upper airway muscles exhibited inspiratory activity during non-REM sleep while rats breathed ambient air. Curled up postures promoted inspiratory activity of genioglossus and sternothyroid muscles, an effect enhanced by CO2 breathing but reduced by hypoxic breathing. During REM sleep, genioglossus and sternothyroid muscles lost their activity but the sternohyoid muscles retained their inspiratory activity. We conclude that the genioglossus and sternothyroid muscles contribute to upper airway patency during non-REM sleep, an effect CO2 augments but hypoxia reduces. The sternohyoid muscles have at least two functions during both sleep states: they contribute to maintenance of upper airway patency and to rib cage fixation, thereby optimizing the ventilatory action of the diaphragm.

Sleep-waking pattern and body temperature in hypoxia at selected ambient temperatures.

We studied the effect of mild hypoxia (15% O2) and low ambient temperature (Ta = 15 degrees C) on the rat's sleep-waking pattern (SWP) and maximum-minimum core temperature (max-min Tb). Mild hypoxia at neutral Ta (29 degrees C) disrupted the SWP in the same way as low Ta during normoxia: both affected the pattern of frequency of state changes (P less than 0.01), not the pattern of epoch durations. Mild hypoxia and low Ta together caused a degree of disruption of the SWP which was the sum of each alone, i.e., additive. Although both mild hypoxia and low Ta significantly depressed max-min Tb, low Ta exerted a greater effect than mild hypoxia. Together they further depressed max-min Tb in an additive way. We conclude that mild hypoxia disrupts the rat's SWP independent of central thermoregulatory mechanisms at neutral Ta, that the effects of mild hypoxia and low Ta on the SWP are additive at the stimulus levels used, and that Ta, not inspired O2, determines Tb.

Sleep-waking patterns of intact and carotid sinus nerve-transected rats during hypoxic-CO2 breathing.

Two groups, each consisting of six male rats, breathed 21% O2-4% CO2 or 10% O2-4% CO2, respectively, before and after carotid sinus nerve (CSN) transection. Polygraphic recording techniques served to monitor sleep and wakefulness. The effects of these gas mixtures on the sleep-waking pattern (SWP) were studied. The SWPs of the intact and CSN-transected rats breathing 21% O2-4% CO2 were no different from rats breathing air. While breathing 10% O2-4% CO2, the greatest alteration in the rat's SWP, compared with breathing 10% O2 devoid of CO2, was in the pattern of frequency of change of states, an effect unchanged by CSN transection. We conclude that CO2 added to air did not affect the rat's SWP. However, a hypoxic-CO2 gas mixture radically altered all parameters of the SWP, an effect which was centrally mediated.