Effect of Sleep Deprivation on Righting Reflex in the Rat Is Partially Reversed by Administration of Adenosine A1 and A2 Receptor Antagonists

This study was performed with approval from the Institutional Animal Care and Use Committee at our institution (University of Chicago, Chicago, Illinois). Male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 250–300 g were anesthetized with intraperitoneal ketamine (70 mg/kg) and xylazine (6 mg/kg). Five stainless steel screws (Small Parts Inc., Miami Lakes, FL) were implanted through the skull to serve as dural electroencephalographic electrodes, and two electromyographic electrodes were implanted in the neck musculature. In rats scheduled to undergo intracranial microinjection of 8-CPT, bilateral 24-gauge stainless steel guide cannulae were then placed into the basal forebrain using stereotactic coordinates derived from Paxinos and Watson11and our earlier work: anterior/posterior −0.4, medial/lateral ±2.0, dorsal/ventral +8.0 mm from bregma. At the conclusion of surgery, a 31-gauge stainless steel stylet was placed into each guide cannula to maintain patency. All rats were then allowed to recover for 7 days in a temperature (21°–24°C)– and light-controlled room with ad libitum  access to food and water. Lights were turned on at 6:00 am and off at 6:00 pm every day.

Five separate experiments were performed in five separate groups of rats. In each group, rats underwent four trials, consisting of all combinations of a behavioral intervention (24-h period of ad libitum  activity or total sleep deprivation beginning and ending at 12:00 noon), followed by administration of a specific dose of adenosine antagonist(s) or vehicle. The five groups differed on the basis of the dose and drug administered:

• Group 1 (n = 12): microinjection of low-dose 8-CPT (0.25 ng)

• Group 2 (n = 12): microinjection of high-dose 8-CPT (0.50 ng)

• Group 3 (n = 15): intraperitoneal administration of low-dose ZM241385 (5 mg/kg)

• Group 4 (n = 15): intraperitoneal administration of high-dose ZM241385 (10 mg/kg)

• Group 5 (n = 10): combined administration of low-dose 8-CPT and ZM241385 (0.25 ng and 5 mg/kg, respectively)

Sleep deprivation was achieved by the disk-over-water method.12Specifically, rats were placed on a 45-cm-diameter disk suspended over a pan of water with continuous computerized electroencephalographic and electromyographic monitoring. When sleep onset was detected, the computer rotated the disk at a rate of 3 revolutions/min, causing the rat to wake up and walk to avoid falling into the water. When the rat awakened, rotation stopped. This method has previously been validated as able to produce near-total sleep deprivation without excessive physical exertion.13Rats receiving ad libitum  activity were exposed to the same environment, except that a platform was placed over the wheel to eliminate the water hazard and effects of wheel rotation.

Immediately after the behavioral intervention (12:00 noon), rats were given either an adenosine antagonist or vehicle. In the first two groups (1 and 2), the adenosine A1 antagonist 8-CPT was microinjected into the basal forebrain. After removal of the stylet from the guide cannula implanted during surgery, a 31-gauge stainless steel injection cannula (Plastics One, Roanoke, VA) was inserted so that the tip extended 1.0 mm past the tip of the guide cannula. After warming to 37°C, 8-CPT (Sigma Chemicals, St. Louis, MO) dissolved in 0.2 μl artificial cerebrospinal fluid (145 mm Na⁺, 1.2 mm Ca²⁺, 2.7 mm K⁺, 1.0 mm Mg²⁺, and 150 mm Cl⁻, pH 7.4), or artificial cerebrospinal fluid alone was injected via  a 10-μl Hamilton syringe through PE-20 tubing attached to the injection cannula. The injection was made over 30 s, and the inner cannula left in place for 30 s after each injection. Each rat received bilateral injections for a total volume of 0.4 μl. Volumes and infusion rates were calculated to minimize tissue damage and limit diffusion of drug from the injection site.14Each group received a different dose of 8-CPT. The first group received a 2.5-μm solution (0.25 ng total in 0.4 μl), and the second group received a 5-μm solution (0.5 ng total in 0.4 μl). After injection, rats were returned to their cages for a 15-min incubation period to allow drug diffusion, and those undergoing sleep deprivation were kept awake by gently shaking the cage when sleep was observed.

In the second two groups (3 and 4), the adenosine A2a antagonist ZM241385 was administered. ZM241385 (Sigma Chemicals) was dissolved in a 1:1 mixture of Alkamuls EL-620 (Rhone-Poulenc, Collegeville, PA) and dimethyl sulfoxide (Fisher Scientific, Fair Lawn, NJ). The resulting mixture was then diluted 1:4 in 0.9 normal saline. Because this mixture was too viscous to be administered via  microinjection, it was administered via  intraperitoneal injection. One hour before the end of the behavioral intervention (11:00 am), rats were given either ZM241385 or the Alkamuls–dimethyl sulfoxide–saline vehicle. The higher dose of ZM241385 was 10 mg/kg body weight, and the lower dose was 5 mg/kg. The final injection volume for all rats was 2 ml. Rats were then returned to their cages after injection, and those undergoing sleep deprivation were kept awake by gently shaking the cage.

In the fifth group, rats received both antagonists together or both vehicles. Forty-five minutes before the end of the behavioral intervention, rats were given ZM241385 (5 mg/kg) or vehicle via  intraperitoneal injection. Immediately after the end of deprivation, rats were then given 8-CPT (0.25 ng) or vehicle via  bilateral microinjection into the basal forebrain. Rats were then returned to their cages for an additional 15-min incubation period, and those undergoing sleep deprivation were kept awake by gently shaking the cage.

After drug administration and incubation, rats were given isoflurane, and the time to LORR/spontaneous movement and recovery were measured as follows. Rats were placed in a 13 × 13 × 23-cm clear plastic anesthesia chamber fitted with a gas inlet and outlet. A compressed oxygen tank and a standard isoflurane vaporizer (Cyprane Ltd., Keighley, England, United Kingdom) were used to direct a constant 3-l/min flow of oxygen containing 1.1% isoflurane (Abbott Laboratories, Chicago, IL) through the box. The isoflurane concentration was chosen to allow detection of accelerated LORR in sleep-deprived rats.6Isoflurane concentrations were monitored continuously using a Puritan-Bennett Datex model 254 airway gas monitor (Puritan Bennett, Carlsbad, CA) calibrated before each use with a reference gas containing 1.5% isoflurane (Biochem Int., Waukesha, WI). The presence or absence of righting reflex and attempts to self-right were tested by rotating the box every 10 s (or after cessation of spontaneous movement) and observing the rat to verify that no righting attempts or movements in response to repeated positioning on their back or side were observed. When loss of all attempts to self-right (despite repositioning or box rotation) was achieved, rats were removed from the chamber and allowed to recover while spontaneously breathing room air. Rats were gently prodded every 15 s to determine the onset of recovery, defined as the return of righting reflex. An investigator blinded to the deprivation and antagonist history of the rat recorded the time from initial exposure to isoflurane to LORR and from discontinuation of the anesthetic to recovery.

After recovery, rats were returned to their temperature- and light-controlled colony. After a 5- to 7-day recovery period, the above protocol was repeated in random order until each rat had received every possible combination of behavioral intervention and drug (sleep deprivation and vehicle, sleep deprivation and antagonist, ad libitum  activity and vehicle, ad libitum  activity and antagonist). When all treatments had been completed, rats were killed via  intraperitoneal injection of 400 mg/kg sodium pentobarbital. Those with intracranial cannulae were perfused transcardially with 0.9% NaCl followed by 4% formalin in 1.25% NaCl. After perfusion, rats were decapitated, and the brain was extracted and stored in a solution of 4% formalin–30% sucrose for 48 h. Coronal brain sections (40 μm) were cut on a freezing microtome, mounted, and stained with cresyl violet. The tip of the injection cannula track was then localized by light microscopy.

Electroencephalographic/electromyographic data were recorded on a Grass model 78 polygraph (Grass-Telefactor, West Warwick, RI) and relayed to a computer for digital recording. Data were divided into 30-s epochs and scored as waking, nonrapid eye movement, or rapid eye movement sleep using an automated scoring system previously validated against visual and behavioral methods.12,13Portions of the electroencephalogram/electromyogram were also scored visually to verify the reliability of the automated system. Definitions of sleep stages have been presented in detail previously.15 

We performed five experiments in five separate groups of rats, testing two doses of 8-CPT, two doses of ZM241385, and one dose of both antagonists given together. Rats in each group served as their own controls, receiving all four combinations of sleep deprivation/ad libitum  activity and drug/placebo. Data collected included weights; ages; rapid eye movement, nonrapid eye movement, and total sleep times for rats during sleep deprivation; and cumulative duration of wheel rotation for all groups. The times to LORR and to recovery were recorded by an observer blinded to the deprivation and microinjection history of the rat. All values were expressed as mean ± SD. Repeated-measures analysis of variance was used to determine the significance of our observed outcomes, treating condition (ad libitum  vs.  sleep deprivation), and treatment (placebo vs.  drug) as repeated factors for both drugs (8-CPT and ZM241385) and both doses (high and low). Post hoc  comparisons were made using the Bonferroni adjustment. Random-effects mixed models were used to examine the effect of age and weight on time to LORR and to recovery. All analyses were performed using Stata version 8 (Stata Corp., College Station, TX).

Although completion of the four trials required as many as 21 days for some rats, average weights, ages, amount of wheel rotation, and sleep frequencies were similar within and between all groups. For all rats, the wheel rotated between 14 and 20% of the time, resulting in approximately 90% wakefulness during the 24-h behavioral intervention. Analysis of variance revealed no significant effect of weight, age, or sequence of treatments on the amount of sleep during deprivation or the amount of wheel rotation. For rats with intracranial cannulae, the average location of the 8-CPT injection sites in millimeters was as follows: (mean ± SD) anterior/posterior −0.32 ± 0.06, medial/lateral 1.9 ± 0.42 (L), 2.0 ± 0.36 (R), dorsal/ventral 8.6 ± 0.25 (L), 8.7 ± 0.24 (R). Figure 1depicts the location of individual injection sites.

In all experiments, rats given vehicle after 24 h of sleep deprivation lost their righting reflex with isoflurane significantly sooner than after the same duration of ad libitum  activity (figs. 2–4). The time to recovery was also prolonged. This effect of sleep deprivation on LORR in untreated rats was similar in magnitude to findings reported previously.6 

In rats given 8-CPT only, no difference in time to LORR or to recovery was seen in rested animals that received vehicle, low (0.25 ng) doses of 8-CPT, or high (0.5 ng) doses of 8-CPT (fig. 2). In sleep-deprived rats, basal forebrain microinjection of 8-CPT significantly delayed isoflurane-induced LORR in a dose-dependent manner. Rats receiving the lower dose of 8-CPT demonstrated a slight, nonsignificant increase in time to LORR when compared with those receiving the artificial cerebrospinal fluid vehicle. At the higher dose, 8-CPT significantly increased the time to LORR (426.5 ± 92 vs.  264.8 ± 53 s [mean ± SD]; F^1,33= 23.16, P = 0.001; fig. 2). Time to recovery was also reduced in a dose-dependent manner, with the difference becoming significant at the higher dose (74.6 ± 18 vs.  115.0 ± 17 s; F^1,33= 22.03, P < 0.02; fig. 2). Sleep-deprived rats treated with the high dose of 8-CPT still lost their righting reflex sooner than rested rats receiving vehicle (426.5 ± 92 vs.  693.4 ± 112 s; F^1,33= 63.14, P = 0.0001). Figure 2represents a composite of low- and high-dose 8-CPT groups, where vehicle data from both groups are averaged together.

In rats given ZM241385 only, rested rats receiving vehicle, low (5 mg/kg) doses, or high (10 mg/kg) doses also demonstrated no differences in time to LORR or recovery (fig. 3). As with 8-CPT, ZM241385 prolonged the time to LORR in a dose-dependent manner, reaching significance at the higher dose (596 ± 114 vs.  340.6 ± 76 s; F^1,42= 29.46, P = 0.001; fig. 3). Time to recovery was also significantly reduced at the higher dose (107.7 ± 50 vs.  157.3 ± 52 s; F^1,42= 13.72, P = 0.0012; fig. 3). As with rats receiving 8-CPT, deprived rats receiving the higher dose of ZM241385 lost their righting reflex sooner than rested rats (596 ± 114 vs.  892.1 ± 176.2 s; F^1,42= 39.42, P = 0.0001). Figure 3represents a composite of low and high dose ZM241385 groups, where vehicle data from both groups are averaged together.

In rats given both 8-CPT and ZM241385, no effect of antagonist administration in rested rats was seen (fig. 4). In sleep-deprived rats, coadministration of 8-CPT and ZM241385 significantly prolonged the time to LORR over rats that received vehicle only (591 ± 201.8 vs.  267.9 ± 82.6 s; F^1,27= 29.19, P < 0.033; fig. 4). Although time to recovery was slightly lower in rats that received antagonist, the difference was not significant.

Observations that anesthetic requirements vary with time of day16and that sleep deprivation enhances some measures of anesthetic potency6suggest that mechanisms involved in the homeostatic regulation of sleep may interact with and potentially modify the anesthetized state. In this article, we report that blockade of both adenosine A1 and A2a receptors in the rat partially prevented sleep deprivation from potentiating isoflurane-induced LORR. Specifically, we found in sleep-deprived rats that basal forebrain microinjection of the adenosine A1 receptor antagonist 8-CPT increased the time to LORR with isoflurane and reduced the time to recovery in a dose-dependent manner (fig. 1). Systemic administration of the adenosine A2a receptor antagonist ZM241385 had a similar effect (fig. 2). When the two antagonists were administered together in doses too low to have a significant effect separately, a similar prolongation of time to LORR was noted (fig. 3). These findings suggest that modulation of both adenosine A1 and A2a receptors partly uncouple the link between sleep homeostatic mechanisms and anesthetic potency and raise the possibility that adenosine may mediate such a link.

Our observations are supported by existing human and animal data. In humans, systemic administration of adenosine potentiates hypnosis induced by intravenous anesthetics10and reduces intraoperative anesthetic requirements.17In rats and cats, extracellular adenosine concentrations in the basal forebrain increase progressively with prolonged sleep loss and return to baseline levels with recovery sleep.18These changes in endogenous adenosine concentration also alter sleep behavior. Administration of exogenous adenosine reuptake inhibitors (or adenosine itself) into the basal forebrain increases sleep, whereas the adenosine A1 receptor antagonist 8-CPT produces the opposite effect.18 

Although how the brain generates and maintains either the sleep or waking state is incompletely understood, adenosine may modulate the arousal state by altering interactions between specific neuronal cell groups active during wakefulness such as the basal forebrain (wake-active), and sleep-active cell groups such as the ventrolateral preoptic nucleus (VLPO).8Projections from the VLPO to the basal forebrain have led to hypotheses that sleep-active cell groups, when activated, may exert their effects by inhibiting the activity of wake-active neurons.19In an in vitro  model, for example, adenosine administration to the VLPO acts on γ-aminobutyric acid–mediated synaptic inputs to “disinhibit” VLPO sleep-active neurons.20Behaviorally, sleep-inducing effects have been observed when adenosine is administered via  microdialysis both to sleep-active and wake-active cell groups.18,21 

Existing data also support a relation between increased adenosine concentrations and changes in anesthetic potency. Basal forebrain administration of the adenosine A1 receptor antagonist 8-CPT increases the discharge rate of cholinergic neurons known to be involved in arousal.22This effect of adenosine on brain cholinergic activity may plausibly allow it to modify anesthetic action. Direct administration of cholinergic agents into the pontine reticular nucleus, for example, inhibits spinal motoneuron excitability in cats23and reduces halothane minimum alveolar concentration (MAC) in rats.24In addition, halothane decreases pontine acetylcholine release,25and pontine administration of an adenosine A1 agonist both decreases acetylcholine release and alters recovery from halothane anesthesia.26Adenosine-induced modulation of brain cholinergic activity may thus have partly accounted for our observations.

Our finding that both adenosine A1 and A2a receptor antagonists blocked the effect of sleep deprivation on anesthetic potency suggests an effect mediated by both receptor subtypes. This possibility is consistent with previous reports implicating both receptors in the sleep-promoting effects of adenosine. In cats, delivery of the A1 receptor agonist N6-cyclo-hexyl-adenosine to the basal forebrain depressed the activity of wake-active neurons, whereas the A1 antagonist 8-CPT had the opposite effect, and the A2a agonist CGS-16284 had no effect.8In rats, increases in adenosine A1 and decreases in A2a receptor messenger RNA with sleep deprivation have been observed.27These results suggest that in the basal forebrain, adenosine acts via  an A1 receptor–specific mechanism on sleep homeostasis.

Adenosine A2 receptors, however, have also been associated with sleep regulation. Existing data demonstrate in rats that subarachnoid administration of the adenosine A2a receptor antagonist KF17837 blocks the sleep-inducing effects of prostaglandin D2 and that direct microinjection of the adenosine A2a receptor agonist CGS-21680 into the ventrolateral preoptic area (VLPO) increases sleep.9,28Furthermore, although administration of both A1 and A2a agonists into the pontine reticular formation of the rat increases sleep, only the A2a effect is inhibited by atropine.29These data suggest a specific A2a effect, likely localized apart from the A1 site on the ventral surface of the rostral basal forebrain.9 

Our rationale for administering A1 and A2a antagonists via  different routes was thus based in part on the strong possibility that effects of adenosine on sleep homeostasis were likely to occur via  different receptors and at different sites. In addition, we were unable to reliably administer ZM241385 via  microinjection because of the viscosity of the Alkamuls–dimethyl sulfoxide–saline vehicle. Our finding that simultaneous administration of both agents in doses too low to be effective alone significantly prolonged time to LORR further suggests that both receptor subtypes have important roles in the effect of adenosine on sleep deprivation and may interact physiologically in their effects on anesthetic potency as well.

We were only able to partially reverse the effect of sleep deprivation on LORR. At the higher dose of either antagonist, sleep-deprived rats continued to lose their righting reflex sooner than if they had not been deprived. Several possible reasons exist for this incomplete reversal. First, the doses of antagonists that we used, although physiologically significant, may not have been adequate. Second, neither antagonist would have blocked adenosine effects on other receptor subtypes. 8-CPT may have blocked A1 effects of adenosine, for example, but would not have prevented A2a receptor effects. Third, our incubation periods (12–15 min for 8-CPT, 60 min for ZM241385) may not have captured the peak effect of our intervention. Finally, effects of sleep deprivation unrelated to changes in adenosine receptor activity may also modulate anesthetic potency. The endogenous sleep modulator oleic acid amide, for example, not only accumulates in the cerebrospinal fluid of sleep-deprived rats and cats, but also enhances γ-aminobutyric acid type A activity in vitro  30and potentiates the activity of benzodiazepines.31 

Our study has several important limitations. First, we did not measure adenosine concentrations and can only infer the relation among sleep deprivation, increased adenosine concentrations, and anesthetic action that our data suggest. It is possible, for example, that sleep deprivation alters the transduction of the adenosine signal downstream from the adenosine receptor, or the number of adenosine receptors in addition to adenosine levels. Clarifying this issue would require the measurement of adenosine concentrations during sleep deprivation, recovery sleep, and general anesthesia. Second, it is possible that both adenosine antagonists induced a nonspecific, general increase in resistance to anesthetic action. We believed such a possibility was unlikely, however, because we observed no effect in rested rats (when compared to vehicle) at the doses we tested. Third, our study design did not measure anesthetic effects at steady state. Although it was therefore possible that we were measuring a pharmacokinetic and not a pharmacodynamic effect of adenosine antagonism on anesthetic potency, we thought it was unlikely, given the magnitude of the pharmacokinetic change that would have had to occur to produce our results.

We did not use MAC as our endpoint for anesthetic action for several reasons. First, determination of MAC can require a prolonged period of anesthetic titration. This may have caused us to miss peak levels of drug effect. Because rats may discharge accumulated sleep debt while anesthetized,32the duration of anesthesia required to determine MAC may have introduced variability in the “dose” of sleep deprivation. Also, MAC involves a painful stimulus, which may be perceived differently in sleep-deprived subjects.33In addition, MAC is unchanged by spinal cord transaction in rats34and thus may represent effects of anesthetics primarily on spinal cord reflex pathways. Although successful righting is in part also spinally mediated, righting attempts occur despite transections as high as the postcollicular level.35 

Our study involved a rat model of sleep homeostasis. Although this model has been used extensively to model human sleep behavior, differences between rats and humans in the behavioral response to sleep deprivation or anesthesia may limit ready generalization of our results. Nevertheless, we believe that our data support a potential relation between preoperative sleep adequacy and perioperative anesthetic management. Both anecdotal reports36and clinical trials37link sleep apnea to enhanced respiratory depressant effects of sedatives. Such clinical reports suggest an important possible role for sleep deprivation in potentiating sensitivity to anesthetic drugs and underscore the importance of diagnosing sleep apnea in the perioperative period. Because a 24-h period of sleep deprivation in the rat cannot be correlated to an equivalent duration of sleeplessness in humans, it is not possible to extrapolate how much sleep deprivation is required to clinically alter anesthetic action. However, reducing sleep to less than 5 h in children undergoing noninvasive procedures with conscious sedation significantly increased periprocedure nursing care requirements.38Studies such as these suggest that sleep deprivation in humans can adversely affect anesthetic care and perioperative management.

In conclusion, we report an ability of both the adenosine A1 receptor antagonist 8-CPT and the A2a receptor antagonist ZM241385 to partially reverse the effect of sleep deprivation on isoflurane-induced LORR in the rat. Although neither antagonist altered LORR in rested rats, both increased the time to isoflurane-induced LORR in sleep-deprived rats and shortened the time to recovery. Our results suggest that one possible link between sleep deprivation and changes in anesthetic potency may be fluctuations in regional adenosine concentrations induced by sleep deprivation. These findings support the hypothesis that neuronal networks active during sleep may have a significant role in generating the reduced responsiveness characteristic of general anesthesia. Further elucidating the relation between sleep homeostatic regulatory mechanisms and anesthetic action may help to clarify the mechanisms of general anesthesia and may allow anesthesiologists to understand some of the sources of variability in anesthetic effect.