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Albumin enhances sleep in the young rat
Physiology & Behavior, Vol. 64, No. 3, pp. 261–266, 1998 © 1998 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/98 $19.00 1 .00
PII S0031-9384(98)00074-2
Albumin Enhances Sleep in the Young Rat ´ L, JR,* L. KAPA ´ S† AND J. M. KRUEGER1‡ F. OBA *Department of Physiology, A. Szent-Gyo¨rgyi Medical University, Szeged, Hungary; †Department of Biological Sciences, Fordham University, Bronx, NY; and ‡Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA Received 1 August 1997; Accepted January 28, 1998 ´ L, F., JR., L. KAPA ´ S AND J. M. KRUEGER. Albumin enhances sleep in the young rat. PHYSIOL BEHAV 64(3) 261–266, OBA 1998.—Rats 4 to 7 days after weaning received intraperitoneal (i.p.) injections of vehicle (baseline day), and either serum (2 mL of lyophilized rabbit serum), 140 mg of rat albumin, or hyperosmotic NaCl (experimental day). Injections were given 1 h before light onset. Sleep-wake activity and cortical brain temperature were recorded during the subsequent 12-h light period. The intensity of non-rapid eye movement sleep (NREMS) was characterized by the power density values of the electroencephalogram slow-wave activity. The sera and albumin preparations enhanced both NREMS and slow-wave activity for 5 to 6 h starting during Hour 2 after light onset. Rapid eye movement sleep (REMS) tended to decrease. Modest (0.6°C maximum deviation) biphasic changes were observed in cortical brain temperature with initial decreases for 3 h followed by rises between Hours 3 and 9 of the light period. There were no differences in the sleep responses to albumin between male and female rats. Albumin also enhanced NREMS in young rats on a protein-rich diet. A significant negative correlation was found between the NREMS promoting activity of albumin injections and the body weight of the rats. NaCl solution with the same osmolarity as that of the albumin solution failed to alter sleep. I.p. albumin injection elicited significant increases in the concentrations of cholecystokinin-like immunoreactivity in the plasma. Sleep-promoting materials (hormones) in the albumin fraction, the calorigenic or nutritional value of proteins, the release of somnogenic cytokines by albumin, or endogenous humoral mechanisms stimulated by proteins (e.g., cholecystokinin or the somatotropic axis) might mediate the enhanced sleep after albumin. © 1998 Elsevier Science Inc. Sleep
Proteins
Plasma
Albumin
Cholecystokinin (CCK)
VARIOUS endogenous substances promote sleep. Some of these substances are found in body fluids such as the blood. For example, the delta sleep-inducing peptide (DSIP) was isolated from dialysates of blood (24). The plasma carries various hormones and immunostimulants which may also influence sleep: systemic growth hormone (9), prolactin (29), steroid hormones (21,35), chorionic gonadotropin, or luteotropic hormone (36) can alter sleep. We report here that concentrated preparations of whole sera and the albumin fraction of the serum also possess sleep-promoting activity in young rats.
were weaned at the age of 21 days. The surgeries were carried out between Days 21 and 24 of life. Using pentobarbital anesthesia (50 mg/kg), jewelry screws were implanted over the frontal and parietal cortices and the cerebellum for recording the electroencephalogram (EEG), and a thermistor was placed over the parietal cortex to measure cortical brain temperature (Tcort). The body weight of the rats was 31–74 g (mean 6 SE: 46.8 6 1.0 g) at the time of the surgery and 45–105 g (mean 6 SE: 70.9 6 2.3 g) at the termination of the experiments. After the surgeries, the rats were housed in individual Plexiglas recording cages placed in the recording room. In the room, the ambient temperature was maintained at 26 6 1°C, and the same light-dark cycle was provided as that in the environmental room where the rats were bred. Standard rat food and water were continuously available. The rats were connected to light flexible recording tethers made from flatband computer cables. The tethers were attached to electronic commutators, thus the rats were free to move around. Before recording, the rats were allowed 4 to 6 days for recovery and habituation to the environment.
METHODS
Materials Rat albumin was obtained from Sigma, and a radioimmunoassay kit for unsulfated CCK26 –33 was from Peninsula Laboratories, Inc., Belmont, CA. Animals The surgery, the experimental conditions, and recording procedures were similar to those reported previously in studies on young rats (28). Sprague–Dawley rats were bred in an environmental room at an ambient temperature between 22 and 24°C, and with a 12 h:12 h light/dark cycle (light onset: 0830 hours). The rats
Recordings The EEG, Tcort, and motor activity (signals generated in electromagnetic transducers activated by the movements of the recording tethers) were collected by computers (64-Hz sampling rate).
1 Requests for reprints should be addressed to J. M. Krueger, 205 Wegner Hall, Washington State University, Pullman, WA 99164-6520. E-mail: [email protected]
261
´ L, KAPA ´ S, AND KRUEGER OBA
262 The states of vigilance were determined visually for consecutive 8-s epochs according to standard criteria: non-rapid eye movement sleep (NREMS): high-amplitude EEG slow waves, lack of body movement, declining Tcort upon entry; rapid eye movement sleep (REMS): highly regular theta activity in the EEG, general lack of body movements with occasional twitches, and a rapid increase in Tcort at onset; wakefulness: EEG activities similar to, but often less regular and with lower amplitude than those in REMS, frequent body movements, and a gradual increase in Tcort after arousal. The percentage time spent in each state of vigilance over 1-h periods, and for the 12-h light period was determined. The Tcort values were averaged for 1-h periods. Power density spectra were calculated by fast Fourier transformation of the EEG for consecutive 8-s epochs in the frequency range 0.25–20 Hz for 0.25-Hz bands. The power density values for the 0.25– 4 Hz (delta) frequency range were integrated and used as an index of EEG slow-wave activity (SWA). Mean SWA during NREMS was determined by using SWA values obtained in uninterrupted periods of artifact-free NREMS epochs in each hour of recording. The percentage changes between the power density values on Day 1 (baseline day) and Day 2 (experimental day) were calculated for each hour and for each rat. Experimental Protocol The effects of i.p. administration of rat albumin on sleep. The animals (Group 1, n 5 47; 24 males and 23 females) were injected with isotonic NaCl on Day 1 and with rat albumin (140 mg in 0.5 mL of isotonic NaCl) on Day 2, 1 h before light onset. The recording started at light onset and continued for 12 h; recording from six of these animals started immediately after the injections. Out of the 47 animals, eleven rats were provided with a protein rich diet for 2 days before the recordings and also during the 2 days of recording (in addition to the food pellets, boiled eggs were placed in the cages every morning and evening). For control, twelve of their litter-mates were fed the same standard diet as the rest of the rats in the albumin group. The effects of i.p. administration of rabbit serum on sleep (Group 2). Twenty rats (6 males and 14 females) were injected i.p. with isotonic NaCl on Day 1. On Day 2, the animals received rabbit serum i.p. The serum was obtained by taking blood from the ear of unanesthetized rabbits. For each dose, 2 mL of rabbit serum was lyophilized and dissolved in 0.5 mL of isotonic NaCl. The rats received the injections 1 h before light onset. The recordings started at light onset and continued for 12 h. The effects of i.p. administration of hypertonic NaCl solution on sleep. To test if the actions of serum and albumin on sleep are due to the osmotic activity of the solutions, the effects of a NaCl solution of an equal osmotic concentration were also studied. The osmolarity of the albumin solution (as determined by the depression of the freezing point) corresponded to that of a NaCl solution containing 1.3 g NaCl in 100 mL of water. Seven rats (Group 3, three males and four females) received isotonic NaCl on Day 1 and hypertonic NaCl solution i.p. on Day 2 1 h before light onset. Recordings started at dark onset and lasted for 12 h. The effects of i.p. Administration of albumin on plasma cholecystokinin (CCK)-like immunoreactivity (CCK-LI). Eight groups of male rats (45–50 g) were injected with isotonic NaCl and eight groups of rats with 140 mg of rat albumin, i.p. One group of either treatment was sacrificed by decapitation 30 min (n 5 6, each group), 60 min (n 5 6), 80 min (n 5 7), 100 min (n 5 7), 120 min (n 5 6), 140 min (n 5 6), 160 min (n 5 7), and 180 min (n 5 7) postinjection. The trunk blood was collected and centrifuged. CCK-LI was measured in triplicate by radioimmunoassay. According to the manufacturer (Peninsula Laboratories, Inc., Belmont,
CA, USA), the antiserum revealed crossreactivities as follows: unsulfated CCK 26 –33: 100%, sulfated CCK 26 –33: 78%, human gastrin I 100%, human pancreatic polypeptide: , 0.1%, and vasoactive intestinal peptide: 0%. The plasma concentrations of CCK-LI measured at the various time points were pooled as follows: 30 and 60 min postinjection 5 Hour 1; 80, 100, and 120 min postinjection 5 Hour 2; and 140, 160, and 180 min postinjection 5 Hour 3. Statistical Analysis The differences in NREMS, REMS, SWA during NREMS, and Tcort between the baseline day and the experimental day were evaluated by two-way ANOVA for repeated measures. The treatment-effects (differences between the 2 days), and the time effects (variations during the 12-h recording period) were the factors of the ANOVA. Due to malfunction of several thermistors, the results of the Tcort were calculated from 27 rats in Group 1. Two-way ANOVA for independent samples (factors: treatment and time) was used to compare CCK-LI in the control and albumin-injected rats. Two-way ANOVA was also used to determine whether the sleep states differed between the rats injected with albumin and hypertonic NaCl solution on both the baseline and the experimental days (group effect: independent samples; time effects: repeated measures). An a-level of p , 0.05 was considered significant in all tests. RESULTS
The Effects of Albumin In Groups 1 and 2, after control injections, the expected diural variation in sleep was observed; e.g., the time spent in NREMS was relatively high at the beginning of the light period and declined continuously across the 12-h recording [time effect in ANOVA; Group 1: F(11, 506) 5 16.723, p , 0.001); Group 2: F(1, 209) 5 15.167, p , 0.001]. The percentages and the distributions of the sleep states on the baseline day corresponded to those reported by Alfo¨ldi et al. (2) for young rats. In Group 1, albumin injection significantly promoted NREMS. After albumin injection, NREMS started to increase above the control level in Hour 2 of the recording and was elevated for 4 to 5 h (Fig. 1). Sleep was not altered during the hour preceding light onset as determined in 6 rats [mean 6 SE, NREMS after isotonic NaCl: 29.9 6 4.4; NREMS after albumin: 33.1 6 4.2; REMS after isotonic NaCl: 10.0 6 2.8; and REMS after albumin: 7.5 6 2.3]. The promotion of sleep after albumin treatment, therefore, had a latency of about 2 h. I.p. injection of albumin failed to enhance REMS; in fact, a tendency to a suppression in REMS was observed in several groups (Table 1). These changes in REMS were small (Fig. 1) and barely reached the level of statistical significance. In Group 1, there were no differences between the male and female rats in the NREMS responses to albumin (Table 1). Also, the albumin stimulated NREMS responses in the rats maintained on the normal diet and in the rats that were provided with proteinrich diet were similar. Promotion of NREMS by albumin, however, depended on the body weight of the animals. After pooling the data obtained in the rats injected with albumin or rabbit serum preparations together, a correlation was calculated between the body weight at the end of the recordings (independent variable) and the changes in the duration of NREMS during the 12-h recording period (dependent variable). A significant negative correlation (20.591, p , 0.001) was obtained, indicating that albumin was less effective in enhancing NREMS in rats with larger body weights than in the smaller rats. Albumin did not promote NREMS in rats of 90 g or above.
ALBUMIN STIMULATES SLEEP
263 min-induced changes were the function of time. Indeed, SWA during NREMS was enhanced in Hour 2 of the recording, then declined toward or below the baseline values (Fig. 1). Injection of albumin was followed by biphasic changes in Tcort [treatment effect: F(1, 26) 5 9.784, p , 0.004, time effect: F(11, 286) 5 11.427, p , 0.001, interaction: F(11, 286) 5 18.133, p , 0.001]. Tcort was significantly below baseline in Hours 1 and 2 of the recording, then Tcort increased from Hours 3 to 8. The maximum deviation from baseline was 0.6°C (Fig. 1). The albumininduced variations in Tcort did not correlate with the time course of the changes in NREMS. REMS, however, tended to decrease during the period of hyperthermia. The Effects of Rabbit Serum; Group 2 The sleep, SWA, and Tcort alterations elicited by i.p. rabbit serum were similar to those found after i.p. injection of albumin. The duration of NREMS increased whereas REMS did not change during the 12-h light period (Table 1). The Effects of Hypertonic NaCl; Group 3 Injection of hyperosmotic saline solution did not alter NREMS (Table 1.), SWA, or Tcort (data not shown). Although REMS tended to decrease, the changes did not reach the level of statistical significance. Statistical comparisons between NREMS in the rats injected with albumin (Group 1) and hypertonic NaCl supported that albumin enhanced NREMS [group effect: F(1, 52) 5 12.028, p , 0.001]. NREMS varied across the 12-h recording period [time effect: F(11, 572) 5 5.147, p , 0.001], and the difference between the two groups also varied with the time [group 3 time interaction: F(11, 572) 5 2.397, p , 0.007]. there were no intergroup differences in NREMS on the baseline days and in REMS on either the baseline or experimental day. The Effects of Albumin on Plasma CCK-LI The plasma concentration of CCK-LI varied significantly during the 3-h postinjection period [F(2,98) 5 5.768, p , 0.05]. I.p. injection of albumin elicited a significant a rise in CCK-LI [F(1, 98) 5 5.891, p , 0.05]. This increase was already obvious at 30 min postinjection [1.6 6 0.12 ng/mL vs. 2.09 6 0.17 ng/mL] and persisted in the samples collected 60 min after the injection resulting in a significant difference (Student’s t-test) between the control and the albumin groups in postinjection Hour 1 (Fig. 2). Concentrations of CCK-LI in the rats injected with albumin gradually approached the control values in postinjection Hour 2, and the difference between the groups vanished in Hour 3. DISCUSSION
FIG. 1. Effects of an i.p. injection of 140 mg of albumin on the slow-wave activity (SWA) in the EEG during NREMS, on the durations of NREMS and REMS, and on cortical brain temperature (Tcort) in young rats (n 5 47). Open symbols: i.p. vehicle; closed symbols: i.p. albumin. For SWA, the percent difference from baseline (vehicle) is provided for each recording hour. I.p. injections were performed 1 h before light onset, and the recording occurred during the 12-h light period.
Albumin also significantly affected NREMS-associated SWA (Fig. 1) [treatment effect: F(1, 46) 5 4.175, p 5 0.047]. SWA varied across the 12-h light period [F(11, 506) 5 10.238, p , 0.001]. The significant interaction between the time and treatment factors [F(11, 506) 5 5.125, p , 0.001] indicated that the albu-
The results indicate that preparations of serum and albumin selectively promote NREMS in the young rat. Sera contain various growth factors, endogenous immunostimulants, and hormones which may enhance sleep. Albumin is a general carrier protein for many hormones, therefore the albumin preparation may contain these hormones in relatively high concentrations. For example, an opioid-like hibernation trigger substance has been isolated from the plasma of hibernating animals that is bound to albumin (3). Steroid hormones bind to albumin, and steroid hormones or their metabolites, like pregnenolone, promote NREMS (21,35). Further, contamination of the albumin preparation by some exogenous sleep-promoting material may also be the cause of increased sleep. Bacterial cell wall pyrogens, endotoxin or muramyl peptides, enhance NREMS and elicit fever (16). Albumin in high concentrations, however, may be intrinsically pyrogenic. Systemic administration of decontaminated bovine plasma albumin elicits fe-
´ L, KAPA ´ S, AND KRUEGER OBA
264 TABLE 1
EFFECTS OF INTRAPERITONEAL PROTEIN INJECTION ON SLEEP (% RECORDING TIME, MEAN 6 SE) DURING THE 12-H LIGHT PERIOD
Group
1
2 3
Albumin Albumin, female Albumin, male Albumin, S. diet Albumin, HP Diet Serum Hypertonic NaCl
n
NREMS Control
EXP
p
REMS Control
EXP
p
47 23 24 12 11 20 7
46.7 6 0.65 46.5 6 0.97 46.8 6 0.87 44.3 6 1.26 45.8 6 1.05 46.9 6 0.82 45.5 6 2.07
53.4 6 1.10 54.7 6 1.78 52.2 6 1.25 54.9 6 2.49 54.6 6 2.96 49.2 6 0.75 42.6 6 2.62
,0.001 ,0.001 ,0.001 ,0.001 ,0.009 ,0.001 ns
12.9 6 0.42 12.9 6 0.55 12.9 6 0.63 12.8 6 0.71 13.2 6 0.87 13.1 6 0.67 13.9 6 1.09
11.7 6 0.60 12.5 6 0.80 11.2 6 0.91 10.3 6 1.16 10.4 6 1.12 12.3 6 0.51 12.5 6 1.08
,0.038 ns ,0.042 ,0.049 ,0.025 ns ns
p, two-way ANOVA for repeated measures, treatment effects; S, standard rat food; HP, high protein (boiled egg) diet 1 standard rat food; ns, not significant.
ver in rabbits (23). The mechanism of the albumin-induced fever is not known, one possibility is that high concentrations of albumin can activate macrophages eliciting the production of endogenous pyrogens, i.e., cytokines such as interleukin-1 and tumor necrosis factor. Both interleukin-1 (17) and tumor necrosis factor (12) are somnogenic. Tcort, in fact, tended to increase in our rats, and this may result from fever elicited by cytokines. Some features of the sleep and temperature responses to albumin were, however, different from those induced by cytokines. Thus, the rises in Tcort were small and biphasic. After albumin, the most prominent enhancements in NREMS occurred prior to the rise in Tcort, whereas the development of fever usually precedes the increments in NREMS after the injection of exogenous or endogenous pyrogens. NREMS is associated with decreases in Tcort and body temperature in the rat (30). Although brain temperature changes coupled to sleep states persist during fever (37) the magnitude of these changes is many-fold less than the rise in Tcort during fever and it is unlikely that the enhanced NREMS delayed fever in our exper-
FIG. 2. Concentrations of plasma CCK-like immunoreactivity following i.p. injections of isotonic saline (controls, open columns) or 140 mg of albumin (closed columns) in young rats. Sample sizes in the control and albumin groups were as follows. Hour 1: n 5 12 rats sacrificed 30 min (n 5 6) and 60 min (n 5 6) postinjection; Hour 2: n 5 20 rats sacrificed at 80 min (n 5 7), 100 min (n 5 7), and 120 min (n 5 6) postinjection; Hour 3: n 5 20 rats sacrificed at 140 min (n 5 6), 160 min (n 5 7), and 180 min (n 5 7) postinjection. Asterisk (p) denotes significant differences between the control and the albumin groups (Student’s t-test following ANOVA).
iments. Finally, pyrogens stimulate NREMS predominantly during the dark period in the rat (20,34), whereas albumin promoted NREMS during the light period. In addition to cytokines, therefore, other mechanisms should also be considered to explain the effects of albumin on sleep. The modest increases in Tcort may result from the thermogenic effects of nutrients. The thermogenic action of a meal depends on the caloric value (39). The hyperthermia following protein ingestion lasts for several hours and is attributed, in part, to the stimulation of cellular metabolism by amino acids released from protein. Heat load stimulates NREMS in the young rat (28). Heat is regarded as an input for physiological sleep regulation (22,31). Because the onset of postprandial sleep is associated with the peak of the meal-induced thermogenesis, it is proposed that the rise of body temperature may act as a stimulus for enhanced sleep after food intake (39). In our experiments, however, the changes in Tcort and NREMS did not correlate, and, therefore, it is not likely that the rise in body temperature plays a major role in the albumininduced enhancements in NREMS. In contrast, the tendency to a suppression of REMS, which occurred during the period of hyperthermia, might be related to the changes in Tcort. A close relationship has been suggested between protein metabolism and NREMS. Sleep is linked to enhanced protein anabolism (1), and, in particular, the occurrence of NREMS is associated with increased rates of cerebral protein synthesis in the rat (32) and monkey (25). During fasting, body proteins are initially spared at the expense of lipids. This period is characterized by enhancements in NREMS (7,8). NREMS decreases during the subsequent phase of fasting when protein utilization increases. Protein anabolism and the duration of the sleep cycles correlate in prepubertal children who are on total parenteral nutrition (33). Nicolaidis and Danguir (26) suggest that sleep depends on the availability of nutrients in the body. Meal size correlates with the duration of postprandial sleep; this correlation is particularly strong about 2 h after eating when the nutrients are already absorbed. Rats infused with a highly nutritive solution (proteins 1 carbohydrates 1 lipids) exhibit enhanced NREMS and REMS but infusion of pure amino acid solutions stimulates only REMS (5). Similarly to our findings in young rats, infusion of amino acids acutely stimulates deep NREMS and suppresses REMS in humans (18). In addition to the general nutritional value of proteins, particular amino acids may have a special importance in the sleep-promoting effects of albumin and serum. For example, increases in NREMS and EEG SWA and decreases in REMS were observed after administration of tryptophane in humans (38). Arginine stimulates the somatotropic axis (11) and growth hormone-
ALBUMIN STIMULATES SLEEP
265
releasing hormone enhances NREMS (10,27). The duration of sleep is also enhanced in transgenic mice with excess growth hormone (19). Gastrointestinal hormones are assumed to play a key role in the mechanisms of postprandial sleep. For example, insulin enhances NREMS (6). CCK is another gastrointestinal hormone with sleeppromoting activity. In rats, CCK is released in response to proteins in the gastrointestinal tract. The plasma concentration of CCK-LI rose after i.p. albumin in our experiments. CCK suppresses motor activity (4), selectively stimulates NREMS in rats (14) and rabbits (15), and decreases brain and body temperature (13,14). CCK release, therefore, may also explain the initial, slight decreases in Tcort after albumin injection. We found a negative correlation between the albumin-induced enhancement in NREMS and the body weight of the rats. It is possible that the sleep-promoting effects of albumin are associated with the nutritional status of the young rats. Signs of malnutrition, however, were not observed. Each rat gained significant weight between the surgery and the recording, and the albumin-induced changes in sleep did not differ between the rats on normal diet and the ones provided with boiled egg. Nevertheless, albumin failed to promote sleep in young rats with body weight above 90 g. Also, enhancements in sleep were not found in adult rats (approx. 300 g
body weight) which were injected with a dose 4 times larger than that used in the young rats (unpublished observations). When calculating the injected quantity of protein with respect to the body weight, however, the dose of albumin was smaller in the adult rats than in the young rats. The negative correlation between the promotion of NREMS and the body weight of the young rats may also be interpreted as a manifestation of dose-response relationships because each rat received 140 mg of albumin irrespective of body weight. In conclusion, increases in the duration and intensity of NREMS were observed in young rats in response to a single injection of albumin or sera. The mechanism of the albumininduced sleep promotion is not known. The nutritional or caloric value of the injected protein, or endogenous humoral mechanisms, such as cytokine and CCK release and activation of the somatotropic axis, may be involved in the effects of albumin on sleep. ACKNOWLEDGEMENTS
We thank J. Gardi for the determination of the osmolarity of the solutions, and I. Ponicsa´n and Sz. Toth for the technical assistance. This work was supported by the Hungarian Science Foundation, (OTKA16080), the Ministry of Welfare (ETT 627/1996 – 04), and the National Institutes of Health (NS-30514, NS-27250, and NS-31453).
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thionine elicit selective increases in REM sleep in rabbits. Brain Res. 490:292–300; 1989. Oba´l, F., Jr.; Rubicsek, G.; Alfo¨ldi, P.; Sa´ry, G.; Oba´l, F. Changes in the brain and core temperatures in relation to the varioius arousal states in the light and dark periods of the day. Pflu¨gers Arch. 404:73–79; 1985. Oba´l, F., Jr. Thermoregulation and sleep. Exp. Brain Res. 8S:157–172; 1984. Ramm, P.; Smith, C. T. Rates of cerebral protein synthesis are linked to slow wave sleep in the rat. Physiol. Behav. 48:749 –753; 1990. Salzarulo, P.; Fagioli, I. Sleep for development or development for waking? Some speculations from a human perspective. Behav. Brain Res. 69:23–27; 1995. Shoham, S.; Krueger, J. M. Muramyl dipeptide-induced sleep and fever: Effects of ambient temperature and time of injections. Am. J. Physiol. 255:R157–R165; 1988. Steiger, A.; Traschel, L.; Guldner, J.; Hemmeter, U.; Rothe, B.; Rup-
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PII S0031-9384(98)00074-2
Albumin Enhances Sleep in the Young Rat ´ L, JR,* L. KAPA ´ S† AND J. M. KRUEGER1‡ F. OBA *Department of Physiology, A. Szent-Gyo¨rgyi Medical University, Szeged, Hungary; †Department of Biological Sciences, Fordham University, Bronx, NY; and ‡Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA Received 1 August 1997; Accepted January 28, 1998 ´ L, F., JR., L. KAPA ´ S AND J. M. KRUEGER. Albumin enhances sleep in the young rat. PHYSIOL BEHAV 64(3) 261–266, OBA 1998.—Rats 4 to 7 days after weaning received intraperitoneal (i.p.) injections of vehicle (baseline day), and either serum (2 mL of lyophilized rabbit serum), 140 mg of rat albumin, or hyperosmotic NaCl (experimental day). Injections were given 1 h before light onset. Sleep-wake activity and cortical brain temperature were recorded during the subsequent 12-h light period. The intensity of non-rapid eye movement sleep (NREMS) was characterized by the power density values of the electroencephalogram slow-wave activity. The sera and albumin preparations enhanced both NREMS and slow-wave activity for 5 to 6 h starting during Hour 2 after light onset. Rapid eye movement sleep (REMS) tended to decrease. Modest (0.6°C maximum deviation) biphasic changes were observed in cortical brain temperature with initial decreases for 3 h followed by rises between Hours 3 and 9 of the light period. There were no differences in the sleep responses to albumin between male and female rats. Albumin also enhanced NREMS in young rats on a protein-rich diet. A significant negative correlation was found between the NREMS promoting activity of albumin injections and the body weight of the rats. NaCl solution with the same osmolarity as that of the albumin solution failed to alter sleep. I.p. albumin injection elicited significant increases in the concentrations of cholecystokinin-like immunoreactivity in the plasma. Sleep-promoting materials (hormones) in the albumin fraction, the calorigenic or nutritional value of proteins, the release of somnogenic cytokines by albumin, or endogenous humoral mechanisms stimulated by proteins (e.g., cholecystokinin or the somatotropic axis) might mediate the enhanced sleep after albumin. © 1998 Elsevier Science Inc. Sleep
Proteins
Plasma
Albumin
Cholecystokinin (CCK)
VARIOUS endogenous substances promote sleep. Some of these substances are found in body fluids such as the blood. For example, the delta sleep-inducing peptide (DSIP) was isolated from dialysates of blood (24). The plasma carries various hormones and immunostimulants which may also influence sleep: systemic growth hormone (9), prolactin (29), steroid hormones (21,35), chorionic gonadotropin, or luteotropic hormone (36) can alter sleep. We report here that concentrated preparations of whole sera and the albumin fraction of the serum also possess sleep-promoting activity in young rats.
were weaned at the age of 21 days. The surgeries were carried out between Days 21 and 24 of life. Using pentobarbital anesthesia (50 mg/kg), jewelry screws were implanted over the frontal and parietal cortices and the cerebellum for recording the electroencephalogram (EEG), and a thermistor was placed over the parietal cortex to measure cortical brain temperature (Tcort). The body weight of the rats was 31–74 g (mean 6 SE: 46.8 6 1.0 g) at the time of the surgery and 45–105 g (mean 6 SE: 70.9 6 2.3 g) at the termination of the experiments. After the surgeries, the rats were housed in individual Plexiglas recording cages placed in the recording room. In the room, the ambient temperature was maintained at 26 6 1°C, and the same light-dark cycle was provided as that in the environmental room where the rats were bred. Standard rat food and water were continuously available. The rats were connected to light flexible recording tethers made from flatband computer cables. The tethers were attached to electronic commutators, thus the rats were free to move around. Before recording, the rats were allowed 4 to 6 days for recovery and habituation to the environment.
METHODS
Materials Rat albumin was obtained from Sigma, and a radioimmunoassay kit for unsulfated CCK26 –33 was from Peninsula Laboratories, Inc., Belmont, CA. Animals The surgery, the experimental conditions, and recording procedures were similar to those reported previously in studies on young rats (28). Sprague–Dawley rats were bred in an environmental room at an ambient temperature between 22 and 24°C, and with a 12 h:12 h light/dark cycle (light onset: 0830 hours). The rats
Recordings The EEG, Tcort, and motor activity (signals generated in electromagnetic transducers activated by the movements of the recording tethers) were collected by computers (64-Hz sampling rate).
1 Requests for reprints should be addressed to J. M. Krueger, 205 Wegner Hall, Washington State University, Pullman, WA 99164-6520. E-mail: [email protected]
261
´ L, KAPA ´ S, AND KRUEGER OBA
262 The states of vigilance were determined visually for consecutive 8-s epochs according to standard criteria: non-rapid eye movement sleep (NREMS): high-amplitude EEG slow waves, lack of body movement, declining Tcort upon entry; rapid eye movement sleep (REMS): highly regular theta activity in the EEG, general lack of body movements with occasional twitches, and a rapid increase in Tcort at onset; wakefulness: EEG activities similar to, but often less regular and with lower amplitude than those in REMS, frequent body movements, and a gradual increase in Tcort after arousal. The percentage time spent in each state of vigilance over 1-h periods, and for the 12-h light period was determined. The Tcort values were averaged for 1-h periods. Power density spectra were calculated by fast Fourier transformation of the EEG for consecutive 8-s epochs in the frequency range 0.25–20 Hz for 0.25-Hz bands. The power density values for the 0.25– 4 Hz (delta) frequency range were integrated and used as an index of EEG slow-wave activity (SWA). Mean SWA during NREMS was determined by using SWA values obtained in uninterrupted periods of artifact-free NREMS epochs in each hour of recording. The percentage changes between the power density values on Day 1 (baseline day) and Day 2 (experimental day) were calculated for each hour and for each rat. Experimental Protocol The effects of i.p. administration of rat albumin on sleep. The animals (Group 1, n 5 47; 24 males and 23 females) were injected with isotonic NaCl on Day 1 and with rat albumin (140 mg in 0.5 mL of isotonic NaCl) on Day 2, 1 h before light onset. The recording started at light onset and continued for 12 h; recording from six of these animals started immediately after the injections. Out of the 47 animals, eleven rats were provided with a protein rich diet for 2 days before the recordings and also during the 2 days of recording (in addition to the food pellets, boiled eggs were placed in the cages every morning and evening). For control, twelve of their litter-mates were fed the same standard diet as the rest of the rats in the albumin group. The effects of i.p. administration of rabbit serum on sleep (Group 2). Twenty rats (6 males and 14 females) were injected i.p. with isotonic NaCl on Day 1. On Day 2, the animals received rabbit serum i.p. The serum was obtained by taking blood from the ear of unanesthetized rabbits. For each dose, 2 mL of rabbit serum was lyophilized and dissolved in 0.5 mL of isotonic NaCl. The rats received the injections 1 h before light onset. The recordings started at light onset and continued for 12 h. The effects of i.p. administration of hypertonic NaCl solution on sleep. To test if the actions of serum and albumin on sleep are due to the osmotic activity of the solutions, the effects of a NaCl solution of an equal osmotic concentration were also studied. The osmolarity of the albumin solution (as determined by the depression of the freezing point) corresponded to that of a NaCl solution containing 1.3 g NaCl in 100 mL of water. Seven rats (Group 3, three males and four females) received isotonic NaCl on Day 1 and hypertonic NaCl solution i.p. on Day 2 1 h before light onset. Recordings started at dark onset and lasted for 12 h. The effects of i.p. Administration of albumin on plasma cholecystokinin (CCK)-like immunoreactivity (CCK-LI). Eight groups of male rats (45–50 g) were injected with isotonic NaCl and eight groups of rats with 140 mg of rat albumin, i.p. One group of either treatment was sacrificed by decapitation 30 min (n 5 6, each group), 60 min (n 5 6), 80 min (n 5 7), 100 min (n 5 7), 120 min (n 5 6), 140 min (n 5 6), 160 min (n 5 7), and 180 min (n 5 7) postinjection. The trunk blood was collected and centrifuged. CCK-LI was measured in triplicate by radioimmunoassay. According to the manufacturer (Peninsula Laboratories, Inc., Belmont,
CA, USA), the antiserum revealed crossreactivities as follows: unsulfated CCK 26 –33: 100%, sulfated CCK 26 –33: 78%, human gastrin I 100%, human pancreatic polypeptide: , 0.1%, and vasoactive intestinal peptide: 0%. The plasma concentrations of CCK-LI measured at the various time points were pooled as follows: 30 and 60 min postinjection 5 Hour 1; 80, 100, and 120 min postinjection 5 Hour 2; and 140, 160, and 180 min postinjection 5 Hour 3. Statistical Analysis The differences in NREMS, REMS, SWA during NREMS, and Tcort between the baseline day and the experimental day were evaluated by two-way ANOVA for repeated measures. The treatment-effects (differences between the 2 days), and the time effects (variations during the 12-h recording period) were the factors of the ANOVA. Due to malfunction of several thermistors, the results of the Tcort were calculated from 27 rats in Group 1. Two-way ANOVA for independent samples (factors: treatment and time) was used to compare CCK-LI in the control and albumin-injected rats. Two-way ANOVA was also used to determine whether the sleep states differed between the rats injected with albumin and hypertonic NaCl solution on both the baseline and the experimental days (group effect: independent samples; time effects: repeated measures). An a-level of p , 0.05 was considered significant in all tests. RESULTS
The Effects of Albumin In Groups 1 and 2, after control injections, the expected diural variation in sleep was observed; e.g., the time spent in NREMS was relatively high at the beginning of the light period and declined continuously across the 12-h recording [time effect in ANOVA; Group 1: F(11, 506) 5 16.723, p , 0.001); Group 2: F(1, 209) 5 15.167, p , 0.001]. The percentages and the distributions of the sleep states on the baseline day corresponded to those reported by Alfo¨ldi et al. (2) for young rats. In Group 1, albumin injection significantly promoted NREMS. After albumin injection, NREMS started to increase above the control level in Hour 2 of the recording and was elevated for 4 to 5 h (Fig. 1). Sleep was not altered during the hour preceding light onset as determined in 6 rats [mean 6 SE, NREMS after isotonic NaCl: 29.9 6 4.4; NREMS after albumin: 33.1 6 4.2; REMS after isotonic NaCl: 10.0 6 2.8; and REMS after albumin: 7.5 6 2.3]. The promotion of sleep after albumin treatment, therefore, had a latency of about 2 h. I.p. injection of albumin failed to enhance REMS; in fact, a tendency to a suppression in REMS was observed in several groups (Table 1). These changes in REMS were small (Fig. 1) and barely reached the level of statistical significance. In Group 1, there were no differences between the male and female rats in the NREMS responses to albumin (Table 1). Also, the albumin stimulated NREMS responses in the rats maintained on the normal diet and in the rats that were provided with proteinrich diet were similar. Promotion of NREMS by albumin, however, depended on the body weight of the animals. After pooling the data obtained in the rats injected with albumin or rabbit serum preparations together, a correlation was calculated between the body weight at the end of the recordings (independent variable) and the changes in the duration of NREMS during the 12-h recording period (dependent variable). A significant negative correlation (20.591, p , 0.001) was obtained, indicating that albumin was less effective in enhancing NREMS in rats with larger body weights than in the smaller rats. Albumin did not promote NREMS in rats of 90 g or above.
ALBUMIN STIMULATES SLEEP
263 min-induced changes were the function of time. Indeed, SWA during NREMS was enhanced in Hour 2 of the recording, then declined toward or below the baseline values (Fig. 1). Injection of albumin was followed by biphasic changes in Tcort [treatment effect: F(1, 26) 5 9.784, p , 0.004, time effect: F(11, 286) 5 11.427, p , 0.001, interaction: F(11, 286) 5 18.133, p , 0.001]. Tcort was significantly below baseline in Hours 1 and 2 of the recording, then Tcort increased from Hours 3 to 8. The maximum deviation from baseline was 0.6°C (Fig. 1). The albumininduced variations in Tcort did not correlate with the time course of the changes in NREMS. REMS, however, tended to decrease during the period of hyperthermia. The Effects of Rabbit Serum; Group 2 The sleep, SWA, and Tcort alterations elicited by i.p. rabbit serum were similar to those found after i.p. injection of albumin. The duration of NREMS increased whereas REMS did not change during the 12-h light period (Table 1). The Effects of Hypertonic NaCl; Group 3 Injection of hyperosmotic saline solution did not alter NREMS (Table 1.), SWA, or Tcort (data not shown). Although REMS tended to decrease, the changes did not reach the level of statistical significance. Statistical comparisons between NREMS in the rats injected with albumin (Group 1) and hypertonic NaCl supported that albumin enhanced NREMS [group effect: F(1, 52) 5 12.028, p , 0.001]. NREMS varied across the 12-h recording period [time effect: F(11, 572) 5 5.147, p , 0.001], and the difference between the two groups also varied with the time [group 3 time interaction: F(11, 572) 5 2.397, p , 0.007]. there were no intergroup differences in NREMS on the baseline days and in REMS on either the baseline or experimental day. The Effects of Albumin on Plasma CCK-LI The plasma concentration of CCK-LI varied significantly during the 3-h postinjection period [F(2,98) 5 5.768, p , 0.05]. I.p. injection of albumin elicited a significant a rise in CCK-LI [F(1, 98) 5 5.891, p , 0.05]. This increase was already obvious at 30 min postinjection [1.6 6 0.12 ng/mL vs. 2.09 6 0.17 ng/mL] and persisted in the samples collected 60 min after the injection resulting in a significant difference (Student’s t-test) between the control and the albumin groups in postinjection Hour 1 (Fig. 2). Concentrations of CCK-LI in the rats injected with albumin gradually approached the control values in postinjection Hour 2, and the difference between the groups vanished in Hour 3. DISCUSSION
FIG. 1. Effects of an i.p. injection of 140 mg of albumin on the slow-wave activity (SWA) in the EEG during NREMS, on the durations of NREMS and REMS, and on cortical brain temperature (Tcort) in young rats (n 5 47). Open symbols: i.p. vehicle; closed symbols: i.p. albumin. For SWA, the percent difference from baseline (vehicle) is provided for each recording hour. I.p. injections were performed 1 h before light onset, and the recording occurred during the 12-h light period.
Albumin also significantly affected NREMS-associated SWA (Fig. 1) [treatment effect: F(1, 46) 5 4.175, p 5 0.047]. SWA varied across the 12-h light period [F(11, 506) 5 10.238, p , 0.001]. The significant interaction between the time and treatment factors [F(11, 506) 5 5.125, p , 0.001] indicated that the albu-
The results indicate that preparations of serum and albumin selectively promote NREMS in the young rat. Sera contain various growth factors, endogenous immunostimulants, and hormones which may enhance sleep. Albumin is a general carrier protein for many hormones, therefore the albumin preparation may contain these hormones in relatively high concentrations. For example, an opioid-like hibernation trigger substance has been isolated from the plasma of hibernating animals that is bound to albumin (3). Steroid hormones bind to albumin, and steroid hormones or their metabolites, like pregnenolone, promote NREMS (21,35). Further, contamination of the albumin preparation by some exogenous sleep-promoting material may also be the cause of increased sleep. Bacterial cell wall pyrogens, endotoxin or muramyl peptides, enhance NREMS and elicit fever (16). Albumin in high concentrations, however, may be intrinsically pyrogenic. Systemic administration of decontaminated bovine plasma albumin elicits fe-
´ L, KAPA ´ S, AND KRUEGER OBA
264 TABLE 1
EFFECTS OF INTRAPERITONEAL PROTEIN INJECTION ON SLEEP (% RECORDING TIME, MEAN 6 SE) DURING THE 12-H LIGHT PERIOD
Group
1
2 3
Albumin Albumin, female Albumin, male Albumin, S. diet Albumin, HP Diet Serum Hypertonic NaCl
n
NREMS Control
EXP
p
REMS Control
EXP
p
47 23 24 12 11 20 7
46.7 6 0.65 46.5 6 0.97 46.8 6 0.87 44.3 6 1.26 45.8 6 1.05 46.9 6 0.82 45.5 6 2.07
53.4 6 1.10 54.7 6 1.78 52.2 6 1.25 54.9 6 2.49 54.6 6 2.96 49.2 6 0.75 42.6 6 2.62
,0.001 ,0.001 ,0.001 ,0.001 ,0.009 ,0.001 ns
12.9 6 0.42 12.9 6 0.55 12.9 6 0.63 12.8 6 0.71 13.2 6 0.87 13.1 6 0.67 13.9 6 1.09
11.7 6 0.60 12.5 6 0.80 11.2 6 0.91 10.3 6 1.16 10.4 6 1.12 12.3 6 0.51 12.5 6 1.08
,0.038 ns ,0.042 ,0.049 ,0.025 ns ns
p, two-way ANOVA for repeated measures, treatment effects; S, standard rat food; HP, high protein (boiled egg) diet 1 standard rat food; ns, not significant.
ver in rabbits (23). The mechanism of the albumin-induced fever is not known, one possibility is that high concentrations of albumin can activate macrophages eliciting the production of endogenous pyrogens, i.e., cytokines such as interleukin-1 and tumor necrosis factor. Both interleukin-1 (17) and tumor necrosis factor (12) are somnogenic. Tcort, in fact, tended to increase in our rats, and this may result from fever elicited by cytokines. Some features of the sleep and temperature responses to albumin were, however, different from those induced by cytokines. Thus, the rises in Tcort were small and biphasic. After albumin, the most prominent enhancements in NREMS occurred prior to the rise in Tcort, whereas the development of fever usually precedes the increments in NREMS after the injection of exogenous or endogenous pyrogens. NREMS is associated with decreases in Tcort and body temperature in the rat (30). Although brain temperature changes coupled to sleep states persist during fever (37) the magnitude of these changes is many-fold less than the rise in Tcort during fever and it is unlikely that the enhanced NREMS delayed fever in our exper-
FIG. 2. Concentrations of plasma CCK-like immunoreactivity following i.p. injections of isotonic saline (controls, open columns) or 140 mg of albumin (closed columns) in young rats. Sample sizes in the control and albumin groups were as follows. Hour 1: n 5 12 rats sacrificed 30 min (n 5 6) and 60 min (n 5 6) postinjection; Hour 2: n 5 20 rats sacrificed at 80 min (n 5 7), 100 min (n 5 7), and 120 min (n 5 6) postinjection; Hour 3: n 5 20 rats sacrificed at 140 min (n 5 6), 160 min (n 5 7), and 180 min (n 5 7) postinjection. Asterisk (p) denotes significant differences between the control and the albumin groups (Student’s t-test following ANOVA).
iments. Finally, pyrogens stimulate NREMS predominantly during the dark period in the rat (20,34), whereas albumin promoted NREMS during the light period. In addition to cytokines, therefore, other mechanisms should also be considered to explain the effects of albumin on sleep. The modest increases in Tcort may result from the thermogenic effects of nutrients. The thermogenic action of a meal depends on the caloric value (39). The hyperthermia following protein ingestion lasts for several hours and is attributed, in part, to the stimulation of cellular metabolism by amino acids released from protein. Heat load stimulates NREMS in the young rat (28). Heat is regarded as an input for physiological sleep regulation (22,31). Because the onset of postprandial sleep is associated with the peak of the meal-induced thermogenesis, it is proposed that the rise of body temperature may act as a stimulus for enhanced sleep after food intake (39). In our experiments, however, the changes in Tcort and NREMS did not correlate, and, therefore, it is not likely that the rise in body temperature plays a major role in the albumininduced enhancements in NREMS. In contrast, the tendency to a suppression of REMS, which occurred during the period of hyperthermia, might be related to the changes in Tcort. A close relationship has been suggested between protein metabolism and NREMS. Sleep is linked to enhanced protein anabolism (1), and, in particular, the occurrence of NREMS is associated with increased rates of cerebral protein synthesis in the rat (32) and monkey (25). During fasting, body proteins are initially spared at the expense of lipids. This period is characterized by enhancements in NREMS (7,8). NREMS decreases during the subsequent phase of fasting when protein utilization increases. Protein anabolism and the duration of the sleep cycles correlate in prepubertal children who are on total parenteral nutrition (33). Nicolaidis and Danguir (26) suggest that sleep depends on the availability of nutrients in the body. Meal size correlates with the duration of postprandial sleep; this correlation is particularly strong about 2 h after eating when the nutrients are already absorbed. Rats infused with a highly nutritive solution (proteins 1 carbohydrates 1 lipids) exhibit enhanced NREMS and REMS but infusion of pure amino acid solutions stimulates only REMS (5). Similarly to our findings in young rats, infusion of amino acids acutely stimulates deep NREMS and suppresses REMS in humans (18). In addition to the general nutritional value of proteins, particular amino acids may have a special importance in the sleep-promoting effects of albumin and serum. For example, increases in NREMS and EEG SWA and decreases in REMS were observed after administration of tryptophane in humans (38). Arginine stimulates the somatotropic axis (11) and growth hormone-
ALBUMIN STIMULATES SLEEP
265
releasing hormone enhances NREMS (10,27). The duration of sleep is also enhanced in transgenic mice with excess growth hormone (19). Gastrointestinal hormones are assumed to play a key role in the mechanisms of postprandial sleep. For example, insulin enhances NREMS (6). CCK is another gastrointestinal hormone with sleeppromoting activity. In rats, CCK is released in response to proteins in the gastrointestinal tract. The plasma concentration of CCK-LI rose after i.p. albumin in our experiments. CCK suppresses motor activity (4), selectively stimulates NREMS in rats (14) and rabbits (15), and decreases brain and body temperature (13,14). CCK release, therefore, may also explain the initial, slight decreases in Tcort after albumin injection. We found a negative correlation between the albumin-induced enhancement in NREMS and the body weight of the rats. It is possible that the sleep-promoting effects of albumin are associated with the nutritional status of the young rats. Signs of malnutrition, however, were not observed. Each rat gained significant weight between the surgery and the recording, and the albumin-induced changes in sleep did not differ between the rats on normal diet and the ones provided with boiled egg. Nevertheless, albumin failed to promote sleep in young rats with body weight above 90 g. Also, enhancements in sleep were not found in adult rats (approx. 300 g
body weight) which were injected with a dose 4 times larger than that used in the young rats (unpublished observations). When calculating the injected quantity of protein with respect to the body weight, however, the dose of albumin was smaller in the adult rats than in the young rats. The negative correlation between the promotion of NREMS and the body weight of the young rats may also be interpreted as a manifestation of dose-response relationships because each rat received 140 mg of albumin irrespective of body weight. In conclusion, increases in the duration and intensity of NREMS were observed in young rats in response to a single injection of albumin or sera. The mechanism of the albumininduced sleep promotion is not known. The nutritional or caloric value of the injected protein, or endogenous humoral mechanisms, such as cytokine and CCK release and activation of the somatotropic axis, may be involved in the effects of albumin on sleep. ACKNOWLEDGEMENTS
We thank J. Gardi for the determination of the osmolarity of the solutions, and I. Ponicsa´n and Sz. Toth for the technical assistance. This work was supported by the Hungarian Science Foundation, (OTKA16080), the Ministry of Welfare (ETT 627/1996 – 04), and the National Institutes of Health (NS-30514, NS-27250, and NS-31453).
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