Brain Research, 557 (1991) 149-153 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391169004
Inhibition of growth hormone-releasing factor suppresses both sleep and growth hormone secretion in the rat E Obfil Jr. 1'2, L. Payne 1, L. Kapfis 1, M. Opp I and J. M. Krueger 1 l Department of Physiology and Biophysics, University of Tennessee, Memphis (U.S.A.) and 2Department of Physiology, A. Szent-Gy6rgyi, Medical University, Szeged (Hungary) (Accepted 2 April 1991)
Key words: Growth hormone-releasing factor; Growth hormone-releasing hormone; Growth hormone; Sleep
To study the possible involvement of hypothalamic growth hormone-releasing factor (GRF) in sleep regulation, a competitive GRF-antagonist, the peptide (N-Ac-Tyr~,D-Arg2)-GRF(1-29)-NH2, was intracerebroventricularly injected into rats (0.003, 0.3, and 14 nmol), and the EEG and brain temperature were recorded for 12 h during the light cycle of the day. Growth hormone (GH) concentrations were determined from plasma samples taken at 20-min intervals for 3 h after 14 nmol GRF-antagonist. The onset of non-rapid eye movement sleep (NREMS) was delayed in response to 0.3 and 14 nmol GRF-antagonist, the duration of NREMS was decreased for one or more hours and after 14 nmol EEG slow wave amplitudes were decreased during NREMS in postinjection hour 1. The high dose of GRF-antagonist also suppressed REMS for 4 h, inhibited GH secretion, and elicited a slight biphasic variation in brain temperature. These findings, together with previous observations indicating a sleep-promoting effect for GRF, support the hypothesis that hypothalamic GRF is involved in sleep regulation and might be responsible for the correlation between NREMS and GH secretion reported in various species. INTRODUCTION A positive correlation b e t w e e n growth h o r m o n e ( G H ) secretion and the onset of d e e p non-rapid eye m o v e m e n t sleep ( N R E M S ) characterized by intense electroencephalographic ( E E G ) slow-wave activity has been r e p o r t e d in m a n y m a m m a l s (reviewed in ref. 22) including the rat 8-1°. Nevertheless, G H release and sleep may occasionally dissociate from one a n o t h e r 26. It was p r o p o s e d therefore several years ago that stimulation of G H secretion and p r o m o t i o n of N R E M S represent two i n d e p e n d e n t outputs of a c o m m o n mechanism 23. Through this c o m m o n mechanism, G H secretion and N R E M S are normally synchronized. H o w e v e r , one of the two outputs might be blocked i n d e p e n d e n t l y without interfering with the activity of the o t h e r function and thus dissociation m a y occur. Since the hypothalamic releasing factor for pituitary G H secretion, (growth hormonereleasing factor, G R F , also known as growth hormonereleasing h o r m o n e : G H R H ) , also p r o m o t e s sleep 5'19-21, especially N R E M S , hypothalamic G R F neurons m a y be part of the c o m m o n mechanism linking G H secretion and sleep 21. If e n d o g e n o u s G R F is involved in sleep regulation, then blocking G R F actions should suppress sleep. We
r e p o r t here that intracerebroventricular (i.c.v.) administration of a competitive G R F - r e c e p t o r antagonist, the p e p t i d e (N-Ac-Tyr1,D-Arg2)-GRF(1-29)-NH2, in fact, suppresses both sleep and G H secretion in the rat. MATERIALS AND METHODS
Animal surgery Male Sprague-Dawley rats weighing between 300 and 350 g were used. Rats were anesthetized using ketamine-xylazine anesthesia (87 and 13 mg/kg) and stainless steel jewelry screws were implanted into the skull over the parietal and occipital cortices and over the cerebellum. A thermistor (model 4018, Omega Engineering, Stamford, CT) placed over the parietal cortex served to measure brain (cortical) temperature (Tbr). A guide cannula was implanted into one of the lateral cerebral ventricles. The position of the cannula was verified during implantation by a sudden drop in resistance against the inflow of physiological saline. The placement of the cannula and the drainage of the lateral ventricle was verified by means of the drinking response elicited by i.c.v, injection of angiotensin (200 ng, 2 ~ui) a few days before and the day after the sleep and hormone experiments. Angiotensin injected i.c.v, elicits drinking in about 2 min via the stimulation of preoptic structures6. Finally, Trypan blue was injected into the cannula, and the ventricular system was checked for staining after sacrifice. Data were used only from those rats in which the angiotensin tests were positive and the postmortem examination of the brain confirmed the proper position of the cannula.
Sleep experiments The rats were housed in individual cages placed in environmental
Correspondence: J.M. Krueger, University of Tennessee, Memphis, Department of Physiology and Biophysics, 894 Union Avenue, Memphis, TN 38163, U.S.A.
150 chambers (Hotpack 352600, Philadelphia, PA, 2 cages per chamber). A 12-12 h light-dark cycle (light on from 00.80 to 20.00 h) was provided and ambient temperature was regulated at 24 _+ 1 °C. Food and water were continuously available. The rats were habituated to the experimental conditions for 5-7 days. During this period they were connected to the recording tethers which were attached to electronic swivels (Plastics One, Roanoke, VA). Artificial cerebrospinal fluid (aCSF) ts was injected daily between 07.50 and 08.00 h to habituate the rats to handling. The recording started with light onset at 08.00 and continued for 12 h. Some of the rats received i.c.v, aCSF on day 1 and GRFantagonist on day 2 whereas the order of the injections was reversed for the others. Ultrasonic motion detectors were used to record movements of the rats. Cables from the swivels and the motion detectors were connected to Grass 7D polygraphs in an adjacent room. The polygraphs recorded EEG, Tbr, and body movement for each animal. Additionally, the EEG for each rat was band-pass filtered with the 0.5-3.5 Hz (slow waves, delta), and 4.0--7.5 Hz (theta) frequency bands rectified by a Buxco 24 data logger (Buxco Electronics, Sharon, CT). The ratio of theta-to-delta activity was also simultaneously displayed with the EEG, Tbr, and movements on the paper chart. The polygraph records were visually scored in 12-s epochs for duration of the states of vigilance (wakefulness, (W); rapid-eye movement sleep, (REMS); NREMS) for hourly intervals. NREMS is associated with high-amplitude EEG slow waves, low theta-to-delta ratios, lack of body movements, and declining Tbr upon entry. REMS is distinguished by EEG voltages lower than in NREMS, high theta-to-delta ratios, general lack of body movement with occasional twitches, and a rapid increase of Tbr at onset. W is characterized by EEG amplitudes similar to those in REMS, midlevel theta-to-delta ratios, frequent body movements, and a gradual increase in Tbr after arousal. The duration of the states of vigilance was expressed as percent of time for each hour postinjection. The latency to NREMS or REMS was determined as the time from the onset of the recording to the first 1-min episode of uninterrupted NREMS and REMS, respectively. To determine whether the injections elicited obvious behavioral effects, e.g., abnormal behavioral patterns, the rats were watched for about 20 min after the injections through a closed-circuit television system with cameras installed in each recording chamber. The filtered and rectified EEG amplitude values recorded by the data logger were also used to characterize EEG slow wave activity; such activity is thought to be indicative of NREMS intensity 1. The average EEG voltages in the delta range were calculated for the 10 1-min periods of uninterrupted NREMS with the highest amplitudes for each rat and for each of the first 3 postinjection hours. TbrS were recorded at 10-min intervals by a data collecting system (Acrosystems 400, Beverly, MA). Using these values, the average Tbrs were calculated for each hour postinjection for each rat. The GRF-antagonist (Bachem, Torrance, CA) was dissolved in aCSE The doses tested were as follows: 0.003 nmol (n = 7), 0.3 nmol (n = 6), and 14 nmol (n = 11). The concentration of the solution was such that the volume injected was 4/~1 for each group of rats. The groups of rats were independent with the exception of 3 rats which received two doses each several days apart. Results from these animals were similar to those obtained from the other rats. Accordingly, the volume of the aCSF injection was also 4/al on the baseline day. The i.c.v, injections were delivered in about 1 min, and the injection cannula was left in the guide tube for another min.
exteriorized at the dorsum of the neck. Physiological saline containing 500 IU/ml heparin was used to fill and flush the catheter once a day. For 2-3 days, the rats were habituated to the experimental conditions similar to those of the sleep studies. The rats were housed in environmental cages. Each morning between 07.00 and 07.15 h, a Silastic extension tube was connected to the intracardial catheter and routed through the side of the recording chamber. In this way, blood samples were taken without disturbing the freely moving animals. The habituation procedure also included i.c.v, injections of aCSF immediately before light onset at 07.55 h. Blood samples, 0.1 ml each, were withdrawn every 20 min for 3 h following i.c.v, administration of either aCSF or GRF-antagonist. GH secretion after administration of aCSF or GRF-antagonist was determined on consecutive days; the order of the two injections varied with the animals. The samples were centrifuged, the red blood cells were resuspended in physiological saline and reinjected when the next blood sample was taken. The plasma was stored at -20 °C until GH levels were determined by RIA. GH was measured in duplicate samples using the reagents kindly provided by the NIDDK (Bethesda, MD) through the NHPP (University of Maryland School of Medicine) for rat GH-RIA. The inter- and intra-assay coefficients of variation were below 10%. The results were expressed in terms of the rat G H reference preparation-2 (rGH RP-2).
Statistical analysis Friedman's test for k-related samples was used to test for differences between the values obtained after aCSF and GRFantagonist across the recording period (12 h for the sleep and Tbr studies, and 9 samples in 3 h in the hormone experiments). The Friedman's test was also applied to reveal significant variations in GH concentrations during the 3 h following the injection of aCSF or GRF-antagonist. Significant alterations in the duration of the states of vigilance were identified by means of the Wilcoxon matched-pairs signed-ranks test in postinjection hour 1. The significance of the effects of GRF-antagonist on the latencies of NREMS and REMS, and on E E G slow wave amplitudes during NREM were also examined using the Wilcoxon-test. Each statistical test was two-tailed. An a level of P < 0.05 was accepted as indicating significant departure from control values. RESULTS
Effects on sleep-wake activity I.c.v. i n j e c t i o n o f 0.003 o r 0.3 n m o l G R F - a n t a g o n i s t did not alter significantly the duration of the states of vigilance across However,
12 h r e c o r d i n g
in r e s p o n s e t o 0.3 n m o l G R F - a n t a g o n i s t ,
s i g n i f i c a n t r e d u c t i o n in N R E M S
w a s o b s e r v e d in p o s t i n -
j e c t i o n h o u r 1, a n d N R E M S w a s b e l o w t h e b a s e l i n e level for a n o t h e r 2 h (Fig. 1) t h o u g h t h i s l a t t e r d e c r e a s e w a s n o t significant. T h e d e c r e a s e in N R E M S o c c u r r e d in s p i t e o f t h e fact t h a t in t h i s g r o u p o f r a t s t h e c o n t r o l v a l u e s f o r the duration of NREMS postinjection
w e r e r e l a t i v e l y l o w in t h e first EEG
slow w a v e
during NREMS were not altered after the administration
GH assay Plasma GH concentrations after i.c.v, injections of 14 nmol GRF-antagonist were determined. Seven rats were used in these experiments. Each rat had a cannula in the lateral cerebral ventricle; 4 of these rats had been previously involved in testing the sleep effects of the GRF-antagonist. Rats were anesthetized using ketamine-xylazine anesthesia, then were implanted with a Silastic intracardial catheter via the right jugular vein. The catheter was
o f e i t h e r 0.003 o r 0.3 n m o l G R F - a n t a g o n i s t A m a r k e d r e d u c t i o n in N R E M S
( T a b l e I).
w a s o b s e r v e d in t h e
r a t s i n j e c t e d w i t h 14 n m o l G R F - a n t a g o n i s t .
d e c r e a s e d b y a b o u t 5 0 % in p o s t i n j e c t i o n h o u r 1 (Fig. 1). Although duration of NREMS b a s e l i n e v a l u e in p o s t i n j e c t i o n
t e n d e d to a p p r o a c h t h e hour
2, it w a s a g a i n
151 GRF- ANTAGONIST
T T ' T ' T T I I
. , ;
/ .... 20.
i "'"'" ~I
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 01234s1~ I ' 8 9 ~ 0 II 9 ~ N ~ 01234s6789~n~
PO6TINJECTION HOURS Fig. 1. Effects of 3 doses of OR_F-antagonist on non-rapid eye
movement sleep (NREMS), rapid eye movement sleep (REMS) and brain temperature of rats. Each dose was injected intracerebroventricularly at time 0 at the beginning of daylight hours. Rats were then recorded from for the next 12 h. Control recordings (broken lines) were obtained from the animals after injection of artificial cerebrospinal fluid. The GRF-antagonist reduced NREMS and at the highest dose also reduced REMS (see text for details).
significantly suppressed in postinjection hour 3. The changes in N R E M S were mirrored by significant increases in W. R E M S was suppressed for 4 h. The reductions in N R E M S and R E M S and increases in W were significant across the 12-h recording period. This dose of GRF-antagonist also significantly reduced the amplitudes of E E G slow waves during N R E M S in postinjection hour 1 (Table I). The occurrences of the first episodes of N R E M S were significantly delayed after the i.c.v, administration of 0.3 and 14 nmol GRF-antagonist. The latency from the i.c.v. injection to the first episode of R E M S was significantly increased after the high dose of the GRF-antagonist
TABLE I Mean (+ S.E.M.) latencies of the occurrences of the first episodes of NREMS and REMS, and mean EEG slow wave amplitudes during NREMS in hour 1 after the administration of CSF or GRF-antagonist Latency (rain) NREMS
aCSF GRF-ant. 0.003 nmol aCSF GRF-ant. 0.3 nmol aCSF GRF-ant. 14nmol
7 12.9 + 2.33
45.7 + 2.93
15.9+3.76 60.4+13.12 6 13.2 + 1.86 47.9 + 7.23 19.2 _+2.72* 68.4 _+8.02 11 16.4 + 3.70 46.0 + 4.83 27.4 + 3.80* 105.4 _+19.18"
EEG-Slow wave amplitudes (#V)
162 + 13.8 170+17.8 145 + 19.1 142 _+17.9 171 + 7.4 139 _+7.5*
Fig. 2. Effects of 14 nmol of GRF-antagonist administered intracerebroventricularly on plasma levels of growth hormone (GH). After control injections of artificial cerebrospinal fluid (broken line), there was a spontaneous release of GH 80-140 min after injection. In contrast, after the GRF-antagonist (solid line), this surge in plasma GH levels was not observed.
(Table I). Qualitative increases in activity occurred after the injection of 14 nmol GRF-antagonist; animals displayed long periods of vigorous exploration, grooming and eating. Effects on brain temperature
I.c.v. injection of 0.003 and 0.3 nmol GRF-antagonist did not affect the courses of Tbr across the 12-h recording period (Fig. 1). In contrast, the 12-h Tbr curves after i.c.v, injections of 14 nmol GRF-antagonist differed significantly from that observed after aCSF in hours 2 - 4 postinjection. However, these differences were very small. Effects on G H secretion
G H plasma concentrations increased 80-140 min after i.c.v, injection of aCSF in each rat although the timing of the G H secretory pulse varied among the individual rats (Fig. 2). In response to 14 nmol GRF-antagonist, there were no significant variations in G H concentrations during the 3-h sampling period (Fig. 2). The G H concentrations after 14 nmol GRF-antagonist were significantly less than corresponding values obtained after injections of a C S E DISCUSSION The discovery of the GRF-antagonist activity of the peptide (N-Ac-Tyrl,D-Arg2)-GRF(1-29)-NH2 resulted from an experiment by Robberecht et al. 24 aiming to test the effects of various G R F analogs on cyclic A M P production in rat anterior pituitary homogenates. (NAc-Tyrl,D-ArgE)-GRF(1-29)-NH2 fails to stimulate ade-
152 nylate cyclase activity and competitively inhibits the effects of exogenous G R E In contrast, the peptide does not modify the vasoactive intestinal peptide-stimulated anterior pituitary adenylate cyclase activity indicating that the effect is specific for GRF-receptors 24. The GRF-antagonist activity of (N-Ac-Tyrl,o-Arg2)-GRF(1 29)-NH 2 was also demonstrated in vivo; after peripheral administration, the peptide suppresses spontaneous GH secretion z'H'~2, and attenuates somatic growth in immature rats lz'17. The effect of the GRF-antagonist seems to be specific for GH since prolactin release is not affected 11. I.c.v. injection of a small dose of the GRFantagonist was reported to block a central action of GRF, the activation of the ultrashort negative feedback loop, whereby GRF inhibits its own secretion 1~. Finally, the present findings indicated that after i.c.v, administration of 14 nmol GRF-antagonist, the concentration of the peptide reaching the pituitary somatotrophs was high enough to suppress endogenous GH secretion. Each measure used to characterize NREMS in this study evidenced a suppression by the GRF-antagonist; the latencies to the first episodes of NREMS increased and the duration of NREMS decreased for at least 1 h after 0.3 and 14 nmol GRF-antagonist, and the E E G slow wave amplitudes decreased after 14 nmol GRF-antagonist. These changes in NREMS mirrored those obtained in response to a comparable dose range of GRF (0.01, 0.1 and 1.0 nmol/kg, i.e., approx. 0.03, 0.3 and 3.0 nmol/ rat) 21. I.c.v. administration of GRF enhances NREMS duration for 1 h (or longer after the high dose) and increases the EEG slow wave amplitudes during NREMS. Besides inhibiting NREMS, i.c.v, injection of 14 nmol GRF-antagonist also suppressed REMS. In agreement with this finding, i.c.v, administration of GRF promotes REMS in rats and rabbits zl. The effects of GRF in rats on REMS, however, are less consistent than those on NREMS and the increases in REMS in response to GRF occur only in postinjection hour 2 in rabbits. It was speculated, therefore, that GRF is directly involved in NREMS regulation by acting on somnogenic structures in the preoptic region, whereas the promotion of REMS might be mediated through GH or somatostatin released by GRE Both GH 4'14'z5 and somatostatin 3 increase REMS without promoting NREMS in various species. The assumption that endogenous GRF is more closely linked to the regulation of NREMS than to REMS was also indicated by the differences in the responsiveness of these sleep states to the GRF-antagonist in the present experiment. Thus, i.c.v. injection of 0.3 nmol GRF-antagonist elicited a reduction in NREMS without affecting REMS. The high dose of the GRF-antagonist strongly suppressed both NREMS and REMS; this effect on REMS might be related to the inhibition of GH secretion.
It has been proposed that the GRF released in the median eminence inhibits further GRF secretion through an ultrashort negative feedback loop ~3'15. I.c.v. injections of low doses of the GRF-antagonist, e.g., doses comparable to the smallest dose in the present experiment, selectively inhibit the GRF receptor-mediated negative feedback without reaching the pituitary somatotrophs in a concentration necessary to block stimulation of GH by endogenous GRF 11 . As a result, instead of suppressing GH secretion, low doses of the GRF-antagonist stimulate GH release. By analogy, if the ultrashort feedback loop also worked on the GRF-containing neurons mediating enhancement of NREMS, a small dose of the GRF-antagonist (0.003 nmoi) should increase NREMS. However, in this study there was a tendency for suppression of NREMS after i.c.v, administration of 0.003 nmol GRF-antagonist. Although this finding might indicate a lack of the GRF-mediated ultrashort negative feedback in the regulation of NREMS, it is noted that the beginning of the light period, when rats sleep most of the time, is not a favorable period for revealing sleep-promoting properties of exogenously administered substances. It is assumed that the concentrations of the endogenous sleep substances are already high at this time of the day and, therefore, the exogenously administered sleep-promoting substances often fail to induce significant increases in NREMS 7. I.c.v. injection of 14 nmol GRF-antagonist elicited a 0.2-0.3 °C rise followed by a slight decrease in Tbr during the first and second portions of the light period, respectively. The significance and the mechanism of the Tbr variations currently are not clear. It is possible that the rise in Tbr was related to an enhanced motor activity though the period of increased Tbr was longer than that of increased W. An increase in the average GH concentration was observed 80-140 min after the i.c.v, administration of aCSF though the exact timing of the occurrence of the GH pulse varied greatly among the rats. Since GH and sleep were recorded in separate experiments, the variations in GH concentration cannot be directly compared to sleep-wake activity. However, considering the substantial sleep duration found in postinjection hour I in each group of rats, GH secretion seemed to be significantly delayed. In fact, in a previous study describing the correlation between sleep and plasma GH concentrations, GH secretion followed sleep onset with a long time lag of 70 min TM. The time lag between sleep onset and GH secretion had a high intra-individual constancy and a great inter-individual variability. The mechanism of the delay of GH secretion with respect to sleep onset is not clear. Nevertheless, the findings that i.c.v. injection of GRF-antagonist suppressed sleep together with the previous observation that i.c.v, administration of GRF promotes sleep support the suggestion that hypothalamic GRF is involved in sleep regulation.
Acknowledgements. We thank Sandy Johnson, Gail Richmond, and Donna Maxwell for assisting in these studies. This research was
supported, in part, by the National Institutes of Health (NS-27250).
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