INTRODUCTION

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PITUITARY GROWTH HORMONE (GH) secretion is stimulated and inhibited by two hypothalamic neurocrines, GH-releasing hormone (GHRH) and somatostatin, respectively. The metabolic and proliferative actions of the somatotropic axis are mediated, in part, by GH itself and, in part, by insulin-like growth factor I (IGF-I) released from the liver and produced locally in the tissues. The somatotropic axis is also a major humoral mechanism regulating sleep-wake activity. GHRH promotes sleep, particularly non-rapid eye movement sleep (NREMS), in both animals (12, 24, 25) and humans (18, 22, 34), and inhibition of GHRH is associated with decreases in sleep (27, 28). The sleep-promoting activity of GHRH is independent from the stimulation of GH secretion (26). Nevertheless, GH also seems to enhance sleep, possibly via some metabolic actions (20). It is anticipated, therefore, that somatostatin, which suppresses both GH and GHRH, alters sleep.

The aim of our experiments was to study the effects of somatostatin on sleep. Instead of somatostatin, however, an octapeptide analog of somatostatin, octreotide (SMS-201995, Sandostatin) was injected systemically. Out of the five somatostatin receptors, octreotide is a potent agonist at two, the SSTR₂ and SSTR₅ receptors; the inhibition of the somatotropic axis is attributed to stimulation of SSTR₂ receptors (see Ref. 30 for review). Octreotide is 20-75 times more potent than somatostatin in inhibiting GH secretion, and it is highly resistant to degradation (3, 29). The half-life of octreotide is 45-110 min, whereas somatostatin is broken down in a few minutes (10, 29). Octreotide is, therefore, used instead of somatostatin to suppress GH secretion in clinical practice. Previous articles describing the effects on sleep of intracerebral or systemic administration of somatostatin or octreotide in rats and humans described diverse findings: no alterations in sleep, selective increases in REMS, and inhibition of sleep were all reported (5-7, 13, 15, 19, 34). Our results indicate that the previous seemingly controversial reports are not necessarily exclusive.

	METHODS

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Animals. Male Sprague-Dawley rats weighing 320-350 g were used. The rats were anesthetized with ketamine-xylazine (87 and 13 mg/kg, respectively), and stainless-steel jewelry screws for electroencephalograph (EEG) recording were implanted over the frontal [~1 mm posterior (P) and 1 mm lateral (L) from the bregma] and parietal (P: 5.5 mm, L: 4 mm) cortices and over the cerebellum. A thermistor placed over the parietal cortex served to measure brain cortical temperature (T_c).

Recording. The rats were housed in individual Plexiglas cages. The cages were placed in recording rooms with a 12:12-h light-dark cycle (lights on from 0830 to 2030) and with an ambient temperature regulated at 26°C. The rooms were sound attenuated. Food and water were continuously available. The rats were kept in conditions identical to those in the recording rooms for at least 1 mo before the operation. After the surgery, the rats were connected to the recording tether and habituated to the experimental condition for another 10 days.

The tethers were attached to commutators. The motor activity of the rats was assessed by recording potentials generated in electromagnetic transducers attached to the tethers. Cables from the commutators and electromagnetic transducers were connected to polygraphs in an adjacent room. The signals from the polygraphs were digitized (64-Hz sampling rate) and collected by a computer and stored on a VHS format video tape. For scoring, the EEG, T_c, and motor activity signals were restored on the computer screen. In addition, power density values were calculated by fast-Fourier transformation for consecutive 8-s epochs in the frequency range 0.25-20 Hz for 0.25 Hz-bands and integrated for 0.5 Hz bins; the spectral resolution was 0.125 Hz. The power density spectra were also displayed on the computer screen. The states of vigilance were determined over 8-s epochs by the usual criteria: NREMS (high-amplitude EEG slow waves, lack of body movements, declining T_c on entry); REMS (highly regular theta activity in the EEG, general lack of body movements with occasional twitches, and a rapid rise in T_c 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 T_c after arousal). The percentage of time spent in each state of vigilance over 1-h periods and for the light and dark periods were determined. The 8-s T_c values were averaged for 1-h periods. Mean power density spectra were calculated for 8-s uninterrupted periods of artifact-free NREMS in each hour. The power density values for the 0.25- to 4-Hz (delta) frequency range were integrated and used as an index of EEG slow-wave activity (SWA) during NREMS to characterize sleep intensity in each recording hour during the light period. SWA was not analyzed at night because rats are often awake for periods longer than 1 h at night.

Treatments. Octreotide (Sandostatin, Sandoz, Basel, Switzerland, octreotide acetate diluted in physiological saline) was injected subcutaneously in the back of the neck in a volume of 0.1 ml/100 g in doses as follows: 1 µg/kg (n = 8), 10 µg/kg (n = 9), and 200 µg/kg (n = 9). A group of rats (n = 8) received an acidic control solution as described by Danguir and De Saint-Hilaire-Kafi (7) (acetic acid = 3.2 mg, sodium chloride = 7 mg, sodium acetate = 1.8 mg, in 1 ml distilled water; pH = 4). Each rat was recorded from for 23 h on two consecutive days: a baseline day when physiological saline was injected and an experimental day when octreotide or the control solution was administered. In each group, approximately one-half of the rats received physiological saline on day 1 and octreotide on day 2, whereas the order of the baseline day and experimental day was reversed for the rest of the animals. The rats were injected 10-15 min before light onset. The recording started at light onset and continued for 23 h (12-h light + 11-h dark).

Two groups of rats were included in the experiments with repeated injections: one group (n = 10) received octreotide, and the other (n = 7) was injected with the control solution. Octreotide (10 µg/kg) and the control solution was administered twice a day, 10-15 min before light and dark onset. Both groups of rats received physiological saline for 5 days. Sleep-wake activity was recorded for 11 h in the light and in the dark period on three days: on day 5 of physiological saline injection (baseline) and on days 1 and 5 of the administration of octreotide or the control solution.

Determination of GH. To study the changes in plasma GH concentrations after 200 µg/kg octretotide, blood samples were collected in one group of rats (n = 7) on two consecutive days. Physiological saline was injected on one day and octreotide on the other day. The rats were implanted with a cardiac catheter introduced into the right atrium via the external jugular vein 4 days before blood sampling. The rats were housed in environmental chambers. Silastic tubing was connected to the cardiac catheters, and the tubing was routed out of the chambers through holes in the wall. Thus the rats could move freely, and they were not disturbed by the sampling. Octreotide or physiological saline was injected via the catheter 10-15 min before light onset. Blood sampling started 30 min after light onset and continued for 5 h at 30-min intervals. Each blood sample was 200 µl. The blood was immediately centrifuged, and the plasma was stored at 20°C until assay. The red blood cells were reconstituted in physiological saline and reinjected. GH was determined in a single radioimmunoassay in triplicates. The intra-assay coefficient of variation was less than 7%. The immunoreagents (rat GH antiserum: GH-S-5; iodination grade rat GH: GH-I-6; and standard: rat GH RP-2) were provided by The National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases.

Statistics. The hourly values of the states of vigilance, SWA, and T_c during the 12-h light and 11-h dark periods were compared by means of two-way analysis of variance (ANOVA) for repeated measures between the baseline day and the experimental days in each group. The treatment effect (experimental vs. baseline) and the time effect (variations across the individual hours) were the two factors of the ANOVA. In general, the F statistics are only provided for the treatment-effect and for the interactions between the treatment and the time factors when statistically significant differences are noted. Significant variations in time are not discussed. (NREMS, REMS, and SWA vary significantly in time: NREMS and SWA are high in the morning and decline toward the end of the light period, whereas REMS peaks in the afternoon). There are large interindividual variations in SWA. Deviations from baseline are, therefore, depicted in Figs. 1 and 3 for SWA (light periods), but the absolute values were used in the intragroup statistics. In the case of repeated administrations of octreotide and the control solution, the Student-Newman-Keuls-test was applied to identify the day when the states of vigilance differed from the other recording days. ANOVA for repeated measures was also used to compare GH concentrations between the baseline day and the octreotide day. Intergroup differences were evaluated by means of ANOVA in sleep among the groups injected with various doses of octreotide or the control solution (time factor: repeated measures; group-factor: independent samples). SWA was not compared between groups. Significant changes in sleep were determined by means of the paired t-test in hour 1 postinjection. An -level of P < 0.05 was considered to be significant in all tests.

	RESULTS

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Single injection of octreotide. Subcutaneous injection of octreotide elicited prompt vigorous scratching aiming at the site of the injection. The control solution also elicited scratching, which was, therefore, attributed to skin irritation due to the low pH of the solutions. The rats often drank water during the first 5 min after octreotide; drinking was not observed after the control solution. Scratching and drinking declined rapidly, and the behavior of the rats looked normal 5-10 min postinjection.

Alterations in sleep-wake activity were not found after administration of the control solution (Table 1, Fig. 1). NREMS and SWA were also normal after 1 µg/kg octreotide. REMS, however, enhanced significantly [F(1,8) = 10.861, P < 0.05] during the light period. The increases in REMS started 2-3 h postinjection and persisted during the rest of the day, although they were very small when the individual hours were considered. In response to 10 µg/kg octreotide, NREMS was significantly suppressed in hour 1 postinjection; thereafter, values of NREMS were closer to baseline values. Calculated for the 12-h light period, NREMS or SWA did not differ between the baseline and the octreotide days. Statistically significant, albeit modest, increases were found in REMS throughout the light period [F(1,8) = 14.099, P < 0.05]. Sleep was greatly altered after 200 µg/kg octreotide. NREMS was significantly suppressed in hour 1 postinjection, whereas a tendency to enhanced NREMS was observed starting in hours 3-4 and continuing thereafter in the light period. As a result of the biphasic changes, the total time in NREMS did not change, but the distribution of NREMS across the 12-h postinjection differed significantly between the baseline day and the octreotide day [time × treatment interaction: F(11,88) = 2.388, P < 0.05]. SWA during NREMS was greatly enhanced in response to 200 µg/kg octreotide [F(1,8) = 35.524, P < 0.05]. The changes in SWA varied with the time [treatment × time interaction: F(11,88) = 4.607, P < 0.05]; in fact, increases in SWA were only noted between hours 3 and 7 postinjection. REMS also increased [F(1,8) = 12.985, P < 0.05]. Unlike after the smaller doses of octreotide, enhancements in REMS only tended to occur in the last portion of the light period.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Mean (±SE) values of the non-rapid eye movement sleep (NREMS) intensity characterized by electroencephalograph (EEG) slow-wave activity (SWA) during NREMS (A), states of sleep [NREMS (B) and rapid eye movement sleep (REMS; C)], and brain cortical temperature (Tc; D) during the 12-h light period on the baseline day when physiological saline was subcutaneously injected ( in B-D) and on the experimental day ( in B-D) when the rats received a control solution (Contr Sol; n = 8) or octreotide (Oct) in doses of 1 (n = 8), 10 (n = 9), or 200 µg/kg (n = 9) 10-15 min before light onset. Instead of absolute values, the %differences are depicted between baseline and experimental values for SWA in each hour and in each group.

Comparisons of 12-h NREMS and REMS time after the three doses of octreotide failed to detect significant differences. The alterations in SWA, however, differed among the three doses [F(11,264) = 4.079]; the changes in SWA after 200 µg/kg octreotide were different from those after 1 or 10 µg/kg octreotide (Student-Newman-Keuls test).

Injection of the various doses of octreotide at the onset of the light period failed to alter sleep at night (Table 1). T_c was also not influenced after the administration of octreotide.

Plasma concentrations of GH were determined after injection of physiological saline (baseline) and 200 µg/kg octreotide. The mean (±SE) GH concentrations in the 10 samples taken in 5 h were significantly less after octreotide (26.7 ± 5.88 ng/ml) than after physiological saline (50.6 ± 5.88 ng/ml, t-test). ANOVA also indicated significant differences between the effects of the two injections [F(1,6) = 7.325, P < 0.05]. Plasma GH concentrations varied with time [F(9,54) = 2.182, P < 0.05], and the differences in GH concentrations between the baseline and the octreotide days also changed with time [treatment × time interaction: F(9,54) = 3.466, P < 0.05]. Plasma GH concentrations were very low for 2 h after octreotide. Between hours 2.5 and 4, however, rises in plasma GH were observed in each rat, resulting in a modest hump in the mean GH concentration curve (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Mean (±SE) concentrations of growth hormone (GH) in the plasma in a group of rats (n = 7) that received physiological saline () on the baseline day and 200 µg/kg octreotide () on the experimental day. Physiological saline or octreotide was subcutaneously injected 10-15 min before light onset, and blood sampling started 30 min after light onset.

Repeated administration of octreotide. Repeated administration of the control solution at light and dark onset did not alter sleep (Table 2). The baseline values of the states of vigilance did not differ between the control group and the octreotide group.

In the rats injected with octreotide, a decline was observed in NREMS between the baseline day and day 5 of octreotide administration during both the light and dark periods [light: F(2,18) = 17.053, P < 0.05; dark: F(2,18) = 8.570, P < 0.05] (Fig. 3 and Table 2). NREMS was significantly suppressed in hour 1 postinjection during the light period on day 1 of octreotide injection. The baseline values of NREMS were recovered for the rest of the light period. The decrease in NREMS was somewhat attenuated, although it remained significant in postinjection hour 1 on day 5. Thereafter, however, NREMS stayed below baseline throughout the light period. NREMS time, calculated for the 11-h light period, was significantly less on day 5 than either on the baseline day or on day 1 (Student-Newman-Keuls test). At night, the decreases in NREMS were already significant on day 1, and they became marked on day 5 (Student-Newman-Keuls test). The rats spent little time in NREMS during the first hour of the dark period on the baseline day, and further suppressions could not be observed. The decreases in NREMS occurred in the middle of the night on both recording days after octreotide injections. Comparisons between the control and the octreotide groups indicated significantly less NREMS after octreotide in both the light and the dark periods on day 5 [F(1,165) = 7.424, P < 0.05].

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Mean (±SE) values of the NREMS intensity characterized by EEG SWA during NREMS (A), the states of sleep [NREMS (B) and REMS (C)], and Tc (D) during the light and the dark period on the baseline day when physiological saline was subcutaneously injected (), on day 1 (), and on day 5 () of repeated octreotide administrations (n = 10). Physiological saline (baseline) and octreotide (10 µg/kg) were subcutaneously injected twice a day, 10-15 min before light and dark onset. The rats were recorded for 11 h in both the light and the dark periods. SWA, depicted as %deviation from baseline values for days 1 and 5 of octreotide administrations, was only analyzed during the light period. Horizontal black bar, dark period.

EEG SWA (the intensity of NREMS) also varied significantly during the light period among the three recording days in the group injected with octreotide [F(2,18) = 5.606, P < 0.05]. SWA was normal on day 1 and declined below baseline on day 5 (Student-Newman-Keuls test).

Significant differences were also found in REMS during the light period among the three recording days [F(2,18) = 5.298, P < 0.05]. REMS was enhanced on day 1 of octreotide injection, whereas octreotide failed to promote REMS on day 5 (Student-Newman-Keuls test). REMS decreased significantly at night on day 5 [F(2,18) = 5.728, P < 0.05].

T_c was not altered by the repeated administration of octreotide.

	DISCUSSION

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Both NREMS and REMS were altered in response to octreotide injection in the rat. REMS was consistently, albeit modestly, enhanced. The increases in REMS occurred several hours after administration of octreotide and did not vary significantly with the dose. These findings corroborate the articles by Danguir and collaborators reporting increases in REMS in response to intracerebral infusion of somatostatin (5) or systemic administration of 150-200 µg/kg octreotide in rats (6, 7). Active immunization against systemic somatostatin does not alter REMS (11). In contrast, depletion of brain somatostatin by means of cysteamine decreases REMS (5). An antiserum to somatostatin administered into the brain stem blocks the REMS-promoting activity of a locally applied muscarinic agonist, carbachol (8). It was suggested, therefore, that octreotide enhances REMS via intracerebral action sites, perhaps in the brain stem.

At variance to reports by Danguir's group, however, the effects of octreotide on sleep were not selective for REMS in our experiments. Injection of octreotide elicited a prompt suppression in NREMS that was followed by increases when the large dose was administered. The immediate sleep-suppressive effects of somatostatin were among the first behavioral actions of this peptide described more than 20 years ago (15, 31). Both intracerebroventricular (15) and systemic (31) administrations of somatostatin inhibit NREMS. In addition, Havlicek et al. (15) also observed a prompt suppression in REMS in response to somatostatin injection. Rats exhibit little REMS at the beginning of the light period, and, therefore, immediate decreases in REMS after octreotide might remain unnoticed in our experiments. Instead of short time blocks, Danguir (5) reported NREMS time in 24-h periods during somatostatin infusion. Only REMS was studied in experiments where the authors analyzed sleep in shorter (2 h) time blocks after systemic octreotide injections (6, 7). The changes in NREMS could, therefore, have been missed in those studies. The reports greatly vary on the effects of systemic somatostatin on sleep in human subjects. Alterations in sleep were not observed in one study (19), a tendency to an increase in the frequency of REMS was found in another experiment (34), whereas sleep was significantly deteriorated in response to somatostatin in elderly subjects (13). Compared with the dose of the highly potent somatostatin analog octreotide used for systemic administrations in the rat, relatively small doses of somatostatin acting for 2-3 min were injected in humans. In addition, Frieboes et al. (13) also suggest that a strong GHRH activity blocks the somatostatin-induced arousal in young subjects. The age-related decreases in hypothalamic GHRH may increase the responsiveness of the elderly to the sleep-suppressive effects of somatostatin.

Subcutaneous injection of octreotide elicited scratching of the site of injection. Local erythema and itching are, in fact, known side effects of the octreotide preparation used. Scratching lasted for a few minutes and vanished by the time the recording was started (10 min postinjection). The control solution evoked scratching comparable to that elicited by the highest dose of octreotide without altering sleep-wake activity. The octreotide-induced changes in sleep are, therefore, attributed to somatostatin-like effects. Five somatostatin receptors have been cloned (termed as SSTR₁ to SSTR₅ receptors) (reviewed in Refs. 30 and 33). Octreotide has high affinity for SSTR₂ and SSTR₅ receptors and modest affinity for SSTR₃ receptors, whereas it does not bind SSTR₁ and SSTR₄ receptors. In conclusion, the SSTR₂ and SSTR₅ receptors are the most likely candidats to mediate the effects of octreotide on sleep. As discussed above, despite the inconsistencies in the previous findings, the majority of the octreotide-induced sleep alterations reported herein (suppression in NREMS and promotion of REMS) were previously also observed after both systemic and intracerebroventricular administration of somatostatin. This indicates intracerebral action sites for the octreotide-induced changes in sleep. Somatostatin inhibits both hypothalamic GHRH release and pituitary GH secretions. Although the functional significance of the various somatostatin receptors is largely unknown, suppression of the somatotropic axis is attributed to a stimulation of SSTR₂ receptors (30), and the hypothalamus is rich in SSTR₂ receptors (36). The SSTR₂ receptors are also expressed in the pituitary (35). It is likely, therefore, that the inhibition of GH secretion is mediated by both hypothalamic and pituitary action sites after systemic administration of octreotide.

GHRH and GH promote sleep independently from one another. Somatostatin inhibits both GHRH and GH. The sleep-promoting activity of GH is assumed to be indirect, mediated via some metabolic actions of GH or IGF-I (20). Depending on the species and the age of the subject, chronic alterations in pituitary GH secretions may be associated with changes in NREMS (2). Transgenic mice with excess GH production sleep more than normal mice (20). Administration of an antiserum to GH is followed by modest and persistent decreases in NREMS (unpublished observations), and NREMS is also chronically suppressed in transgenic mice deficient in the entire somatotropic axis (37). Lamberts et al. (21) report that repeated injections of 5-20 µg/kg octreotide twice a day suppress the GH release and result in a progressive decline in the GH content of the pituitary in the rat. The persistent decreases in NREMS and SWA on day 5 of octreotide injections correspond to those sleep alterations that are anticipated in a condition of true GH deficiency. In contrast, REMS failed to increase, and the acute inhibition of NREMS was attenuated in hour 1 postinjection on day 5 of octreotide injection. The attenuation of these responses may result from a desensitization of the somatostatin receptors mediating them. In fact, administration of octreotide for longer than 5 days also causes desensitization of the somatostatin receptors in the pituitary in normal rats (21).

Inhibition of GHRH is another candidate for mediating suppression in NREMS after octreotide. Promotion of sleep is attributed to a neurotransmitter-like activity of GHRH modulating the function of the somnogenic structures in the anterior hypothalamus/preoptic region (26). Administration of GHRH is followed by prompt increases in NREMS (12, 25), whereas an antagonist to GHRH elicits immediate decreases in NREMS lasting for ~1 h (27). An enhanced activity of the GHRH neurons after the octreotide-induced inhibition could also explain the reboundlike increases in the intensity of NREMS (SWA) after 200 µg/kg octreotide. There are two GHRH-containing neuronal pools in the hypothalamus: one in the arcuate nucleus and another around the ventrolateral rim of the ventromedial nucleus (32). The former seems to have a dominant role in the stimulation of pituitary GH secretion, whereas the latter has been implicated in sleep regulation. The problem with the involvement of GHRH in the octreotide-induced alterations in sleep arises from the reported distribution of somatostatin receptors. Histologically documented interactions between somatostatinergic and GHRHergic neurons exist in the arcuate nucleus and, perhaps, at the level of the median eminence (reviewed in Ref. 4). Somatostatin receptors, however, have not been found in the GHRH-containing cells around the ventromedial nucleus, which are the putative sleep-promoting neurons. Nevertheless, there are somatostatin receptors in the hypothalamus/preoptic region that may control via interneurons the activity of the GHRH neurons around the ventromedial nucleus or may directly inhibit the GHRH-responsive somnogenic structures in the basal forebrain. The role of GHRH, therefore, remains to be determined in the sleep-suppression elicited by octreotide. Alternative mechanisms might also be considered. Among the hormones that are inhibited by somatostatin, an NREMS-promoting property is attributed to insulin (9). Octreotide is far less potent in inhibiting insulin secretion than GH release (3). Insulin deficiency decreases NREMS particularly during the second portion of the light period (17), and the sleep changes might develop relatively slowly. Somatostatin-containing cells (16) and somatostatin receptors (35) mediating unknown physiological processes occur throughout the central nervous system and may also be involved in the suppression of sleep after octreotide administration. Finally, both somatostatin and octreotide may bind to and inhibit µ-opioid receptors (30). µ-Receptors promote NREMS (14).

The initial suppressions in NREMS were followed by a tendency to increased NREMS time and significant enhancements in NREMS intensity after 200 µg/kg octreotide. It is interesting to note that REMS failed to increase during the period of enhanced SWA; instead, stimulation of REMS was delayed to 10-12 h postinjection when the enhancements of SWA were over. This pattern of sleep enhancement, i.e., initial increases in NREMS, particularly SWA, followed by increases in REMS, are characteristic of the sleep rebound elicited by short-term sleep deprivation in the rat (28). It is assumed that the activation of the mechanisms generating NREMS suppresses REMS, causing the delay in the enhancements in REMS after sleep deprivation. The 10 µg/kg octreotide, however, also suppressed sleep as effectively as the 200 µg/kg, and significant rebound was not noted. Sleep deprivation, therefore, cannot explain the late enhancements in NREMS. As indicated by the concentration of plasma GH, the inhibition of the somatotropic axis has declined by the time SWA increases. The enhancements in NREMS, therefore, might reflect a withdrawal of the octreotide-induced stimulation of somatostatin receptors. An acute withdrawal of somatostatinergic activity can trigger discharges of GHRH (23). The concentrations of GH in the plasma, however, failed to reveal larger than normal GH surges after the octreotide-induced inhibition in our experiments. In addition, as discussed above, we cannot conveniently link the initial suppressions in NREMS to the somatotropic system. Although the mechanism of the late increases in NREMS remains to be determined, the analogy between these changes and the recovery sleep after sleep deprivation deserves special attention. Sleep deprivation is stressful, and stress is associated with increased somatostatin release in the rat (1).

In conclusion, the long-acting somatostatin analog octreotide elicits changes in both NREMS and REMS. It is likely that the persistent suppressions in NREMS result from GH deficiency after repeated administrations of octreotide. The role of the somatotropic axis is, however, not clear in the acute sleep alterations elicited by somatostatinergic stimulation, and mechanisms independent from GHRH and GH should also be considered.