Electroencephalographic analysis of the sleep-inducing actions of cholecystokinin

Nenropeptides 3: 129-1~8, 1982

ELECTROENCEPEALOGRAPHIC ANALYSIS OF TEE SLEEP-INDUCING ACTIONS OF CBOLECYSTOKININ

Jose A. Rojas-Ramirez,

133 , Jacqueline N. Crawleg', and Wallace B. Mendelson

1 2Department of Pharmacology, Faculty of Medicine, N.A. University of Mexico Clinical Neuroscience Branch, National Institute of Mental Health, ethesda, MD* 3 Adult Psychiatry Branch, National Institute of Mental Health, St. Elizabeth's Hospital, Washington, DC

*Present address: Neurobiology Program, Central Research C Development Department, E.I. du Pont de Nemours 6 Company, ABSTRACT Cortical EEG analysis of the effects of cholecystokinin on sleep were performed in rats treated intraperitoneally with 1,10, or 20 ug/kg CCK8. Latency to the first episode of non-REM sleep was significantly reduced from 29 minutes to 16 minutes. No change in total waking time, total non-REM sleep, or total REM sleep was seen at any dose over the first hour after CCK administration. Locomotor activity was significantly reduced by 10 ug/kg CCK8 during the first ten minutes only. The temporal separation between the sleep-latency reduction and the behavioral inactivity induced by CCK indicate that these effects are distinguishable phenomena. Sleep does not appear to be a primary cause for the satiety-related behavioral effects of CCK. (CCK)

INTRODUCTION Cholecystokinin (CCK) is a gut hormone secreted in response to ingested food (Roupt, 1980; Mutt, 1979; Rehfeld et al. 1979). The recent discovery of the cholecystokinin octapeptide (CCK8) in brain (Beinfeld, et al. 1980, 1981a, 1981b, 1982; Dockray et al. 1977; Emson et al. 1982; Greenwood et al. 1981; Handelmann et al. 1981; Bokfelt et al. 1980; Innis et al. 1979, Larsson and Rehfeld 1979; Muller et al. 1977; Rehfeld 1978; Rehfeld et al. 1979; Straus and Yalow 1979; Vanderhaeghen et al. 19801, as

129

well as CCK receptors regionally distributed in brain (Bays et al. 1980; Saito et al. 1980) has stimulated renewed interest in the behavioral actions of CCK (Morley, 1982). In particular, the role of CCK in mediating feeding behaviors is presently under intense investigation. Exogenously administered CCK reduces total food intake in fasted rats (Antin et al. 1975; Bernstein et al. 1975; Blass et al. 1979; Gibbs et al. 1973; Gosnell and Hsiao, 1981; Hsiao et al. 1979; Levine and Morley 1981; McLaughlin and Baile 1980; Mueller and Hsiao, 1979; Nemeroff et al. 1978; Schneider et al. 1979; Smith and Gibbs 1975; Smith et al. 1981), mice (Koopmans et al. 1979; McLaughlin and Baile 1981; Parrott and Batt 1980; Schneider et al. 1979), sheep, (Della-Fera and Baile 1979; Della-Fera et al. 1981), pigs (Parrott and Baldwin 1980), monkeys (Falasco et al. 1979) and humans (Kissileff, H.R. 1981; Stacher et al. 1979; Sturdevant et al. 1976). CCK triggers behaviors associated with the syndrome of satiety in rats and mice, including grooming and resting (Antin et al. 1975), and reduced exploration and social interactions (Crawley et al. 1981a, 1981b, 1981c). Feelings of fullness, relaxation, and drowsiness have been reported in human studies of the role of CCK in inducing the satiety syndrome (Stacher et al. 1979). CCK effects on satiety, exploratory, and relaxation are initiated at a peripheral CCK receptor. Sensory feedback from h gastrointestinal tract is relayed via the vagus nerve6'g6 to brain regions thought to mediate appetitive behaviors. The association of CCK with behavioral inactivity and self-reported drowsiness suggested that CCK might be inducing episodes of sleep. Reports of CCK prolongation of hexobarbital-, pentobarbital-, and ethanol-induced behavioral sedation also suggested a CCK action on sleep (Katsuura and Itoh, 1982; Zetler 1980). Using a video-tracking animal behavior monitor with computer analysis of fifteen parameters of exploratory and social behaviors in a novel environment (Crawley et al. 1982), we found that the most striking effects of CCK8 in mice and rats is the occurrence of long pauses of behavioral inactivity (Crawley et al. 1981a,b,c). These pauses begin within two minutes of intraperitoneal CCK8 administration. They are most prevalent during the first ten minutes after CCK treatment. Behavioral inactivity during the first few minutes of access to a novel environment is unusual in mice and rats, as this period is generally characterized by constant, active exploration (Crawley et al. 1981a,b,c).

The possibility that CCK-induced long pauses of total inactivity are actually short periods of sleep is addressed in this report. Cortical EEG analyses of rats treated with several doses of CCK were performed over our after CCK an eight hour period, with concentration on the first l! administration. Doses chosen included one low dose which is slightly active in this system in rats (1 ugfkg), and one high dose with robust behavioral effects on both feeding and exploration (10 ugfkg). Another dose which was higher than normally used in behavioral studies (20 ug/ kg) was chosen, to control for the longer session length required by sleep studies, since CCK8 has a relatively short half life in vivo. The time course of EEG changes was then compared against the timecourse of decreased behavioral activity, to examine the hypothesis that aspects of the satiety syndrome are a function of CCK-induced alterations in the sleep-waking cycle. 130

METHODS

CCK8 effects on sleep and locomotor activity were studied in 60 male Sprague-Dawley rats, 250-350 g. Stainless steel screw electrodes, O-80, l/8 inch, were implanted under chlclral hydrate anesthesia 5 days before testing, in the bifrontal and fronto-occipital cortical areas. Electromyograph electrodes, 0.005 inch teflon-coated steel wire, were implanted into the nuchal musculature. Sulfated CCK8, (Bachem, Torrance, CA), 1, 10, or -20 ug/kg or saline was administered intraperitoneally to rats, whose EEGs were individually recorded in a lighted cage with food and water available ad libitum. Sleep-waking parameters were recorded from 08:OO to 16:OO hours using a Model 78 Grass polygraph with paper speed of Waking, non-RRM sleep and REM sleep were visually identified and lOmm/sec. Sleep EEG measured using 30 second epochs. (Mendelson, et al., 1974). data were analyzed by One Way Analysis of Variance for independent groups.

TABLE

1 CCK8 (~G/KG)

SAL!NE INTERMITTANT

1

10

20

WAKING

TOTAL

MIN/FIRST

TOTAL

MIN/8

HOUR

15.1

+ 2.8

317,9

f 9.0

9.1

f 1.5

13.1 + 2.5

17.4 f 2.9

316.3

* 9.8

311.9 f 13.6

317.0 f 12.7

26.2

f 2.8

19.0 f 2.2

25.8 5 3.6

28.8 * 205

288.8

f 7.2

27844 * 12.6

291.9 + 10.0

289.2 + 5,3

0.35

+ 0.3

0.41 f 0.4

0.14 * 0.1

0.14 + 0.1

27.5

f 3.6

33.4 * 2.9

25.2 f 3.1

28.7 2 4.9

LATENCY TO NON-REF; SLEEP (MINUTES)

28,9

* 5.6

27.6 ? 3,3

16.6 + 2.9*

15.9 f 2.7-

LATENCY TO (MINUTES)

99.1

+ 19.4

302.0 + 10.2

130.0 + 25.3

132.9 + 24.7

RON-REtl

HOURS

SLEEP

TOTAL

MIN/FIRST

TOTAL

MIN/8

HOUR

HOURS

REV SLEEP TOTAL

MIN/FIRST

TOTAL

MIN/8

HOUR

HOURS

REM

SLEEP

Table. 1 Sleep EEG parameters after intraperitoneal administration of cholecytokinln or saline. Waking, non-REM sleep, and REM sleep are presented as cumulative minutes as analyzed from EEG electrodes implanted in the frontal and frontooccipital cortex. Latency to Non-REM sleep and latency to REM sleep represent minutes from drug administration to the first episode of Non-REM and REM sleep. + S.E.M. N= 11 for each group *p
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Locomotor activity was separately studied in 16 rats. Vertical movements were automatically recorded during the first 120 minutes. Type 160 FC, Ebtron-Produkter, Sweden. on an IR Electronic Mobility Meter, CCK 10 ug/kg or saline was intraperitoneally administered at the beginning of ahe session in a paired design. Number of movements per ten minutes were recorded for 120 minutes after drug administration. Locomotor data were analyzed by t-test for matched pairs. RESULTS CCKg 10 ug/kg i.p. significantly reduced latency to the first episode of non-REM sleep and produced a small decrease in length As seen in Table 1, there was no change in total of REM sleep periods. amount of time spent awake or asleep during the first hour or during the Figure 1 illustrates entire eight hour session after CCKg administration. an acceleration of the occurrence the major CCK effect on sleep patterns, sleep episodes, from 29 minutes after saline administration to of non-REM Figure 3 demonstrates a significant 17 minutes after CCKg administration. reduction in motor activity during the first ten minutes of locomotor activity measurement in a separate group of rats. No significant differences in locomotor activity were seen between 20 and 120 minutes after CCK administration. %I

LATENCY TO NON-REM SLEEP I

-

CK c

Figure pg/kg *p <

1. i.p. 0.025.

I CCK II

CCK 20

: i’fiqlkql

Mean latency to Non-REM sleep after Data are expressed as mean minutes

132

cholecystokinin + S.E.M. N=l

for

1,lO or 20 each group

DISCUSSION

CCK significantly decreased the latency to the first episode of non-REH sleep in rats during the daytime phase of their sleep-wake cycle. There was no significant difference in total amount of time spent awake, total non-REM sleep, or total REM sleep time during the first hour after intraperitoneal CCK administration. Since CCK8 has a half-life of several minutes in plasma, the peptide is most likely to show effects within the first hour of EEG record. lhe highest dose used, which induced profound behavioral inactivity (Crawley et al. 1981a), also had no effect on total sleep time. The small decrease in mean length of REM sleep periods during the first hour after CCK administration was not sufficient to affect total REM sleep time or total awake time during the first hour after CCK administration, when REM represented only Z-3% of the hour session.

LOCOMOTOR ACTIVITY 0-a

SALINE CCK

10~9/kg i.p.

t

Figure 2. Vertical movement of rats during the first 120 minutes after treatment with saline or cholecystokinin 10 ug/kg i.p. N=8 for each group pXO.01 for t=lO minutes only. CCK-treated rats at all other time points were not significantly different from saline controls. The period of behavioral inactivity in this study persisted for only the first ten minutes after CCK administration. By twenty minutes, CCK-treated rats showed locomotor activities similar to those of saline-treated controls. Previous studies showed that major effects on exploration occur during the first five minutes after CCK administration (Crawley et al. 1981a,b,c). Major effects on feeding are measurable within the first fifteen minutes after CCK administration (Levine and Morley, 1981; Mueller and Hsiao, 1979; Nemeroff et al. 1978). However, the latency to non-REM sleep after CCK treatment averaged around 16 minutes, with a

133

of 3-34 minutes. Only two animals exhibited a period of non-REM during the first ten minutes. Therefore, a comparison of time courses for the satiety-related behaviors and the sleep latency reduction induced by CCK indicates that these effects are temporally separable. Since the episodes of non-REM sleep began considerably after the episodes of behavioral inactivity, it appears reasonable to conclude that sleep is not a primary cause for the satiety-related behavioral effects of CCK.

range

The decrease in latency to sleep indicates that CCK accelerated the time course of falling asleep. The decreased latency was seen in rats ;;;;~dyw~:ha:~ uglkg CCK8 i.p., which effectively reduce5 exploration 1981a,b) and food intake (Morley 1982; Smith and Gibbs 1975). It was not seen at 1 ug/kg, a dose with effects on feeding and exploration of novel objects (Crawley 1981a). Decreased latency to sleep would be consistent with another interpretation of the behavioral effects of CCK, i.e. an increased tendency toward habituation. It is interesting to speculate that the reduced food intake and reduced exploratory behavior5 after CCK treatment represent a faster habituation to critical or novel environmental stimuli. The reduction in sleep latency could then reflect the rapid development of "disinterest" in the test chamber used for the EEG study. Habituation might then be followed by a faster induction of the normal sleep seen in rats during the daytime hours of their sleep-wake cycle. Alternatively, CCK may play a role in sleep regulation in a time frame which occurs after the effects on feeding and satiety-related behaviors. Since the behavioral edfggts previously reported for CCK are the sensory feedback loop to the initiated at a peripheral receptor ' brain may include secondary, long-term actions which influence sleep.

In conclusion, these data suggest that the reductions in feeding and exploration after systemic CCK administration are not a result of sleep-induction. The major EEG parameter affected by CCK appears to be . latency to non-REM sleep. Previously observed time courses for the CCK-inhibition of feeding behavior and CCK-induction of long pauses of behavioral inactivity precede the time course observed for episodes of non-REM sleep. The temporal separation of the satiety-related behavioral effects of CCK and it5 effect5 on sleep-latency prompt5 further investigation of these actions as independent phenomena.

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