Effect of beta-casomorphin on neonatal sleep in rats

Pepndes. Vol 11, pp 1--4 e Pergamon Press plc, 1990 Pnnted m the U S A

0196-9781/90 $3 00 + 00

Effect of Beta-Casomorphin on Neonatal Sleep in Rats T O M I T A I R A , L E E N A A. H I L A K I V I , J O U N I A A L T O * A N D I L K K A H I L A K I V I

University of Helsinki, Department of Physiology, Siltavuorenpenger 20 J SF-O0170 Helsinki, Finland *Fmnish Co-Operative Dairies' Association, Laboratory of Cell Biology Kalevankatu 56 SF-00181 Helsinki, Finland R e c e i v e d 31 July 1989

TAIRA, T , L A. HILAKIVI, J. AALTO AND I HILAKIVI Effectofbeta-casomorphm on neonatalsleep m rats PEPTIDES 11(1) 1-4, 1990.--The effects of bowne beta-casomorphm(1-7) (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) on neonatal sleep m rats were studmd. The pups recewed mtraperitoneal injectmns of beta-casomorphm(1-7) (1 mg, 5 mg, 10 mg, 50 mg, or 100 mg/kg) or a corresponding volume of sodmm chloride. In any of the doses used, beta-casomorphm(1-7) had no effect on waking. Only 100 mg/kg caused significant changes in sleep the percentage of quiet state of the total recording time (TRT) increased and the percentage of active sleep decreased Beta-casomorphm(1-7) did not cause slgmficant respiratory depressmn Naloxone pretreatment (1 mg/kg IP) reversed the effects of beta-casomorphm(1-7) on sleep, a finding which suggests that opmte p.-receptors are involved m medmtlng the sleep effects of beta-casomorphm Beta-casomorphm(1-7)

Movement sensitive mattress

BETA-CASOMORPHINS are milk-derived 4--7 amino acid-containing peptides having opioid activity. In rats they cause analgesia (7,12), as well as apnea (14) reversed by naloxone and naltrexone: these effects are mediated via ix-receptors. In dogs they have been shown to participate in regulating the release of insuhn, somatostatin and pancreatic polypeptide (21-23). Beta-casomorphins are cleaved from beta-casein in the gastrointestinal (GI) tract (8), though it is still unclear whether they are released m vivo in amounts that are physiologically significant (18,24). It has been shown, though, that oral milk infusions cause analgesia reversed by naloxone in rats (4). Besides their opioid action, tyrosine-containing beta-casomorphins are believed to interact with other transmitter systems in the central nervous system (CNS), especially with dopaminergic neurons (20). The presence of opioid-hke material in milk is intriguing from the point of view that newborns are known to be more sensitive to the analgesic and respiratory-depressant effect of morphine than more mature ammals (6). The development of morphine sensitivity and the subsequent development of relative morphine insensitivity are most likely related to the ontogenic development of opiate receptors in the brain (1). Apart from their possible role as neurotransrmtters and neuromodulators, beta-casomorphins may also influence neural maturation, since prenatal and postnatal admmistratmn of opmte receptor agonlsts can alter the development of various neural pathways (13). Aside from their suggested role m the infant-mother relationship (19), the effects of beta-casomorphins on behavior are not known. Because milk is the only source of nutrition for newborn mammals, we were interested whether the most common behavior

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Neonatal

Rat

Sleep-wake behawor

during the early postnatal period, sleep, is affected by betacasomorphms. An addiuonal reason for using newborn ammals is that the possible effects on the central nervous system of betacasomorphlns are seen more readily because of the immature blood-brain barrier METHOD

Ammals Seven-day-old male rats (n = 51) of the W~star strain, housed m their home cage with httermates and a nursing mother, served as subjects. The llluminatmn cycle in the animal room was 12 hr (lights on 7 a.m.-7 p.m.), the ambient temperature 22-24°C and the relative humidity 50-55%. Rat dams had free access to food (R3 pellets, Ewos, Sodertalje, Sweden) and water.

Materials The pups received beta-casomorphin(1-7) (Tyr-Pro-Phe-ProGly-Pro-Ile) in the doses of 1 ( n = 6 ) , 5 (n= 6), 10 (n = 8), 50 ( n = 7 ) , 100 ( n = 5 ) mg/kg, or naloxone (1 mg/kg) ( n = 6 ) or naloxone in combination with 100 mg/kg beta-casomorphin(1-7) (n = 6). All drugs were obtained from Sigma Chemmal Co., St. Louis, MO. Control animals (n = 7) received 0.9% sodium chloride (saline) Naloxone was given 5 minutes before and betacasomorphin just prior to the recordings of the sleep-wake behavior. All drugs were diluted in saline, and they were administered lntraperitoneally (0.1 ml/10 g of body weight).

SCSB Recordings The sleep-wake patterns of the rat pups were recorded by

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TAIRA, HILAKIVI, AALTO AND HILAKIVI

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FIG 1 Effect of beta-casomorphm(l-7) on the states of wakefulness and sleep m 7-day-old rat pups The recording time was 1 hr Each bar represents the mean -+SEM of 5-8 rats Statistically slgmficant differences (Dunnett's test) as compared with the sahne controls **p<0 01

using a movement-sensitive mattress [Static Charge Sensitive Bed, SCSB, see (15)] Movements of a pup on the mattress induce static charge redistribution in the conducting layers inside the mattress, which are further transduced into potential differences [for details, see (11)] The potentials were processed by a preamplifier and a filtering unit (Blorec BA-8R, Finland) and recorded by an inkwriting polygraph (Nihon-Kohden, ME 95D, Japan). The unfiltered signal (0.3-60 Hz) reflects total body movements, and another output signal filtered from it (0.3-2 Hz) reflects respiratory movements. We also used a small plezoceramlc transducer (Siemens-Elema 230, Sweden) attached to the animal's abdominal skin to record respiratory movements. The scoring criteria for using the SCSB in recording the sleep of newborn rats were introduced by HilakiVl and Hllaklvl (15) Waking. The animal is considered to be awake when it is moving actively or shows other body activities. On SCSB recording this can be seen as h~gh voltage, high frequency activity both in movement (M) and the respiratory (R) channels. The respiration sensor shows a high amplitude, irregular breathing pattern Quwt state. During the quiet state, the rat is mostly stall, though startle responses and changes m body posture occur intermittently. Neck muscle tone is maintained and respiration is regular. The SCSB recording shows no activity in the M channel except for occasional 1-2 sec long medium voltage bursts Both the R channel and the respiratmn sensor show regular respiration. Active sleep. In active sleep, trunk muscle tone is lost and the animal is in a recumbent position. Twitches occur in the whiskers and extremities, and respiration is irregular with varying amplitudes. SCSB recordings show repetitive, short-lastmg activity peaks m the M channel The R channel as well as the sensor tracing reveal irregular, low voltage and medmm voltage actwlty. The SCSB recordings were scored visually m 20-sec epochs (paper speed 5 mm/sec) accordmg to the criteria described above [for methodological details see (15)]. During the recordings the rat pup was kept on the SCSB mattress (63 × 36 × 2 cm) under a Plexiglas cylinder (diameter 20 cm, height 20 cm). The temperature in the recording apparatus

(SCSB + Plexlglas cylinder) was maintained at 30°C. The recordings lasted for 60 minutes and they were carried out between 10 a.m. and 4 p.m. Two rat pups were recorded simultaneously on separate SCSB mattresses. Respiratory frequencies were calculated from respiration tracings obtained by the plezoceramic transducer during the quiet state 10 minutes after the beginning of each recording session, lasting 1 minute

Stattsttcal Analysts One-way analysis of variance and Dunnett's test for betweengroup comparisons were used for statistical analysis of the data RESULTS

In all of the doses used, beta-casomorphxn(l-7) had no effect on waking The highest dose of beta-casomorphin (100 mg/kg) increased the percentage of quiet state of the total recording time (TRT) from 38% (control) to 78%, F(5,33)=7.75, p < 0 0001 (Dunnett's test. p < 0 01), and decreased the percentage of active sleep from 44% (control) to 9%, F(5,33)=6.55, p<0.0002 (Dunnett's test: p<0.01), (Fig. 1). Beta-casomorphin 50 mg/kg tended to have similar effects on the quiet state and active sleep, but the changes were not statistically significant Naloxone (1 mg/kg) alone increased the percentage of waking of the TRT from 17% (control) to 30% (Dunnett's test p<0.05), but had no effect on the quiet state or active sleep Naloxone was able to completely reverse the effects of beta-casomorphln(l-7) (100 mg/kg) on quiet state and active sleep (Fig. 2). The percentages of behavioral states after an injection of 100 mg/kg of beta-casomorphln(1-7) alone or in combination with naloxone were 12% and 32% (waking) (Dunnett's test: p < 0 01), 78% and 20% (quiet state) (p<0.01), 9% and 48% (active sleep) (p<0 01), respectively. The percentages of waking, quiet state and active sleep in the saline-treated animals were 18%, 38% and 44% (Fig. 2). Beta-casomorphin(1-7) also tended to decrease respiratory frequency slightly, though this effect was not statistically significant (Fig 3)

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FIG 3 Effect of beta-casomorphm(1-7) (100 mg/kg) on respiratory frequency (__+SEM) m 7-day-old rats QUIET STATE

ACTIVE SLEEP

FIG 2 Effects of naloxone (1 mg/kg) and beta-casomorphm(1-7) (100 mg/kg) on the states of wakefulness and sleep m 7-day-old rat pups The recording ume was 1 hr Each bar represents the mean +-_SEM of 5-7 rats Statistically slgmficant differences (Dunnett's test) *p<0 05, **p<0 01

DISCUSSION

Beta-casomorphins, including 4-7 amino acids, have been detected in gastrointestinal (GI) tract in humans after the ingestion of milk (24). The finding of beta-casomorphins in the GI tract raises the possibility that beta-casomorphlns participate in the physiological control of intestinal functions via intestinally located optate receptors (5,24). In order to elicit CNS effects, betacasomorphlns should first penetrate the GI bamer and then the blood-brain barrier. In newborn calves, beta-casomorphm immunoreactive material can be detected m plasma after the ingestion of milk (25). This implies that at least in newborn calves, a fraction of beta-casomorphins, derived from food materials, can reach the systemic c~rculatmn At present, it is not known to what extent beta-casomorphms can penetrate into the CNS. The penetration of peptldes through the blood-bram barrier occurs either by transmembrane diffusion, m which case the degree of passage depends on the lipid solubdity and the molecular weight of the peptlde, or by a specific saturable transport system, as suggested by Banks et al. (3). Beta-casomorphms are suggested to be transported into CNS by this system (2) The transport system xs saturable and competmve, which may explain why a relauvely large dose of beta-casomorphln is generally needed to produce CNS effects when admxmstered peripherally. The dose of beta-casomorphm(1-7) needed to produce significant changes in sleep was high (100 mg/kg). This indeed suggests that beta-casomorphm(1-7) does not easily pass the blood-brain bamer. The fact that only a moderate decrease m the respiratory rate, induced by beta-casomorphm(1-7), was observed although beta-casomorphln(1--7) is a potent respiration-depressant agent when admlmstered mtracerebroventncularly (14) also indicates that when given peripherally, beta-casomorphin does not easily

penetrate through the blood-brain barrier. The fact that 100 mg/kg beta-casomorphin(1-7) caused significant alterations in sleep but not in respiration shows that sleep regulation mechanisms are more sensitive to the effects of beta-casomorphln than are respiratory functions. In adult rats and cats ix-receptor ligands cause insomnia and REM (acuve) sleep suppression (16,17). In this study only active sleep suppression was seen, since beta-casomorphin(l-7) did not reduce total sleep time. Evidently, ix-receptors were involved m the effects of beta-casomorphln(1-7), since active sleep suppression was prevented by naloxone, a ix-receptor antagonist. This counteracting effect of naloxone was to be expected, because beta-casomorphlns are ix-oriented compounds m the brain of the newborn rat (26). Beta-casomorphins have been shown to elicit an increase in lnsuhn release m dogs (21) Since peripheral lnsuhn administration promotes slow wave sleep in rats (10), it is possible that increased insulin secreuon was partially responsible for the increase m qmet state found m this study The poss~bdlty that changes in the behavioral states were caused by an increase in somatostatin release (20) is unlikely, because the administration of somatostatin brings about a selective increase of acUve sleep in rats (9), the effect opposite to that caused by the administration of beta-casomorphln. Blass and Fitzgerald (4) found that oral milk mfusmn reduces the distress vocalization of rat pups when they are separated from their mother They describe this as ecologically reasonable, as increased vocalization while the mother is away from the nest could reveal the nest's location to predators. Th~s may also explain why the mother usually feeds milk to her pups before leaving the nest Therefore, it can be hypothesized that one of the physiological roles of beta-casomorphln is to mediate the calming effect of milk As long as no determmatmns of beta-casomorphms m the brain are available, it is difficult to evaluate the biological functions of these substances. This study showed that beta-casomorphin(1-7) caused behavioral effects after systemic admlmstration

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TAIRA, HILAKIVI, AALTO AND HILAKIVI

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15 Hilaklvl, L , Hllaklvi, I Sleep-wake behavior of newborn rats recorded with movement sensitive method Behav Brain Res 19 241-248, 1986 16 Khazan, N , Colasann, B Protracted rebound in rapid eye movement sleep nine and electroencephalogram voltage output in morphine dependent rats upon withdrawal J Pharmacol Exp Ther 183 23-30, 1972 17 King, C , Masserano, J M , Copp, E , Byrne, W L Effects of beta-endorphm and morphine on the sleep-wakefulness behavior of cats Sleep 4 259-262, 1981 18 Petrllh, P , Picone, D , Caporale, C , Addeo, F , Aurlcchlo, S , Marlno, G Does casomorphin have a functional role 9 FEBS Lett 169 53-56, 1984 19 Panksepp, J , Normansell, L , Swly, S , Rossl, J ,III, Zolovlclc, A J Casomorphln reduce separation distress In chicks Peptldes 5 829-831, 1984 20 Rauca, C , Matthles, H The effect of beta-casomorphin on the apomorphlne-induced turning after nlgral lesions m rats Neuropharmacology 25 1137-1140, 1986 21 Schusdlarra, V , Schlck, R , Holland, A , de la Fuente, A , Specht, J , Mayer, V , Brantl, V , Pfelffer, E F Modulation of insulin release by ingested oploid-hke substances in dogs Dlabetologla 24 113-116, 1983 22 Schusdlarra, V , Holland, A , Schlck, R , de la Fuente, A , Kher, H , Mayer, V , Brantl. V , Pfelffer, E F Effects of beta-casomorphln on somatostatln release In dogs Endocrinology 112 1948-t951, 1983 23 Schusdlarra, V , Schlck, R , Holland, A , de la Fuente, A , Specht, J , Mayer, V , Brantl, V , Pfeiffer, E F Effects of opiate-acnve substances on pancreatic polypepnde levels m dogs Peptldes 4 205-210, 1983 24 Svedberg, J , De Haas, J , Lelmenstolc, G , Paul, F , Teschemacher, H Demonstration of beta-casomorphin lmmunoreacnve materials in vitro digests of bovine rmlk and m small intestine contents after milk ingestion m adult humans Peptldes 6 825-830, 1985 25 Umbach, M , Teschemacher, H , Praetorms, K . Hlrschhauser, R , Bostedt, H Demonstration of a beta-casomorpbm lmmunoreacnve material m the plasma of newborn calves after milk intake Regul Pept 12 223-230, 1985 26 Volterra, A , Restant, P , Brunello, N , Galll, C L , Racagm, G Interaction of beta-casomorphlns with multiple oplmd receptors In vitro and in vlvo studies in the newborn rat brain Dev Brain Res 30 25-30, 1986