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Beta-endorphinmodulatestheacuteresponsetoasocialconflictinmalemicebutdoesnotplayaroleinstress-inducedchangesinsleep.

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Brain Research 978 (2003) 169–176www.elsevier.com/ locate/brainres

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b -Endorphin modulates the acute response to a social conflict in malemice but does not play a role in stress-induced changes in sleep

*Lobke M. Vaanholt, Fred W. Turek, Peter MeerloDepartment of Neurobiology and Physiology, Northwestern University, Evanston, USA

Accepted 9 April 2003

Abstract

b-Endorphin is an endogenous opioid peptide that is released during stress and has been associated with many physiological functions.In this experimentb-endorphin deficient mice were used to study the role of endorphins in the acute physiological and behavioralresponses to a social conflict, as well as their role in social stress-induced changes in sleep. Adult maleb-endorphin deficient and wildtype mice were subjected to the stress of a 1 h social conflict with an aggressive dominant conspecific. After the conflict, theb-endorphindeficient mice had higher corticosterone levels but the peak increase in body temperature was not different from that in wild type animals.In fact, body temperature returned to baseline levels faster in theb-endorphin deficient mice. During their interaction with the aggressiveconspecific several of theb-endorphin deficient mice showed clear signs of counter aggression whereas this was not seen in any of thewild type mice. Overall, theb-endorphin deficient mice and wild type mice had fairly similar sleep patterns under baseline conditions andalso showed similar amounts of NREM sleep, REM sleep and EEG slow-wave energy after the social conflict. In addition, no differenceswere found in the sleep patterns of mice that showed counter aggression and mice that did not. In conclusion, the results suggest thatb-endorphin modulates the acute endocrine, thermoregulatory and behavioral response to a social conflict but the data do not support amajor role forb-endorphin in the regulation of sleep or social stress-induced alterations in sleep. 2003 Elsevier Science B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Stress

Keywords: Social conflict; Aggression; Opioid; Glucocorticocoid; Body temperature; REM sleep

1 . Introduction exhibits projections within the brainstem to the lateralreticular nucleus. In addition to these central systems,

b-Endorphin is an endogenous opioid that acts as a b-endorphin producing cells are found in the anterior andneuropeptide within the central nervous system and func- intermediate lobes of the pituitary gland, from wheretions as a hormone in the periphery upon release in the endorphins are released into the circulation[15].blood stream[1,6]. In the brain,b-endorphin producing b-Endorphin has various functions but is especially wellcells are found in the hypothalamic arcuate nucleus and the known as an analgesic compound and a factor thatcaudal nucleus tractus solitarius, which together have modulates the physiological and behavioral response toextensive projections throughout the brain[15]. The neu- stressors[14,22,24,29,32,for reviews see1,6,18,25].Al-rons of the arcuate nucleus innervate other hypothalamic though few studies have addressed this issue, stress-in-regions, structures of the limbic system, and various areas duced release ofb-endorphin might also affect subsequentin the brain stem. Also the nucleus tractus solitarius sleep. In agreement with this, human beings receiving

opioids often feel sleepy while the rapid eye-movement(REM) phase of their sleep is actually inhibited[5]. In

*Corresponding author. Department of Molecular Neurobiology, Uni-cats, microinjections ofb-endorphin and other opioids inversity of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands.the brain have variable effects on slow wave sleep orTel.: 131-50-363-2334; fax:131-50-363-2331.

E-mail address: p.meerlo@biol.rug.nl(P. Meerlo). non-rapid eye-movement (NREM) sleep, depending on the

0006-8993/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0006-8993(03)02805-1

170 L.M. Vaanholt et al. / Brain Research 978 (2003) 169–176

brain area and the receptor types that are activated, but a cycle with lights on from 9:00 to 21:00. Food and waternumber of studies report increased signs of NREM sleep were provided ad libitum.[9,27]. In cats, similar to opioid effects in humans,b-endorphin may cause a strong reduction in REM sleep 2 .2. Experiment 1: body weight, aggression and[16]. corticosterone

Interestingly, some of the reported effects of exogenousb-endorphin administration mimic the effects of social In the first experiment, 14 wildtype and 16b-endorphindefeat stress on sleep in rodents. In particular, mice rapidly deficient mice were subjected to a social interaction withovercome the arousal that is induced by a social conflict an aggressive male. The aggressive males were animals ofand show an increase in NREM sleep afterwards, while the same strain (C57BL/6J) that were older and heavierREM sleep, on the other hand, is strongly suppressed[20]. than the experimental animals. They had been trained toIt is known that a social conflict in rodents activates central fight and protect their territory by housing them with aand peripheral opioid systems and causes an increase in female and regularly exposing them to young males theb-endorphin levels[8,13,22,30,31],particularly in defeated week prior to the experiment[19,20].animals as compared to dominants[8,13]. In the present The experimental animals were placed in the home cagestudy, experiments were performed withb-endorphin of the aggressor for a period of 1 h. The interaction tookdeficient mice to determine the role of endorphins in the place during the 6th hour of the light phase, conforming tophysiological and behavioral response to a social conflict our previous study in mice[20]. Prior to the conflict, bodyand, in particular, the role ofb-endorphins in sleep and weights of the experimental animals were measured.social stress-induced changes in sleep. Ifb-endorphins During the conflict, the number of clinches was recordedwould facilitate NREM sleep and inhibit REM sleep after and, since some of the experimental animals displayedsocial defeat stress, one would expect a prolonged period counter aggression towards the resident male, we also keptof wakefulness but a less pronounced decrease in REM a record of this. Afterwards, the experimental animals weresleep after a conflict inb-endorphin deficient mice. decapitated and their trunk blood was collected in pre-

chilled (08C) tubes with EDTA as anticoagulant. Theblood was centrifuged at 48C for 15 min at 2600g and thesupernatant was stored at280 8C for later analysis.

2 . Materials and methods Corticosterone levels were determined by radioimmunoas-say (ICN Biomedicals, Costa Mesa, USA).

2 .1. Animals and housing2 .3. Experiment 2: body temperature and sleep

The b-endorphin deficient mice were originally pro-duced by introducing a point mutation into the pro- For sleep recordings, 14 wildtype and 15b-endorphinopiomelanocortin gene so that it translates to a truncated deficient mice were implanted with permanent electrodesprohormone lacking the entire C-terminal amino acid to record cortical EEG and neck muscle EMG as describedregion encodingb-endorphin[28]. b-Endorphin deficient previously[20,21]. In brief, two screws in the skull servedmice have a normal birth weight and development into as EEG electrodes, one placed above the right hemisphere,adulthood. However, after the onset of puberty they attain 2 mm from the midline and 1 mm anterior of bregma, and10–15% more weight than wild type mice. As expected, one placed on the left hemisphere, 3 mm from the midlineopioid function is reduced as reflected in a decrease in and 1 mm anterior of lambda. Two insulated stainless-steelopioid-induced analgesia[29]. Otherwise, stress reactivity wires served as EMG electrodes and were inserted underand overt behavior appears to be normal in adultb- the neck muscles. The EEG and EMG electrodes wereendorphin deficient mice[29]. attached to a connector that was cemented on the skull

For the present study, male and female homozygous with dental acrylic. In addition to the head implant, awild type C57BL/6J andb-endorphin deficient mice (stock transponder for body temperature measurements was

tm1Lowname C57BL/6-Pomc ) were obtained from Jackson placed in the abdominal cavity (Model PDT4000; Minimit-Laboratory (Bar Harbor, ME, USA). With these mice a ter, Sunriver, Oregon, USA). After at least 2 weeks ofbreeding colony was started. Homozygousb-endorphin recovery from surgery, the mice were hooked up to thedeficient mice were crossed with homozygous wildtypes to recording equipment via a recording cable and a swivel,obtain heterozygous offspring. This offspring was crossed which allowed free movement throughout the cage. After 3and the second generation was then genotyped and used in days of habituation to the recording tether, EEG and EMGthe experiments described hereafter. In all experiments signals were recorded and fed into an amplifier (Grass3–4-month-old males were used. The animals were in- model 12; Grass Instrument Division, Astro-med, Westdividually housed from about 2 weeks before the start of Warwick, RI, USA). The EEG signal was amplified 10 000the experiment and were kept under a 12 h-light–12 h-dark times, high-pass filtered at 1 Hz (26 dB, 6 dB/octave) and

L.M. Vaanholt et al. / Brain Research 978 (2003) 169–176 171

low-pass filtered at 30 Hz (26 dB, 6 dB/octave). The 3 . ResultsEMG signal was amplified 5000 times, high-pass filtered at3 Hz and low-pass filtered at 100 Hz. The signals were 3 .1. Experiment 1: body weight, aggression andthen converted to digital format and stored at 102.4 Hz corticosteroneresolution. The signals were collected on an IBM computersystem with specialized software for acquiring and pro- At an adult age of 4 months, theb-endorphin deficientcessing sleep data in rodents (Multisleep; Actimetrics, mice were about 10% heavier than wild type miceEvanston, IL, USA). (27.760.5 g versus 30.960.4 g, respectively;t54.88;

Baseline recordings were made from the beginning of P,0.001). When the mice were transferred to the cage ofthe light phase until the end of the dark phase. During the an aggressive dominant conspecific, all experimental ani-6th hour of the light phase of the following (experimental) mals were readily attacked. The number of clinches theday animals were subjected to a 1-h social conflict as experimental animals were involved in did not differdescribed above. During this conflict animals remained between wildtypes andb-endorphin deficient mice (Fig.attached to the recording system. EEG, EMG and body 1). However, whereas all of the wild type mice weretemperature were recorded for an 18-h recovery period, submissive, as indicated by freezing or fleeing, eight out ofuntil the end of the following dark phase. sixteenb-endorphin deficient mice initially showed clear

By visual inspection of the EEG and EMG signals, 10-s signs of counter aggression, including tail rattling, initia-epochs were classified as wakefulness, NREM sleep orREM sleep[20]. In addition, the EEG signal was subjected

to spectral analysis by fast Fourier transformation and, forall NREM epochs, the EEG deltapower was calculated,that is, the spectral density in the delta or slow wave range(1–4 Hz). The amount and amplitude of slow waves in theEEG is considered to be an indication of NREM sleepintensity[3,7,12].To correct for interindividual differencesin the strength of the EEG signal, the delta power values ofall animals were normalized by expressing them relative totheir average NREM sleep delta power during the 24-hbaseline recording. The accumulation of NREM sleep deltapower was calculated per 6-h interval and expressed aspercentage of the total 24 h NREM sleep deltapowerduring baseline[20]. The normalized accumulated delta-power is referred to as slow wave energy (SWE). Bodytemperature was also measured every 10 s. For statisticalanalysis, average temperature was calculated for 6- and12-h blocks. The deviation from baseline was calculatedfor the first 6 h after the conflict.

2 .4. Statistics

For statistical analysis of body weight and corticosteronelevels, two-samplet-tests were applied. Also the average24- and 12-h light /dark values of body temperature duringbaseline were analyzed with two-samplet-tests. In addi-tion, for 1- and 6-h values of body temperature and sleepdata, repeated measures ANOVA was performed with afactor genotype (wildtype vs.b-endorphin deficient) and afactor time (1-h blocks and 6-h blocks). For body tempera-ture and sleep after the conflict, the deviations from thecorresponding baseline values were calculated. WhenANOVA revealed an overall effect of genotype or a

Fig. 1. Number of clinches during a 1-h social conflict with an aggressivegenotype3time interaction, separate blocks were testedconspecific (top) and the corticosterone level at the end of the conflict

with two-samplet-test. Within each genotype, data col- (bottom) in wild type mice (n514, grey bars) andb-endorphin deficientlected on the experimental day were compared with mice (n516, black bars). The figure shows the average values (6S.E.M.).corresponding baseline values using a pairedt-test. Significant difference between genotypes: *,P,0.005.

172 L.M. Vaanholt et al. / Brain Research 978 (2003) 169–176

tion of clinches, and chasing the resident cage owner. The mice, we expressed the experimental values during andoutcome of the fights in which experimental animals after the conflict as deviations from the baseline. Thecounter-reacted was not always clear, but at the end of the increase in temperature during the 1-h conflict was not1-h interaction most of them displayed the typical freezing significantly different betweenb-endorphin deficient miceand avoidance behavior suggesting submission. The occur- and wild type animals. However, when the hour of therence of counter aggression in theb-endorphin deficient conflict and the remaining 6 h of the light phase weremice was not related to their body weight. When a analyzed, there was a significant genotype3time inter-comparison was made within theb-endorphin deficient action (F 52.90; P50.016). In theb-endorphin de-5,125

group between animals that showed counter aggression ficient mice, the elevation of body temperature did notduring the conflict and the ones that did not, contrary to persist as long as in the wild type mice (t-test,P,0.05 forwhat one might expect, the mice that showed counter the last 2 h of the light phase). Thus, although the initialaggression were slightly lighter than animals that did not rise in body temperature did not differ between theb-show counter aggression (P50.099). endorphin deficient and wild type mice, the temperature

At the end of the 1-h social conflict, the corticosterone returned to baseline levels faster in theb-endorphinlevels in theb-endorphin deficient mice were on average deficient mice.slightly but significantly higher compared to the levels in In this experiment, fourb-endorphin deficient micethe wild type mice (Fig. 1; t52.29; P50.027). Important- showed clear signs of counter aggression as against elevenly, within the group ofb-endorphin deficient mice, the animals that did not. However, the temperature response tocorticosterone levels did not differ between the animals the conflict was similar in both groups. The increase inthat showed counter aggression and the ones that did not. temperature above baseline during the 1-h conflict, was not

significantly different between mice that showed counter3 .2. Experiment 2: body temperature and sleep aggression and mice that did not and when the hour of the

conflict and the remaining 6 h of the light phase wereFig. 2 shows 1-h values of body temperature under tested, there was no significant genotype3time interaction.

baseline conditions and after a 1-h social conflict. Under The amount of REM and NREM sleep, as well as thebaseline conditions, the average 24-h temperature was not accumulated NREM sleep SWE for 6-h intervals aredifferent between the wild type andb-endorphin deficient shown inFig. 3. Under baseline conditions, there were nomice. Also, there was no significant difference for the 12-h major differences in the sleep patterns betweenb-en-light phase value but there was a trend for slightly lower dorphin deficient and wild type mice. Neither NREM nortemperatures in theb-endorphin deficient mice during the REM sleep patterns differed between the genotypes.12-h dark phase (t51.81; P50.082). However, a slight difference in the temporal pattern of

During the conflict, body temperature increased and accumulated SWE was found between wildtype andb-gradually returned to baseline during the remainder of the endorphin deficient mice under baseline conditions (F 53,81

light phase. Although the baseline values did not differ 3.150,P50.029). For separate 6-h block this only reachedsignificantly betweenb-endorphin deficient and wild type statistical significance for the second half of the light phase

Fig. 2. Average hourly values of body temperature measured in wild type mice (left) andb-endorphin deficient mice (right) under baseline conditions (s)and on the experimental day (d) when the animals were subjected to the stress of a 1-h social conflict during the 6th hour of the light phase. Dark barsabove the graph indicate the dark phase. Significant differences: B, relative to baseline (pairedt-test,P,0.05); *, relative to wild type (two-samplet-testfor deviations from baseline, see text).

L.M. Vaanholt et al. / Brain Research 978 (2003) 169–176 173

Fig. 3. Cumulative NREM sleep SWE, NREM sleep and REM sleep per 6-h interval in wild type mice (left) andb-endorphin deficient mice (right) underbaseline conditions (s) and on the experimental day (d) when the animals were subjected to a 1-h social conflict during the 6th hour of the light phase.Data are expressed as averages (6S.E.M.). Significant differences: B, relative to baseline (pairedt-test, P,0.05); *, relative to wild type (two-samplet-test).

when the accumulated SWE was slightly higher in the wild this was compensated during the remainder of the lighttype mice (t52.12; P50.043). phase (data not shown; totals for second half of the light

After the conflict, there was an initial short lasting phase depicted inFig. 3). In fact, NREM sleep wasdecrease in NREM sleep the hour after the interaction but increased above baseline levels during the first half of the

174 L.M. Vaanholt et al. / Brain Research 978 (2003) 169–176

dark phase in theb-endorphin deficient mice, but not the deficient mice confirms an earlier report[29]. Crosswild type mice (Fig. 3). The changes in NREM sleep after fostering studies suggested that the increase in body weightthe conflict, expressed as deviations from baseline, were resulted from a change in the maternal behavior of thenot different between wild type mice andb-endorphin homozygoteb-endorphin deficient mother, rather than adeficient mice. change in the pup itself (Jackson Lab.). To prevent the

During the remainder of the light phase after the occurrence of a difference in weight due to maternalconflict, the accumulation of NREM sleep delta power or effects, we used heterozygote parents for the breeding,SWE was significantly increased above baseline levels in resulting in nests with mixed genotype. Nevertheless, aboth genotypes (Fig. 3). In theb-endorphin deficient mice, difference in body weight of about 10% was found.SWE was still increased compared to baseline during the Therefore, the fact thatb-endorphin deficient mice have anfirst half of the dark phase but then slightly decreased increased body weight cannot simply be explained by abelow baseline in the second half of the dark phase. change in the behavior of the homozygous mothers.Overall, the changes in NREM sleep SWE after the Alternatively, the lack ofb-endorphin may affect growthconflict relative to baseline were not different between in the offspring via different pathways. An increase in foodwild type mice andb-endorphin deficient mice. intake byb-endorphin deficient mice might seem a plaus-

Social defeat caused a strong initial suppression of REM ible explanation for their elevated body weight. This,sleep during the remainder of light phase in both geno- however, is not supported by the literature since, intypes, which was followed by a significant increase during general, opioid agents enhance feeding and opioid antago-the following dark phase (Fig. 3). Overall, the changes in nists decrease feeding[17,24]. Perhaps, the lack ofb-REM sleep after the conflict were not different between endorphin during development causes a change in metabo-wild type mice andb-endorphin deficient mice. lism rather than food intake, which results in the increase

In the group ofb-endorphin deficient mice, four mice in body weight. The latter is weakly supported by theshowed clear signs of counter aggression during the finding of a trend towards a lower body temperature duringconflict. To determine whether these mice had different the active phase under baseline conditions inb-endorphinsleep patterns after the conflict as compared to mice that deficient mice compared to wild types.did not show counter aggression, we analyzed both groups The display of counter aggression by theb-endorphinseparately. However, no differences in NREM sleep, deficient mice during the conflict was somewhat unex-NREM sleep SWE, or REM sleep were found. pected. With the protocol we used, experimental animals

rarely display such counter aggression when they arefacing the heavier and territorial cage owner. Perhaps the

4 . Discussion counter aggression in theb-endorphin deficient mice wasdue to their attenuated analgesia[29]. It is not excluded

After a social conflict,b-endorphin deficient mice had that theb-endorphin deficient mice experienced more painhigher corticosterone levels than wild type animals while when receiving bites from the dominant male and this maythe peak temperature increase was not different between have elicited defensive aggression and counter attacks.the genotypes. In fact, body temperature returned to Alternatively, it could be that theb-endorphin deficiencybaseline levels faster in theb-endorphin deficient mice. affected aggressive behavior directly. It is known from theDuring the interaction with an aggressive dominant con- literature thatb-endorphin levels increase during a socialspecific, several of theb-endorphin deficient mice showed conflict, particularly in the submissive animals[8,13]. Thisclear signs of counter aggression whereas this was not seen increase ofb-endorphin in a defeated animal may functionin any of the wild type mice. Overall, theb-endorphin to suppress aggression and stimulate submissive behaviordeficient mice and wild type mice had fairly similar sleep in these individuals, thereby preventing further attackspatterns under baseline conditions and they showed similar from the dominant[11].changes in the amount of NREM sleep, REM sleep and In addition to the occurrence of counter aggressiveaccumulated SWE in response to a social conflict. In behavior, theb-endorphin deficient mice also had smalladdition, no differences were found in the sleep patterns of but significant alterations in their physiological response tomice that showed counter aggression and mice that did not. the social interaction. At the end of the conflict,b-en-Thus, these data do not support our hypothesis thatb- dorphin deficient mice had significantly higher corticos-endorphin facilitates NREM sleep and suppresses REM terone levels. This difference in corticosterone responsesleep after a social conflict. Although the complete lack of may be specific for social stress since Rubinstein et al.[29]endorphins from early development onwards might result did not find a difference in the corticosterone levels afterin mechanisms that compensate for this deficiency[10,29], restraint or ether stress. Such stimulus-specific modulationthe present study demonstrates that the typical sleep of corticosterone release underscores the complex role ofpattern after social defeat stress can still occur in the endogenous opioids in stress (for review see[25]). None-absence ofb-endorphin. theless, the results are in line with the finding that, of the

Our finding of an increased body weight inb-endorphin various opioids,b-endorphin in particular seems to have

L.M. Vaanholt et al. / Brain Research 978 (2003) 169–176 175

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