Effects of hypophysectomy on the sleep of neonatally Monosodium Glutamate-treated rats

0361~9230/88 $3.00 + .oO

Brain Research Bullerin , Vol. 2 1,pp. 897-903.B Pergamon Press plc, 1988. hinted in the U.S.A.

Effects of Hypophysectomy on the Sleep of Neonatally Monosodium Glutamate-Treated Rats JING

XING

ZHANG,’

JEAN-LOUIS

VALATX

AND MICHEL

JOUVET

Laboratoire de MtGdecine Expkrimentale, INSERM U5.2, CNRS WA1195 UniversitP Claude Bernard, 8 Avenue Rockefeller, 69373 Lyon-Cedex 08, France Received

17 November

1987

ZHANG, J. X., J.-L. VALATX AND M. JOUVET. Eficts ofhypophysectomy on the sleep of neonatally monosodium rats. BRAIN RES BULL 21(6) 897-903, 1988.-Monosodium Glutamate (MSG), known to induce neuronal cell degeneration of the arcuate nucleus of the hypothalamus, was subcutaneously injected (4 mg/g body wt.) at postnatal days 1 to 5 or 1 to 10 in female rats. Hypophysectomy was performed at 45-60 days of age. Sleep parameters were continuously recorded for at least 7 days. Results indicated that hypophysectomized (HYPX) NaCl-treated rats showed an increase of Slow Wave Sleep (SWS) (+29.%) and a decrease of Paradoxical Sleep (PS) (-36.7%) durations. In MSGtreated rats, hypophysectomy did not alter SWS durations but it increased PS durations as MSG dosing increased. It was concluded that arcuate nucleus neurons seemed to be not critically involved in sleep production mechanisms.

glutamate-treated

Monosodium glutamate Female Rats

Hypophysectomy

Slow wave sleep

Paradoxical sleep

Circadian rhythms

METHOD

advances in sleep physiology suggested the existence of endogenous sleep factors. Several neuropeptides could have a specific role in the regulation of the sleep wake cycle (14,22). In rats, intraventricular injections of derivatives of Pro-Opio-Melano-Cortin (POMC): desacetyl-aMSH and Corticotropin-like Lobe Intermediate Peptide (CLIP), respectively enhance the duration of Slow Wave Sleep (SWS) and Paradoxical Sleep (3). POMC perikaryons are almost exclusively located in the hypophysis and in the arcuate nucleus of the hypothalamus. If they play a major role in the sleep regulation, the lesion of these neurons should provoke a pronounced sleep deficit. For this purpose, Monosodium Glutamate (MSG) has been used for its neurotoxic effect. Since the first work of Olney (20), MSG neonatally injected is known to cause a neuronal cell degeneration mainly in the arcuate nucleus (17,18). In a previous publication Olivo et al. (19) showed that an increase of the total sleep duration was observed in the MSG-treated rats. This result could be interpreted as a compensatory effect of the remaining POMC cells in the hypophysis. The aim of the present work was to investigate the effects of the lack of both structures (arcuate nucleus and hypophysis) on the sleep waking cycle of hypophysectomized (HYPX) MSG-treated rats. RECENT

Six litters of 10 female OFA pups, supplied by IFFACREDO (France) at the first postnatal day were divided into three groups: Group 1 (MSG x 5) received a daily SC injection of MSG (4 mg/g b.wt.) at postnatal days 1 to 5; Group 2 (MSG x 10) received MSG at the same dosage at postnatal days 1 to 10, and Group 3 received a daily injection of O.% NaCl. All the rats were weaned at day 30. Under ether anesthesia, hypophysectomy was performed by a transaural route (IFFA-CREDO) at 45 or 60 days of age in 12 animals from each group. Hypophysectomized (HYPX) animals were housed in a room at a constant warm temperature (28? 1°C) with food (standard pellets EXTRALABO) and 5% glucosed water ad lib, conditions which allowed to keep them in a good health status for over 6 months. Twenty-seven animals were implanted with 4 cortical (EEG) and 4 neck muscle (EMG) electrodes under pentobarbital anesthesia (HYPX: 30 mg/kg IP; NaCl: 50 mg/kg IP). After a ten day habituation period, continuous polygraphic recordings were performed for at least seven days in a room at the same warm temperature and on an alternating 12

‘Present address: Department of Physiology, College of Medicine of ANHUI, HOFF1 Popuiat Republic of China. 2Requests for reprints should be addressed to Dr. J. L. Valatx.

897

X9X

ZHANG,

VALATX AND JOUVET

(9)

wt 300 250 zoo

150100

1

0

20

40

60 SO 100 age days

f2g

MSG-5

Ot

60

, age

1

80

f

.

igo days

l

.

12Q

MSG-10

lO$iM---80

80

age

days

age

days

FIG. I. Evolution of body weight as a function of age fin days). Upper teft diagram: Curve f representsthe body weight of NaCI-treated rats; Curve 2=rats receiving 5 daily injections of MSG; Curve 3=rats injected with MSG for IO days after birth. In spite of fat ace~m~atioo~ MSGtreated animals weighed less than control rats. Upper right diagram: Body weight of hypophysectomized (HI and nonhypophysectomi~ed (C)-NaCl-treated rats. Bottom diagrams: curves of body weight of nonhypophysectomized (C) and hypophysectomized (H) animak treated with S (MSG-5) or 10 injections of MSG (MSG-10). Note there was no difference between control-(C) and hypophysectomized (W)-MSGlO-treated rata, and so, the MSG treatment prevented the effect of hypophyaectomy on the weight’s curve.

hr light-dark schedule (lights on at 7.00 hr). WYPX rats were recorded between 3 and 28 weeks after hypophysectomy. Sleep records were armlyzed every 30 seconds visually and/or by an on-line sleep analyzer device (5). Records were classified into three stages: I) wakefuh~ss characterized by a low amp&&de fast activity on &onto-parietaf derivations,

associated with high EMG activity; 2) Slow Wave Sleep (SWS) with spindles activity (10-1s Kz) and high amplitude slow waves (0.5-S Hz) and 3) Paradoxical Sbep (PS) characterized by a low amplitude theta (6-S Hz) activity associated with muscular atonia and rapid eye movements. Hypnograms were stored on a magnetic tape memory mass and

SLEEP DURATIONS

IN HYPX MSG-TREATED

RATS

899

TABLE

1

FOOD AND WATER CONSUMPTION, URINE VOLUME AND ADRENAL WEIGHT IN NaCl AND MSG-TREATED RATS WITHOUT (CONT) OR WITH HY~PHYSECTOMY (HYPX)

Watert

Food*

NaCI

CONT

HYPX

CONT

HYPX

CGNT

HYPX

11.60 21.45

4.2? rtO.88

53.10 28.72

38.70 25.47

5.80 r1.83

23.60’ ~3.12

25.46 27.91

6.20’ 22.40

20

20

20

20

20

20

8

8

11.78 20.48

3.371 21.47

40.76 -r-8.72

35.32 26.85

23.91” 28.27

17.00 k3.48

12.53 23.81

6.20’ k1.74

20

20

20

20

20

20

4

4

7.64a 20.54

5.29 21.29

54.72 k6.75

37.62’ k1.16

22.40a + 12.62

21.72 21.54

18.58 r7.52

6.44’ 21.71

20

20

20

20

10

4

8

n MSG-IO**

Adrenaisg

HYPX

nq MSG-S#

urine Volt

CONT

n

10

*Food intake is expressed as g/24 hr *SD. tTap water with YTO glucose is expressed as ml124 hr *SD. #Urine volume is expressed as m1124hr *SD. §Adr~n~s=adren~ weight {mg)/l~ g body weight *SD. Vn=number of measurements. #MSG5=daily SC injection of Monosodium Glutamate from 1 to 5 postnatal days. **MSH-lO=daily SC injection of Monosodium Glutamate from 1 to 10 postnatal days. r-Test significance: MSG versus NaCI: $
further statistically computerized with specific homemade softwares for time series analysis (23), variance of analysis and Student t-test. For all the rats, food and water consumption, and urine volume were checked every day at 9.00 hr before and during the recording period. At the end of the experiment, animals were anesthetized and the brain was transcardially perfused with a fixative solution, removed and stored at -80°C for further immunohistochemical processing (7). Complete ablation of the hypophysis was checked. Adrenals were removed and their weights served as functional controls of hypophysectomy. RESULTS

For a better understanding of the sleep alterations, some of the physiological observations are summarized in Fig. 1 and Table 1. Developmental weight curves showed the MSG-treated rats weighed less, although fat deposit was much greater than in control animals (Fig, IA). Hy~physe~tomy stopped the NaCl-treated rat’s growth while it only slowed down the weight gain of MSG x S-treated rat and apparently had no effect on the weight of MSG x 10 rats (Fig. 1). Food consumption was lower in MSG x 10 rats (7.64+0.54 Vs. 11.6rt1.45 p
Adrena/ Weight (Table I) Given that body weights were very different, the ratio of adrenal weight over body weight was used for intergroup comparisons. This index was very low in hypophysectomized rats but there was no si~~cant difference between HYPX rats regardless of treatment. Sleep Durations (Table 2)

In nonhypophysectomized (non-HYPX) rats, MSG caused an increase of SWS duration (MSG x 5: -l-16.% p
ZHANC.

VALATX

AND JOUVET

FIG. 2. Circadian rhythms of Paradoxical Sleep (left panels) and Slow Wave Sleep (right panels) of hy~pbys~ctom~2~d rats receiving either NaCl (top panels) or 5 (MSG-5) (middle panels) or 10 injections MSG(MSG-10) (bottom panels). Sleep durations (ordinates) are expressed in minutes. Thick black lines indicate the dark period of the day. Note hypophysectomy provoked a 4 hour shift in the PS increase at the onset of the light phase, while there was no delay in the SWS increase.

of

but the light-on increase remained delayed by 4 hours. Hypaphyseetomy provoked a dissociation between SWS and FS rhythms. DISCUSSION

more pronounced in males, b) using PS as a measure, ferna& seem to be less sensitive to MSG than males. Such a sex difference in the MSG effects has been previously observed for other hormonal levels (6,tg).

Present results raise at least three main questions:

In a previous work (19) neonatal administration of MSG {4 injections) to male rats caused an increase of SWS and PS durations. fn the present study, with the same dose, we observed in fern&es an increase of SWS and a decrease of PS durations, With a higher dosage ($0 injections) fenafe PS duration was similar to that of NaCl-treated rats. Neuronal degeneration of the axcuate nucleus seemed however to be the same in both sexes. These results indicate 1) a dissociation of the effects of MSG on SWS and PS durations. SWS alterations were similar in both sexes while PS changes were

In our ~oudi~o~s~ ~~physe~torny caused a large increase in SWS duration in control rats which has not been observed in previous work (23). The only difference in the experimental protocols was the addition of 5% glucose in the d~~ing water in the present experiment. This effect was not observed in non~~~phy~~tomized rats whether the glucase was absorbed by intravenous infusion (8) or in the drinking water (present data). However, when &~cose was infused with insulin, Danguir and Nicolaidis (8) observed an increase in sleep duration. Moreover intracerebroventricular infusion of‘insufin caused a selective increase in SWS dura-

SLEEP DURATIONS

IN HYPX MSG-TREATED

RATS

901

TABLE 2 SLEEP DURATIONS*

IN MSG- AND NaCI-TREATED

RATS WITHOUT (CONT) OR WITH HYPOPHYSECTOMY

Slow Wave Sleep

Paradoxical Sleep 24 hr

NaCl nt MSG-5$ n MSG-108 n

Night

CUNT

HYPX

CONT

HYPX

754.1 8z 270.00

181.21 k22.86

255.85’ +33.71

400.14 229.06

498. i02 T54.20

15

27

15

27

15

27

679.41’ 280.88

644.4& -1-70.90

238.00’ k49.26

225.38” 243.71

441.17b k43.81

419.02? 1t55.25

34

36

34

36

34

36

256.8@ ~56.00

278.24” +51.06

389.00 k88.36

388.67’ k54.28

28

80

28

80

HYPX

CONT

HYPX

CONT

HYPX

96.66 211.24

65.372 213.37

34.13 218.78

33.70 k12.30

62.53 2 13.52

26.402 *7.70

581.78 228.60

15

27

15

27

15

27

76.76’ t2.5.70

64.30] k9.87

27.78 211.94

23.61c 28.02

34

36

34

36

34

36

32.39 + 15.40

39.30 213.70

59.77 213.86

49.86( -t16.76$

28

80

28

80

80

48.76” 40.38’ 2 18.67 f 10.02f

Day

HYPX

CONT

28

Night

24 hr

Day

CONT

101.39 89.1jc k20.24 k23.51

(HYPX)

645.8@ 666.91’ r 108.44 k73.94 28

80

*Sleep durations are expressed as minutes &SD. ?n=number of recording days. $MSG-5=Daily SC injection of Monosodium Glutamate from 1 to 5 postnatal days. $MSG-lO=Daily SC injection of Monosodium Glutamate from 1 to 10 postnatal days. t-Test significance: MSG versus N&l: apcO.05; ‘@
tion (9). Hypophysectomy, which is known to induce hypoglycemia, makes subjects hypersensitive to insulin (12). The SWS increment in HYPX rats could thus be explained. In MSG groups. The SWS durations of HYPX rats were not significantly different from those of non-HYPX animals. In other words, hypophysectomy no longer increased SWS duration in MSG-treated rats. If insulin were a sleep modulator, this result could be explained by the profound reduction in the insulin-specific binding capacity of the median eminence induced by MSG treatment (24). The comparison between HYPX rats shows an increase in the PS duration as MSG dosing increases. This augmentation in HYPX MSG females (-l-48%), which is greater than that observed in non-HYPX MSG males (+31%) (191, suggests that ablation of the hypophysis leads to a disinhibition of PS mechanisms in female rats. Some preliminary data have shown that adrenalectomy done in HYPX-NaCl-treated rats reversed the decrease of the PS quantity induced by hypophysectomy. In this condition, the amount of PS reached the level of non-HYPX animals. Adrenocortical steroids are known to activate neuronal activity in numerous regions of the brain and increase wakefulness (10). One could thus speculate that, after hypophysectomy, brain glucocorticoid receptors become more sensitive to the remaining level of corticosterone. As for insulin receptors (24), MSG treatment might also reduce brain corticoid receptors and make PS mechanisms insensitive to adrenocortical steroids. Plasma corticosterone level has been demonstrated to be higher in females (6), and this might explain why a higher MSG dosage is needed in females to reach the same level of results as in the males.

Hypothalamic lesions induced by MSG, i.e., the almost complete degeneration of POMC and VIP perikarya in the arcuate nucleus associated with hypophysectomy, did not dramatically alter the PS duration. MSG treatment however spared the rrMSH neurons located in the dorso-lateral hypothalamus (so-called (~2 group) (13,25). Moreover, the perikarya immunoreactive to crMSH antiserum appeared more numerous around the fomix in MSG-treated animals (Fig. 3) which might compensate for the AN deficit. These cells are not however immunoreactive to beta-endorphin nor ACTH antisera (16). In fact, this cYMSH-like peptide does not seem to be a POMC derivative. Some authors have demonstrated that these perikarya contain a peptide, different from MSH, sharing some common epitopes recognized by MSH antiserum (16,21). The same cells are also reactive to salmon MCH (Melanin-Concentrating-Hormone) and hGRFl-37 antisera (11). The role of these peptides in sleep regulation is still unknown. According to Chastrette and Cespuglio (31, two POMC derivatives (CLIP and des-acetyl-uMSH) seem to have sleep enhancing effects in rats. Given that sleep parameters of hypophysectomized MSG x lo-treated rats, i.e., in the absence of POMC derivatives, were not very different from control animals, our results suggest that POMC peptides are not critically involved in the basic sleep production mechanisms. These peptides may however play a role in sleep modulation related to stress situations (4). Indeed, MSG treatment causes impairments in stress responses (2), and for this reason sleep deprivation studies in MSG-treated rats are in progress to provide arguments in favor of this hypothesis.

902

ZHANG.

VALATX

AND JOUVET

SLEEP DURATIONS

IN HYPX MSG-TREATED

RATS

903

ACKNOWLEDGEMENTS

This work was supported by INSERM (U52), CNRS (UA 1195) and DRET (grant 84-160). We are grateful to Mrs. L. Leger, L. Pam-Pagan0 and Mr. K. Kitahama for their helpful assistance for immunochemistry. We thank Mr. J. Carew for revision of the English text and Miss A. Sidrot for typing the manuscript.

REFERENCES 1. Andersson, K.; Fuxe, K.; Eneroth, P.; Blake, C. A.; Agnati, L. F.; Gustafsson, J. A. Effects of androgenic and adrenocortical steroids on hypothalamic and preoptic catecholamine nerve terminals and on the secretion of anterior pituitary hormones. In: Fuxe, K.; Gustafsson, J. A.; Wetterberg, L., eds. Steroid hormone regulation of the brain. London: Pergamon Press; 1981:117-133. 2. Badillo-Martinez, D.; Nicotera, N.; Butler, P. D.; Kirchgessner, A. L.; Bodnar, R. J. Impairments in analgesic, hypothermic and glucoprivic stress responses following neonatal monosodium glutamate. Neuroendocrinology 38:438-446; 1984. 3. Chastrette, N.; Cespuglio, R. Influence of proopiomelanocortin derived peptides on the sleep-waking cycle of the rat. Neuroscience 62:365-370; 1985. 4. Chastrette, N.; Clemens, H.; Prtvautel, H.; Cespuglio, R. Proopiomelanocortin components: differential sleep-waking regulation? In: Inoue, S., ed. Sleep peptides: Basic and clinical approaches. Tokyo: Japan Scientific Societies Press; 1987:in press. 5. Chouvet, G.; Louailles, G.; Thomasset, D.; Valatx, J. L.; Emptoz, H. Analyse automatique en ligne des Ctats de vigilance chex l’animal de laboratoire. In: Court, L.; Trochetie, S.; Coucet, J., eds. Le traitement du signal electrophysiologie exptrimentale et clinique du systtme nerveux central. Fontenay-aux-roses: C.E.A.; 1986409421. 6 Conte-Devolx, B.; Giraud, P.; Castanas, E.; Boudouresques, F.; Orlando, M.; Gillioz, P.; Oliver, C. Effect of neonatal treatment with monosodium glutamate on the secretion of MSH, /3-endorphine and ACTH in rat. Neuroendocrinology 33:207-211; 1981. 7. Coons, A. H.; Leduc, E. H.; Connolly, J. M. Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. Exp. Med. 162:49960; 1955. 8. Danguir, J.; Nicolai’dis, S. Intravenous infusion of nutrients and sleep in the rat: an ischymetric sleep regulation hypothesis. Am. J. Physiol. 238:E307-E312; 1980. 9. Danguir, J.; Nicolaldis, S. Chronic intracerebroventricular infusion of insulin causes selective increase of slow wave sleep in rats. Brain Res. 30:97-103; 1984. 10. Feldman, S. Electrophysiological effects of adrenocortical hormones on the brain. In: Fuxe, K.; Gustafsson, J. A.; Wetterberg, L., eds. Steroid hormone regulation of the brain. London: Pergamon Press; 1980: 175-190. 11. Fellman, D.; Bugnon, C.; Risold, P. Y. Unrelated peptide immunoreactivities coexist in neurons of the rat lateral dorsal hypothalamus: human growth hormone-releasing factorl-37-, salmon melanin concentrating hormone- and o-melanotropin-like substances. Neurosci. Lett. 74:275-280; 1987. 12. Fulton, J. F. Textbook of physiology. London: W.B. Saunders Company; 1955.

13. Guy. J.; Leclerc, P.; Vaudry, H.; Pelletier, G. Identification of alpha-melanocyte-stimulating-hormone (alpha-MSH) neurons in the rat hypothalamus. Brain Res. 199:135-146; 1980. 14. Jouvet, M. Indolamines and sleep-inducing factors. Exp. Brain Res. (Suppl. 8):81-94; 1984. 15. Kawakami, F.; Terubayashi, H.; Ikamura, I.; Fukui, K.; Ibata, N.; Yanaihara, N.; Zimmerman, E. A.; Shiota, K.; Nakayama, R. Influences of Monosodium-l-Glutamate upon peptide neurons of the hypothalamus of the rat. Biomed. Res. (Suppl. 4):75-81; 1983. 16. Khachaturian, H.; Akil, H.; Brownstein, M. J.; Olney, J. W.; Voigt, K. H.; Watson, S. J. Further characterization of the extra-arcuate alpha-melanocyte-stimulating-hormone-like material in hypothalamus: biochemical nd anatomical studies. Neuropeptides 7:291-313; 1986. 17. Kizer, J. S.; Nemeroff, C. B.; Youngblood, W. Neurotoxic amino acids and structurally related analogs. Endocrinology 29:301-318; 1978. 18. Nemeroff, C. B.; Konkol, R. J.; Bissette, G.; Youngblood, W.; Martin, P.; Brazeau, P.; Rone, M. S.; Prange, A. J.; Breese, G. R.; Kizer, J. S. Analysis of the disruption in hypothalamicpituitary regulation in rats treated neonatally with Monosodium L-Glutamate (MSG): Evidence for the involvement of tuberoinfundibular c‘holindrgic and dopaminergic systems in neuroendocrine function. Endocrinoloav 101:613-622: 1977. 19. Olivo, M.; Kitahama, K.; Vrdatx, J. L.; Jouvet, M. Neonatal monosodium glutamate dosing alters the sleep-wake cycle of the mature rat. Neurosci. Lett. 67:186-190; 1986. 20. Olney, J. W. Glutamate-induced neuronal necrosis in the infant mouse hypothalamus. J. Neuropathol. Exp. Neural. 30:75-90; 1971. 21. Saper, C. B.; Akil, H.; Watson, S. J. Lateral hypothalamic innervation of the cerebral cortex: Immunoreactive staining for a peptide resembling but immunochemically distinct from pituitaryiarcuate a-melanocyte stimulating hormone. Brain Res. BuIl. 16:107-120; 1986. 22. Ursin, R. Endogenous sleep factors. Exp. Brain Res. (Suppl. 8):11%132; 1984. 23. Valatx, J. L.; Chouvet, G.; Jouvet, M. Sleep-waking cycle of the hypophysectomized rat. Prog. Brain Res. 42:115-120; 1975. 24. Van Houten, M.; Nance, D. M.; Gauthier, S.; Posner, B. L. Origin of insulin-receptive nerve terminals in rat median eminence. Endocrinology 113:139>1399; 1983. 25. Watson, S. J.; Akil, H. The presence of two alpha-MSH positive cell groups in rat hypothalamus. Eur. J. Pharmacol. 58:101-103; 1979. 26. Zhu, M. Y.; Wang, X. Y.; Zhang, D. X.; Wan, X. X. Effects of lesion of hypothalamic arcuate nucleus region on brain P-endorphin, 5-hydroxytryptamine and norepinephrine content and acupuncture analgesia in rats. Acta Physiol. Sinica 36:42-48; 1984.