Hypothalamic injection of prolactin or its antibody alters the rat sleep-wake cycle

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Physiology & Behavior, Vol. 55, No. 6, pp. 1015-1019, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0031-9384/94 $6.00 + .00

Hypothalamic Injection of Prolactin or its Antibody Alters the Rat Sleep-Wake Cycle

R A C H I D A R O K Y , 1 J E A N L O U I S V A L A T X , L U C E P A U T - P A G A N O A N D M I C H E L J O U V E T Laboratoire de M~decine Exp~rimentale, INSERM U 52,CNRS UA 1195, UniversiM Claude Bernard,

8, Avenue Rockefeller, 69373 Lyon Cedex 08, France

Received 8 June 1993

ROKY, R., J. L. VALATX, L. PAUT-PAGANO AND M. JOUVET. Hypothalamic injection ofprolactin or its antibody alters the rat sleep-wake cycle. PHYSIOL BEHAV 55(6) 1015-1019, 1994.--Several studies have suggested an interaction between prolactin and the sleep-wake cycle. In this study ovine prolactin (oPRL) and anti-prolactin antibody were microinjected into the rat dorsolateral hypothalamus, which contains prolactin-like immunoreactive neurons. Results indicate that during the light period, prolactin injection induced an increase in paradoxical sleep duration, whereas it caused a decrease when injected during the dark period. Anti-prolactin antibody injection during the dark period also decreased paradoxical sleep duration. There was no effect of oPRL or antibody on slow wave sleep duration irrespective of injection time. These results suggest that prolactin injection may have an inhibitory effect on hypothalamic prolactin neurons.

Prolactin Paradoxical sleep Dorsolateral hypothalamus Circadian rhythm Rat brain

SEVERAL studies demonstrate that prolactin (PRL) can induce behavioral effects such as lordosis (12), grooming, yawning (18), active avoidance behavior (7), and reduction of the responsive- ness to electrical foot shock (7). These effects may be mediated by an action on brain neurotransmission through central prolactin receptors (21,38). Indeed, recent studies indicate that PRL alters brain level of catecholamine (11,17), GABA (19), acetylcholine (40), opioid (23), VIP (27,30), and pro-opiomelanocortin (32).

Moreover, several studies demonstrate that cerebral administra- tion of PRL can influence the activity of hypothalamic neurons (4,11,14,41).

The involvement of PRL in sleep regulation was suggested by the observation that injections of PRL in the pontine cat increase paradoxical sleep (PS) (16). In hypoprolactinemic mu- tant rats, on the other hand, the circadian rhythm of PS was reversed, whereas slow wave sleep (SWS) remained unchanged (36). Moreover, Obal et al. have found that SC injections of ovine prolactin (oPRL) enhance PS duration in rabbits (22). We recently have observed the same sleep alteration in the rat after SC or ICV administration of oPRL during the light period.

However, nocturnal injection produced a decrease in PS dura- tion, suggesting that PRL may play a role in the regulation of the PS rhythm. Recently, the existence of a central neuronal population containing (24,31,33) and synthesizing (8,35) a PRL-like substance has been demonstrated. In our laboratory, we have found that PRL-like perikarya are exlusively located in the dorsolateral hypothalamus (24). The present study was undertaken to try to directly influence these neurons by in situ

injection of oPRL or anti-oPRL antibody and observe the ef- fects on sleep regulation.

METHOD

Twenty male OFA Sprague-Dawley rats weighing 250 g (IFFA-CREDO, France) were anesthetized with ketamine (150 mg/kg, IP) and implanted with four EEG electrodes over the parietal and occipital cortex and two EMG electrodes in the dor- sal neck muscles. A permanent stainless steel guide cannula (o.d.

= 0.64 mm, i.d. = 0.34 mm) was placed in the dorsolat44eral hypothalamus 2 mm above the PRL perikarya (Fig. 1) (1.3 mm to the midline, 3.3 mm posterior to bregma, and 8 mm ventral to the dural surface) according to the Paxinos and Watson atlas (25).

A removable mandrel was used to close the cannula. The elec- trodes and cannula were fixed to the skull with dental cement.

After surgery, animals were individually housed in a Plexiglas cage at a constant ambient temperature (23 _ I°C) and a 12 h - 12 h light-dark schedule (lights on at 0700 h, lights off at 1900 h) with water and food ad lib. A cable, through a swivel coupler, was routed to an eight-channel polygraph. After 10 days of post- operative recovery, rats were continuously recorded (24 h/24 h) for 2 days as a baseline control recording.

In situ injections were performed with a very small diameter glass needle (o.d. = 145/zm, i.d. = 75/~m) linked by a catheter to a Hamilton syringe. Rats received 0.2 #1 of either the vehicle or active substance over a l-rain period. The needle was removed after an additional 1 rain following the end of the injection. An-

1 To whom requests for reprints should be addressed.

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FIG. 1. Photomontage of two coronal sections showing on the left, pro- lactin immunoreactive neurons at the level of the perifornical region of the dorsolateral hypothalamus, and on the right, the shape and size of the cannula site at the same level (cresyl violet stained). V, third ventri- cle; f, fornix; arc, arcuate nucleus (×50).

imals received o P R L (1 or 10 ng) or polyclonal antibody raised against o P R L in the rabbit. The antibody was diluted at 1:10 in 0.9% NaC1 and glycerol. To test the specific action of the anti- body, we used normal rabbit serum as a control. For habituation to injection, rats received a saline injection at 1900 h, followed 48 h later with a vehicle + normal rabbit serum. The o P R L antibody was mjected 3 days later at 1900 h. The o P R L injection was performed at 1900 or 1200 as previously de- scribed (29).

States of vigilance were visually scored in 30-s epochs and classified as wake (low-amplitude and high-frequency E E G with frequent body m o v e m e n t s ) , S W S (spindles and cortical slow waves of progressively increasing amplitude and diminution of electromyogram amplitude), and PS ( E E G similar to that in wakefulness but more regular with a neck muscle atonia).

Data were stored and computed with h o m e - d e v e l o p e d soft- ware. M e a n values of sleep duration and numbers of episodes were compared by analysis of variance ( A N O V A ) and the Stu- d e n t ' s t-test.

At the end of the experiment, a histological control of the cannula position was performed for each animal. Data from two animals were not included in the analysis because the cannula was not in the dorsolateral hypothalamus.

RESULTS Effect of Diurnal oPRL Injection

A dose of 10 n g injected at 1200 h elicited a significant in- crease in PS duration ( + 2 0 % , p < 0.05) for the 7 h postinjection (Table 1). This effect appeared 1 h after the injection [Fig.

2(A,B)]. The n u m b e r of PS episodes was not significantly in- creased. The effect was no longer present during the following light period. Ovine prolactin at 1 n g produced a trend toward an increase in PS duration ( + 9 . 6 % ) [Fig. 2(C,D)]. For the two doses, wake (W) and S W S durations were not significantly changed.

"FABLE 1

EFFECT OF DIURNAL INJECTION OF OVINE PROLACTIN (oPRL~

CONT oPRL l0 ng -PRI. ! ng

w

d 157 ± 17.7 155,9 _+_ 15.2 16~).1 ± 27.2 Ep.nb 68.9 ± 11.9 71.3 _+ 10.4 69.1 ± 8.9 SWS

d 213 ± 17.2 206.6 +_ 10.4 203.2 + 28.8 Ep.nb 68.6 _+ 12.3 71,2 + 10.9 7(I.3 -+ 9.5 PS

d 44.9 _+ 7.6 53,9 +_ 8,5* 49.2 + 8,1 Ep.nb 2 5 . 7 _ + 6 30,1 ± 8.4 26.9_+ 5.1 Duration (d) expressed in minute (mean ± SEM) and episode number (Ep.nb) (mean ± SEM) of wake (W), slow wave sleep (SWS) and par- adoxical sleep (PS) after 10 ng (n = 9) or 1 ng (n = 7) of ovine prolactin (oPRL) dorsolateral hypothalamic injection at 12 h. CONT = saline in- jection.

* p < 0.02.

Effect of Nocturnal oPRL Injection

A dose of 10 n g of o P R L elicited a significant decrease in PS duration (Table 2). This effect appeared 1 h after the injection, reached a m a x i m u m 5 h later [Fig, 3(B)], and remained signifi- cantly decreased for the 7 h postinjection ( - 3 2 . 6 % , p < 0.02).

The n u m b e r of PS episodes was also significantly decreased.

There was no overall change in PS duration during the last 5 h of the dark period or during the following 12 h of the light period.

The W and S W S durations were not significantly changed.

Nocturnal Antibody Injection

There was no significant difference b e t w e e n the results ob- tained after saline or normal rabbit serum (Table 3). Results fol- lowing antibody injections were compared to those after the rab- bit serum. A n t i b o d y injections at 1900 h also provoked a decrease in PS duration. This effect appeared 4 h after the injection and lasted throughout the night ( - 3 0 . 2 % , p < 0.01) (Table 3)

T A B L E 2

EFFECT OF NOCTURNAL INJECTION OF OVINE PROLACTIN (oPRL)

Saline oPRL 10 ng

W

d 297.7 ± 32.5 289.2 ± 31.8 Ep.nb 47.1 _+ 14.6 45.9 ± 14.4 SWS

d 117.9 ± 28.1 113.7 ___ 28.3 Ep.nb 68.6 ± 12.3 45.6 _ 14.8 PS

d 22.4 _+ 7.5 15.1 ± 7.5*

Ep.nb 16.7 + 4.1 10.5 _+ 3.7t Duration (d) and episode number (Ep.nb) of W, SWS and PS after 10 ng (n = 9) of ovine prolactin (oPRL) dorso-lateral bypothalamic injection at 12 h.

For futher detail see Table 1.

* p < 0.05; tP < 0.005.

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FIG. 2. Duration of paradoxical sleep (mean _-!- SEM) after diurnal ovine prolactin (oPRL) injections at the level of the dorsolateral hypothalamus throughout the 7 h postinjection. Solid line: oPRL, dashed line: saline. Cu- mulative duration on the left (A,C), hourly duration on the right (B,D). (A,B) oPRL 10 ng at 1200 h, n = 9.

(C,D) oPRL 1 ng at 1200 h, n = 7. *p < 0.05; **p < 0.02.

( - 3 0 . 2 % , p < 0.01), with a high level of significance in the 12th h postinjection (just before the light period) [Fig. 4(B)]. The cu- mulative durations of W and S W S were not significantly altered.

However, there was a significant change at the 12th h post- injection. Wake increased by 50%, whereas SWS decreased by 52%.

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FIG. 3. Duration of paradoxical sleep (mean _+ SEM) after oPRL injection at 1900 h at the level of dorsolateral

hypothalamus throughout a 24-h period (n = 11). Solid line: oPRL, dashed line: saline. Cumulative duration on

the left (A), two hourly duration on the right (B). *p < 0.05.

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FIG. 4. Duration of paradoxical sleep (mean _+ SEM) after anti-oPRL antibody (Ab) injections at 1900 h at the level of the dorsolateral hypothalamus (n = 12). For further details see Fig. 3.

DISCUSSION

A neuronal population containing a PRL-like substance has recently been described in the laterodorsal part of the hypothal- amus (24). To influence this system, oPRL was infused in the vicinity of these neurons. Present results show that hypothalamic o P R L administration had the same effect as SC or ICV applica- tion. The PS duration decreased when injected during the night period and increased when injected during the light period (29).

These results suggest that o P R L acts locally on either non-PRL neurons surrounding the injection site or PRL neurons through autoreceptors. Several mechanisms may be considered to explain the observed o P R L effect on sleep. First, it has been shown that PRL alters the release of some brain factors known to modify sleep regulation such as dopamine (I1,17), pro-opiomelanocortin (32), and VIP (27,30). However, these effects are not in accor- dance with the specific action of PRL on PS. Second, oPRL in- jections could exert an inhibitory effect on PRL-containing

T A B L E 3

EFFECT OF NOCTURNAL INJECTION OF ANTI-PRL ANTIBODY

Saline CONT Ab

W

d 505 _ 41.4 493.2 _+ 50.3 527.1 _+ 49.6 Ep.nb 72.5 _+ 21.4 83.8 _+ 16.0 72.0 _+ 19.4 SWS

d 184.6 _+ 36.9 196.0 _+ 43.9 171.8 _+ 47.9 Ep.nb 73.7 _+ 22.0 83.7 _+ 16.9 72.0 _+ 19.7 PS

d 30.1 _+ 7.6 30.8 + 12.5 21.0 _+ 9.6*

Ep.nb 23.7 _+ 7.8 24.3 +_ 8.3 15.3 + 6.1t Duration (d) and episode number (Ep.nb) of W, SWS, and PS through the night after anti-PRL antibody injection at 1900 h in the dorso-lateral hypothalamus (n = 12). CONT = rabbit preimmun serum. For further detail see Table 1.

*p < 0.05.

t p < 0.005.

neurons through PRL autoreceptors located on these neurons.

Such an inhibitory action of PRL has been reported by Clemens et al. (4), who demonstrated a decrease in the firing rate of neu- rons located in the dorsolateral part of the hypothalamus after local PRL infusion. A similar effect produced by o P R L antiserum may be explained by the fact that antiserum may penetrate into PRL neurons, bind to this protein, and block its synaptic release.

Indeed, it has been demonstrated that antibodies raised against peptides such as vasopressin, corticotropin factor (1,2), or sub- stance P (3) are specifically taken up by neurons that synthesize these peptides and temporally blocked the physiological effect.

Opposing effects during the light and dark period suggest that PRL acts on the regulation of the circadian rhythm of PS. It has recently been found that PRL-like-containing neurons project to the suprachiasmatic nucleus (24,37) and raphe dorsalis (24) nu- clei, the lesion of which produces an alteration of PS and SWS rhythms (5,15). However, this hypothesis seems unlikely because present results show that only PS duration was altered after o P R L injections. Such a dissociation between PS and SWS rhythms suggests that the systems responsible for PS and SWS rhythms are different. This dissociation has already been observed after the lesion of hypothalamic paraventricular nucleus (26) and in hypoprolactinemic mutant rats (36). In the mutant animal, the decrease of plasma PRL was associated with a 50% diminution of the pineal melatonin content (9). Moreover, pinealectomy causes a lack of the PS rhythm without changes of SWS (20).

Furthermore, it has been shown that the lateral hypothalamus directly projects to the pineal gland (10).

In conclusion, our results suggest that the prolactin injection may have an inhibitory effect on hypothalamic PRL neurons that, in turn, could act on the regulation of PS rhythm. New experi- ments, particularly specific inhibition of the central PRL synthe- sis by injection of strands of PRL antisense mRNA, are in pro- gress for a better understanding of the mechanism by which prolactin acts on sleep regulation.

ACKNOWLEDGEMENTS

This work was supported by INSERM U52, CNRS UA 1195 and DRET (Grant No. 89203). We thank Markus Schmidt for correcting the English.

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