Sleep-inducing function of noradrenergic fibers in the medial preoptic area

0361-9230/93 $6.00 + .OO Copyright 0 1993 Pergamon Press Ltd.

Brain Research Bullefin, Vol. 32, pp. 153-158, 1993 Printed in the USA. All rights reserved.

Sleep-Inducing Function of Noradrenergic Fibers in the Medial Preoptic Area V. MOHAN KUMAR,’

R. SHARMA, S. WADHWA AND S. K. MANCHANDA

Department of Physiology and Anatomy, All India Institute of Medical Sciences, New Delhi-l 10029, India

Received 22 June 1992; Accepted 8 March 1993 MOHAN KUMAR, V., R. SHARMA, S. WADHWA AND S. K. MANCHANDA. Sleep-inducing function of noradrenergic fibers in the medial preoptic area BRAIN RES BULL 32(2) 153-158, 1993.-The aim of the investigation was to find out the

role of noradrenergic (NE) terminals of the medial preoptic area (mPOA), in the regulation of sleep-wakefulness. Studies were conducted on free-moving adult male rats with chronically implanted cannulae in the mPOA. Sleep-wakefulness was assessed on the basis of EEG, EMG, and EOG recordings along with behavioral observations. Lesioning of catecholamine terminals (with 6-hydroxydopamine) in the mPOA produced an increase in quiet wakefulness. Prevention of NE fiber destruction, by pretreating the rats with imipramine, prevented this effect. This demonstrated that the increased quiet wakefulness produced by 6-OHDA was the result of NE fiber destruction. Changes in sleep-wakefulness were also assessed after microinjection of NE into the mPOA, in normal and ventral noradrenergic bundle (VNA)-lesioned rats. NE administration induced sleep in VNA-lesioned rats, and arousal in normal rats. The findings suggest that the NE terminals in the mPOA, projecting via VNA, play a role in the induction of sleep. Noradrenaline Medial preoptic area Desmethylimipramine Catecholamines

Sleep-wakefulness

6-Hydroxydopamine

Brain stem

METHOD

LESIONS in the preoptic area (POA) produce not only reduction in sleep but also disruption in the sleep-wake cycle (3,2 1,22,25). Low frequency electrical stimulation of the POA induced EEG synchronization and sleep. On the other hand, high frequency stimulation of the medial areas of the POA produced EEG desynchronization and arousal (4,9,27,28,32). Local application of NE at the mPOA also produces EEG desynchronization and behavioral arousal in rats (9,19,20). The POA receives afferent noradrenergic (NE) projections from the brain stem, mainly through VNA (2,30). The lesion in the VNA bundle by 6OHDA, in cats, has been shown to increase deep slow wave sleep and REM sleep (23). This may be taken to suggest that the NE terminals in the mPOA stimulate the arousal mechanism (9,19,32). It was suggested, on the basis of the action ofthe blockers, that the (Y,postsynaptic receptors are the target area of action of NE (20). Still, it is difficult to presume the role of NE fibers on the basis of a study where the drugs are locally applied through chronically implanted cannulae. The study of changes in sleep-wakefulness, after the destruction of NE fibers in the mPOA, would further clarify the role played by the NE terminals in the mPOA in normal sleep-wakefulness. The specific role of postsynaptic NE receptors could be investigated by studying the changes in sleep-wakefulness, after application of NE at the mPOA in the VNA-lesioned rats.

Male Wistar rats, weighing between 150 and 250 g, were used for the study. They were housed in separate cages in an animal room having controlled temperature (26 -t 2°C) and light-on period from 0500 to 1900 h. Food and water were provided ad lib. Experiments were separately conducted in two studies. NE

Fiber Lesion Study

This study was conducted to find out the changes in sleepwakefulness after the degeneration of NE fibers in the mPOA. Experiments were conducted in three groups of animals in which substances were injected into the mPOA through chronically implanted cannulae. The effects upon sleep-wakefulness were studied, in one group ofanimals, after the injection of 6-OHDA, which destroys the catecholamine (CA) fibers (17,30). Sleepwakefulness was assessed in another group of animals also, which were pretreated with desmethylimipramine (DMI) before the administration of 6-OHDA. Dopamine (DA) fibers were selectively destroyed by this procedure. Animals that received saline administration served as the control group. Bilateral guide cannulae were chronically implanted in the mPOA (A 6.8, L 0.6, H 1.8) according to Konig and Khpper atlas (13) along with EEG, EMG, and EOG electrodes in all the animals as described earlier ( 19,20). Sleep-wakefulness was

I To whom requests for reprints should be addressed.

153

154

MOHAN KUMAR

assessed in each animal for a period of 24 h by recording EEG, EMG, and EOG on a polygraph (Grass Model 7B). The behavior of the animal was also noted simultaneously. The record was divided into epochs of 30 s and visually scored using the criteria of Panksepp et al. (23). The entire record was analyzed and classified into five different stages of sleep-wakefulness. The active awake stage (W 1) was characterized by a desynchronized EEG, high voltage EMG, and eye movement artifacts in EOG. Behavioral observations showed that the animal was actively moving around in the cage. Quiet wakefulness (stage W2) was characterized by a d~ynchroniz~ EEG without body movement artifacts and absence of REM indicators in EMG and EOG. Records showing intermittent bursts of spindle or slow wave activity, interspersed with desynchronized EEG, low EMG, and little or no EOG activity, were classified as light slow wave sleep (stage SWS 1). Deep slow wave sleep (stage SWS 2) was characterized by continuous slow wave activity and complete bodily quiescence characterized by low EMG and low EOG activity. During REM sleep (PS) the cortical EEG was desynchronized. There was total neck muscle atonia and presence of rapid eye movement artifacts in the record. The rats were divided into three groups. After the basal recording of sleep-wakefulness, one group of rats received an injection of 8 pg of 6-OHDA (dissolved in 1~1of saline containing 0.2 mg/ml ascorbic acid) in the mPOA (group 1). The second group of rats was treated with DMI (25 mg/kg, IP), 30 min before injection of the same dose of 6-OHDA. The third group of rats received an injection of 1 ~1of normal saline at the mPOA immediately after the recording of sleep-wakefulness for 24 h. All the intracerebral injections were given at the rate of 0. I PI/ min. Sleep-wakefulness was recorded for an additional 24 h in all these animals, starting after 2 days of administration of drugs and saline at the mPOA. The study was completed, and injection sites and the degree of fiber degeneration histologically confirmed, in six animals of each group. Animals were sacrificed under ether anesthesia prior to removing the brain for histochemical localization of CA fibers according to the glyoxylic acid method (29). The values for each phase of sleep-wakefulness of different animals in each group, prior to intracerebral injection, were statistically compared using one-way analysis of variance. The same test was also applied for finding out the significance of the variation of the pooled readings of various phases of sleep-wakefulness from different groups. The values of different phases of

TABLE I SUBSTANCES

INJECTED

Substance

Time of

Injected at mPOA

Injection

I.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Saline Saline NE NE NE NE NE NE NE NE

Amount Injected at mPOA

at mPOA

0.2 pcl 0.2 pl I .5 fig in 0.2 ~1 saline I .5 pg in 0.2 ~1 saline 3 pg in 0.2 ~1 saline 3 pg in 0.2 pl saline 1.5 gg in 0.2 ~1 saline 1.5 pg in 0.2 ~1 saline 3 gg in 0.2 fil saline 3 pg in 0.2 pi saline

12-14 22-24 12-14 12-14 12-14 12-14 22-24 22-24 22-24 22-24

h h h h h h h h h h

Substance Injected at VNA

6-OHDA 6-OHDA 6-OHDA 6-OHDA

DMI+G-OHDA

6-OHDA

SALINE

ET At

100 90 80 70 T

l-----

60

A

50

E

40 30 20 10 0 Wl w2 Sl 52 PS m

WI w2 Sl $2 PS

BEFORE INJECTION

0

Wl w2 Sl s2 PS AFTER INJECTION

FIG. I. The percentage of time (mean + SE) of the total recording time spent in various phases of sleep-wakefulness before and after injection of various substances into the mPOA. The total recording time (24 h) is shown in percentage along y axis. Postinjection values are compared with preinjection values. **I, -C0.0 1.level of significant change compared to its own preinjection values, and increase as compared to saline injection.

sleep-wakefulness, before and after intracerebral injections, were compared in each group, by using paired t-test (two tailed, p < 0.01).

This study was conducted to find out the effect of injection of NE at the mPOA in rats whose NE terminals were degenerated. Electrodes for assessment of sleep-wakefulness and cannulae for injection at the mPOA were chronically implanted as described earlier. The animals were divided into 10 groups, in four of which 6-OHDA (8 pg in I ~1 saline with 0.2 m&ml ascorbic acid) was injected at the VNA bundle (A 1, L IS, V 1.6), in order to destroy the catecholamine fibers. lntracerebral injections were given in all the animals after 3 days of postoperative recovery. Recovery from operative trauma was assessed on the basis of return of rectal temperature, normal food and water intake, and locomotor activity to the preoperative level. The study was conducted in six animals of each group. Animals were habituated to the recording room and cage for at least 1 h before the experiment. Food and water were provided in the recording cage. The experimental procedure included uninterrupted recording of EEG, EMG, and EOC for 30 min before and 60 min after the intracerebral injection, as described earlier {19,20). The details of substances injected in the different groups of animals are indicated in Table 1. After the experiment, the animals were sacrificed under ether anesthesia to remove the brain for hi&chemical localization of CA in the mPOA and brain stem. The site of injector cannulae was also confirmed in the histological sections. Sleep-wakefulness was assessed on the basis of the EEG, EMG, and EOG records and behavioral observations. The 90min polygraphic record was divided into 5-min periods, and the awake period. within each ofthese time bins, were tabulated for statistical analysis. Firstly, the preinjection data obtained from each group was analyzed by the nonparametric two-way analysis of variance (Friedman test). in the second step, the mean preinjection and all the postinjection data of each group were analyzed by using the same test. The next analysis was done only on those

INDUCTION

155

OF SLEEP BY NE FIBERS

FIG. 2. Bar diagram shows the effect of injection of saline in the mPOA in normal rats during day (A) and night(B). Postinjection values are compared with preinjection values. *p < 0.05, **p -C0.01, level of significant change as compared to preinjection values. x-Axis indicates the time in minutes. The time before injection is shown as negative values. y-Axis shows the epoch time (5 min).

groups whose postinjection readings showed temporal variation. The preinjection mean readings were compared with postinjection readings, obtained during each S-min period after the injection, by the multiple range test (Friedman test). Lastly, the drug-injected groups were compared with the saline-injected group. The drug effects in the normal rats were also compared with the VNA-lesioned rats using the Wilcoxon matched pair signed rank test. RESULTS

NE Fiber Lesion Study All the phases of sleep-wakefulness, before injection, did not show significant variation among different animals of the same

group (Fig. I). Nor was there any significant variation among the animals of all the groups. Administration of saline, or 6OHDA along with DMI, did not produce alterations in any of the phases of sleep-wakefulness. 6-OHDA treatment, on the other hand, produced a significant increase in W2. This increase was atso statistically significant when compared with the saline injection (Fig. I). CA and DA fiber degenerations were confirmed histologically in the 6-OHDA treated animals. NE Replacement Study Preinjection data from different animals in each group did not show any significant variation. Injection of NE in the mPOA in normal sleeping animals, during the day, produced dose-dependent arousal, compared to the saline control (Figs. 2 and 3).

156

MOHAN

VNA

4

LE!iXONEO

KUMAR

ET AL.

ANIMALS

RECORDING

MB

-1)

(-in)

NE

(qq)

in

o.zjlI

SaIti)

FIG. 3. Bar diagram shows the effect of injection of NE in the mPOA in normal (left hand side) and VNA lesioned (right hand side) rats on awake period during day. The awake periods in VNA-lesioned rats are compared with identical periods in normal rats. *p -c0.05,**p i 0.01,***p < 0.00I,levelof significant change as compared to normal rats. x-Axis indicates the time in minutes. The time before injection is shown as negative values. y-Axis shows the epoch time (5 min).

During the induced awake period, the rats showed normal exploratory, licking, and scratching activities. Injection of NE in normal rats, during the night (when they are normally awake), produced no change, as they remained awake after the injection also, as expected (Fig. 4). In contrast to the effect observed in the normal animals, injection of NE (1.5 pg and 3 pg) in the VNA-lesioned rats, during the day, failed to produce arousal (Fig. 3). The difference was significant from 10 min onwards after the 3 fig NE injection, but it was identical to that observed in the saline control group (Fig. 2). On the other hand, injection of NE (1.5 and 3 pg) in the VNA-lesioned awake animals, induced sleep during the night. This effect was significantly different from that seen in normal animals treated with NE or saline (Figs. 2 and 4). Higher doses of the drug produced higher magnitude and earlier onset of change. After injection of 6-OHDA at the VNA, no fluorescence was seen in the mPOA and VNA, in histological sections, as compared with the normal. DISCUSSION

6-OHDA injection in the mPOA, which destroyed the catecholamine terminals, produced an increase in quiet wakefulness. Pretreating the animals with DMI (which can restrict the lesion to the DA terminals alone) caused no alteration in sleep-wake-

fulness. As the alterations in sleep-wakefulness were obtained only after destruction of NE and DA fibers (in the mPOA), but not after DA fibers alone, it can be inferred that it is the lesion of NE fibers that produce an increase in quiet wakefulness. In other words, the NE fiber terminals in the mPOA contribute to the induction of sleep. These results are contrary to the findings of Panksepp et al. (23) in cats, in which an increased somnolence was reported after injection of 6-OHDA into the trajectory of the VNA. The lesion of VNA bundle, however, is not comparable to the lesion of NE terminals at the mPOA, as the VNA supplies NE fibers to several other areas of the hypothalamus and basal forebrain, apart from the mPOA. Moreover, in their study, these authors do not rule out damage to the dorsal noradrenergic bundle because of a widespread decrease in the NE content of the brain. It may be recalled that the dorsal noradrenergic bundle has been shown to be involved in the arousal mechanism ( 11,15). Increased wakefulness observed (in normal animals) after injection of NE in the mPOA (19,20) may appear contrary to expectation in the light of increased quiet wakefulness obtained on destruction of NE fibers. The apparent contradiction probably resulted from the presynaptic action of NE. In an area innervated by NE fibers, locally applied NE could act on both postsynaptic and presynaptic receptors (25). A presynaptic site of action of hypothalamically injected NE in normal animals was suggested in an earlier study (5). The presynaptic terminals have predom-

INDUCTION

157

OF SLEEP BY NE FIBERS

NORM

At4IMAt.S

f

lo

20

30

40

50

61D

lo

20

3O

_-

NE

(3~1gin

40

50

60

RIZORDI?KI TIME t&it

0.2~11 saline)

FIG. 4. Bar diagram shows the effect of injection of NE in the mPOA in normal (left hand side) and VNA lesioned (right hand side) rats on awake period during night. *p -z 0.05, **p < 0.01, ***p < 0.001, level of significant change as compared to

normal rats.

inantly a2 receptors (14,26), the activation of which produces reduced release of NE from the terminals. Clonidine, an a2 NE agonist, had been shown to have powerful inhibitory action on NE cells of the locus coeruleus (6,7). In the present study, NE injection at the mPOA could induce sleep in the VNA-lesioned animals. The presynaptic NE receptors were not available at the mPOA in these rats, as the NE terminals had already degenerated by the time of local injection. Therefore, the response elicited in these animals must be due primarily to the action of NE on the postsynaptic receptors. Based on these findings, we may come to the conclusion that the arousal produced in the normal animals is due to the action of NE on the presynaptic terminals. Thus, it suggests a paradox in which injection of NE, in the normal animals, produces a decreased action of NE on the postsynaptic receptors. Such elf-inhibition would, of course, explain the seemingly paradoxical effects reported in normal animals. It also supports the present findings in which lesioning of NE terminals in the mPOA produces an increase in wakefulness. Local injection of neurotransmitte~ at a higher dose might produce a depola~~tion blockade (24), stimulation of recurrent

inhibitory circuit ( 18), and activation of dual receptors located at a single synapse (8,3 1). Some of these effects were indeed shown to occur within the rat hypothalamus ( 18). So, comparatively lower doses and volume of NE were used in this study (5,9,19,20). Lesions in the mPOA produce an increase in wakefulness (1,20,21). Electrical stimulation of this area at low frequency induces sleep, whereas at high frequency it produces arousal (4). Local injection of NE in normal animals produced effects similar to high-frequency stimulation. But the present findings show that the NE fibers are involved in hypnogenesis at the level of the mPOA. Thus, the NE fibers, ascending via the VNA to the mPOA, has a role that is different from that of the dorsal noradrenergic bundle (lo- f2,i 5,16). The findings of this study are also in line with the classical concept of the role of the preoptic area in hypnogenesis (9,27,28,32). ACKNOWLEDGEMENTS The study was supported by Indian Council of Medical Research. Help rendered by K. R. Sundaram, Department of B~ostatisti~, AIIMS is acknowledged.

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