GABAergic Neurons in PrepositusHypoglossi Regulate REM Sleep by ItsAction on Locus Coeruleus in Freely

Moving RatsSATVINDER KAUR, R.N. SAXENA AND BIRENDRA N. MALLICK*

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 India

KEY WORDS picrotoxin; REM-OFF neurons; REM sleep duration per episode; sleep-wakefulness; stimulation

ABSTRACT GABA in locus coeruleus modulates REM sleep. Apart from the pres-ence of interneurons, locus coeruleus also receives GABAergic inputs from prepositushypoglossi in the medulla, where the presence of REM-ON-like neurons have beenreported. Therefore, it was hypothesized that GABAergic projections from prepositushypoglossi to locus coeruleus may modulate REM sleep. The experiments were con-ducted on chronic rats prepared for recording EEG, EOG, and EMG in freely movingconditions. Bipolar stimulating electrodes were implanted in prepositus hypoglossibilaterally, while chemitrodes were implanted bilaterally in locus coeruleus. The pre-positus hypoglossi were bilaterally stimulated (3 Hz, 250 msec, 100 mA) for 8 h in thepresence and absence of picrotoxin (0.25 mg/250 nl) microinjection bilaterally in locuscoeruleus, followed by poststimulation recording for 4 h. It was observed that stimula-tion of prepositus hypoglossi alone significantly increased REM sleep primarily byincreasing the REM sleep duration per episode. However, when it was stimulated in thepresence of picrotoxin in LC, REM sleep decreased, predominantly due to decreasedREM sleep duration per episode. The results of this study suggest that GABAergicinputs from prepositus hypoglossi act on locus coeruleus and regulate REM sleep,possibly by inhibition of REM-OFF neurons. Synapse 42:141–150, 2001.© 2001 Wiley-Liss, Inc.

INTRODUCTION

The neural mechanism for the control of rapid eyemovement (REM) sleep is not yet known. A reciprocalinhibition between the REM-OFF and the REM-ONneurons for such regulation was proposed (Hobson etal., 1975; Sakai, 1988). Recent evidence suggests thatin the locus coeruleus (LC) cholinergic induction ofREM sleep is mediated by GABA (Mallick and Kaur,1999) possibly by inhibition of the REM-OFF neurons.The role of GABA in LC may be supported by thepresence of GABA-ergic neurons, terminals (Iijima andOhtomo, 1988; Jones 1991; Ford et al., 1995; Peyron etal., 1995), as well as GABAergic receptors (Olpe et al.,1988; Luque et al., 1994) in LC. GABA levels increasedin LC during REM sleep (Nitz and Siegel, 1997) andmicroinjection of GABA-antagonist, picrotoxin, de-creased (Kaur et al., 1997) while microinjection ofGABA into LC increased (Mallick et al., 2001) REMsleep. Iontophoretic application of GABA inhibited LCneurons (Gervasoni et al., 1998). Also, since GABAergic

neurons decreased c-fos expression during REM sleepdeprivation, it is likely that their activity reduced dur-ing REM sleep deprivation (Maloney et al., 1999).

Although GABA in LC has been proposed to be themost likely candidate to inhibit the REM-OFF neuronsduring REM sleep (Mallick et al., 2001), its sourceneeded investigation. The LC receives GABA-ergic in-puts from local interneurons (Iijima and Ohtomo, 1988)as well as from distant areas, viz., prepositus hypoglos-sus (PrH) (Aston-Jones et al., 1991), paragigantocellu-laris (Mugnaini and Oertel, 1985), and the preopticarea (Singewald and Philippu, 1998; Peyron et al.,1995). Although classically cholinergic neurons were

Contract grant sponsors: Council of Scientific and Industrial Research andDepartment of Science and Technology, India.

*Correspondence to: B.N. Mallick, School of Life Sciences, Jawaharlal NehruUniversity, New Delhi 110 067 India. E-Mail: remsbnm@yahoo.com

Present address for R.N. Saxena: Dept. of Zoology, University of Delhi 110 007.

Received 29 November 2000; Accepted 18 May 2001

Published online 00 Month 2001; DOI 10.1002/syn.1109

SYNAPSE 42:141–150 (2001)

© 2001 WILEY-LISS, INC.

proposed to be REM-ON type, the possibility of noncho-linergic REM-ON neurons, which could also be GABAer-gic, have also been proposed (Sakai and Koyama, 1996;Mallick et al., 1999). Besides, it has also been reportedthat some neurons in the medulla including PrH in-crease firing during REM sleep, the REM-ON neurons(Sakai, 1991). Hence, GABAergic neurons from PrHare one of the most likely candidates to act on theneurons in LC for the regulation of REM sleep. There-fore, this study was conducted with two objectives: 1) toinvestigate if the GABAergic neurons in the PrH mayinfluence REM sleep, and 2) whether the action of thePrH GABAergic neurons is mediated through the LC.

MATERIALS AND METHODS

Male albino wistar rats (200–250 g) were surgicallyimplanted with EEG, EOG, and EMG electrodes forrecording bipolar sleep-wakefulness (S-W) and REMsleep as mentioned in detail previously (Alam and Mal-lick, 1990). In brief, for recording bipolar EEG twostainless steel screw electrodes were fixed in the skull(2 mm anterior to and 4 mm lateral to bregma). Flex-ible insulated (except at the tip) wires were connectedbilaterally on either side of dorsal cervical neck mus-cles and to the muscles near the external canthus ofeyes for recording bipolar EMG and EOG, respectively.Another screw electrode was fixed on the midline overthe frontal sinus to serve as animal ground. A pair ofguide cannulae (chemitrode) targeting LC bilaterallywas implanted at AP 9.8 mm, L 1.0 mm, H 7.0 mm(Paxinos and Watson, 1982) as previously (Kaur et al.,1997). Two thin (125 mm) straight insulated wires,joined along their length, keeping the tips dorsoven-trally separated by about 1 mm, were used as bipolarstimulating electrodes. These stimulating electrodeswere implanted in the PrH bilaterally at coordinatesAP 10.8 mm, L 0.3–0.5 mm, H 7.5 mm through drillholes made in the skull. The stimulating and recordingelectrodes were connected to two separate 9-pin femaleplugs and fixed on the skull with dental acrylic. Atleast 4 days were allowed for the rat to recover fromsurgical trauma. Meanwhile, the rats were also habit-uated to the recording chamber before recording.

Recording

Experiments were conducted on 12 rats where thesame animal served as control for itself. Bipolar EEG,EMG, and EOG were recorded simultaneously on threechannels of a Grass polygraph. On the first day, base-line sleep-wakefulness-REM sleep was recorded for12 h (between 10 AM–11 PM). The second-day recordingwas continued when PrH was bilaterally stimulated for8 h (3 Hz, 250 msec, 100 mA) with rectangular pulsesobtained from an S8800 Grass stimulator connectedthrough SIU5 isolation unit (Grass, USA) followed bypoststimulation recording for 4 h. In two rats record-

ings were done on the third day without any treatmentto compare if the stimulation of PrH had any lastingeffect on S-W. On the fourth day LC was bilaterallyinfused with 250 nl of 0.1% picrotoxin (1.6 mM) andrecording continued for 8 h, when PrH was bilaterallystimulated with the same parameters followed by 4 hpoststimulation recording. The stimulus current wasmonitored on an oscilloscope at regular intervalsthroughout the experiment. The stimulation did notcause any unusual behavior or signs of fear or stress.The stimulation artifact was either absent or minimal,which did not interfere with sleep scoring. However,stimulation of PrH at higher current strength (150–200 mA) produced increased artifacts on the poly-graphic recording, making it difficult to discriminateS-W-REM sleep states, while the lower (50 mA) currentstrength did not affect S-W-REM sleep significantly.

Microinjection accompanied by stimulation

Two injector cannulae (30-gauge) were connected totwo 1 ml Hamilton syringes through thin polythelenetubings. Both the syringes were fitted into WPI make(SP210iw) infusion pump. The cannulae were filledwith 0.1% (1.6 mM) picrotoxin and inserted into the LCbilaterally through each of the guide cannulae. Afteran hour of acclimatization, 250 nl of 0.1% picrotoxinwas injected into the LC bilaterally at 100 nl/min usingthe infusion pump without disturbing the animal. Themicroinjection was followed by 8 h stimulation of thePrH followed by 4 h poststimulation recording as men-tioned above.

At the end of the experiment, under deep anesthesiathe stimulation sites were lesioned electrolytically byusing anodal current 500 mA for 20 sec (Grass DCLM5lesion maker). To determine the site and spread ofinjection of picrotoxin in the LC, 2% pontamine skyblue was microinjected in the same way as that ofpicrotoxin injection. The rat brains were perfused with4% paraformaldehyde containing 2% potassium ferro-cyanide and the stimulation as well as the picrotoxininjection sites were histologically identified. The stim-ulation and injection sites were reconstructed and plot-ted as in Figures 1 and 2, respectively. Some of therandomly selected sections were processed for GAD-immunostaining (for stimulation site at PrH) and TH-immunostaining (for injection site at LC) to establishthe presence of GAD1ve GABAergic (Fig. 3) and TH-positive noradrenergic (Fig. 4) cells at the stimulationand injection sites, respectively.

Data analysis

The histological localization of the stimulation sitesrevealed that out of 12 rats, in four rats the PrH wasstimulated bilaterally and in five the stimulation wasunilateral. In three animals none of the stimulationsites was found to be in the PrH and those were takenas nonspecific stimulation control.

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Fig. 1. Coronal sections of rat brain stem through PrH as per theatlas of Paxinos and Watson showing the reconstruction of lesionedsites at the tip of stimulating electrode are shown in this figure. Thesites where stimulation was found to increase REM sleep (hatched)

were in PrH, while the stimulation sites (filled) which did not increaseREM sleep were away from PrH. Abbreviations of the anatomicalterms: 4V, fourth ventricle; PrH, prepositus hypoglossi; MVe, vestib-ular nucleus.

GABA FROM PrH REGULATES REM SLEEP DURATION 143

Fig. 2. Schematic reconstruction diagram of coronal sections of therat brain stem through LC as per the atlas of Paxinos and Watson,showing the anatomical location and spread of the picrotoxin in LC.The sites in LC (hatched) were effective in significantly decreasingREM sleep when microinjected with picrotoxin, while those sites

which did not decrease REM sleep were away from LC and aremarked as ineffective (filled). Abbreviations of the anatomical terms:4V, fourth ventricle; Me5, trigeminal nerve; LC, locus coeruleus; Rpn,raphe pontis nucleus; PnC, pontis reticular nucleus (caudal).

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The polygraphic records were first scanned visuallyto mark obvious disturbances, if any. Based on stan-dard criteria (Singh and Mallick, 1996; Timo-Iaria,1970) the records were then scored in bins of 10-secepoch and subdivided into Active awake (AW), Quietawake (QA), Slow sleep (S1), Deep sleep (S2), and REMsleep. Total REM sleep, number of REM sleep episodesper hour, and the average duration of REM sleep perepisode were calculated and statistically compared us-ing one-way ANOVA (using statistical package BIO-STAT). The levels of significance were calculated ap-plying Neuman Keuls post-hoc test.

RESULTS

The percent time spent in sleep-wakefulness-REMsleep states in the normal, control, during stimulationof PrH alone, and during stimulation of PrH in thepresence of picrotoxin in the LC are shown in Table I.

Effect of bilateral stimulation of PrHon S-W-REM sleep

Bilateral stimulation of PrH significantly increasedREM sleep as compared to the baseline [P , 0.01;F(1,6) 5 24.19] during the stimulation period, while itreturned to baseline during the poststimulation period(Table I). As compared to baseline the increase in REMsleep during the stimulation period was due to a sig-nificant increase in both the frequency of generation of

REM sleep per hour [P , 0.05; F(1,6) 5 3.8] (Fig. 5a)as well as in the mean duration of REM sleep perepisode [P , 0.01; F(1,6) 5 21.73] (Fig. 6a). Boththese increases returned to baseline level during thepoststimulation period. The stimulation significantlydecreased AW [P , 0.05; F(1,6) 5 5.71] and increasedS2 [P , 0.05; F(1,6) 5 2.22] during the stimulationperiod as compared to the baseline (Table I). However,during the poststimulation period there was a signifi-cant decrease in S2 as compared to the baseline [P ,0.01; F(1,6) 5 29.18]. An hour-wise analysis revealedthat the frequency of generation of REM sleep in-creased significantly in the first and third hour only(Fig. 5b), while the mean duration of REM sleep perepisode significantly increased for every hour duringthe entire period of stimulation (Fig. 6b). However,during the poststimulation period both of these de-creased significantly in the third and fourth hour ascompared to the baseline (Figs. 5b, 6b).

Unilateral stimulation of PrH or stimulation of areasaway from the PrH did not affect REM sleep signifi-cantly (Table I).

Effect of stimulation of PrH in presence ofmicroinjection of picrotoxin in LC

Stimulation of PrH in the presence of picrotoxin inLC did not increase REM sleep as compared to thebaseline [P , 0.01; F(1,6) 5 12.03]. However, as

Fig. 3. The photomicrographs ofthe histological section through PrHimmunostained for anti-GAD areshown at different magnifications.a: The lesion mark at the tip of thestimulating electrode as shown byan asterisk was in prepositus hypo-glossi (PrH) surrounded by GAD-positive GABA-ergic neurons. b:The magnified photomicrograph ofthe area marked in a is shown here.c: A magnified view of the areamarked in b is shown here. The ar-rows mark some of the GAD positiveneurons. Abbreviations: Cbl, lobuleof cerebellum; PrH, prepositus hy-poglossi; V, fourth ventricle. Scalebar in a 5 200 mm; b 5 80 mm, c 520 mm.

GABA FROM PrH REGULATES REM SLEEP DURATION 145

compared to baseline it increased QA [P , 0.05;F(1,6) 5 4.61] and decreased S2 [P , 0.05; F(1,6) 54.21]. The decrease in REM sleep was due to a signif-icant reduction in the mean duration (Fig. 6a) of REMsleep per episode [P , 0.01; F(1,6) 5 11.21], while thefrequency of generation of REM sleep per hour re-mained unaffected (Fig. 5a). During the poststimula-tion period all the parameters were comparable to thebaseline.

An hour-wise analysis showed that during stimula-tion the frequency of induction of REM sleep per hourdecreased significantly for the second and fourth hour

only (Fig. 5b); however, the mean duration per episodedecreased significantly for the first three hours and thefifth hour (Fig. 6b).

Comparison with values when GABA andpicrotoxin were individually microinjected

into the LC

The results of this study were also compared with theresults of our studies reported recently where undersimilar conditions picrotoxin (Kaur et al., 1997) andGABA (Mallick et al., 2001) were microinjected into theLC and the effects on REM sleep were studied. The

Fig. 4. Photomicrograph of a representative histo-logical section through LC is shown in (a). The sites andspread of bilateral pontamine sky blue injection (ar-rows) where picrotoxin was microinjected are seen. In(b) a section at higher magnification through LC fromanother experiment where picrotoxin could prevent thePrH stimulation-induced increase in REM sleep isshown. This section was immunostained with TH-anti-body. Dense TH-positive neurons (marked by arrows) inthe immediate vicinity of the tip of the cannula tract (ct)are seen. Faint blue coloration of pontamine sky blue(mostly washed off while immunostaining) may also beseen at the injection site. Abbreviation: V, fourth ven-tricle.

TABLE I. Mean (6SEM) percent sleep-wakefulness-REM sleep

Groups AW QA S1 S2 REM sleep

Baseline 35.69 6 1.99 15.05 6 1.91 18.22 6 1.29 26.03 6 0.37 4.73 6 0.67PrH STIM S 23.2 6 3.26* 19.17 6 2.83 15.09 6 4.05 35.30 6 6.45* 9.89 6 0.77**

PS 36.75 6 6.11 16.52 6 1.58 22.05 6 4.0 18.59 6 0.80** 3.56 6 0.19PrH STIM 1 PICRO S 33.05 6 5.09 26.04 6 4.63* 18.07 6 1.49 20.96 6 3.69* 1.63 6 0.39**

PS 29.01 6 6.20 20.71 6 5.47 18.29 6 2.54 25.06 6 8.16 3.91 6 0.81STIM away from PrH S 27.06 6 4.48 21.90 6 6.22 18.26 6 0.72 27.67 6 13.95 5.56 6 1.06

PS 30.47 6 5.35 27.58 6 5.05 19.70 6 1.68 17.89 6 7.93 4.83 6 0.81

*P , 0.05 as compared to baseline and **P , 0.01 as compared to baseline. S, stimulation; PS, poststimulation.

146 S. KAUR ET AL.

PrH stimulation-induced increase in REM sleep wassignificantly more as compared to that of after micro-injection of GABA alone in the LC [F(1,7) 5 7.58; P ,0.05] (Fig. 7a). However, the effect of PrH stimulationassociated with picrotoxin microinjection in LC onREM sleep was comparable to that of picrotoxin injec-tion alone in the LC (Fig. 7a). The increase in REMsleep duration per episode on stimulation of PrH wascomparable to microinjection of GABA alone in LC.Similarly, the decrease in REM sleep duration per ep-isode on stimulation of PrH in presence of picrotoxin inLC was comparable to that of microinjection of picro-toxin alone in the LC (Fig. 7b).

DISCUSSION

Bilateral stimulation of PrH significantly increasedREM sleep throughout the stimulation period. Thisenhanced REM sleep was due to an increased meanduration of REM sleep per episode throughout, while

the frequency of initiation of REM sleep per hour in-creased significantly for the initial 3 h only. Thechanges returned to baseline during the poststimula-tion period. The PrH stimulation-induced increase inREM sleep was prevented if the stimulation was deliv-ered in the presence of picrotoxin in the LC. The stim-ulation did not cause any abnormal behavioral changein the animal. A lower current intensity (50 mA) did notaffect REM sleep significantly. The observed effectswere not caused by tissue damage due to electrodeimplantation in the brain or due to the applied currentfor the following two reasons. One, only 0.025 mC/pulsewas delivered, which was significantly lower than thecurrent intensity (0.5 mC/pulse) reported to induce tis-sue damage (Lilly, 1961; Babb et al., 1977), and, two,the S-W-REM sleep during the poststimulation periodand that recorded the following day after stimulationwere comparable to that of the baseline.

Fig. 5. The effects of bilateral PrH stimulation alone (STIM) andin the presence of picrotoxin in LC (STIM 1 PICRO) on the mean(6SEM) frequency of initiation of REM sleep per hour, as compared tobaseline (BSLN) and their respective poststimulation (PS and PS 1PICRO) values are shown. The results for the entire recording periodare shown in A while the same every hour are shown in B. Signifi-cance levels, as compared to baseline values, are marked by asterisks.*P , 0.05; **P , 0.01.

Fig. 6. The effects of bilateral stimulation of PrH alone (STIM)and in presence of picrotoxin in LC (STIM 1 PICRO) on the mean(6SEM) duration of REM sleep per episode as compared to baseline(BSLN) and respective poststimulation (PS and PS 1 PICRO) valuesare shown. The results for the entire recording period is shown in Awhile that for every hour during stimulation and post-stimulationperiod are shown in B. Significance levels are shown in Figure 5.

GABA FROM PrH REGULATES REM SLEEP DURATION 147

The stimulation parameters (3 Hz, 250 msec, 100 mAfor 8 h) were selected based on our previous study(Singh and Mallick, 1996) where the LC was stimu-lated at 2 Hz, 300 msec, and 200 mA for 8 h. However,in this study a relatively lesser strength but higherfrequency was used so that the stimulation was ex-pected to be more effective while the spread of currentwas expected to reduce. The PrH neurons are reportedto stain for GAD (Mugnaini and Oertel, 1985) and thestimulation site showed GAD1ve neurons (Fig. 3). Thissuggested that GABA neurons were likely to have beenstimulated. It may be argued that since the strengthand frequency of stimulation were low, it is unlikelythat the spread of stimulation could be far. Althoughthe exact distance of the spread of stimulation cannotbe commented upon, it may at least be said that theeffects were likely to be specific to stimulation of thePrH neurons because stimulation of areas adjacent toPrH did not affect REM sleep significantly. Also, sincethe stimulation did not significantly increase wakeful-ness, it is unlikely that the stimulation had spread intothe midbrain reticular formation (Huttenlocher, 1961;Kasamatsu, 1970; Manohar et al., 1972). Additionally,the stimulus did not spread too far may also be argued

by the fact that LC was approximately 1 mm away fromthe stimulation site and it has been reported that stim-ulation of LC under similar conditions reduced REMsleep (Singh and Mallick, 1996). Possibilities of theeffects caused by stimulation of non-GABAergic neu-rons present around the stimulating area or due tostimulation of nerve fibers passing through the region(whose perikarya may be located outside the stimulat-ing sites) cannot be ruled out.

It was observed in this study that mild bilateralelectrical stimulation of PrH increased REM sleep aswell as S2, but decreased AW. The stimulation effect onREM sleep was opposite to that of injection of theGABA antagonist, picrotoxin (Kaur et al., 1997) butsimilar to that of injection of GABA (Mallick et al.,2001) in the LC. Although the stimulation effects onAW and S2 were opposite to that of microinjection ofGABA in LC (Mallick et al., 2001), the effects werecomparable to those after microinjection of picrotoxinin the preoptic area (Ali et al., 1999) where some of theGABAergic sleep-active cells are reported to be present(Gallopin et al., 2000). The PrH stimulation-inducedeffects were more pronounced on REM sleep (109%increase) as compared to on S2 (34% increase) and onAW (26% decrease) stages. This supports our hypothe-sis that GABAergic neurons in the PrH are primarilyinvolved in the regulation of REM sleep. Since sleepand wakefulness are related but opposite phenomenaand normally REM sleep follows deep sleep, it is notunusual that alteration in one of the phenomena isreflected in the other phenomenon as well. Neverthe-less, the phenomenon in which the maximum effectwas exerted, REM sleep in this case, should be consid-ered as the primary effector. A word of caution, how-ever, is that a possible role of these neurons in modu-lating other states of sleep-wakefulness cannot beconclusively ruled out from the results of this study.

The consistent increase in duration of REM sleep perepisode through the stimulation period may be sup-ported by our previous findings that the REM sleepduration per episode is maintained by GABA in LC(Mallick and Kaur, 1999). Stimulation of LC reducedREM sleep in rats due to a reduction in the frequencyof generation of REM sleep (Singh and Mallick, 1996),while stimulation of LDT in cats increased REM sleep,which was due to an increase in REM sleep durationper episode (Thakkar et al., 1996). Hence, it is possiblethat the LDT might be influencing the PrH neurons formodulating REM sleep.

The results of this study showed that the PrH stim-ulation-induced increase in REM sleep could be pre-vented by microinjection of picrotoxin into the LC. Insimplistic terms it suggests that GABAergic neuronsfrom PrH synaptically relay to the LC and inducedREM sleep. If there was no synaptic relay but PrHoriginated fibers were only passing through the LCarea, picrotoxin (a chemical) in LC would not have been

Fig. 7. The histograms show the percent change in total (mean 6SEM) REM sleep (A) and REM sleep (mean 6 SEM) duration perepisode (B) in baseline (BSLN) and under different experimentalconditions. The values of microinjection of GABA (Mallick et al., 2001)and picrotoxin (PICRO) (Kaur et al., 1997) in LC have been takenfrom data of our recent reports for comparison. BSLN: baseline with-out stimulation; STIM: stimulation of PrH; STIM 1 PICRO: PrHstimulation in presence of picrotoxin in LC; PICRO: microinjection ofpicrotoxin in LC. **P , 0.01 as compared to baseline; $P , 0.05 ascompared to GABA microinjection in the LC.

148 S. KAUR ET AL.

able to prevent the effect. It may be argued that if theGABAergic neurons in PrH were influencing REMsleep by relaying to some area in the brain other thanthe LC, then picrotoxin injection into the LC would nothave blocked the PrH stimulation-induced increase inREM sleep while picrotoxin away from LC would nothave been ineffective. Thus, circumstantial evidencesupports that at least some GABAergic inputs fromPrH to LC (Aston-Jones et al., 1991) are involved in theregulation of REM sleep; however, it may need to beconfirmed experimentally using a direct and more con-servative approach.

A reduction in duration of REM sleep per episode, asobtained in this study, is similar to that of picrotoxininjection alone into the LC (Kaur et al., 1997). Thestimulation of PrH increased REM sleep primarily byincreasing the duration of REM sleep per episode.Thus, the results of this study also support a previousreport that GABA and picrotoxin alone in the LC in-creased and reduced, respectively, REM sleep durationper episode (Mallick et al., 2001). Under identical con-ditions saline injection into LC was ineffective (Kaur etal., 1997) and if the picrotoxin injection was away fromthe LC, the PrH stimulation-induced effects could notbe blocked. These suggest that the effects of picrotoxininjection into the LC were due to specific blockage ofGABA-A receptors in the LC. The dose of picrotoxinwas one-sixth the dose of GABA and the doses were thesame as used in preceding studies (Mallick et al., 2001).

It may be said that PrH stimulation should haveflooded LC with GABA, which in turn competed withpicrotoxin and, therefore, PrH stimulation should haveprevented the effects of picrotoxin in LC. However,since picrotoxin noncompetitively inhibits the GABA-Areceptors by binding to a site different from that of theGABA binding site (Olsen et al., 1991), increased levelsof GABA, caused by PrH stimulation, could not com-pete with the effect of picrotoxin in the LC. Thus, theresults of this study support our model (Mallick et al.,2001) that persistence GABA in LC modulates theREM-OFF neurons in LC and maintains the durationof REM sleep. This may be supported by the report thatPrH stimulation inhibited the noradrenergic LC neu-rons and the response was blocked by i.v. injection ofpicrotoxin (Ennis and Aston-Jones, 1989). Neverthe-less, for further confirmation it needs to be shown invivo, although that is difficult, if behaviorally identifiedREM-OFF neurons in LC are also hyperpolarized byGABAergic inputs coming from PrH and that modu-lates REM sleep.

In addition to GABAergic neurons from PrH project-ing to LC, they also project to occulomotor nucleus andregulate eye movements (Fuchs et al., 1985; McCreaand Baker, 1985). It is possible that the control of eyemovements through such a GABA-mediated pathwaymay be responsible for REM sleep associated eye move-ments. Also, projections from the PrH to the pedun-

culo-pontine tegmentum (PPT) (Higo et al., 1989),which are triggered by eye-movement related informa-tion (McCarley et al., 1978; Nelson et al., 1983), havebeen postulated to play a role in the generation of thePGO waves during REM sleep. Thus, in addition to theprojection of GABAergic neurons from PrH to LC, theirprojections to PPT may also help in regulating REMsleep because REM-ON and REM-OFF neurons in PPTand LC, respectively, are mutually connected for theregulation of REM sleep (Mallick et al., 2001).

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