Brain Research 955 (2002) 1–7www.elsevier.com/ locate/brainres

Research report

D iurnal and nocturnal rodents show rhythms in orexinergic neuronsa a,b,c a,b ,*´Gladys S. Martınez , Laura Smale , Antonio A. Nunez

aDepartment of Psychology, Michigan State University, East Lansing, MI 48824-1117,USAbNeuroscience Program, Michigan State University, East Lansing, MI 48824-1117,USAcDepartment of Zoology, Michigan State University, East Lansing, MI 48824-1117,USA

Accepted 15 July 2002

Abstract

Orexin (ORX) A and B (hypocretins) are excitatory neuropeptides produced by neurons of the lateral hypothalamus that have beenimplicated in the regulation of the sleep–wake cycle. In rats, Fos (the product of the cfos gene) expression shows daily rhythms in areasinvolved in sleep and wakefulness and orexinergic neurons show elevated Fos expression during the night. The present study directlycompared the daily pattern of Fos expression in orexinergic neurons in diurnal (A. niloticus; grass rats) and nocturnal (R. norvegicus; labrats) rodents. Animals kept on a 12:12 light–dark cycle were perfused at six different Zeitgeber times (ZT), with lights on at ZT 0: 1, 5,13, 17, 20 and 23. In both nocturnal and diurnal rodents orexinergic neurons showed rhythms in Fos expression, with more Fos seenduring the active phase of each species. In the diurnal species, Fos expression in cells of the lateral hypothalamus that do not produceORX was elevated at ZT 20, a time when these animals sleep, and was low at ZT 13, a time of peak of activity. These results providefurther evidence for a link between activity in orexinergic neurons and wakefulness and that in grass rats, other neurons of the lateralhypothalamus may work in an antagonistic fashion with respect to orexinergic neurons to regulate wakefulness in this diurnal species. 2002 Elsevier Science B.V. All rights reserved.

Theme: Autonomic, limbic and other systems

Topic: Biological rhythms and sleep

Keywords: Orexin; Nile grass rat; Sleep–wake cycle; Rhythm

1 . Introduction (LC), the dorsal and medial raphe nuclei, and thetuberomammillary nucleus (TMN) [27]. Cholinergic neu-

Orexin (ORX) A and B, also known as hypocretin 1 and rons in the lateral tegmental nucleus and the pedun-2, have been recently implicated in the regulation of the culopontine nucleus, which mediate the cortical excitationsleep–wake cycle [15,27]. ORX-producing neurons send characteristic of REM and also REM atonia [13], aresubstantial projections to areas of the brain thought to play among the targets of ORX-ir neurons [27]. Other brainimportant roles in the control of vigilance and sleep. All regions thought to be involved in the support of wakeful-the monoaminergic cell groups, which have wake-depen- ness (e.g. the paraventricular nucleus of the thalamus,dent activity and provide diffuse cortical activation [32], PVT) [22] or in the facilitation of sleep (e.g. the lateralreceive projections from ORX-immunoreactive (ORX-ir) preoptic area [33]) also receive substantial projectionsneurons; ORX-ir fibers terminate in the locus coeruleus from ORX-ir neurons [27].

Several studies have confirmed the role of ORX in theregulation of sleep and arousal. Studies in narcoleptic dogsshowed that this condition is caused by the disruption ofone of the two ORX receptors genes, the ORX receptor2*Corresponding author. 118 Linton Hall, East Lansing, MI 48824-(ORX R) gene [17]. Behavioral and electroencephalog-1044, USA. Tel.:11-517-353-9066; fax:11-517-353-3355. 2

E-mail address: nunez@msu.edu(A.A. Nunez). raphic studies found that prepro-orexin knockout mice

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03264-X

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exhibit a syndrome of sleep dysregulation similar to human middle of the light and dark phases of the illuminationand canine narcolepsy [7]. Additionally, recent studies cycle (ZT 1, ZT 5, ZT 13, ZT 17, ZT 20 and ZT 23).found deficiencies of ORX-A in the cerebrospinal fluid(CSF) in human narcolepsy [20]. Further, ORX-A in-creases the firing rate of LC neurons in vitro [11,12], 2 . Materials and methodsdecreases the amount of REM sleep [5], and stronglyexcites serotonergic neurons in the dorsal raphe [6]. Animals used in this study were thirty adult maleIntracerebroventricular (i.c.v.) administration of ORX-A at Sprague–Dawley rats (lab rats) obtained from Charlesthe onset of the normal sleep period increases the propor- River Laboratories and thirty adult male grass rats from thetion of time spent awake and decreases the proportion of breeding colony established in this laboratory and derivedparadoxical sleep without a significant decrease in deep from the original group caught in Kenya in 1993 [14].slow wave sleep [11]. I.c.v. administration of ORX-A at Animals were singly housed and allowed free access tothe onset of the normal sleep period produces a dose- food (Harlan Teklad 8640 rodent diet) and water. Alldependent increase in the proportion of time awake; high animals were kept in a 12:12-h LD cycle with a dim reddoses produced also a reduction in paradoxical sleep and light on at all time. All experiments were performed indeep slow wave sleep [28]. I.c.v. administration of ORX-A compliance with the Michigan State University All-Uni-and -B also induces expression of Fos (the product of the versity Committee on Animal Use and Care, in accordancecfos gene) in nuclei where ORX fibers project, including with the standards in the NIHGuide for the Care and Usethe LC [9]. Fos expression in ORX-A neurons correlates of Laboratory Animals. Measures were taken to minimizepositively with wakefulness and negatively with non-REM pain or discomfort of the animals used in these experi-and REM sleep in rats [10] and in cats, Fos expression in ments.orexinergic neurons peaks during active wakefulness [36].

Daily rhythms in sleep and wakefulness are present in 2 .1. Immunocytochemistrymost mammals. In the rat, brain areas involved in the onsetand maintenance of sleep exhibit greater neural activity Animals (n56 per sampling time for each species) were(i.e. enhanced Fos expression) during the light than during perfused at ZTs 1, 5, 13, 17, 20 and 23. A light tight foilthe dark phase of the cycle [22,26,33]. In contrast, brain hood was placed over the heads of animals sacrificedareas involved in the support of wakefulness and arousal during the dark phase to prevent acute effects of light onshow more activity during the dark phase of the cycle, Fos expression. They were given an overdose of Nembutalwhen rats are active [22,24]. In the unstriped Nile grass rat (0.5 cc for grass rats and 1.0 cc for lab rats) and were(Arvicanthis niloticus; here referred to as grass rats), a transcardially perfused with 0.01 M phosphate buffereddiurnal rodent, the sleep cycle is reversed. In contrast with saline (PBS) followed by paraformaldehyde–lysine–so-rats, these animals sleep during the dark phase of the cycle dium periodate (PLP). Brains were removed and postfixedand are awake during the day with peaks of activity also at for 2 h in PLP before transferring them to 20% sucrose.dawn and dusk [18,23]. Brain areas involved in sleep in Brains were sectioned at 30mm thickness into cryoprotec-grass rats also show rhythms of Fos expression concordant tant in three alternate sets and stored in the freezer. Allwith their sleep pattern: Fos expression in the ventrolateral incubations were done in PBS with 0.3% Triton-X-100preoptic area (VLPO) is high at Zeitgeber time (ZT) 17 (PBS–TX). One set of tissue was washed for 1 h in 0.1 M(with ZT 0 as the beginning of the light phase of a 12:12 PBS and preincubated in 5% normal donkey serum (NDS,light–dark cycle) when these animal are likely to be Jackson Immunoresearch Labs.), for 1 h. Sections weresleeping, and is low at ZT 1 and ZT 13 when they are then rinsed and incubated in the primary antibody (rabbitawake [24]. anti Fos, Santa Cruz 1:25,000) with 3% NDS for 24 h at

Daily rhythms in the activity of ORX-A neurons were 48C. Tissue was then washed, placed in the secondaryreported in a study with rats showing that Fos expression antibody (donkey anti rabbit IgG, Jackson Immunoresearchin these neurons peaked at night [10]. In addition, prepro- Labs., 1:200) with 3% NDS at 48C for 1.5 h., rinsed again,ORX mRNA and ORX-A in the pons and in the preoptic / and incubated in avidin–biotin complex solution (Vectoranterior hypothalamic area increase during the dark hours ABC elite kit) for 1.5 h. Tissue was rinsed in acetateand decrease during the day in rats, suggesting a role of buffer and Fos reaction product was visualized using aORX in the regulation of the sleep–wake cycle [35]. DAB nickel solution in acetate buffer reacted with hydro-

To investigate variations in the activity of ORX-ir gen peroxide. After this, tissue was rinsed for 10 min inneurons during the light–dark cycle in diurnal and noctur- PBS–TX, washed in PBS for 30 min and preincubated innal rodents we used double immunocytochemistry (ICC) normal horse serum (NHS, Vector Labs.) for 1 h. Sectionsstaining for Fos and ORX-B. Animals were taken at six were rinsed again for 10 min and incubated in goatdifferent times of day in order to obtain information about anti-orexin-B (Santa Cruz 1:100,000) for 48 h. After threeFos expression in orexinergic neurons at the beginning and 10-min washes, the tissue was incubated in the secondary

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(horse anti goat, Vector Labs., 1:200) for 1.5 h, rinsed, Fos-ir cells, ORX-ir cells, and double-labeled cells. Allincubated in ABC (Elite kit, Vector Labs.), and washed labeling was confirmed by examining the tissue with high-again in PBS. ORX reaction was then visualized using power oil immersion lens (1003 magnification). CellsDAB in Trizma buffer reacted with hydrogen peroxide. were counted in one side of the brain for each of the threeTissue was then rinsed, mounted onto gelatin-coated slides, areas and counts were used to determine the total numberdried, dehydrated, and cover-slipped. Antisera specificity of Fos-ir cells, ORX-ir cells and the proportion of ORX-irwas determined in a control experiment in which the neurons containing Fos. All counts were made withoutprimary antiserum was omitted. knowledge of the ZT at which the animals were perfused.

Two-way ANOVAs within species were used to evaluate2 .2. Data analysis the effects of ZT and area (medial, lateral, and central) on

the percentage of ORX-ir cells containing Fos, the numberA camera lucida was used to draw a horizontally- of cells expressing Fos but not ORX-ir, and the number of

oriented (1200mm3700 mm for grass rats and 2150 cells containing ORX. When necessary, data were trans-mm3750 mm for lab rats in order to cover relatively the formed (square root and log transformations) prior to thesame region in both species) rectangle that covered the 2-way ANOVA. Nonparametric one-way ANOVAs (Krusk-dorsal and lateral hypothalamus (LH), with the fornix al–Wallis test) were used to evaluate the individual effectslocated in the central region of the rectangle just above the of ZT or area on the number of ORX-ir cells in grass rats.lower edge (see Fig. 1b). This rectangle was divided Comparisons among individual means used the Fisher’svertically in three regions of the same size in order to get protected least significant difference (LSD) following thethe medial, central and lateral areas of this region. One parametric ANOVAs, and nonparametric multiple com-section per animal was selected with the mammil- parisons following the Kruskal–Wallis tests. Differenceslothalamic tract, the third ventricle and the fornix as were considered significant whenP,0.05. In all cases, thelandmarks and a camera lucida drawing was made of data are presented as mean6S.E.M.

Fig. 1. (a) ORX-immunoreactivity and Fos-immunoreactivity in grass rats as seen under high-power oil immersion lens (1003 magnification). The imagewas edited with AdobePHOTOSHOP5.0 (Adobe Systems, San Jose, CA, USA). Double arrow, Orexin-ir neurons; arrowhead, Fos-ir nucleus; single arrow,Fos-ir orexin-ir neurons (b) Rectangle used to count cells in one section per animal. Cells were counted within an area defined using three landmarks:fornix (f), third ventricle (3V), and mammillothalamic tract (mt). Dark dots represent ORX-ir cells in the lateral hypothalamus of a grass rat.

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3 . Results each other (i.e. central.lateral.medial, see Fig. 2, bottompanel)

Representative examples of single and double-labeledORX-ir cells in grass rats are shown in Fig. 1a. Similar 3 .2. Fos expression inside and outside ORX-ir cellsprofiles were found in lab rats (not shown here). Nolabeled cells were seen in the control sections.

3 .2.1. Lab ratsIn lab rats, a two-way ANOVA revealed a significant

main effect of ZT (F56.747, df55, P#0.001) and area3 .1. Distribution of ORX-ir cells in the lateral(F517.69, df52, P#0.001) on the percentage of ORX-irhypothalamusneurons expressing Fos. The interaction was close to beingsignificant (P50.059). The significant main effect of ZT isFor lab rats, a two-way ANOVA for the number of cellsdepicted in Fig. 3a. Collapsing the data across areas,containing ORX (both single- and double-labeled) revealedcomparisons among the different ZTs showed that Fosa significant effect of area (F5127.1, df52, P,0.001),expression within ORX-ir neurons was significantly higherwith no effect of ZT or a significant interaction. Pairwiseat ZT 1 than at ZTs 5, 13, and 17. The percentage ofcomparison showed significant differences between theORX-ir neurons was also higher at ZT 20 compared to ZTsthree areas, with the number of cells in the central area5, 13, and 17 and at ZT 23 compared to ZT 13. Com-higher than the numbers in the medial and lateral areasparison among areas showed significantly higher (P,0.05)(P,0.05; Fig. 2, top panel).percentage of ORX-ir cells containing Fos in the medialIn the case of grass rats, a parametric two-way ANOVA(mean6S.E.M.510.6361.84) and central (mean6S.E.M.5for the number of cells containing ORX was not performed7.0761.76) areas than in the lateral regionbecause lack of homogeneity of variance was evident even(mean6S.E.M.52.3461.04). Inspection of the patterns ofafter standard transformations of the data. A nonparametricFos expression in the three areas revealed more varianceone-way ANOVA (Kruskal–Wallis test) failed to detect awithin some ZTs (data not shown here) and overallsignificant effect of ZT on this measure (data not shown).relatively few double-labeled neurons in the lateral region,The same nonparametric analysis showed a significantwhich may explain the trend for a significant interaction.effect of area, with the three areas being different form

Considering the data on Fos expression in cells notcontaining ORX, a two-way ANOVA showed a significanteffect of both ZT (F58.183, df55, P,0.0001) and area(F525.66, df52, P,0.0001) and no interaction (P50.4).Pair comparisons showed that the number of cells showingonly Fos cells at ZT 13 was significantly lower than thenumbers seen at ZTs 1, 5, 20 and 23. At ZT 17, thenumber of Fos positive cells not containing ORX wasalso significantly lower than those at ZTs 1, 5 and 20(P,0.05; Fig. 3b). Pairwise comparisons showed significant(P,0.05) differences between the three areas, withgreater number of only-Fos cells in the medial area(mean6S.E.M.590.4616.37) than in the central(mean6S.E.M.552.93617.79) and lateral areas(mean6S.E.M.533.03619.78).

3 .2.2. Grass ratsIn grass rats, a two-way ANOVA for the proportion of

orexin neurons expressing Fos revealed a significant maineffect of ZT (F53.234, df55, P50.01) with no effect ofarea (P50.12) or a significant interaction (P50.99). Paircomparisons made to further probe the main effect of ZT(see Fig. 3c) showed that the percentage was significantlygreater in animals perfused at ZT 13 than in those perfusedat ZTs 17 and 20 (P,0.05). Animals perfused at ZT 1 hada higher percentage of ORX-ir neurons expressing Fosthan those perfused at ZT 20. In addition, animals perfusedFig. 2. Mean (6S.E.M.) number of ORX-ir cells (both single and doubleat ZT 20 had lower percentage of double labeled cells thanlabeled) in the three regions in lab rats (top panel) and grass rats (bottom

panel). See text for statistical comparisons. those perfused at ZT 23 (P,0.05).

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Fig. 3. Percentage (mean6S.E.M.) of ORX-ir cells that contained Fos-ir nuclei in lab rats (a) and grass rats (c) at six different zeitgeber times (ZTs). In (b)and (d) the effects of ZT on Fos expression in neurons not labeled for ORX are presented. Note that in grass rats there is an inverse pattern of Fosexpression in ORX-ir cells (d) compared to other neurons (c) at ZTs 13 and 20, a feature absent in lab rats. Lights were on from ZT 0 to ZT 12. See text forstatistical comparisons.

A two-way ANOVA performed to evaluate the effects of rats exhibited a daily rhythm in the expression of Fos in aZT and area on the number of cells expressing Fos but not pattern that corresponds to the activity patterns of thiscontaining ORX-ir revealed a significant effect of ZT species. Although we did not record activity in this(F52.395, df55, P50.04) and area (F57.249, df52, experiment, previous studies with grass rats have shownP50.015) without a significant interaction. Pair compari- daily patterns of activity with two peaks, one around thesons showed that the number of Fos-only cells in animals time of lights-on and another around the time of lights-offperfused at ZT 20 was significantly greater than the [4]. Fos expression in orexinergic neurons in grass ratsnumber in animals perfused at ZTs 13 and 23 (P,0.05). In followed this pattern: it was high at ZT 1, a time whenaddition, significantly fewer Fos-only cells were seen in these animals exhibit a peak on activity, and increased atanimals perfused at ZT 13 compared to those perfused at ZT 13, a time when these animals show a second peak inZT 17 (P,0.05; Fig. 2d). The number of cells expressing activity. Fos expression decreased at ZT 17 and ZT 20,Fos but not ORX-ir was significantly (P,0.05) higher in times in which these animals sleep [24].the medial (mean6S.E.M.569.90629.84) and central Interestingly, the pattern of Fos expression in ORX-ir(mean6S.E.M.542.23615.77) areas than in the lateral neurons is similar to what has been reported for anotherarea (mean6S.E.M.523.90611.64). system involved in the support of wakefulness. Thus, in

grass rats, Fos expression in histaminergic cells of theventral tuberomammillary nucleus (VTM) shows a rhythm

4 . Discussion that features a dramatic reduction at ZT 17 compared withthe other ZTs sampled in that study (ZTs 1, 5 and 13) [24].

The distribution of ORX-ir cells is similar in lab rats Fos expression in both the ORX cells (present data) and in[8,9,19,27] and grass rats [21]. However, in the area of the the histaminergic group of the VTM shows a pattern that isLH examined here, grass rats have relatively more ORX-ir 1808 out of phase with the rhythm seen in the VLPO of thiscells in the lateral region and fewer in the medial region diurnal species [22–24]. The VLPO becomes active whenwhen compared to lab rats. animals sleep and is likely to provide inhibitory inputs to

Under a light–dark cycle, orexinergic neurons in grass the ORX and histaminergic cells.

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In grass rats, the pattern of Fos expression typical of cells raises the possibility that the circadian clock in theORX-ir cells was absent among cells that do not express suprachiasmatic nucleus (SCN) directly or indirectly in-ORX-ir. In fact, the expression of Fos in these non-ORX-ir fluences their activity. The presence of direct projectionscells peaked at ZT 20, when Fos expression was very low from the SCN to ORX-ir neurons [2] raises the possibilityin the ORX-ir cells of the region. Further, non-ORX-ir that the SCN regulates sleep via ORX-ir cells in the LHA.cells with Fos were rare at ZT 13, a time when grass rats In addition, circadian regulation may be imposed throughare active and Fos expression is high in ORX-ir cells. This second order projections from the SCN, such as the medialsuggests the presence of neurons in the LH that may work preoptic nuclei and the anterior hypothalamic nuclei that inin an antagonistic fashion with respect to ORX-ir cells. turn project to ORX-ir neurons [3] or via humoral SCNThus, local circuits within the area may interact to control outputs [34]. Since the SCN seems to function in a similarwakefulness in grass rats. way across species ([30,31], but also see [1,16,29]),

The pattern of activity in ORX-ir neurons across the day differences in behavioral rhythms in diurnal and nocturnalin lab rats seems slightly different from that presented in a species may result from differences in the responsivenessprevious study [10]. However, the trend in general is the of brain regions receiving signals from the SCN [25].same: the previous study showed higher number of double- In summary, our results confirm the presence of dailylabeled neurons in the night-time groups (ZTs 15 and 21) rhythms in the expression of Fos in ORX-ir neurons inthan in the daytime groups (ZTs 3 and 9). This pattern is nocturnal species and extend those findings to a diurnalalso present in our study: double-labeled neurons are species. Fos expression in ORX-ir neurons in both noctur-higher in animals perfused at ZTs 20, and 23 than in nal and diurnal rodents increases when the animals areanimals perfused at ZTs 5 and 13. One unexpected finding active. These data support the claim that ORX neuronsin our study was the lack of significant Fos production at activation is important for the promotion of wakefulness.ZT 17, 5 h into the dark and thus during the active phasefor lab rats. As noted above, in the study by Estabrooke etal. [10] Fos was elevated in ORX-ir at ZT 15. We did not

A cknowledgementsmonitor activity or sleep in our study so it is impossible todetermine if the reduced Fos expression at ZT 17 was

This work was supported by grant MH53433 to LS andassociated with an interval of reduced wakefulness ora Fulbright fellowship to GSM.increased display of sleep in our animals during the

nocturnal active period. Another difference between ourresult with lab rats and those of Estabrooke et al. [10]involves differences across regions in the LH. In their R eferencesstudy, a significant rhythm of Fos expression by ORX-ircells was detected only in the medial and perifornical

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