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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
journa l homepage: www.e lsev ier .com/ locate /bbr
Research report
Activation of serotonin 5-HT1B receptor in the dorsalraphe nucleus affects REM sleep in the rat
Jaime M. Montia,∗, Héctor Jantosa, Patricia Lagosb
a Department of Pharmacology and Therapeutics, School of Medicine Clinics Hospital, Montevideo 11600, Uruguayb Department of Physiology, School of Medicine, Montevideo 11800, Uruguay
a r t i c l e i n f o
Article history:Received 17 April 2009Received in revised form 15 August 2009Accepted 18 August 2009Available online 6 September 2009
The effects of CP-94253, a selective 5-HT1B receptor agonist, and of SB 224-289, a selective 5-HT1B receptorantagonist, on spontaneous sleep were studied in adult rats implanted for chronic sleep recordings. The5-HT1B receptor ligands were microinjected directly into the dorsal raphe nucleus (DRN) during the lightperiod of the 12-h light/12-h dark cycle. Infusion of CP-94253 (1–4 mM) into the DRN induced a significantreduction of rapid-eye-movement sleep (REMS) and of the mean duration of REM episodes. On the otherhand, SB 224-289 (0.25–0.5 mM) decreased REMS and the number of REM periods. Pretreatment withSB 224-289 (0.125–0.25 mM) antagonized the CP-94253 (4 mM)-induced reduction of REMS and of themean duration of REM periods. Administration of the GABAA receptor agonist muscimol (1.5 mM), whichby itself did not significantly affect sleep variables, prevented the effect of CP-94253 (4 mM) on REMSsuppression. It is proposed that the suppression of REMS after microinjection of CP-94253 into the DRNis related to the inhibition of GABAergic interneurons that make synaptic contacts with serotonergiccells. The resultant increase of serotonin release at postsynaptic sites involved in the induction andmaintenance of REMS would induce the suppression of the behavioral state.
Although many questions remain about the complicated roleof serotonin (5-HT) and its receptors in regulating sleep andwaking, recent neurochemical, electrophysiological, genetic andneuropharmacological studies have revealed much detailed infor-mation about this process. Based on such approaches it is currentlyaccepted that 5-HT functions to promote waking (W) and to inhibitrapid-eye movement sleep (REMS).
The serotonin-containing neurons of the dorsal raphe nucleus(DRN) and the median raphe nucleus (MRN) provide the principalsource of serotonergic innervation of the telencephalon, dien-cephalon, mesencephalon, and rhombencephalon of laboratoryanimals and man [28,38]. In this respect, 5-HT neurons of theDRN and the MRN innervate brain areas involved in sleep/wakeregulation. These areas include the cholinergic nuclei of the mes-encephalon and the basal forebrain, the dopaminergic neurons ofthe ventral tegmental area and the substantia nigra compacta, thenoradrenergic cells of the locus coeruleus, the GABAergic, histamin-ergic and orexinergic cell aggregates of the hypothalamus and theglutamatergic neurons of the thalamus and the brain stem reticularformation [28].
The inhibitory neurotransmitter �-aminobutyric acid (GABA)plays an important role in the initiation and maintenance of slowwave sleep (SWS) and REMS. GABAergic neurons that project to theDRN are located in several neuroanatomical structures includingthe basal forebrain, the hypothalamus, the mesencephalon and therhombencephalon [24]. In addition, GABAergic interneurons havebeen described in the DRN [11]. GABA has been made responsiblefor the reduction of the activity of 5-HT neurons during SWS andREMS. Accordingly, iontophoretic application of GABA decreasesthe activity of DRN 5-HT neurons and this effect is antagonized bypicrotoxin [16]. Furthermore, Nitz and Siegel [31] have found thatREMS is accompanied by a significant increase in GABA release inthe DRN and that local administration of muscimol into the raphenucleus increases REMS, whereas picrotoxin blocks its occurrence.
The DRN contains 5-HT and non-5-HT neurons. The latterexpress a variety of substances including dopamine, GABA and glu-tamate. In addition, nitric oxide and a variety of neuropeptides havebeen characterized in the DRN, some of them being coexpressed in5-HT cells [9,10,23,36].
The serotonergic cells are present throughout the rostral–caudalextent of the DRN, in all clusters of the nucleus. However, theypredominate along the midline of the rostral, dorsal and ven-tral subdivisions of the DRN [11,25]. GABAergic neurons are alsoabundant throughout the DRN of the rat. Although all DRN sub-nuclei contain GABAergic interneurons, they predominate in thelateral wings of the raphe nucleus [7,11,15]. A relatively small
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number of glutamatergic neurons have been characterized in therostral subdivision of the DRN [34]. Moreover, glutamatergic inputsto the DRN have been described that originate in the medialprefrontal cortex, the amygdala, various hypothalamic areas, theparabrachial nuclei, and the laterodorsal and pedunculopontinetegmental nuclei (LDT/PPT) among others [21].
Allers and Sharp [2] successfully identified the neurochemicaland morphological properties of 5-HT-containing cells and GABA-containing cells in the DRN of urethane-anesthetized rats. Theslow-firing cells were immunoreactive for 5-HT and/or tryptophanhydroxylase and were distributed throughout the rostral–caudalextent of the DRN. Intravenous administration of the serotonin5-HT1A receptor agonist 8-OH-DPAT inhibited the activity of theslow-firing cells. The fast-firing neurons were immunoreactive forglutamic acid decarboxylase (GAD), the enzyme that catalyzes thesynthesis of GABA, and immunonegative for tryptophan hydroxy-lase, and were predominantly located in the lateral subdivisions ofthe DRN.
The 5-HT receptors can be classified into seven classes, des-ignated 5-HT1–7. The 5-HT1 class consists of five (5-HT1A-B-D-E-F)subtypes [18]. The 5-HT1B receptor is linked to the inhibition ofadenylate cyclase and decreased production of cAMP. The activa-tion of 5-HT1B receptor results in membrane hyperpolarization,leading to an inhibition of neuronal firing [30]. The 5-HT1B recep-tor was initially proposed to be located on 5-HT axon terminals(presynaptic autoreceptor) and postsynaptically (outside the DRN)on non-5-HT neurons. Additionally, a high density of 5-HT1B recep-tor mRNA has been detected in the raphe nuclei of the rat [6,13,39].The density of 5-HT1B receptor at the DRN is greatest in the ventro-medial cluster, and it is predominantly expressed by non-5-HT cells[8]. The finding that administration of the selective 5-HT1B recep-tor agonist CP-94253 increases the firing of 5-HT neurons and ofdialysate 5-HT in the DRN, seems compatible with the activationof 5-HT1B receptors located in inhibitory, tentatively GABAergic,interneurons [1,14].
The 5-HT1B receptor is involved in the regulation of the synapticrelease of 5-HT and of other neurotransmitters including acetyl-choline, norepinephrine, dopamine, GABA and glutamate, whichis indicative of its role as auto- and heteroreceptor, respectively.In addition, available evidence tends to suggest that the activa-tion of 5-HT1B receptor expressed by inhibitory interneurons thatmake synaptic contacts with 5-HT cells, indirectly facilitates theirfunctional activity [14].
Few studies has been published on the effect of 5-HT1B recep-tor ligands on sleep variables. Systemic administration of the5-HT1B agonists CGS 12066B or CP-94253 significantly increasedW and reduced SWS and REMS in the rat [4,27]. The mixed �-adrenoceptor/5-HT1A/1B receptor antagonist pindolol preventedthe increase of W and reduction of SWS by CP-94253. However,pindolol failed to prevent the suppression of REMS [27]. Further-more, quantization of spontaneous sleep–waking cycles in 5-HT1Breceptor knockout mice has shown that REMS is increased duringthe light phase [5].
Recently, Gyongyosi et al. [17] studied the effect of CP-94253on sleep and W in rats pretreated with 3,4-methylenedioxy-methamphetamine (MNMA) six months earlier. MNMA lesioned5-HT nerve endings in the striatum and prevented the increaseof active W observed after systemic administration of the 5-HT1Breceptor agonist in the control animals. On the other hand, the CP-94253-induced reduction of SWS and REMS was still present in theMNMA-pretreated rats.
Thus, the limited available evidence tends to indicate that5-HT1B receptor activation facilitates the occurrence of W and neg-atively influences SWS and REMS.
To date, no investigations into the role of 5-HT1B receptorspresent in the DRN in sleep and W have been reported. However,
a role for these receptors in the regulation of the behavioral statemay be anticipated based on the association of 5-HT neurons withbrain regions known to be important in the regulation of W andREMS [28].
The present experiments were undertaken to test the hypoth-esis that stimulation of the 5-HT1B receptor in the DRN shouldnegatively influence REMS occurrence. For this purpose we madeuse of the selective 5-HT1B receptor agonist CP-94253 {3-(1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine} [20].Several doses of CP-94253 were injected into the DRN of ani-mals prepared for chronic sleep recordings. In addition, wetested the potential use of the selective 5-HT1B receptor antago-nist SB 224-289 {5,1′-methyl-5-[[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,67-tetrahydrospiro[furo[2,3-f]indole-3,4′-piperidine]} [35,37] to counteract theCP-94253-induced changes of REMS. We considered also the pos-sibility that the effects induced by CP-94253 are, at least in part,via the inhibition of local GABAergic interneurons. To this purposewe tested the ability of the selective GABAA receptor agonistmuscimol to reverse the CP-94253-induced reduction of REMS.
2. Materials and methods
2.1. Animals
Five different groups of male Wistar rats weighing 320–350 g at the time ofsurgery were used. All experiments were conducted in accordance with the NationalInstitutes of Health (USA) guidelines for the care and use of laboratory animals. Allprocedures were approved by the Institutional Animal Care and Use Committee ofthe Medical School, Montevideo, Uruguay.
2.2. Surgical procedures
All surgical procedures were performed stereotaxically under aseptic con-ditions. Sodium pentobarbital (40 mg/kg) was administered intraperitoneally foranesthesia. In addition, the animals were treated postoperatively for 4 days with theantibiotic penicillin 50 mg/kg. The rats were implanted with Nichrome® electrodes(200 �m diameter) for chronic sleep recordings of electroencephalogram (EEG) andelectromyogram (EMG) activities, through placement on the frontal and the occip-ital cortices for the former, and on the dorsal neck musculature for the latter. Leadsfrom the recording electrodes were routed to a nine pin miniature plug that matesto one attached to a recording cable. A guide cannula (26 gauge) constructed formicroinjection into the DRN was implanted with its tip 2 mm above the DRN (AP7.8; L 0.0; V −5.8) [33]. The recording plug and the cannula were affixed to the skullwith dental acrylic and anchor screws. Drug or vehicle was injected into the DRNwith an injection cannula (29 gauge), which extended 2 mm beyond the guide, in a0.2-�l volume over a 2-min period. On completion of the microinjections, we iden-tified the injection site by the microinjection of Pontamine Sky-blue dye (0.2 �l) intothe DRN. The rats were sacrificied with an overdose of pentobarbital, perfused with4% paraformaldehyde and their brains were removed. Thereafter, the brains werecryoprotected in a solution of sucrose 30%, and cut in 40 �m coronal sections with acryostat. Selected sections were stained with tionin acetate (Merck, Germany). Cor-rectness of the cannula/injection sites was assessed using the atlas of Paxinos andWatson [33]. All the data presented in this report are derived from animals whoseinjection site was within the limits of the DRN.
2.3. Recording and sleep scoring
The animals were housed individually in a temperature-controlled room(23 ± 1 ◦C) under a 12-h light/12-h dark cycle (the lights went on at 06:00 h) andwith food and water provided ad libitum. Ten days after surgery the animals werehabituated to a soundproof chamber fitted with slip-rings and cable connectors, andto the injection procedures. The drugs were always administered during the lightphase of the 12-h light/12-h dark cycle, at approximately 08:00 h. A balanced orderof drug and control injections was always used to merge the effects of both thedrug and the time elapsed during the protocol. Recording was begun 15 min laterand continued for 6 h. The predominant activity of each 10 s epoch was assigned toone of the following categories: W, light sleep (LS), SWS, or REMS. Slow wave sleepand REMS latencies, and the number and mean duration of REM periods were alsodetermined [26].
2.4. Experimental design
The doses of CP-94253 (1–4 mM), SB 224-289 (0.125–0.5 mM) and muscimol(1–1.5 mM) selected for the present study were based on pilot work in our laboratoryand the limited previous research in which administration of these compounds was
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Fig. 1. Schematic drawing of 5-HT1B receptor ligands injection sites in the dorsal raphe nucleus of the rat. Abbreviation: DRN = dorsal raphe nucleus. Microinjection sitescorresponding to: (A) experiment 1 (infusion of CP-94253 into the DRN); (B) experiment 2 (infusion of SB 224-289 into the DRN); and (C) experiment 3 (infusion of CP-94253into animals pretreated with SB 224-289). Sections according to Paxinos and Watson [33].
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employed to determine the actual physiological and pharmacological roles of the5-HT1B receptor. In all experiments at least 3 days were allowed to elapse betweenexperiments to avoid long-lasting and rebound effects on sleep. The effects of CP-94253, SB 224-289 and muscimol were studied in five different groups of animalsaccording to the following experimental paradigms:
Experiment 1 (group 1): CP-94253 hydrochloride (Tocris Bioscience, Ellisville, MO,USA) 1, 2 or 4 mM or vehicle (distilled water) was infused into the DRN (n = 6).Experiment 2 (group 2): SB 224-289 hydrochloride (Tocris Bioscience, Ellisville,MO, USA) 0.125, 0.25 or 0.5 mM or vehicle (distilled water + Tween 80 added) wasinfused into the DRN (n = 7).
Fig. 2. Schematic drawing of muscimol and muscimol-CP-94253 injection sites in the dorsal raphe nucleus of the rat. (A) experiment 4 (infusion of muscimol into the DRN)and (B) experiment 5 (infusion of CP-94253 into animals pretreated with muscimol). Sections according to Paxinos and Watson [33].
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Experiment 3 (group 3): In the third set of experiments 4 mM CP-94253 wasinjected into animals pretreated with 0.125 or 0.25 mM SB 224-289 (n = 6).Experiment 4 (group 4): Muscimol (Sigma, St. Louis, MO, USA) 1 or 1.5 mM orvehicle (distilled water) was infused into the DRN (n = 6).Experiment 5 (group 5): In the fifth set of experiments 4 mM CP-94253 was injectedinto animals pretreated with 1 or 1.5 mM muscimol (n = 6).
The drugs were microinjected into the DRN 20 min apart in the interactionexperiments.
2.5. Statistics
A repeated measures analysis of variance (ANOVA) using dose as the betweensubject-factor was performed, with multiple post hoc comparisons carried out withthe Dunnett multiple comparisons test when the ANOVA indicated significance(P < 0.05).
3. Results
The histological analysis of the injection sites showed that 31 ofthe 35 animals originally included in the study received microin-jections of the 5-HT1B ligands and the GABAA receptor agonist thatwere confined within the limits of the DRN, predominantly its ven-tromedial subregion. The data from the 31 rats are summarizedin the anatomical schematic of Figs. 1 and 2. In those animalswhere the microinjections of the 5-HT1B receptor ligands or theGABAA receptor agonist were not confined within the limits ofthe corresponding neural structure, REMS values remained almostunchanged or showed erratic changes.
As mentioned earlier, CP-94253 and muscimol were dissolvedin distilled water whereas SB 224-289 was dissolved in distilledwater + Tween 80 added. Notwithstanding this, there were no sig-nificant differences in the values of sleep variables determinedusing distilled water or distilled water + Tween 80 added in thecontrol experiments.
3.1. Effects of microinjection of CP-94253
Following the microinjection of 1–4 mM CP-94253 into the DRN,REMS was suppressed during the first 2-h period after treatment
(F(3,15) = 5.45, P < 0.05 and P < 0.01, respectively). The 4 mM dose ofthe 5-HT1B agonist similarly reduced REMS during the second andthe third 2 h of recording (F(3,15) = 5.72, P < 0.01 and F(3,15) = 3.13,P < 0.05, respectively). Values of W, LS, and SWS showed slightbut inconsistent changes that did not attain significance (Fig. 3).Injected into this site, CP-94253 did not significantly modify sleeplatencies and the number of REM periods. However, the mean dura-tion of REM episodes was significantly reduced after the 4 mMdose during the first and the second 2-h period (F(3,15) = 5.45, andF(3,15) = 5.44, P < 0.05, respectively) (Table 1).
3.2. Effects of microinjection of SB 224-289
Treatment with 0.25–0.5 mM SB 224-289 significantly reducedREMS during the second 2-h period (F(3,18) = 4.48, P < 0.05 andP < 0.01, respectively). Values of LS were increased by the 0.5 mMdose during the second 2 h of recording (F(3,18) = 3.24, P < 0.05)(Fig. 4). The number of REM periods was reduced with the wholerange of doses during the second 2-h period (P < 0.05 and P < 0.01,respectively) (Table 2).
3.3. Effects of microinjection of CP-94253 into the DRN in animalspretreated with SB 224-289
Following the administration of CP-94253 (4 mM) into ani-mals of group 3, REMS was reduced during the 6-h recordingperiod (F(5,25) = 3.26, P < 0.05; F(5,25) = 3.41, P < 0.01 and F(5,25) = 2.98,P < 0.05, respectively) whereas the duration of REM periods showeda decrease during the first and the second 2-h periods after treat-ment (F(5,25) = 3.46, P < 0.05 and F(5,25) = 3.22, P < 0.05, respectively).After SB 224-289 (0.125–0.25 mM) was microinjected into the DRN,REMS was not significantly modified. However, the number ofREM periods showed a significant decrease. SB 224-289 preventedthe reduction of REMS and of the mean duration of REM periodsinduced by CP-94253. Of note, the SB 224-289-related reductionof the number of REM periods was also reverted following theassociation of the 5-HT1B ligands (Fig. 5 and Table 3).
Fig. 3. Effects of CP-94253 microinjected into the dorsal raphe nucleus on sleep and waking. Sleep stages are quantified in minutes. Ordinate: time spent in sleep and waking(mean ± S.E.M.). Abscissa: time after injection (h). The doses are indicated in mM. *P < 0.05, **P < 0.01: significant statistical difference with respect to the control vehicle. Localadministration of CP-94253 into the dorsal raphe nucleus selectively suppressed REMS.
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Table 1Effects of CP-94253 administered directly into the dorsal raphe nucleus on sleep latencies and the number and the duration of REM periods.
Group SWS latency (min) REMS latency (min) Number of REM periods Duration of REM periods (min)
All values are the means ± S.E.M. Six animals were in each experimental group. The doses are in mM. Compared with control values: *P < 0.05 (Dunnett Multiple ComparisonsTest). CP-94256 4 mM significantly reduced the mean duration of REM periods during the first and the second 2-h period after administration.
Fig. 4. Effects of SB 224-289 microinjected into the dorsal raphe nucleus on sleep and waking. The doses are indicated in mM. *P < 0.05; *P < 0.01: significant statisticaldifference with respect to the control vehicle. Microinjection of SB 224-289 into the dorsal raphe nucleus induced a reduction of REMS and an increase of light sleep.
Table 2Effects of SB 224-289 administered directly into the dorsal raphe nucleus on sleep latencies and the number and the duration of REM periods.
Group SWS latency (min) REMS latency (min) Number of REM periods Duration of REM periods (min)
All values are means ± S.E.M. Seven animals were in each experimental group. The doses are in mM. Compared with control values: *P < 0.05; **P < 0.01 (Dunnett MultipleComparisons Test). SB 224-289 0.125 mM, 0.25 mM, and 0.5 mM significantly reduced the number of REM periods during the second 2-h period after administration.
3.4. Effects of microinjection of muscimol
Administration of 1–1.5 mM muscimol into the DRN induced nosignificant changes in sleep variables over the 6 h of recording (datanot shown).
3.5. Effects of microinjection of CP-94253 into the DRN in animalspretreated with muscimol
Following the microinjection of CP-94253 (4 mM) into ani-mals of group 5, REMS was significantly reduced during hours1–2, 3–4 and 5–6 postinjection (F(3,15) = 3.48, P < 0.05; F(3,15) = 6.60,P < 0.01 and F(3,15) = 3.31, P < 0.05, respectively). Muscimol (1.5 mM)effectively prevented the reduction of REMS induced by CP-
94253 (4 mM) over the 6 h of recording (Fig. 6). In addition, theGABAA receptor agonist reverted the CP-94253-induced decreaseof the mean duration of REM periods (F(3,15) = 3.79, P < 0.05 andF(3,15) = 3.74, P < 0.05, respectively) (Table 4).
4. Discussion
The present study shows that the microinjection of the 5-HT1Breceptor agonist CP-94253 (1–4 mM) into the DRN during the lightphase caused a reduction of REMS and of the mean duration ofREM periods. With intra-DRN administration of the 5-HT1B recep-tor antagonist SB 224-289 (0.25–0.5 mM) a reduction in time spentin REMS and in the number of REM periods was seen. Pretreat-ment with SB 224-289 (0.125–0.25 mM) prevented the CP-94253
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Fig. 5. Effects of pretreatment with SB 224-289 on the suppression of REMS induced by the microinjection of CP-94253 into the dorsal raphe nucleus. The doses are indicatedin mM. *P < 0.05; **P < 0.01: significant statistical difference with respect to the control vehicle. Pretreatment with SB 224-289 prevented the CP-94253-induced reduction ofREMS.
Table 3Effects of SB 224-289 pretreatment on the CP-94253-induced changes of duration of REM periods.
Group SWS latency (min) REMS latency (min) Number of REM periods Duration of REM periods (min)
All values are the means ± S.E.M. Six animals were in each experimental group. The doses are in nM. Compared with control values: *P < 0.05 (Dunnett Multiple ComparisonsTest). The association of CP-94253 with SB 224-289 reverted the reduction of the mean duration of REM periods induced by the 4 mM dose of the 5-HT1B receptor agonistduring the first and the second 2-h period after treatment.
induced decrease of REMS and of the mean duration of REM periods.Moreover, administration of the GABAA receptor agonist musci-mol (1.5 mM), which by itself did not significantly affect sleepvariables, prevented also the effect of CP-94253 on REMS suppres-sion.
The 5-HT1B receptor is negatively coupled to adenylate cyclase.Thus, the CP-94253-induced suppression of REMS cannot be pos-sibly related to a direct effect of the 5-HT1B agonist on DRN 5-HTneurons. On the other hand, it could be an indirect action dependenton the influence of a modulatory system on serotonergic neurons.
Table 4The effect of muscimol pretreatment on the CP-94253-induced changes of the duration of REM periods.
Group SWS latency (min) REMS latency (min) Number of REM periods Duration of REM periods (min)
All values are the means ± S.E.M. Six animals were in each experimental group. The doses are in mM. Compared with control values: *P < 0.05 (Dunnett’s test). Pretreatmentwith muscimol prevented the CP-94253-induced reduction of the mean duration of REM periods during the first and the second 2-h period after administration.
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Fig. 6. The effect of pretreatment with muscimol on the CP-94253-induced suppression of REMS. The doses are in mM. *P < 0.05; **P < 0.01. Muscimol prevented the CP-94253-induced decrease of REMS.
In this respect, our findings tend to indicate a role for the GABAer-gic system in the 5-HT1B agonist-induced suppression of REMS.It is conceivable that microinjection of CP-94253 into the DRNresulted in a decrease of GABA inhibitory tone on 5-HT neurons [14](Fig. 7). As a consequence of the increment of 5-HT release therewas a suppression of REMS. This conclusion is drawn from the factthat microinjection of muscimol prevented the CP-94253-induceddecrease of REMS.
In could be argued that the effect of CP-94253 on REMS dependsupon the selective activation of presynaptic 5-HT1B autoreceptor.However, activation of the latter would be expected to induce adecrease of 5-HT availability at postsynaptic sites and the facilita-tion of REMS occurrence.
Unexpectedly, microinjection of the 5-HT1B antagonist SB224-289 into the DRN induced also the suppression of REMS. Glu-tamatergic interneurons have been characterized in the DRN [19].Furthermore, glutamatergic inputs to the DRN have been describedthat originate in several neuroanatomical structures including thecerebral cortex, the hypothalamus and the brainstem [21]. Acti-
vation of the 5-HT1B receptor in the DRN inhibits the release ofglutamate [22]. The possibility exists that blockade of 5-HT1B recep-tor located on glutamatergic neurons and terminals in the DRNdisinhibits serotonergic neurons and increases 5-HT release at post-synaptic sites involved in the induction and maintenance of REMS.As a result, REMS would be suppressed. However, to elucidate themechanisms underlying the SB 224-289-induced suppression ofREMS further investigations are needed.
The finding that microinjection of CP-94253 into the DRN sig-nificantly decreases the mean duration of REM periods, whereas SB224-289 reduces the number of REM periods tends to suggest thatthe former inhibits REMS maintenance while the latter disruptsneural mechanisms involved in REMS occurrence.
The effect of intra-DRN microinjection of CP-94253 on sleepvariables differs from that observed after systemic administrationof the compound. In this respect, s.c. or i.p. injection of 5-HT1Breceptor agonists suppresses REMS and, in addition, facilitates theoccurrence of W. Although the process by which activation of 5-HT1B receptor facilitates the state of W is still unknown, it should
Fig. 7. Schematic drawing of the DRN of the rat illustrating the inhibition of a serotonergic neuron by a GABAergic interneuron expressing the 5-HT1B receptor. Localmicroinjection of the 5-HT1B receptor agonist CP-94253 would remove the GABAergic inhibitory influence on the serotonergic neuron and indirectly increase the release of5-HT at postsynaptic sites.
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be mentioned that many of the GABAergic cells in the basal fore-brain, hippocampus, and neocortex on which 5-HT1B receptors areexpressed are hyperpolarized by serotonin released from the DRN[3,12,29,32]. Thus, inhibition of GABAergic neurons following theactivation of 5-HT1B receptors could account, at least in part, forthe facilitatory effect on W. In other words, activation of 5-HT1Breceptors may attenuate GABAergic input and thereby indirectlyincrease the release of acetylcholine and glutamate at cortical andsubcortical sites. On the other hand, 5-HT1B receptor-dependentinhibition of cholinergic and glutamatergic neurons in the LDT/PPTand the medial-pontine reticular formation, respectively, would bedirectly responsible for REMS suppression.
5. Conclusions
The present study led to two major findings: (1) microinjectionof CP-94253 into the DRN induces a significant reduction of REMSand (2) pretreatment with SB 224-289 or muscimol prevents thereduction of REMS.
It is currently accepted that inhibitory and facilitatory neuro-transmitter systems project to the DRN and regulate the activity of5-HT neurons during the sleep–wake cycle. Among the former isthe GABAergic system that can promote REMS by inhibiting 5-HTneurons. On the other hand, the noradrenergic, dopaminergic, his-taminergic, cholinergic and orexinergic systems provide excitatoryafferents to DRN 5-HT cells that would induce the opposite effect.
The finding that direct microinjection of a selective 5-HT1Breceptor agonist into the DRN reduces REMS tends to indicate theexistence of a supplemental mechanism in the control of 5-HTneurons functional activity. In addition to the serotonergic cellsthat conventionally define the boundaries of the DRN, this neu-roanatomical structure contains GABAergic cells. In all probability,5-HT1B receptor expressed by GABAergic interneurons indirectlyfacilitates the activity of 5-HT neurons and the release of the neu-rotransmitter at postsynaptic sites.
Acknowledgement
This study was supported by PEDECIBA, Montevideo, Uruguay.
References
[1] Adell A, Celada P, Artigas F. The role of 5-HT1B receptors in the regulation ofserotonin cell firing and release in the rat brain. J Neurochem 2001;79:172–82.
[2] Allers KA, Sharp T. Neurochemical and anatomical identification of fast andslow firing neurons in the rat dorsal raphe nucleus using juxtacellular labellingmethods in vivo. Neuroscience 2003;122:193–204.
[3] Araneda R, Andrade R. 5-Hydroxytryptamine2, and 5-hydroxytryptamine1A
receptors mediate opposing responses on membrane excitability in rat associ-ation cortex. Neuroscience 1991;40:399–412.
[4] Bjorvatn B, Ursin R. Effects of the selective 5-HT1B agonist, CGS 12066B,on sleep/waking stages and EEG power spectrum in rats. J Sleep Res1994;3:97–105.
[5] Boutrel B, Franc B, Hen R, Hamon M, Adrien J. Key role of 5-HT1B receptors inthe regulation of paradoxical sleep as evidenced in 5-HT1B knock-out mice. JNeurosci 1999;19:3204–12.
[6] Bruinvels AT, Landwehrmeyer B, Gustafson EL, Durkin MM, Mengod G,Branchek TA, et al. Localization of 5-HT1B, 5-HT1D� , 5-HT1E and 5-HT1F
receptor messenger RNA in rodent and primate brain. Neuropharmacology1994;33:367–86.
[7] Charara A, Parent A. Chemoarchitecture of the primate dorsal raphe nucleus. JChem Neuroanatomy 1998;15:111–27.
[8] Clark MS, McDevitt RA, Neumaier JF. Quantitative mapping of tryptophanhydroxylase-2, 5-HT1A, 5-HT1B, and serotonin transporter expression acrossthe anteroposterior axis of the rat dorsal and median raphe nuclei. J CompNeurol 2006;498:611–23.
[9] Clements JR, Beitz AJ, Fletcher TF, Mullet MA. Immunocytochemical localizationof serotonin in the rat periaqueductal gray: a quantitative light and electronmicroscopic study. J Comp Neurol 1985;236:60–70.
[10] Commons KG, Connolley KR, Valentino RJ. A neurochemically distinct dorsalraphe-limbic circuit with a potential role in affective disorders. Neuropsy-chopharmacology 2003;28:206–15.
[11] Day HE, Greenwood DN, Hammack SE, Watkins LR, Fleshner M, Maler SF, etal. 5-HT-1A, �1B adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotoner-gic, �-aminobutyric acidergic, and catecholaminergic cells of the dorsal raphenucleus. J Comp Neurol 2004;474:364–78.
[12] Detari L, Rasmusson DD, Semba K. The role of basal forebrain neurons intonic and phasic activation of the cerebral cortex. Prog Neurobiol 1999;58:249–77.
[13] Doucet E, Pohl M, Fattaccini CM, Adrien J, Mestikawy SE, Hamon M. In situhydridization evidence for the synthesis of 5-HT1B receptor in serotonergicneurons of anterior nuclei in the rat brain. Synapse 1995;19:18–28.
[14] Evrand A, Laporte AM, Chastanet M, Hen R, Hamon M, Adrien J. 5-HT1A and5-HT1B receptors control the firing of serotonergic neurons in the dorsalraphe nucleus of the mouse: studies in 5-HT1B knock-out mice. Eur J Neurosci1999;11:3823–31.
[15] Ford B, Holmes CJ, Mainville L, Jones BE. GABAergic neurons in the ratpontomesencephalic tegmentum: codistribution with cholinergic and othertegmental neurons projecting to the posterior lateral hypothalamus. J CompNeurol 1995;363:177–96.
[16] Gallager DW, Aghajanian GK. Effect of antipsychotic drugs on the firing of dorsalraphe cells. II. Reversal by picrotoxin. Eur J Pharmacol 1976;39:357–64.
[17] Gyongyosi N, Balogh B, Kirilly E, Kitka T, Kantor S, Bagdy G. MDMA treatment 6months earlier attenuates the effects of CP-94,253, a 5-HT1B receptor agonist,on motor control but not sleep inhibition. Brain Res 2008;1231:34–46.
[18] Hannon J, Hoyer D. Molecular biology of 5-HT receptors. In: Monti JM, Pandi-Perumal SR, Jacobs BL, Nutt DJ, editors. Serotonin and sleep: molecular,functional and clinical aspects. Basel: Birkhäuser Verlag; 2008. p. 155–82.
[19] Jolas T, Aghajanian GK. Opioids suppress spontaneous and NMDA-inducedinhibitory postsynaptic currents in the dorsal raphe nucleus of the rat in vitro.Brain Res 1997;755:229–45.
[20] Koe BK, Lebel LA, Fox CB, Macor JE. Binding and uptake studies with[3H]CP-93,129, a radiolabeled selective 5-HT1B receptor ligand. Drug Dev Res1992;25:67–74.
[21] Lee HS, Kim MA, Valentino RJ, Waterhouse BD. Glutamatergic afferent projec-tions to the dorsal raphe nucleus of the rat. Brain Res 2003;963:57–71.
[22] Lemos JC, Pan YZ, Ma X, Lamy C, Akanwa AC, Beck SG. Selective 5-HT1B receptorinhibition of glutamatergic and GABAergic synaptic activity in the rat dorsal andmedian raphe. Eur J Neurosci 2006;24:3415–30.
[23] Lowry CA, Evans AK, Gasser PJ, Hale MW, Staub DR, Shekhar A. Topographicorganization and chemoarchitecture of the dorsal raphe nucleus and themedian raphe nucleus. In: Monti JM, Pandi-Perumal SR, Jacobs BJ, Nutt DJ,editors. Serotonin and sleep: molecular, functional and clinical aspects. Basel:Birkhäuser Verlag; 2008. p. 25–67.
[24] Luppi PH, Gervasoni D, Peyron C, Leger L, Salvert D, Fort P. Role and origin ofthe GABAergic innervation of dorsal raphe serotonergic neurons. In: Monti JM,Pandi-Perumal SR, Jacobs BL, Nutt DJ, editors. Serotonin and sleep: molecular,pharmacological and clinical aspects. Basel: Birhäuser Verlag; 2008. p. 237–50.
[25] Molliver ME. Serotonergic neuronal systems: what their anatomic organizationtells us about function. J Clin Psychopharmacol 1987;7:3S–23S.
[26] Monti JM, Hawkins M, Jantos H, Labraga P, Fernández J. Biphasic effects ofdopamine D-2 receptor agonists on sleep and wakefulness in the rat. Psy-chopharmacology 1988;95:395–400.
[27] Monti JM, Monti D, Jantos H, Ponzoni A. Effects of selective activation of the5-HT1B receptor with CP-94253 on sleep and wakefulness in the rat. Neurophar-macology 1995;34:1647–51.
[28] Monti JM, Jantos H, Monti D. Serotonin and sleep–wake regulation. In: MontiJM, Pandi-Perumal SR, Sinton CM, editors. Neurochemistry of sleep and wake-fulness. Cambridge: Cambridge University Press; 2008. p. 244–79.
[29] Newberry NR, Footitt DR, Papanastassiou V, Reynolds DJ. Action of 5-HT onhuman neocortical neurons in vitro. Brain Res 1999;833:93–100.
[30] Nichols DE, Nichols CD. Serotonin receptors. Chem Rev 2008;108:1614–41.[31] Nitz D, Siegel J. GABA release in the dorsal raphe nucleus: role in the control of
REM sleep. Am J Physiol 1997;273:R451–5.[32] Parnavelas JG. Neurotransmitters in the cerebral cortex. In: Uylings HBM, Van
Eden CG, De Bruin JPC, Corner MA, Feenstra MGP, editors. Progress in brainresearch, vol. 85. Amsterdam: Elsevier; 1990. p. 13–29.
[33] Paxinos G, Watson C. The rat brain in stereotaxic coordinates—the new coronalset. 5th ed. Sidney: Academic Press; 2005.
[34] Roche M, Commons KG, Peoples A, Valentino RJ. Circuitry underlying regulationof the serotonergic system by swim stress. J Neurosci 2003;23:970–9.
[35] Selkirk JV, Scott C, Ho M, Burton MJ, Watson J, Gaster LM, et al. SB-224289—anovel selective (human) 5-HT1B receptor antagonist with negative intrinsicactivity. Br J Pharmacol 1998;125:202–8.
[36] Steinbusch HW. Distribution of serotonin-immunoreactivity in the central ner-vous system of the rat cell bodies and terminals. Neuroscience 1981;6:557–618.
[37] Stenfors C, Ross SB. Failure to detect in vivo inverse agonism of the 5-HT1B
receptor antagonist SB-224289 in 5-HT-depleted guinea-pigs. N-S Arch Phar-macol 2002;365:462–7.
[38] Vertes RP, Linley SB. Efferent and afferent connections of the dorsal and medianraphe nuclei in the rat. In: Monti JM, Pandi-Perumal SR, Jacobs BL, Nutt DJ,editors. Serotonin and sleep: molecular, functional and clinical aspects. Basel:Birkhäuser Verlag; 2008. p. 69–102.
[39] Voigt MM, Laurie DJ, Seeburg PH, Bach A. Molecular cloning and characteriza-tion of a rat-brain cDNA-encoding a 5-hydroxytryptamine1b receptor. EMBO J1991;10:4017–23.
