The effects of local microinjection of selective dopamine D1 and D2 receptor agonists and antagonists into the dorsal raphe nucleus on sleep and wakefulness in the rat

Behavioural Brain Research 339 (2018) 11–18

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The effects of local microinjection of selective dopamine D1 and D2 receptor agonists and antagonists into the dorsal raphe nucleus on sleep and wakefulness in the rat

MARK

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Jaime M. Monti , Héctor Jantos Department of Pharmacology and Therapeutics, School of Medicine Clinics Hospital, University of the Republic, Montevideo 11600, Uruguay

A R T I C L E I N F O

A B S T R A C T

Keywords: Wakefulness REM sleep Serotonin Dopamine D1 receptor Dopamine D2 receptor Dorsal raphe nucleus

The effects of the dopamine (DA) D1 and D2 receptor agonists SKF38393, bromocriptine and quinpirole, respectively, on spontaneous sleep were analyzed in adult rats prepared for chronic sleep recordings. Local administration of the DAergic agonists into the dorsal raphe nucleus (DRN) during the light phase of the light-dark cycle induced a significant reduction of rapid-eye movement sleep (REMS) and the number of REM periods. Additionally, bromocriptine and quinpirole significantly increased wakefulness (W). Opposite, the microinjection into the DRN of the DA D1 and D2 receptor antagonists SCH23390 and sulpiride, respectively, significantly augmented REMS and the number of REM periods. Pretreatment with SCH23390 and sulpiride prevented the effects of SKF38393 and bromocriptine, respectively, on sleep variables. Our results tend to indicate that DAergic neurons located in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) contribute to the regulation of predominantly W and REMS by DRN serotonergic neurons.

1. Introduction A number of neuroanatomical structures located in the brainstem, hypothalamus and basal forebrain (BFB) are implicated in the occurrence of wakefulness (W). In this respect, neural populations containing serotonin [5-HT: dorsal raphe nucleus (DRN)] and dopamine [DA: ventral tegmental area (VTA), substantia nigra pars compacta (SNc)] are found in the brainstem [1]. It has been established that separated activation of each of the arousal systems is followed by the appearance of W [2]. In spite of this, under normal circumstances all the arousal systems contribute to the occurrence of behavioral and electroencephalographic (EEG) arousal. This is mainly determined by the neuroanatomical connections of the W-promoting nuclei. Furthermore, the neurotransmitter systems involved in the regulation of W inhibit neural structures that promote and/or induce non-rapid eye movement sleep (NREMS) and rapid-eye movement sleep (REMS). With respect to the DAergic system, systemic administration of the selective DA D1 receptor agonist SKF38393 produces behavioral arousal, significantly increases W and reduces slow wave sleep (SWS) and REMS. Opposite, the selective DA D1 receptor antagonist SCH23390 induces sedation, lowers W and augments SWS and REMS. Pretreatment with the DA D1 receptor antagonist SCH23390 counteracts the effect of SKF38393 on

sleep and W. Furthermore, i.p. injection of the selective DA D2 receptor agonists bromocriptine and quinpirole has been shown to bring about biphasic effects, such that low doses decrease W and enhance SWS and REMS, whereas large doses cause the opposite actions. Concerning the DA D2 receptor antagonists, i.p. administration of haloperidol and sulpiride reduces values corresponding to W and increases these related to SWS in the rat. Additionally, haloperidol has been shown to dosedependently counteract the effects of bromocriptine [3]. A number of studies have examined the origin of the DAergic innervation of the DRN, and tend to indicate the existence of direct projections from the VTA and SNc to the serotonergic nucleus in the cat and rat [4–9]. Besides, the existence of DA-containing neurons within the rat DRN, frequently identified as a caudal extension of the VTA, has been also recognized [7,10,11,12]. Furthermore, Ferré et al. [13] characterized the presence of a neuronal pool of DA within the DRN by means of the local infusion of amphetamine in the rat. In this respect, the pharmacological agent significantly augmented the extracellular concentration of 5-HT, DA and their main metabolites dihydroxyphenylacetic acid and 5-hydroxyindolacetic acid, respectively. In contrast, there is a lack of correspondence between authors in relation to the presence of DA D1 and D2 receptors in the DRN. Consequently, some authors described light to moderate levels of D1 and D2 receptors

Abbreviations: BFB, basal forebrain; DA, dopamine; DRN, dorsal raphe nucleus; EDS, excessive daytime sleepiness; 5-HT, serotonin; LS, light sleep; REMS, rapid-eye movement sleep; SNc, substantia nigra pars compacta; SWS, slow wave sleep; VTA, ventral tegmental area; W, wakefulness ⁎ Corresponding author. E-mail address: [email protected] (J.M. Monti). https://doi.org/10.1016/j.bbr.2017.11.006 Received 14 August 2017; Received in revised form 3 October 2017; Accepted 6 November 2017 Available online 11 November 2017 0166-4328/ © 2017 Elsevier B.V. All rights reserved.

Behavioural Brain Research 339 (2018) 11–18

J.M. Monti, H. Jantos

latencies and the number and mean duration of REM periods were also ascertained [20].

in the DRN [14–17], while other contended that only D2 receptor is expressed in serotonergic neurons [18,19]. To date, no attempts have been made to characterize the changes on sleep and W induced by the local administration into the DRN of DA D1 and D2 receptor agonists in the normally behaving rat. The present experiments were undertaken to test the hypothesis that activation or blockade of DA D1 or D2 receptor in the DRN would influence W and sleep in the rat. For this purpose we made use of the selective DA D1 and D2 receptor agonists SKF38393, bromocriptine and quinpirole, respectively, and the DA D1 and D2 receptor antagonists SCH23390 and sulpiride, respectively. To this aim, several doses of the agents were injected into the DRN of animals prepared for chronic sleep recordings. In addition, we tested the potential use of SCH23390 and sulpiride to counteract the SKF38393 and bromocriptine-induced changes of sleep variables, respectively.

2.4. Experimental design The doses of SKF38393 (2–8 mM), SCH23390 (1–4 mM), bromocriptine (2–4 mM), quinpirole (3–8 mM) and sulpiride (1–4 mM) chosen for the present study were grounded on pilot work in our laboratory and the limited previous research in which dispensation of these agents was employed to ascertain the actual physiological role of the DA D1 and D2 receptors. In all experiments at least 3 days were allowed to pass between experiments to prevent long-lasting and rebound effects on sleep. The latter was supported by the short to intermediate elimination half life (t½) of the compounds administered in the present study (SKF38393: 1–2 h; SCH23390: 45 min; bromocriptine: 10 h; quinpirole: 1.8 h; sulpiride: 8 h) [24,25]. The effects of the DA D1 and D2 receptor agonists and antagonists were studied in two different groups of rats according to the following experimental patterns: Experiment 1 (group 1): SKF38393 hydrobromide (Tocris Bioscience, Ellisville,MO, USA) 2, 4 or 8 mM (molecular weight 255.31) or vehicle (distilled water) was infused into the DRN (n = 6). Experiment 2 (group 1): SCH23390 hydrochloride (Tocris Bioscience, Ellisville, MO, USA) 1, 2 or 4 mM (molecular weight 324.24) or vehicle (distilled water) was infused into the DRN (n = 6). Experiment 3 (group 1): In the third set of experiments 8 mM SKF38393 was injected into animals pretreated with 4 mM SCH23390. The drugs were microinjected into the DRN 20 min apart in these interaction experiments (n = 6). Experiment 4 (group 2): bromocriptine mesylate (Tocris Bioscience, Ellisville, MO, USA) 2, 3 or 4 mM (molecular weight 654.60) or vehicle (1% aqueous solution of Tween 80) was infused into the DRN (n = 6). Experiment 5 (group 2): quinpirole hydrochloride (Tocris Bioscience, Ellisville, MO, USA) 3, 4 or 8 mM (molecular weight 255.79) or vehicle (distilled water) was infused into the DRN (n = 6). Experiment 6 (group 2): sulpiride (Tocris Bioscience, Ellisville, MO, USA) 1, 2 or 4 mM (molecular weight 341.42) or vehicle (1% aqueous solution of Tween 80) was infused into the DRN (n = 6). Experiment 7 (group 2): in the seventh set of experiments 4 mM bromocriptine was injected into animals pretreated with 4 mM sulpiride. The drugs were microinjected into the DRN 20 min apart in these interaction experiments (n = 6). A 6-h recording was started 15 min after vehicle or drug(s) administration.

2. Material and methods 2.1. Animals Male Wistar rats (320–350 g) were used for experiments. The animals were kept in plastic cages under 12 h light-dark cycles (lights on 0700 h) with food and water accessible ad libitum. Experiments were performed in accordance with the National Institutes of Health (USA) guidelines for the care and use of laboratory animals, and were approved by the Institutional Animal Care Committee of the Medical School, University of the Republic, Montevideo, Uruguay. 2.2. Surgical procedures Details of the surgical procedures have been described previously [20]. The rats were implanted with electrodes for chronic sleep recordings of EEG and electromyogram (EMG) activities, by placement in the left/right frontal cortex, the left/right occipital cortex, and the dorsal neck musculature, respectively. In addition, a guide cannula was implanted with its tip 2 mm above the DRN (AP 7.8; L 0.0: V – 5.8) [21]. The recording plug and the guide cannula were fasten to the skull with dental acrylic and anchor screws. Drug or vehicle was injected into the DRN with a 1 μl syringe that was linked with an injection cannula (29 gauge) which lengthen 2 mm beyond the guide, in a 0.2 μl volume over a 2-min period. Although we verified that the cannula tip was confined within the limits of the DRN, it is not possible to discard the diffusion of the pharmacological agents outside the serotonergic nucleus. In this respect, it is well-known that the diffusion rate of a given drug depends upon a number of factors including its diffusion coefficient, and the characteristics of the brain region where the microinjection is performed [22]. However, it should be taken into consideration that methylene blue microinjected into the central nervous system in a 0.2 μl volume diffuses an average ratio of 520 μm [23]. Because of the small doses and volume employed in the present study we consider that the diffusion of effective concentrations of SKF38393, SCH23390, bromocriptine, quinpirole and sulpiride outside the DRN, if present, was insignificant. Histological verification of cannula/injection sites was accomplished at the end of the experiments.

2.5. Statistics A repeated-measures analysis of variance, with dose as a betweensubjects factor, was performed, with multiple post-hoc comparisons with the Dunnett multiple comparisons test when the analysis of variance revealed significance (P < 0.05). 3. Results The histological analysis of the injection sites indicated that the 12 animals originally included in the study received microinjections of the DA D1 and D2 receptor agonists and antagonists that were confined within the limits of the DRN. As mentioned earlier with respect to the DA D2 receptor agents, quinpirole was dissolved in distilled water whereas bromocriptine and sulpiride were dissolved in distilled water + Tween 80 added. Notwithstanding this, there were no significant differences in the values corresponding to the sleep variables including REMS duration (P = 0.23) and REMS latency (P = 0.08), determined using distilled water or distilled water + Tween 80 added in the control experiments.

2.3. Recording and sleep scoring Ten days after surgery the rats were habituated to a sound-proof chamber fitted with slip-rings and cable connectors, and to the injection procedure. The drugs or vehicle were always administered during the light phase of the 12-h light/12-h dark cycle, at approximately 0.800 h. A balanced order or drug and control injections was used at all times to combine the effects of both the drug and the time elapsed during the protocol. Recording was started 15 min later and continued for 6 h. The predominant activity of each 10 s epoch was consigned to one of the following categories: W, light sleep (LS), SWS, or REMS. SWS and REMS 12

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J.M. Monti, H. Jantos

Table 1 Effects of SKF 38393 microinjected into the dorsal raphe nucleus on sleep and wakefulness during 6-h polysomnographic recordings.

Wakefulness Light sleep Slow wave sleep REM sleep Number of REM periods Mean REM period duration Slow wave sleep latency REM sleep latency

Control

SKF 2

SKF 4

SKF 8

62.8 ± 11.8 43.5 ± 7.4 236.0 ± 12.8 17.7 ± 3.1 8.3 ± 1.3 2.1 ± 0.1 4.7 ± 1.8 51.3 ± 13.5

48.2 ± 7.3 47.6 ± 6.2 247.0 ± 15.0 17.2 ± 3.5 8.2 ± 1.6 2.1 ± 0.1 2.0 ± 0.7 99.5 ± 20.9

64.5 ± 6.8 53.3 ± 4.8 230.0 ± 8.2 12.2 ± 3.2 6.2 ± 1.5 2.0 ± 0.2 3.7 ± 1.5 121.2 ± 19.7

61.5 ± 6.9 58.0 ± 5.7 231.2 ± 11.6 9.3 ± 2.5* 4.7 ± 1.1* 2.0 ± 0.2 2.5 ± 1.6 133.5 ± 15.7*

Sleep stages, mean REM period duration and sleep latencies were quantified in minutes. The doses are indicated in mM. * P < 0.05, significant statistical difference with respect to control. Local administration of SKF 38393 (8 mM) into the dorsal raphe nucleus significantly reduced REM sleep duration and the number of REM periods, and increased REM sleep latency.

REMS and of the number of REM periods, and the increase of REM sleep latency induced by 8 mM SKF38393 over the 6-h of recording (data not shown).

3.1. Effects of the activation and blockade of DA D1 receptors Following the microinjection of SKF38393 (8 mM) into the DRN, REMS [F(3.15) = 2.89, P < 0.05] and the number of REM periods [F(3.15) = 2.63, P < 0.05] were significantly reduced whereas REMS latency [F(3.15) = 2.68, P < 0.05] was increased during the 6-h recording period (Table 1). Furthermore, the 8 mM dose reduced REMS [F(3.35) = 5.13, P < 0.01; F(3.35) = 2.74, P < 0.05, respectively] during the first and the second 2-h of recording. The 4 mM dose of the DA D1 receptor agonist reduced REMS only during the first 2-h of recording [F(3.15) = 4.51, P < 0.01] (Fig. 1). SCH23390 4 mM significantly increased REMS [F(3.35) = 2.97, P < 0.05] and the number of REM periods [F(3.35) = 2.65, P < 0.05] during the 6-h recording period (Table 2). Likewise, the 4 mM dose augmented REMS [F(3.35) = 2.85, P < 0.05] during the first 2-h of the recording period (Fig. 2). Pretreatment with 4 mM SCH23390 prevented the reduction of

3.2. Effects of the activation and blockade of DA D2 receptors Bromocriptine at a dose of 4 mM significantly increased W [F(3.35) = 2.65, P < 0.05] and REMS latency [F(3.35) = 3.53, P < 0.01], and reduced REMS [F(3.35) = 4.48, P < 0.01] and the number of REM periods [F(3.35) = 3.03, P < 0.05] during the 6-h recording phase (Table 3). In addition, the microinjection of bromocriptine 4 mM significantly increased W and reduced SWS during the second 2-h period after treatment [F(3.35) = 3.52, P < 0.01 and F(3.35) = 2.82, P < 0.05, respectively]. Moreover, REMS was significantly decreased during the first, second and third 2-h recording period [F(3.35) = 3.24, P < 0.05; F(3.35) = 2.93, P < 0.05 and F(3.35) = 3.51, P < 0.01, respectively] (Fig. 3).

Fig. 1. Effects of SKF38393 microinjected into the DRN on sleep and W. Sleep stages are quantified in minutes. Ordinate: time spent in sleep and W (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. SKF38393 8 mM reduced REMS values during the first and second 2-h period after treatment. The 4 mM dose of the D1 receptor ligand similarly decreased REMS during the first 2-h period post injection.

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Table 2 Effects of SCH 23390 microinjected into the dorsal raphe nucleus on sleep and wakefulness during 6-h polysomnographic recordings.

Wakefulness Light sleep Slow wave sleep REM sleep Number of REM periods Mean REM period duration Slow wave sleep latency REM sleep latency

Control

SCH 1

SCH2

SCH 4

64.5 ± 12.4 52.5 ± 6.1 225.5 ± 16.7 17.5 ± 4.7 9.7 ± 1.7 1.8 ± 0.2 1.8 ± 0.9 52.0 ± 28.9

66.1 ± 10.0 54.2 ± 4.9 221.3 ± 10.3 18.4 ± 2.2 9.8 ± 0.8 1.9 ± 0.1 4.2 ± 2.0 60.0 ± 18.5

62.2 ± 7.2 46.8 ± 1.7 229.2 ± 6.5 21.8 ± 2.9 10.0 ± 1.8 2.2 ± 0.6 2.8 ± 0.9 59.7 ± 9.3

62.8 ± 8.3 53.5 ± 6.2 212.7 ± 13.7 31.0 ± 5.0* 15.8 ± 2.2* 2.0 ± 0.1 4.2 ± 1.7 39.8 ± 6.1

Sleep stages, mean REM period and sleep latencies were quantified in minutes. The doses are indicated in mM. * P < 0.05 significant statistical difference with respect to control. Local administration of SCH 23390 (4 mM) into the dorsal raphe nucleus significantly increased REM sleep and the number of REM periods.

recording period (Table 5). However, no significant changes could be detected when sleep variables were quantified during 2-h periods (Fig. 5). Pretreatment with sulpiride 4 mM prevented the increase of W and REMS latency and the reduction of REMS and of the number of REM periods caused by bromocriptine 4 mM (data not shown).

Quinpirole 8 mM significantly increased W [F(3.35) = 2.72, P < 0.05] and reduced REMS [F(3.35) = 4.12, P < 0.01] and the number of REM periods [F(3.35) = 3.48, P < 0.01] during the 6-h recording period. A significant reduction of REMS [F(3.35) = 3.59, P < 0.01] and of the number of REM periods [F(3.35) = 2,87, P < 0.05] was observed also following the microinjection of the 4 mM dose of the DAergic agonist. On the other hand, the effect of the 3 mM dose was restricted to a significant decrease of the number of REM periods [F(3.35) = 287, P < 0.05] (Table 4). Treatment with 3–8 mM quinpirole significantly reduced REMS during the second 2-h period postinjection [F(3.35) = 3.10, P < 0.05; F(3.35) = 3.96, P < 0.01 and F(3.35) = 4.17, P < 0.01], respectively. Moreover, the 4 and 8 mM dose induced a similar effect during the first 2-h period [F(3.35) = 3.31, P < 0.05 and F(3.35) = 3.48, P < 0.01] (Fig. 4). Following the microinjection of 4 mM sulpiride into the DRN, REMS [F(3.35) = 2.62, P < 0.05] and the number of REM periods [F(3.35) = 3.00, P < 0.05] were significantly increased during the 6-h

4. Discussion The present study shows for the first time that the administration of the selective DA D1 agonist SKF38393 (8 mM) into the DRN during the light phase of the light-dark cycle, induced a reduction of REMS and of the number of REM periods, whereas REMS latency was increased in the rat. With intra-DRN administration of the D1 receptor antagonist SCH23390 (4 mM) an enhancement of REMS and of the number of REM periods was observed. Pretreatment with SCH23390 (4 mM) averted the SKF38393 (8 mM) induced disruption of REMS. Local

Fig. 2. Effect of SCH23390 microinjected into the DRN on sleep and W. Ordinate and abscissa as in Fig. 1. *P < 0.05. SCH23390 4 mM augmented REMS during the first 2-h of the recording period.

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the sleep-wake cycle. However, the effect of SKF38393 was restricted to a decrease of REMS whereas bromocriptine and quinpirole increased also W. It should be stressed that the compounds used in the present study show a marked affinity for either DA D1 or D2 receptor (Table 6). With regard to bromocriptine, the compound exhibits agonist activity in order of decreasing binding on serotonergic 5-HT1A, 5-HT2A and 5HT2C receptor, antagonist activity on adrenergic α1 receptor, and fully inactivates 5-HT7 receptor with a potency of 80 nM [26–28]. Presently, numerous findings tend to support the influence of the DAergic system on DRN 5-HT neurons. Accordingly, 1) systemic administration of the non-selective DA D1/D2 receptor agonist apomorphine has been shown to increase intracellular 5-HT fluorescence in the DRN, and the effect was averted by haloperidol [29]; 2) local infusion of apomorphine or quinpirole into the DRN dose-dependently augmented 5-HT extracellular concentration [13,19]; 3) the selective lesioning of DA neurons with the neurotoxin 6-OH-DA reduced the spontaneous firing activity of DRN 5-HT cells by 60% [30]. Opposite, systemic injection of apomorphine increased the firing rate of DRN 5HT neurons in behaving cats and anesthetized rats [31]. Concerning DRN DA neurons, it has been established that they modulate behavioral and EEG arousal [32]. In this respect, ontogenetically induced activation of DRN DA cells provokes waking from sleep in mice. Opposite, chemogenetic inhibition of these neurons is followed by the appearance of SWS. Moreover, DA neurons located in the DRN project to the medial prefrontal cortex, striatum, nucleus accumbens and lateral septal nuclei [33]. How can we understand the dissimilar changes of sleep variables observed following the local administration of D1 and D2 receptor agonists into the DRN? Serotonergic cells in the DRN have been grouped into six cell aggregates. The subdivisions include the rostral, ventral, dorsal, lateral, caudal and interfascicular parts of the DRN [34]. Fibers that come from the DAergic system reach mainly the caudal DRN. On the other hand, DA cells located in the serotonergic nucleus are circumscribed to the rostral half of the nucleus [35]. In addition to the 5-HT and DA neurons, cells containing γ-aminobutyric acid (GABA),

Table 3 Effects of bromocriptine microinjected into the dorsal raphe nucleus on sleep and wakefulness during 6-h polysomnographic recordings.

Wakefulness Light sleep Slow wave sleep REM sleep Number of REM periods Mean REM period duration Slow wave sleep latency REM sleep latency

Control

BR 2

BR 3

BR 4

51.0 ± 6.8 44.2 ± 4.4 226.5 ± 4.9 38.3 ± 6.2 16.7 ± 1.6

55.7 ± 3.6 45.0 ± 8.7 226.1 ± 8.6 33.2 ± 2.2 15.7 ± 0.7

64.0 ± 7.3 45.3 ± 5.6 215.2 ± 12.2 35.5 ± 6.4 16.7 ± 2.7

77.3 ± 5.2* 51.0 ± 6.4 212.9 ± 7.8 18.8 ± 4.4** 9.0 ± 2.0*

2.3 ± 0.2

2.1 ± 0.2

2.1 ± 0.1

2.1 ± 0.1

1.3 ± 0.9

2.5 ± 1.8

4.5 ± 3.0

1.3 ± 0.5

21.7 ± 4.2

32.3 ± 5.2

37.8 ± 7.9

67.2 ± 18.4**

Sleep stages, mean REM period duration and sleep latencies were quantified in minutes. The doses are indicated in mM. * P < 0.05. ** P > 0.01; significant statistical difference with respect to control. Local administration of bromocriptine (4 mM) into the dorsal raphe nucleus significantly increased wakefulness and REM sleep latency, and reduced REM sleep duration and the number of REM periods.

administration of the D2 receptor agonists bromocriptine (4 mM) and quinpirole (4 mM) was followed also by a decrease of REMS and of the number of REM periods while W and REMS latency were augmented. Additionally, bromocriptine slightly but significantly reduced SWS values. In contrast, the D2 receptor antagonist sulpiride (4 mM) increased REMS and the number of REM periods and prevented the effects of bromocriptine on W and sleep. Our finding that microinjection of SKF38393, bromocriptine and quinpirole into the DRN is followed by changes of the behavioral state, tends to support the claim that DA D1 and D2 receptors are expressed by neurons located in the DRN. As a result, the DAergic system would be contributing to the regulation of the activity of 5-HT neurons during

Fig. 3. Effects of bromocriptine infused into the DRN on sleep and W. Ordinate and abscissa as in Fig. 1. *P < 0.05; **P < 0.01. Bromocriptine 4 mM reduced REMS during the 6-h recording period. Additionally, the 4 mM dose of the D2 receptor ligand augmented W and decreased SWS during the second 2-h period after administration.

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Table 4 Effects of quinpirole microinjected into the dorsal raphe nucleus on sleep and wakefulness during 6-h polysomnographic recordings.

Wakefulness Light sleep Slow wave sleep REM sleep Number of REM periods Mean REM period duration Slow wave sleep latency REM sleep latency

Control

Quin 3

Quin 4

Quin 8

61.9 ± 10.1 50.7 ± 6.8 218.3 ± 10.8 29.1 ± 6.7 12.4 ± 1.6 2.2 ± 0.2 2.7 ± 1.2 49.8 ± 12.2

74.6 ± 12.5 50.6 ± 6.6 214.6 ± 14.6 20.2 ± 9.0 7.7 ± 2.8* 2.3 ± 0.2 4.0 ± 1.6 58.3 ± 12.7

79.9 ± 14.1 56.2 ± 3.8 208.5 ± 11.1 15.4 ± 7.1** 7.7 ± 3.0* 1.9 ± 0.2 1.4 ± 0.7 76.7 ± 15.8

85.7 ± 15.4* 54.4 ± 6.2 206.5 ± 18.4 13.4 ± 4.5** 6.7 ± 1.8** 1.9 ± 0.1 2.4 ± 1.8 82.4 ± 10.3

Sleep stages, mean REM period duration and sleep latencies were quantified in minutes. The doses are indicated in mM. * P < 0.05. ** P < 0.01 significant statistical difference with respect to control. Local administration of quinpirole (8 mM) into the dorsal raphe nucleus significantly increased wakefulness. REM sleep was reduced after microinjection of the 4 and 8 mM doses, whereas the number of REM periods was decreased after the whole range of doses.

Fig. 4. Effects of quinpirole microinjected into the DRN on sleep and W. Ordinate and abscissa as in Fig. 1. *P < 0.05; **P < 0.01. Treatment with 3–8 mM quinpirole reduced REMS during the second 2-h period postinjection. Furthermore, the 4 and 8 mM dose induced a similar effect on the sleep variable during the first 2-h period.

Table 5 Effects of sulpiride microinjected into the dorsal raphe nucleus on sleep and wakefulness during 6-h polysomonographic recordings.

Wakefulness Light sleep Slow wave sleep REM sleep Number of REM periods Mean REM period duration Slow wave sleep latency REM sleep latency

Control

Sulpiride 1

Sulpiride 2

Sulpiride 4

69.1 ± 11.5 53.0 ± 3.3 218.2 ± 12.8 19.7 ± 3.2 9.5 ± 1.4 2.1 ± 0.1 5.2 ± 3.6 36.5 ± 6.8

75.0 ± 12.3 53.2 ± 6.1 213.3 ± 13.3 18.5 ± 6.5 8.5 ± 2.5 2.1 ± 0.2 3.2 ± 1.4 59.0 ± 18.9

75.5 ± 13.7 47.3 ± 4.6 214.0 ± 16.6 23.2 ± 3.3 11.0 ± 1.5 2.1 ± 0.2 4.3 ± 1.5 53.7 ± 13.6

65.5 ± 8.3 48.0 ± 4.0 216.8 ± 11.0 29.7 ± 4.7* 15.3 ± 2.5* 2.0 ± 0.1 2.0 ± 0.9 52.2 ± 7.7

Sleep stages, mean REM period duration and sleep latencies were quantified in minutes. The doses are indicated in mM. * P < 0.05 significant statistical difference with respect to control. Local administration of sulpiride (4 mM) into the dorsal raphe nucleus significantly increased REM sleep and the number of REM periods.

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Fig. 5. Effect of sulpiride microinjected into the DRN on sleep and W. Ordinate and abscissa as in Fig. 1. Treatment with 1–4 mM sulpiride showed no significant changes of the behavioral state when sleep variables were quantified during 2-h periods.

whole-cell-patch-clamp recordings in vitro. It was found DA D2 agonists reduced the postsynaptic response to a GABAA receptor agonist. Thus, the increase of W and reduction of REMS following the local administration of DA D2 agonists into the DRN, could be related to: 1) the depolarization of 5-HT neurons through the activation of nonselective cationic conductance, and/or 2) the disinhibition of 5-HT cells synaptically related to GABAergic interneurons. It could be proposed the existence of two subpopulations of 5-HT neurons in the DRN that respond differently to the local microinjection of D1 and D2 receptor agonists. One subpopulation of 5-HT cells would express D1 receptor, while a second subpopulation would express D2 receptor. The 5-HT neurons whose direct activation by SKF38393 results in the reduction of REMS would make synaptic contact with only GABAergic REM-off cells located in the ventrolateral periaqueductal gray (vlPAG) and the deep mesencephalic nucleus (DPMe). The activation of the GABAergic REM-off cells by serotonergic neurons would result in the inhibition of sublaterodorsal tegmental nucleus (SLD) glutamatergic cells involved in the induction of REMS [40–42]. On the other hand, the 5-HT neurons that directly or indirectly react to the local administration of bromocriptine and quinpirole would give rise to ascending projections to the thalamus, cerebral cortex and BFB and, in addition, would make synaptic contact with the GABAergic REM-off neurons described above [43,44]. However, further studies are needed in order to test the hypothesis. Nocturnal sleep is frequently disturbed in patients with Parkinsońs disease, and DA receptor agonists may indirectly improve sleep by decreasing the incidence of motor symptoms [45]. Excessive daytime sleepiness (EDS) is also a common symptom in these patients. EDS has been attributed to a DA-deficiency state at the central nervous system level related to the degeneration of dopaminergic cells and the loss of a facilitatory effect on neural structures involved in the occurrence of W, and get also better following the administration of DA agonists [46]. It is proposed that the improvement of disturbed nocturnal sleep and EDS

Table 6 Receptor affinities of dopamine D1 and D2 receptor agonists and antagonists. Receptor

D1

D5

D2

D3

D4

SKF38393 SCH23390 Bromocriptine Quinpirole Sulpiride

1.0 0.2 682.0 1900.0 10000.0

0.5 0.3 496.0 n.a. 10000.0

150.0 1100.0 2.9 4.8 9.8

5000.0 800.0 5.4 24.0 8.1

1000.0 3000.0 328.0 30.0 54.0

Receptor affinity expressed as Ki value; n.a.: not available. From: Bourne [27]; National Institute of Mental Health PDSD Ki Database [28].

glutamate, nitric oxide and a number of neuropeptides occur in the DRN. Furthermore, neurons that express among other glutamate and GABA project to the serotonergic nucleus, and directly or indirectly, through local circuits, regulate the activity of 5-HT neurons [34,36]. Dopamine D1 and D2 receptors are distinguished by their distinct biochemical characteristics. The D1 receptor is coupled to adenylate cyclase, and its stimulation facilitates the activity of the enzyme. Rat brain areas rich in D1 receptors include the caudate-putamen, nucleus accumbens and olfactory tubercle. D1 receptors are also expressed at lower levels in the structures involved in the regulation of W, including the DRN [14–17]. The D2 receptor is the predominant D2-like subtype in the brain, and is present also in the DRN. The D2 receptor is predominantly coupled to the inhibition of adenylate cyclase [37]. However, D2 receptors can depolarize a given neuron through the activation of nonselective cationic conductance. This would require the stimulation of phospholipase C but not an increase in intracellular calcium [38]. Moreover, DA D2 receptors are expressed by GABAergic neurons that synapse with cells located in structures involved in the occurrence of W, including the cerebral cortex, BFB and monoaminergic nuclei of the brainstem. In this respect, Seamans et al. [39] studied the effects of DA on GABAergic inputs to the prefrontal pyramidal neurons using 17

Behavioural Brain Research 339 (2018) 11–18

J.M. Monti, H. Jantos

in PD related to the administration of a DA agonist, could be partly related to the activation of 5-HT neurons involved in the promotion of the behavioral state.

[19]

5. Conclusions

[20]

This study examined the effects of local administration into the DRN of DA D1 and D2 agonists and antagonists, on sleep variables in the rat. Microinjection of D1 and D2 receptor agonists into the DRN significantly reduced REMS. In addition, the D2 receptor agonists induced a significant increase of W. On the contrary, D1 and D2 receptor antagonists augmented REMS. Our findings tend to indicate that the DAergic system contributes to the regulation of W and REMS by DRN serotonergic neurons.

[21] [22] [23]

[24] [25] [26]

Conflict of interest statement

[27]

The authors declare that there are no conflicts of interest.

[28]

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