- Email: [email protected]
Effect of dopamine D4 receptor agonists on sleep architecture in rats
PNP-08772; No of Pages 8 Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
Effect of dopamine D4 receptor agonists on sleep architecture in rats
2Q2
Shunsuke Nakazawa ⁎, Keiko Nakamichi, Hideaki Imai, Junji Ichihara
3
Drug Development Research Laboratories, Sumitomo Dainippon Pharma, Co., Ltd., Osaka, Japan
4
a r t i c l e
5 6 7 8 9
Article history: Received 24 January 2015 Received in revised form 28 April 2015 Accepted 11 May 2015 Available online xxxx
10 Q3 11 12 13 14 15 16 17
Keywords: A-412997 EEG NREM REM Ro 10-5824 Sleep Wakefulness
O
a b s t r a c t
R O
i n f o
T
E
D
P
Dopamine plays a key role in the regulation of sleep–wake states, as revealed by the observation that dopaminereleasing agents such as methylphenidate have wake-promoting effects. However, the precise mechanisms for the wake-promoting effect produced by the enhancement of dopamine transmission are not fully understood. Although dopamine D1, D2, and D3 receptors are known to have differential effects on sleep architecture, the role of D4 receptors (D4Rs), and particularly the influence of D4R activation on the sleep–wake state, has not been studied so far. In this study, we investigated for the first time the effects of two structurally different D4R agonists, Ro 10-5824 and A-412997, on the sleep–wake states in rats. We found that both D4R agonists generally increased waking duration, and conversely, reduced non-rapid eye movement (NREM) sleep duration in rats. The onset of NREM sleep was also generally delayed. However, only the A-412997 agonist (but not the Ro 10-5824) influenced rapid eye movement sleep onset and duration. Furthermore, these effects were accompanied with an enhancement of EEG spectral power in the theta and the gamma bands. Our results suggest the involvement of dopamine D4R in the regulation of sleep–wake states. The activation of the D4R could enhance the arousal states as revealed by the behavioral and electrophysiological patterns in this study. Dopamine D4R may contribute to the arousal effects of dopamine-releasing agents such as methylphenidate. © 2015 Published by Elsevier Inc.
32
C
36 34 33 35
1. Introduction
38
It is well known that many brain functions such as cognition, attention, reward, emotion, and motor control are regulated by the neurotransmitter dopamine (DA) (Nieoullon, 2002). In addition, DA plays a key role in the regulation of sleep–wake states. For example, it is known that DA-releasing agents such as amphetamine have wakepromoting effects in rats (Berridge et al., 2006; Gruner et al., 2009), and a study on knock-out (KO) mice for the dopamine transporter (DAT) suggests that the inhibition of DAT is necessary for this action (Wisor et al., 2001). Moreover, methylphenidate (known as Ritalin®), an inhibitor of DAT, is widely used for the treatment of narcolepsy (Billiard, 2008), a condition in which patients suffer from abnormal sudden sleep attacks. Thus, it appears that an enhancement of DA transmission in the brain promotes waking and reduces sleep states across species. However, the specific mechanisms for these actions of DA are not fully understood.
45 46 47 48 49 50 51 52
R
R
N C O
43 44
U
41 42
E
37
39 40
F
1Q1
Abbreviations: DA, dopamine; DAT, dopamine transporter; D4R, dopamine D4 receptor; Ro 10-5824, 5-[(3,6-dihydro-4-phenyl-1 (2H)-pyridinyl) methyl]-2-methyl-4pyrimidinamine; A-412997, N-(3-Methylphenyl)-4-(2-pyridinyl)-1-piperidineacetamide; EEG, electroencephalogram; EMG, electromyogram; REM, rapid eye movement; NREM, non-rapid eye movement; PFC, prefrontal cortex; TRN, thalamic reticular nuclei. ⁎ Corresponding author at: Drug Development Research Laboratories, Sumitomo Dainippon Pharma, Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan. Tel.: +81 6 6337 5974; fax: +81 6 6337 5109. E-mail address: [email protected] (S. Nakazawa).
Five DA receptors have been characterized to date. These DA receptors are coupled to G-proteins and are classified in two distinct groups, D1-like and D2-like receptors, depending on the coupled G-protein (Beaulieu and Gainetdinov, 2011). D1-like receptors, which are coupled to Gαs, include D1 and D5 receptors, whereas the D2-like receptors, which are coupled to Gαi/o, include D2, D3, and D4 receptors. It is suggested that DA affects sleep–wake states by acting on DA receptors, and by its interaction with other neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA) (Monti and Monti, 2007). The role of D1 and D2 receptors on sleep–wake states has been intensively studied in rodents using genetic KO mice or selective pharmacological agents (Monti and Jantos, 2008). The systemic administration of the D1 receptor (D1R) agonists A68930 and SKF 38393 resulted in an increase in the waking time, and a decrease of rapid eye movement (REM) and non-REM (NREM) sleep time (Trampus et al., 1993). On the other hand, the administration of the D1R antagonist SCH 23390 increased NREM sleep time and decreased the waking and REM sleep time (Monti et al., 1990). D2 receptor (D2R) agonists (e.g. quinpirole) are known to have a complex effect on sleep–wake states in rats, such that low doses of the agonist reduce waking and increase sleep, while high doses have the opposite effect (Monti et al., 1988, 1989). However, Ongini et al. (1993) demonstrated that the relatively selective D2R antagonist raclopride did not have a significant effect on the sleep architecture in rats. Finally, Qu et al. (2010) reported a lower waking duration and higher NREM and REM sleep duration in D2R KO mice than in wild-type mice. Thus, the role of D2Rs on sleep–wake states is still under debate because of the lack of selective D2R agonists, but the
http://dx.doi.org/10.1016/j.pnpbp.2015.05.006 0278-5846/© 2015 Published by Elsevier Inc.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
18 19 20 21 22 23 24 25 26 27 28 29 30 31
53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
129 130
2.3. Analysis of the sleep architecture
139
The sleep stage analysis was conducted off-line using Sleepsign software (KISSEI COMTEC CO., LTD., Japan). The EEG signal was high-pass filtered with a 0.75 Hz filter, and the EMG signal was band-pass filtered between 20 and 50 Hz. The electrographic activity of 10-s epochs was analyzed. Each epoch was automatically registered as “WAKE,” “REM,” or “NREM” based on the waveforms of the EEG and the EMG. We used the same conditions than Murai et al. (2014b): We defined the conditions as “WAKE” when the EMG exceeded the individual threshold. They were defined as NREM when the power of the delta waves (0.75–4 Hz) exceeded the individual threshold, and did not present EMG activity. Finally, they were defined as REM when the power of the theta waves (4–8 Hz) exceeded 40% of the total power of 0.75–80 Hz waves, and there was no EMG response. The duration of WAKE, NREM, and REM and the latencies to the initial NREM and REM were calculated. The onset of NREM and REM sleep was defined as the first occurrence of 18 consecutive (3 min) epochs and 3 consecutive (30 sec) epochs, respectively. The EEG spectral power densities in each frequency band (0.75–4 Hz for delta; 4–8 Hz for theta; 8–12 Hz for alpha; 12–30 Hz for beta; 30–80 Hz for gamma) over the first 2 h post-administration were calculated with a fast Fourier transform (FFT). Then, we calculated changes in spectral power (%) by dividing the spectral powers obtained in the drug treatment condition by those obtained in the saline treatment condition in a within-animal comparison manner. To assess the specific influence of drugs on EEG profiles, changes in spectral power (%) were calculated without separating sleep–wake states (i.e., TOTAL), as well as for each state separately (i.e., WAKE, NREM, and REM).
140 141
2.4. Drugs and experimental design
167
Ro 10-5824 and A-412997 were purchased from Tocris Bioscience (Ellisville, MO, USA). Drugs were freshly dissolved in saline on the day of each experiment. Animals were administered subcutaneously saline, Ro 10-5824, or A-412997 at a volume of 2 mL/kg, 10 min before the light-on cycle (7 p.m.), followed by the continuous recording of EEG/EMGs for the entire light-on period. In the present study, we performed two different four-way crossover experiments, one comparing saline and Ro 10-5824 (1, 3, 10 mg/kg), and the other comparing saline and A-412997 (1, 3, 10 mg/kg). The order of the drug treatment varied pseudo-randomly, and at least 1 week was allowed between experiments for each animal. Drug doses were chosen based on previous in vivo studies (Murai et al., 2014a; Powell et al., 2003; Woolley et al., 2008).
168
Reference
2.5. Statistical analysis
181
Powell et al., 2003 Newman-Tancredi et al., 2008 Moreland et al., 2005
All data were expressed as the mean ± standard error of mean (SEM). The duration of WAKE, NREM, and REM in a total 3 h period, and latencies of NREM and REM sleep were compared statistically using a repeated measures one-way ANOVA, followed by post hoc Dunnett tests. Temporal changes in the duration of WAKE, NREM, and REM, or the power spectral densities in each frequency band were statistically compared using repeated measures two-way ANOVA followed by
182 183
110
2.1. Animals
111 112
117
Seven adult male Wistar rats (6 weeks old) were purchased from Charles River Laboratories, Japan. The animals were housed in an airconditioned room kept at a temperature of 20–26 °C, and a humidity of 40–70%, with a 12:12 h light/dark cycle (lights-on at 7 a.m.). Food and water were given ad libitum. The Institutional Animal Care and Use Committee at Sumitomo Dainippon Pharma, Co. Ltd. reviewed and approved all the experimental procedures for the use of animals.
118
2.2. Surgery and electroencephalogram recording
119
128
Sleep electroencephalogram (EEG) recordings were conducted as previously described (Murai et al., 2014b). Briefly, a radio transmitter (TL11M2-F40-EET; Data Science International, New Brighton, MN, USA) was implanted subcutaneously in the back of animals anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A pair of electrode wires was stereotaxically implanted in the skull in the following locations: one wire in the frontoparietal area (2 mm anterior to bregma and 2 mm left to midline), and the other wire in the parietal area (5 mm posterior to bregma and 2 mm right to midline). Two stainless-steel screws were placed as anchors. The EEG electrodes and
t1:1 t1:2
Table 1 Profiles of the two dopamine D4 receptor agonists used in this study.
105 106
113 114 115 116
120 121 122 123 124 125 126 127
T
103 104
C
101 102
E
99 100
R
97 98
R
95 96
O
93 94
C
91 92
N
89 90
U
87 88
t1:3
Drug
t1:4 t1:5
Binding affinity, Ki (nM)
Agonist activity EC50 (nM) a
Emax (%) a
t1:6 t1:7
Ro 10-5824
5.2 40.7
205 81.3a
36.3 19.0a
t1:8
A-412997
7.9
28.4b
83.0b
t1:9 t1:10
a
O
2. Materials and methods
86
R O
109
84 85
E
107 108
82 83
F
screws were fixed using dental cement. Electromyograms (EMG) were recorded from the dorsal neck muscle using another pair of electrodes. Antibiotics and analgesics were applied after surgery. Animals were allowed at least 2 weeks of recovery in individual plastic cages before EEG/EMG recordings. The light/dark cycle was changed during this period (lights-on: 7:00 p.m–7:00 a.m.). EEG/EMGs were recorded in the home cages in a soundproof box using Dataquest A.R.T. software (Data Science International, New Brighton, MN, USA), at a sampling rate of 500 Hz. Prior to the experiments, animals were recorded once to ensure the validity of the EEG/EMG recordings.
complex actions of the D2Rs are thought to depend on their presynaptic or postsynaptic location (Monti and Monti, 2007). On the other hand, it has been suggested that the activation of D3 receptors (D3Rs) induces somnolence, since pramipexole, which is a D3R-preferring agonist, induces sleep in both rodents and humans (Hauser et al., 2000; Lagos et al., 1998). Taken together, these reports suggest that the wake-promoting effect of dopamine could be mediated by its action on different dopamine receptors. However, little is known about the role of D4 receptors (D4Rs) in the regulation of sleep–wake states. D4Rs are widely expressed in the brain, including prefrontal cortex (PFC), striatum, thalamus, and hypothalamus (Khan et al., 1998), all of which are involved in the regulation of sleep–wake states. It has been reported that the D4R antagonist L-741,741 induces a complex effect on the duration of sleep and wake states in rats (Cavas and Navarro, 2006). On the other hand, the influence of D4R agonists on the sleep architecture has not been studied so far. To clarify the role of the activation of D4R in the regulation of sleep–wake states, in the present study we investigated the effect of two structurally different D4R agonists, Ro 10-5824 and A412997, on the sleep architecture in rats. Ro 10-5824 is a selective partial agonist of D4R (Table 1) that has a high binding affinity (Ki = 5.2nM) and presents a 250-fold selectivity for D4R over D3R, and more than 1,000-fold selectivity over D1, D2, and D5 receptors (Powell et al., 2003). A-412997 is a selective full agonist (Table 1) of D4R that has a high binding affinity (Ki = 7.9nM), with negligible binding affinities for other dopamine receptor subtypes (Moreland et al., 2005). Both compounds have been widely used for the assessment of D4Rs in vivo (Browman et al., 2005; González et al., 2012; Murai et al., 2014a; Powell et al., 2003; Woolley et al., 2008).
P
80 81
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
D
2
Agonist activity was measured in human D4.4R using 35S-GTPγS binding assays. b Agonist activity was measured in rat D4R using Ca2+ flux assays.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
131 132 133 134 135 136 137 138
142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
169 170 171 172 173 174 175 176 177 178 179 180
184 185 186 187 188
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
We first examined the influence of the agonists Ro 10-5824 and A412997 on sleep architecture during the 3 h recoding period. As shown in Fig. 1A, Ro 10-5824 significantly increased the duration of WAKE (F3,18 = 11.38, p = 0.0002) and reduced the duration of NREM (F3,18 = 16.68, p b 0.0001). On the other hand, the duration of REM was not affected (F3,18 = 0.4159, p = 0.7437). The post hoc Dunnett test showed that administration of Ro 10-5824 resulted in a significant increase in the duration of WAKE (p b 0.01) and a decrease in the duration of NREM (p b 0.01), at all doses tested (Fig. 1A). Accordingly, as shown in Fig. 2A, the partial agonist Ro 10-5824 significantly delayed the onset of NREM sleep (F3,18 = 5.816, p = 0.0058), but not the onset of REM sleep (F3,18 = 0.02361, p = 0.9949). Administration of the full agonist A-412997 also resulted in wake promotion, as shown in Fig. 1B and Fig. 2B. This agonist significantly increased the duration of WAKE (F3,18 = 7.413, p = 0.0020) and reduced the duration of NREM (F3,18 = 4.191, p = 0.0205) as well as that of REM (F3,18 = 11.43, p = 0.0002). The increase in the duration of WAKE and the decrease in the duration of both NREM and REM reached statistical significance (post hoc tests, p b 0.01) only at a dose of 10 mg/kg of A412997. Sleep onset for both NREM (F3,18 = 29.33, p b 0.0001) and REM (F3,18 = 53.13, p b 0.0001) were delayed by the administration of A-412997 (Fig. 2B). Sleep latency of NREM was significantly increased
208 209 210 211 212 213 214 215
3.2. Effects of D4R agonists on the spectral power density of EEGs
240
218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239
To characterize further the arousal effects of the agonists Ro 10-5824 241 and A-412997, their influence on the spectral power density of EEG was 242 examined. For easier visualization, in Fig. 4 we show only the 243
C
206 207
E
204 205
R
202 203
R
200 201
N C O
198 199
U
196 197
F
194 195
O
3.1. Effects of Ro 10-5824 and A-412997 on sleep architecture in rats
R O
193
P
3. Results
216 217
D
192
at a dose higher than 3 mg/kg (p b 0.05) of A-412997, while the increase in sleep latency of REM reached statistical significance only at a dose of 10 mg/kg (p b 0.001). Thus, both Ro 10-5824 and A-412997 showed wake-promoting effects in rats, but these two drugs affected REM sleep in a different manner. To assess further the effect of these two drugs, changes in time spent in each of WAKE, NREM, and REM states in every 1 h block were analyzed and are shown in Fig. 3. A significant increase in time spent in WAKE and a decrease in time spent in NREM were clearly seen 1 h after the administration of Ro 10-5824, at all doses tested (p b 0.05), although delayed effects at 3 mg/kg were only found in the 2–3 h period (Fig. 3A). In addition, reduction of the duration of NREM by the administration of Ro 10-5824 at 3 and 10 mg/kg reached a trend, but not significance in the 1–2 h period (p = 0.0609 and p = 0.0698, respectively). No significant effect on time spent in REM was found at any doses or times. The administration of A-412997 at 10 mg/kg caused a significant increase in time spent in WAKE (p b 0.001), and a decrease in time spent in NREM (p b 0.001) within the first hour, although the effects on REM (p b 0.001) were found at 2–3 h (Fig. 3B). Taken together, the administration of both Ro 10-5824 and A412997 resulted in an enhancement of waking states within the first hour. The partial agonist Ro 10-5824 did not influence REM sleep, while the full agonist A-412997 caused a reduction in REM sleep at 2–3 h, and a delay on the onset of REM sleep.
T
191
post hoc Dunnett tests. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., CA, USA). A p value less than 0.05 was considered statistically significant.
E
189 190
3
Fig. 1. Effect of the partial agonist Ro 10-5824 or the full agonist A-412997 on the duration of WAKE, NREM, and REM in a 3 h recording period in rats. Saline, Ro 10-5824 (A), or A-412997 (B) was administered subcutaneously 10 min before the start of recordings (light-on). Each bar represents the mean and SEM of seven rats, expressed as the time (min) spent in each state. *p b 0.05), **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
C
T
E
D
P
R O
O
F
4
244
E
Fig. 2. Effect of the partial agonist Ro 10-5824 or the full agonist A-412997 on the onset of NREM and REM sleep in rats. Saline, Ro 10-5824 (A), or A-412997 (B) was administered subcutaneously 10 min before the start of recordings (light-on). Each bar represents the mean and SEM of seven rats, expressed as the latency (min) to the first occurrence of each state. *p b 0.05, **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
262
4. Discussion
263 264
The present study shows that two structurally distinct dopamine D4 receptor agonists, Ro 10-5824 and A-412997, generally increased waking duration, and conversely, reduced duration of NREM sleep in rats. In addition, the onset of NREM was generally delayed, although the full agonist A-412997, but not the partial agonist Ro 10-5824, influenced the onset and duration of REM sleep. Furthermore, these effects were accompanied with an enhancement of EEG spectral power in the theta
254 255 256 257 258 259
265 266 267 268 269
R
O
252 253
C
250 251
N
248 249
U
246 247
R
260 261
comparison between the saline treatment and the highest dose of the two drugs (i.e., 10 mg/kg). As shown in Fig. 4A, the partial agonist Ro 10-5824 reduced TOTAL EEG spectral power in the delta and increased it in the gamma frequency bands during a 2 h period (F4,24 = 7.946, p = 0.0003). Analysis of EEG profiles in each state revealed that Ro 10-5824 significantly changed EEG power during NREM (F4,24 = 6.647, p = 0.0009), but not during WAKE (F4,24 = 0.5258, p = 0.7178) or REM (F4,24 = 0.9848, p = 0.4346) periods. The partial agonist Ro 10-5824 increased spectral power in the theta, alpha, beta, and gamma frequency bands during the NREM period (Fig. 4A). As shown in Fig. 4B, the full agonist A-412997 produced a greater change in EEG spectral power during a 2 h period (i.e., TOTAL) as well as during each state (i.e., WAKE, NREM, and REM) than Ro 10-5824. In particular, A-412997 enhanced theta and gamma powers during the NREM period (F4,24 = 6.969, p = 0.0007) as well as the WAKE period (F4,24 = 10.46, p b 0.0001). In summary, both Ro 10-5824 and A-412997 generally enhanced theta/gamma powers during the NREM period, but only the full agonist A-412997 induced changes in EEG spectral power during the WAKE and REM periods.
245
and gamma bands. Our results suggest that activation of D4R could in- 270 duce an enhancement of the waking states. This is the first study 271 assessing the effect of D4R agonists on sleep architecture in animals. 272 4.1. Role of D4R in the regulation of sleep–wake states
273
In this study, we found that two chemically distinct dopamine D4R agonists, Ro 10-5824 and A-412997, had a robust effect on arousal. The effect on waking and NREM sleep was clearly observed 1 h after administration of the drugs. This is consistent with the results of other animal studies (Murai et al., 2014a; Powell et al., 2003; Woolley et al., 2008) in which cognitive behavioral experiments were performed 1 h after systemic administration of Ro 10-5824 or A-412997. Both drugs also affected the onset of NREM sleep; however, each compound affected onset and duration of REM sleep differently. This may be due to the different agonist activities of the two compounds, since only A412997, a full agonist of the D4R (Table 1), showed a robust effect on REM sleep, while the partial agonist Ro 10-5824 did not. However, the reported agonist efficacy of Ro 10-5824 was measured using human recombinant systems (Newman-Tancredi et al., 2008; Powell et al., 2003), while efficacy of A-412997 was based on data from rat D4R (Moreland et al., 2005). Therefore, it will be necessary to determine the exact Emax value of the partial agonist Ro 10-5824 in rat D4R, although Ro 10-5824 is widely used as a partial agonist for in vitro and in vivo studies in rodents (González et al., 2012; Powell et al., 2003; Tejas-Juárez et al., 2014). In this study, a significant effect of Ro 10-5824 on sleep parameters was found at all doses tested. On the other hand, the full agonist A-
274
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
5
C
T
E
D
P
R O
O
F
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320
R
N C O
298 299
412997 needed a relatively high dose to produce a statistical significant effect (Figs. 1 and 2). Although we did not compare exposure levels of the study drugs, we assumed that differences in pharmacokinetic parameters such as penetration of the drug in the brain might contribute to the difference in effective doses needed between the two drugs. To verify this, it will be necessary to compare the binding of these two drugs to D4Rs in the brain using specific radiotracers. Unfortunately, there are no agonist radiotracers available for D4Rs, although some promising ligands have been described (Kügler et al., 2011; Leopoldo et al., 2014). Alternatively, we examined the effects of Ro 10-5824 (1, 10 mg/kg) or A-412997 (3, 10 mg/kg) on c-fos mRNA expression levels in the frontal cortex and hippocampus of rats using quantitative RT-PCR as an index of change in neural activity. We found that the two agonists, at the dose that induced arousal, changed c-fos mRNA expression levels in the frontal cortex, suggesting that these agonists at these concentrations activate D4Rs (data not shown). Along with the behavioral parameters mentioned above, we showed the effect of Ro 10-5824 and A-412997 on EEG profiles (Fig. 4). We found an enhancement of the theta/gamma powers during the NREM period with both Ro 10-5824 and A-412997. In addition, only the full agonist A-412997, but not the partial agonist Ro 10-5824, increased the theta/gamma powers during the WAKE period. An enhancement of the theta/gamma powers has been previously reported for wakepromoting agents such as metabotropic glutamate receptor (mGluR2) antagonists or negative allosteric modulators, in rats (Ahnaou et al.,
U
296 297
R
E
Fig. 3. Temporal changes in the duration of WAKE, NREM, and REM in each 1 h block, following subcutaneous administration of saline, Ro 10-5824 (A), or A-412997 (B). Each bar represents the mean and SEM of seven rats, expressed as the time (min) spent in each state. *p b 0.05, **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
2014). Thus, the effect on arousal of D4R agonists that we showed in this study is supported by similar EEG patterns observed for other wake-promoting agents. Interestingly, the full agonist A-412997 produced a broader change in EEG spectral powers than the partial agonist Ro 10-5824. Such a differential effect may account for its different influence on REM sleep. Although this is the first report on the effect of two different D4R agonists on sleep architecture in rats, Cavas and Navarro (2006) have previously reported the effect of a single D4R antagonist on sleep– wake states in rats. In that study, they tested three different i.p. doses of the antagonist (L-741,741), and found a complex effect of this drug on sleep architecture. At a low dose (1.5 mg/kg), they did not find any change in sleep parameters for the entire 3 h recording period following drug injection. A middle dose (3 mg/kg) of L-741,741 significantly reduced total time spent in the active wake state. On the other hand, a higher dose (6 mg/kg) significantly increased total time spent in the active wake state, and reduced total sleep time. REM sleep was not influenced at any of the doses tested. Thus, their findings suggested a biphasic effect of L-741,741 on the duration of sleep and wake states. Our data using D4R agonists showed an opposite action to the intermediate dose of the antagonist L-741,741; therefore, the findings of the two studies have no conflicts in terms of influence on wake time. However, the arousal effect of D4R agonists that we show in our study is inconsistent with the effect of a relatively high dose of the antagonist L-741,741.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
P
R O
O
F
6
357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379
C
E
R
355 356
R
353 354
O
351 352
C
349 350
N
348
We propose three possible explanations for the discrepancy between the effect of the D4R agonists reported here, and those reported by Cavas and Navarro (2006) for a relatively high dose of the antagonist L-741,741. First, it is possible that at higher dose, the antagonist L-741,741 may act as a partial agonist on D4Rs. It is known that some D4R antagonists, for example, the well-known D4R antagonist L745,870, often have weak partial agonist activity in vitro (Gazi et al., 1998, 2000) as well as in vivo (Zawilska et al., 2003). It has been reported that this dual mode of action of D4R antagonists is related to the conditions of the in vitro assay, particularly receptor density (Gazi et al., 1999). Therefore, it is possible that at an intermediate dose, L-741,741 could act as antagonist, but at a higher concentration, it may produce the activation of D4Rs. A second possibility is that the biphasic effect of the D4R antagonist resulted from its action on receptors located pre and postsynaptically, as it has been reported for D2Rs (Monti et al., 1988, 1989). We found a robust dose-dependent effect of two structurally different D4R agonists; however, it is unknown whether D4Rs are located preferentially pre or postsynaptically (Azdad et al., 2003; Gasca-Martinez et al., 2010; Govindaiah et al., 2010). For these reasons, it will be worth testing how the local application of D4R agonists/antagonists in dopaminergic brain areas such as the ventral tegmental area, thalamus, and PFC, could influence sleep architecture in animals. Finally, a third possibility is that methodological differences between the two studies were the most important factor producing the discrepancy. Among these methodological differences, they injected the drug 1 h after lights were on, while we injected the drugs just before lights were on, in a similar manner to the polysomnography studies in humans (Murck et al., 2001; Paterson et al., 2007). In addition, we performed our experiments in a crossover, within-subject comparison manner with 1 week washout periods, which is a similar design to human polysomnography studies, while Cavas and Navarro (2006) used different animals for different doses of L-741,741. All of these methodological differences might have a severe impact on the variability of the sleep parameters to be assessed.
U
346 347
T
E
D
Fig. 4. Changes in spectral power density of EEGs in the delta (0.75–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta (12–30 Hz), and gamma (30–80 Hz) frequency bands over a 2 h period following subcutaneous administration of saline, Ro 10-5824 (A), or A-412997 (B). Spectral power densities in each frequency band in the entire 2 h period (i.e., TOTAL), and during each state (WAKE, NREM, and REM) are expressed as percentage of changes over the saline control (i.e., within-animal comparison). Each bar represents the mean and SEM of seven rats. *p b 0.05, **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
Further studies (e.g., in D4R-KO mice) will be needed to verify the effect of the blockade of D4Rs in the regulation of sleep. Nevertheless, this is the first study showing a robust arousal effect of D4R agonists on sleep architecture in rats.
380 381 382 383
4.2. Possible mechanisms for the wake-promoting effect of the activation of 384 D4Rs 385 The present study showed the wake-promoting effect of the activation of D4Rs after a systemic administration of two different D4R agonists. The distribution of D4Rs has been studied by immunohistochemistry in the rat brain, and D4R-positive cells were widely found in sleep–regulating regions such as the thalamus, PFC, hypothalamus, and striatum (Khan et al., 1998). All these brain areas could be involved in the findings of the present study. Currently, the exact mechanisms for the wake-promoting effects of the activation of D4Rs are unknown. However, our findings suggest that D4Rs, at least in part, might contribute to the arousal effects of DA-releasing agents such as methylphenidate. It is known that D4Rs are involved in the actions of methylphenidate in vitro (Florán et al., 2004), and in vivo (Erlij et al., 2012; Michaelides et al., 2010). Florán et al. (2004) found that activation of D4Rs with the agonist PD 168,077 modulated the release of GABA induced by depolarization. This is similar to the effect observed for methylphenidate in slices of the thalamic reticular nucleus (TRN), where D4Rs are highly expressed in rodents and monkeys (Khan et al., 1998; Mrzljak et al., 1996). The neurons of the TRN are mainly inhibitory GABAergic interneurons that send their projections to the relay nuclei of the thalamus, playing an important role in the regulation of processes such as attention and sleep (Pinault, 2004; Steriade, 2005). Importantly, the action of methylphenidate in the TRN was blocked by a D4R antagonist, supporting the possible involvement of D4Rs in the arousal effect of methylphenidate in this brain area.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
445
Conflict of interest
446 447
All authors are employees of Sumitomo Dainippon Pharma, Co., Ltd. This study was funded by Sumitomo Dainippon Pharma.
448
Contributors
449
452
S.N. and K.N. designed the study. S.N., K.N., and H.I. conducted the experiments. S.N. and K.N. analyzed the data. S.N. and J.I. co-wrote the draft of the manuscript, and all authors reviewed and approved the final manuscript.
453
References
454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472
Ahnaou A, Ver Donck L, Drinkenburg WH. Blockade of the metabotropic glutamate (mGluR2) modulates arousal through vigilance states transitions: evidence from sleep–wake EEG in rodents. Behav Brain Res 2014;270:56–67. http://dx.doi.org/10. 1016/j.bbr.2014.05.003. Azdad K, Piet R, Poulain DA, Oliet SH. Dopamine D4 receptor-mediated presynaptic inhibition of GABAergic transmission in the rat supraoptic nucleus. J Neurophysiol 2003; 90(2):559–65. Baskerville TA, Allard J, Wayman C, Douglas AJ. Dopamine-oxytocin interactions in penile erection. Eur J Neurosci 2009;30(11):2151–64. http://dx.doi.org/10.1111/j.14609568.2009.06999.x. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev 2011;63(1):182–217. http://dx.doi.org/10.1124/pr.110. 002642. Berridge CW, Devilbiss DM, Andrzejewski ME, Arnsten AF, Kelley AE, Schmeichel B, et al. Methylphenidate preferentially increases catecholamine neurotransmission within the prefrontal cortex at low doses that enhance cognitive function. Biol Psychiatry 2006;60(10):1111–20. Billiard M. Narcolepsy: current treatment options and future approaches. Neuropsychiatr Dis Treat 2008;4(3):557–66.
434 435
441 442
450 451
T
432 433
C
430 431
E
428 429
R
426 427
R
424 425
N C O
422 423
U
420 421
F
443 444
The results of the present study suggest the involvement of dopamine D4 receptors in the regulation of sleep–wake states. The activation of these receptors could enhance the arousal state as confirmed by the behavioral and electrophysiological patterns shown in this study. Dopamine D4 receptors may contribute to the arousal effect of dopaminereleasing agents such as methylphenidate in humans.
418 419
O
439 440
417
R O
5. Conclusion
415 416
P
438
413 414
Browman KE, Curzon P, Pan JB, Molesky AL, Komater VA, Decker MW, et al. A-412997, a selective dopamine D4 agonist, improves cognitive performance in rats. Pharmacol Biochem Behav 2005;82(1):148–55. Cavas M, Navarro JF. Effects of selective dopamine D4 receptor antagonist, L-741,741, on sleep and wakefulness in the rat. Prog Neuropsychopharmacol Biol Psychiatry 2006; 30(4):668–78. Erlij D, Acosta-García J, Rojas-Márquez M, González-Hernández B, Escartín-Perez E, Aceves J, et al. Dopamine D4 receptor stimulation in GABAergic projections of the globus pallidus to the reticular thalamic nucleus and the substantia nigra reticulata of the rat decreases locomotor activity. Neuropharmacology 2012;62(2):1111–8. http://dx.doi.org/10.1016/j.neuropharm.2011.11.001. Florán B, Florán L, Erlij D, Aceves J. Activation of dopamine D4 receptors modulates [3H]GABA release in slices of the rat thalamic reticular nucleus. Neuropharmacology 2004;46(4):497–503. Gasca-Martinez D, Hernandez A, Sierra A, Valdiosera R, Anaya-Martinez V, Floran B, et al. Dopamine inhibits GABA transmission from the globus pallidus to the thalamic reticular nucleus via presynaptic D4 receptors. Neuroscience 2010;169(4):1672–81. http://dx.doi.org/10.1016/j.neuroscience.2010.05.048. Gazi L, Bobirnac I, Danzeisen M, Schüpbach E, Bruinvels AT, Geisse S, et al. The agonist activities of the putative antipsychotic agents, L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor. Br J Pharmacol 1998;124(5):889–96. Gazi L, Bobirnac I, Danzeisen M, Schüpbach E, Langenegger D, Sommer B, et al. Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L745,870 and U-101958 at human recombinant D4.4 receptors expressed in CHO cells. Br J Pharmacol 1999;128(3):613–20. Gazi L, Schoeffter P, Nunn C, Croskery K, Hoyer D, Feuerbach D. Cloning, expression, functional coupling and pharmacological characterization of the rat dopamine D4 receptor. Naunyn Schmiedebergs Arch Pharmacol 2000;361(5):555–64. González S, Rangel-Barajas C, Peper M, Lorenzo R, Moreno E, Ciruela F, et al. Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain. Mol Psychiatry 2012;17(6):650–62. http://dx.doi.org/10.1038/mp.2011.93. Govindaiah G, Wang T, Gillette MU, Crandall SR, Cox CL. Regulation of inhibitory synapses by presynaptic D₄ dopamine receptors in thalamus. J Neurophysiol 2010;104(5): 2757–65. http://dx.doi.org/10.1152/jn.00361.2010. Gruner JA, Marcy VR, Lin YG, Bozyczko-Coyne D, Marino MJ, Gasior M. The roles of dopamine transport inhibition and dopamine release facilitation in wake enhancement and rebound hypersomnolence induced by dopaminergic agents. Sleep 2009; 32(11):1425–38. Hauser RA, Gauger L, Anderson WM, Zesiewicz TA. Pramipexole-induced somnolence and episodes of daytime sleep. Mov Disord 2000;15(4):658–63. Khan ZU, Gutiérrez A, Martín R, Peñafiel A, Rivera A, De La Calle A. Differential regional and cellular distribution of dopamine D2-like receptors: an immunocytochemical study of subtype-specific antibodies in rat and human brain. J Comp Neurol 1998; 402(3):353–71. Kügler F, Sihver W, Ermert J, Hübner H, Gmeiner P, Prante O, et al. Evaluation of 18 Flabeled benzodioxine piperazine-based dopamine D4 receptor ligands: lipophilicity as a determinate of nonspecific binding. J Med Chem 2011;54(24):8343–52. http:// dx.doi.org/10.1021/jm200762g. Lagos P, Scorza C, Monti JM, Jantos H, Reyes-Parada M, Silveira R, et al. Effects of the D3 preferring dopamine agonist pramipexole on sleep and waking, locomotor activity and striatal dopamine release in rats. Eur Neuropsychopharmacol 1998;8(2): 113–20. Leopoldo M, Selivanova SV, Müller A, Lacivita E, Schetz JA, Ametamey SM. In vitro and in vivo evaluation of N-{2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[(11) C]methoxybenz-amide, a positron emission tomography (PET) radioligand for dopamine D4 receptors, in rodents. Chem Biodivers 2014;11(9):1298–308. http://dx.doi. org/10.1002/cbdv.201400178. Li D, Sham PC, Owen MJ, He L. Meta-analysis shows significant association between dopamine system genes and attention deficit hyperactivity disorder (ADHD). Hum Mol Genet 2006;15(14):2276–84. Michaelides M, Pascau J, Gispert JD, Delis F, Grandy DK, Wang GJ, et al. Dopamine D4 receptors modulate brain metabolic activity in the prefrontal cortex and cerebellum at rest and in response to methylphenidate. Eur J Neurosci 2010;32(4):668–76. http:// dx.doi.org/10.1111/j.1460-9568.2010.07319.x. Monti JM, Jantos H. The roles of dopamine and serotonin, and of their receptors, in regulating sleep and waking. Prog Brain Res 2008;172:625–46. http://dx.doi.org/10.1016/ S0079-6123(08)00929-1. Monti JM, Monti D. The involvement of dopamine in the modulation of sleep and waking. Sleep Med Rev 2007;11(2):113–33. Monti JM, Hawkins M, Jantos H, D’Angelo L, Fernández M. Biphasic effects of dopamine D2 receptor agonists on sleep and wakefulness in the rat. Psychopharmacology (Berl) 1988;95(3):395–400. Monti JM, Jantos H, Fernández M. Effects of the selective dopamine D-2 receptor agonist, quinpirole on sleep and wakefulness in the rat. Eur J Pharmacol 1989;169(1):61–6. Monti JM, Fernández M, Jantos H. Sleep during acute dopamine D1 agonist SKF 38393 or D1 antagonist SCH 23390 administration in rats. Neuropsychopharmacology 1990; 3(3):153–62. Moreland RB, Patel M, Hsieh GC, Wetter JM, Marsh K, Brioni JD. A-412997 is a selective dopamine D4 receptor agonist in rats. Pharmacol Biochem Behav 2005;82(1):140–7. Mrzljak L, Bergson C, Pappy M, Huff R, Levenson R, Goldman-Rakic PS. Localization of dopamine D4 receptors in GABAergic neurons of the primate brain. Nature 1996; 381(6579):245–8. Murai T, Nakako T, Ikeda K, Ikejiri M, Ishiyama T, Taiji M. Lack of dopamine D4 receptor affinity contributes to the procognitive effect of lurasidone. Behav Brain Res 2014a; 261:26–30. http://dx.doi.org/10.1016/j.bbr.2013.11.036.
D
436 437
A study by Michaelides et al. (2010) revealed that in D4R-KO mice there was a modulation of brain metabolic responses induced by methylphenidate in the PFC, as measured with [18 F]2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG PET). In the PFC, D4Rs are highly expressed in both pyramidal and non-pyramidal neurons (Khan et al., 1998; Mrzljak et al., 1996) and are known to differentially modulate glutamatergic and GABAergic neurotransmission in vitro (Wang et al., 2002, 2003; Zhong and Yan, 2014). Therefore, it is possible that the PFC is another region involved in the regulation of sleep–wake states, through the activation of D4Rs. Overall, we could surmise that the promotion of wake produced by the systemic administration of D4R agonists may be mediated by the action of these agonists on multiple areas, such as the PFC and TRN, resulting in the modulation of the neuronal activity of the thalamocortical circuits as a whole. The effect of methylphenidate mentioned above has been studied and discussed in relation to the regulatory roles of DA and D4Rs in hyperactivity and attention (Erlij et al., 2012). This is because methylphenidate has been widely used for the treatment of attention deficit/hyperactive disorder (ADHD), and because of the strong association of polymorphisms in the DRD4 gene with ADHD (Li et al., 2006). However, whether similar mechanisms could be applied to sleep–wake states is currently unknown. Therefore, the role of D4Rs in the regulation of sleep–wake states should be elucidated by further research studies. In fact, in addition to the PFC, TRN, and thalamus, D4Rs are expressed in the hypothalamus and basal ganglia (Baskerville et al., 2009; Khan et al., 1998), which may contribute to the arousal effect of the activation of D4Rs.
E
411 412
7
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
O
F
Tejas-Juárez JG, Cruz-Martínez AM, López-Alonso VE, García-Iglesias B, Mancilla-Díaz JM, Florán-Garduño B, et al. Stimulation of dopamine D4 receptors in the paraventricular nucleus of the hypothalamus of male rats induces hyperphagia: involvement of glutamate. Physiol Behav 2014;133:272–81. http://dx.doi.org/10.1016/j.physbeh.2014. 04.040. Trampus M, Ferri N, Adami M, Ongini E. The dopamine D1 receptor agonists, A68930 and SKF 38393, induce arousal and suppress REM sleep in the rat. Eur J Pharmacol 1993; 235(1):83–7. Wang X, Zhong P, Yan Z. Dopamine D4 receptors modulate GABAergic signaling in pyramidal neurons of prefrontal cortex. J Neurosci 2002;22(21):9185–93. Wang X, Zhong P, Gu Z, Yan Z. Regulation of NMDA receptors by dopamine D4 signaling in prefrontal cortex. J Neurosci 2003;23(30):9852–61. Wisor JP, Nishino S, Sora I, Uhl GH, Mignot E, Edgar DM. Dopaminergic role in stimulantinduced wakefulness. J Neurosci 2001;21(5):1787–94. Woolley ML, Waters KA, Reavill C, Bull S, Lacroix LP, Martyn AJ, et al. Selective dopamine D4 receptor agonist (A-412997) improves cognitive performance and stimulates motor activity without influencing reward-related behaviour in rat. Behav Pharmacol 2008;19(8):765–76. http://dx.doi.org/10.1097/FBP.0b013e32831c3b06. Zawilska JB, Rosiak J, Berezińska M, Nowak JZ. L-745,870 suppresses the nighttime serotonin N-acetyltransferase activity in chick retina: in vivo evidence for agonist activity at D4-dopamine receptors. J Neural Transm 2003;110(3):219–27. Zhong P, Yan Z. Distinct physiological effects of dopamine D4 receptors on prefrontal cortical pyramidal neurons and fast-spiking interneurons. Cereb Cortex 2014. http://dx. doi.org/10.1093/cercor/bhu190.
N
C
O
R
R
E
C
T
E
D
P
Murai T, Nakamichi K, Shimizu I, Ikeda K. Lurasidone suppresses rapid eye movement sleep and improves sleep quality in rats. J Pharmacol Sci 2014b;126(2):164–7. Murck H, Frieboes RM, Antonijevic IA, Steiger A. Distinct temporal pattern of the effects of the combined serotonin-reuptake inhibitor and 5-HT1A agonist EMD 68843 on the sleep EEG in healthy men. Psychopharmacology (Berl) 2001;155(2):187–92. Newman-Tancredi A, Heusler P, Martel JC, Ormière AM, Leduc N, Cussac D. Agonist and antagonist properties of antipsychotics at human dopamine D4.4 receptors: Gprotein activation and K + channel modulation in transfected cells. Int J Neuropsychopharmacol 2008;11(3):293–307. Nieoullon A. Dopamine and the regulation of cognition and attention. Prog Neurobiol 2002;67(1):53–83. Ongini E, Bonizzoni E, Ferri N, Milani S, Trampus M. Differential effects of dopamine D-1 and D-2 receptor antagonist antipsychotics on sleep–wake patterns in the rat. J Pharmacol Exp Ther 1993;266(2):726–31. Paterson LM, Wilson SJ, Nutt DJ, Hutson PH, Ivarsson M. A translational, caffeine-induced model of onset insomnia in rats and healthy volunteers. Psychopharmacology (Berl) 2007;191(4):943–50. Pinault D. The thalamic reticular nucleus: structure, function and concept. Brain Res Brain Res Rev 2004;46(1):1–31. Powell SB, Paulus MP, Hartman DS, Godel T, Geyer MA. RO-10-5824 is a selective dopamine D4 receptor agonist that increases novel object exploration in C57 mice. Neuropharmacology 2003;44(4):473–81. Qu WM, Xu XH, Yan MM, Wang YQ, Urade Y, Huang ZL. Essential role of dopamine D2 receptor in the maintenance of wakefulness, but not in homeostatic regulation of sleep, in mice. J Neurosci 2010;30(12):4382–9. http://dx.doi.org/10.1523/JNEUROSCI.493609.2010. Steriade M. Sleep, epilepsy and thalamic reticular inhibitory neurons. Trends Neurosci 2005;28(6):317–24.
U
559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
R O
8
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
Effect of dopamine D4 receptor agonists on sleep architecture in rats
2Q2
Shunsuke Nakazawa ⁎, Keiko Nakamichi, Hideaki Imai, Junji Ichihara
3
Drug Development Research Laboratories, Sumitomo Dainippon Pharma, Co., Ltd., Osaka, Japan
4
a r t i c l e
5 6 7 8 9
Article history: Received 24 January 2015 Received in revised form 28 April 2015 Accepted 11 May 2015 Available online xxxx
10 Q3 11 12 13 14 15 16 17
Keywords: A-412997 EEG NREM REM Ro 10-5824 Sleep Wakefulness
O
a b s t r a c t
R O
i n f o
T
E
D
P
Dopamine plays a key role in the regulation of sleep–wake states, as revealed by the observation that dopaminereleasing agents such as methylphenidate have wake-promoting effects. However, the precise mechanisms for the wake-promoting effect produced by the enhancement of dopamine transmission are not fully understood. Although dopamine D1, D2, and D3 receptors are known to have differential effects on sleep architecture, the role of D4 receptors (D4Rs), and particularly the influence of D4R activation on the sleep–wake state, has not been studied so far. In this study, we investigated for the first time the effects of two structurally different D4R agonists, Ro 10-5824 and A-412997, on the sleep–wake states in rats. We found that both D4R agonists generally increased waking duration, and conversely, reduced non-rapid eye movement (NREM) sleep duration in rats. The onset of NREM sleep was also generally delayed. However, only the A-412997 agonist (but not the Ro 10-5824) influenced rapid eye movement sleep onset and duration. Furthermore, these effects were accompanied with an enhancement of EEG spectral power in the theta and the gamma bands. Our results suggest the involvement of dopamine D4R in the regulation of sleep–wake states. The activation of the D4R could enhance the arousal states as revealed by the behavioral and electrophysiological patterns in this study. Dopamine D4R may contribute to the arousal effects of dopamine-releasing agents such as methylphenidate. © 2015 Published by Elsevier Inc.
32
C
36 34 33 35
1. Introduction
38
It is well known that many brain functions such as cognition, attention, reward, emotion, and motor control are regulated by the neurotransmitter dopamine (DA) (Nieoullon, 2002). In addition, DA plays a key role in the regulation of sleep–wake states. For example, it is known that DA-releasing agents such as amphetamine have wakepromoting effects in rats (Berridge et al., 2006; Gruner et al., 2009), and a study on knock-out (KO) mice for the dopamine transporter (DAT) suggests that the inhibition of DAT is necessary for this action (Wisor et al., 2001). Moreover, methylphenidate (known as Ritalin®), an inhibitor of DAT, is widely used for the treatment of narcolepsy (Billiard, 2008), a condition in which patients suffer from abnormal sudden sleep attacks. Thus, it appears that an enhancement of DA transmission in the brain promotes waking and reduces sleep states across species. However, the specific mechanisms for these actions of DA are not fully understood.
45 46 47 48 49 50 51 52
R
R
N C O
43 44
U
41 42
E
37
39 40
F
1Q1
Abbreviations: DA, dopamine; DAT, dopamine transporter; D4R, dopamine D4 receptor; Ro 10-5824, 5-[(3,6-dihydro-4-phenyl-1 (2H)-pyridinyl) methyl]-2-methyl-4pyrimidinamine; A-412997, N-(3-Methylphenyl)-4-(2-pyridinyl)-1-piperidineacetamide; EEG, electroencephalogram; EMG, electromyogram; REM, rapid eye movement; NREM, non-rapid eye movement; PFC, prefrontal cortex; TRN, thalamic reticular nuclei. ⁎ Corresponding author at: Drug Development Research Laboratories, Sumitomo Dainippon Pharma, Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan. Tel.: +81 6 6337 5974; fax: +81 6 6337 5109. E-mail address: [email protected] (S. Nakazawa).
Five DA receptors have been characterized to date. These DA receptors are coupled to G-proteins and are classified in two distinct groups, D1-like and D2-like receptors, depending on the coupled G-protein (Beaulieu and Gainetdinov, 2011). D1-like receptors, which are coupled to Gαs, include D1 and D5 receptors, whereas the D2-like receptors, which are coupled to Gαi/o, include D2, D3, and D4 receptors. It is suggested that DA affects sleep–wake states by acting on DA receptors, and by its interaction with other neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA) (Monti and Monti, 2007). The role of D1 and D2 receptors on sleep–wake states has been intensively studied in rodents using genetic KO mice or selective pharmacological agents (Monti and Jantos, 2008). The systemic administration of the D1 receptor (D1R) agonists A68930 and SKF 38393 resulted in an increase in the waking time, and a decrease of rapid eye movement (REM) and non-REM (NREM) sleep time (Trampus et al., 1993). On the other hand, the administration of the D1R antagonist SCH 23390 increased NREM sleep time and decreased the waking and REM sleep time (Monti et al., 1990). D2 receptor (D2R) agonists (e.g. quinpirole) are known to have a complex effect on sleep–wake states in rats, such that low doses of the agonist reduce waking and increase sleep, while high doses have the opposite effect (Monti et al., 1988, 1989). However, Ongini et al. (1993) demonstrated that the relatively selective D2R antagonist raclopride did not have a significant effect on the sleep architecture in rats. Finally, Qu et al. (2010) reported a lower waking duration and higher NREM and REM sleep duration in D2R KO mice than in wild-type mice. Thus, the role of D2Rs on sleep–wake states is still under debate because of the lack of selective D2R agonists, but the
http://dx.doi.org/10.1016/j.pnpbp.2015.05.006 0278-5846/© 2015 Published by Elsevier Inc.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
18 19 20 21 22 23 24 25 26 27 28 29 30 31
53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
129 130
2.3. Analysis of the sleep architecture
139
The sleep stage analysis was conducted off-line using Sleepsign software (KISSEI COMTEC CO., LTD., Japan). The EEG signal was high-pass filtered with a 0.75 Hz filter, and the EMG signal was band-pass filtered between 20 and 50 Hz. The electrographic activity of 10-s epochs was analyzed. Each epoch was automatically registered as “WAKE,” “REM,” or “NREM” based on the waveforms of the EEG and the EMG. We used the same conditions than Murai et al. (2014b): We defined the conditions as “WAKE” when the EMG exceeded the individual threshold. They were defined as NREM when the power of the delta waves (0.75–4 Hz) exceeded the individual threshold, and did not present EMG activity. Finally, they were defined as REM when the power of the theta waves (4–8 Hz) exceeded 40% of the total power of 0.75–80 Hz waves, and there was no EMG response. The duration of WAKE, NREM, and REM and the latencies to the initial NREM and REM were calculated. The onset of NREM and REM sleep was defined as the first occurrence of 18 consecutive (3 min) epochs and 3 consecutive (30 sec) epochs, respectively. The EEG spectral power densities in each frequency band (0.75–4 Hz for delta; 4–8 Hz for theta; 8–12 Hz for alpha; 12–30 Hz for beta; 30–80 Hz for gamma) over the first 2 h post-administration were calculated with a fast Fourier transform (FFT). Then, we calculated changes in spectral power (%) by dividing the spectral powers obtained in the drug treatment condition by those obtained in the saline treatment condition in a within-animal comparison manner. To assess the specific influence of drugs on EEG profiles, changes in spectral power (%) were calculated without separating sleep–wake states (i.e., TOTAL), as well as for each state separately (i.e., WAKE, NREM, and REM).
140 141
2.4. Drugs and experimental design
167
Ro 10-5824 and A-412997 were purchased from Tocris Bioscience (Ellisville, MO, USA). Drugs were freshly dissolved in saline on the day of each experiment. Animals were administered subcutaneously saline, Ro 10-5824, or A-412997 at a volume of 2 mL/kg, 10 min before the light-on cycle (7 p.m.), followed by the continuous recording of EEG/EMGs for the entire light-on period. In the present study, we performed two different four-way crossover experiments, one comparing saline and Ro 10-5824 (1, 3, 10 mg/kg), and the other comparing saline and A-412997 (1, 3, 10 mg/kg). The order of the drug treatment varied pseudo-randomly, and at least 1 week was allowed between experiments for each animal. Drug doses were chosen based on previous in vivo studies (Murai et al., 2014a; Powell et al., 2003; Woolley et al., 2008).
168
Reference
2.5. Statistical analysis
181
Powell et al., 2003 Newman-Tancredi et al., 2008 Moreland et al., 2005
All data were expressed as the mean ± standard error of mean (SEM). The duration of WAKE, NREM, and REM in a total 3 h period, and latencies of NREM and REM sleep were compared statistically using a repeated measures one-way ANOVA, followed by post hoc Dunnett tests. Temporal changes in the duration of WAKE, NREM, and REM, or the power spectral densities in each frequency band were statistically compared using repeated measures two-way ANOVA followed by
182 183
110
2.1. Animals
111 112
117
Seven adult male Wistar rats (6 weeks old) were purchased from Charles River Laboratories, Japan. The animals were housed in an airconditioned room kept at a temperature of 20–26 °C, and a humidity of 40–70%, with a 12:12 h light/dark cycle (lights-on at 7 a.m.). Food and water were given ad libitum. The Institutional Animal Care and Use Committee at Sumitomo Dainippon Pharma, Co. Ltd. reviewed and approved all the experimental procedures for the use of animals.
118
2.2. Surgery and electroencephalogram recording
119
128
Sleep electroencephalogram (EEG) recordings were conducted as previously described (Murai et al., 2014b). Briefly, a radio transmitter (TL11M2-F40-EET; Data Science International, New Brighton, MN, USA) was implanted subcutaneously in the back of animals anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A pair of electrode wires was stereotaxically implanted in the skull in the following locations: one wire in the frontoparietal area (2 mm anterior to bregma and 2 mm left to midline), and the other wire in the parietal area (5 mm posterior to bregma and 2 mm right to midline). Two stainless-steel screws were placed as anchors. The EEG electrodes and
t1:1 t1:2
Table 1 Profiles of the two dopamine D4 receptor agonists used in this study.
105 106
113 114 115 116
120 121 122 123 124 125 126 127
T
103 104
C
101 102
E
99 100
R
97 98
R
95 96
O
93 94
C
91 92
N
89 90
U
87 88
t1:3
Drug
t1:4 t1:5
Binding affinity, Ki (nM)
Agonist activity EC50 (nM) a
Emax (%) a
t1:6 t1:7
Ro 10-5824
5.2 40.7
205 81.3a
36.3 19.0a
t1:8
A-412997
7.9
28.4b
83.0b
t1:9 t1:10
a
O
2. Materials and methods
86
R O
109
84 85
E
107 108
82 83
F
screws were fixed using dental cement. Electromyograms (EMG) were recorded from the dorsal neck muscle using another pair of electrodes. Antibiotics and analgesics were applied after surgery. Animals were allowed at least 2 weeks of recovery in individual plastic cages before EEG/EMG recordings. The light/dark cycle was changed during this period (lights-on: 7:00 p.m–7:00 a.m.). EEG/EMGs were recorded in the home cages in a soundproof box using Dataquest A.R.T. software (Data Science International, New Brighton, MN, USA), at a sampling rate of 500 Hz. Prior to the experiments, animals were recorded once to ensure the validity of the EEG/EMG recordings.
complex actions of the D2Rs are thought to depend on their presynaptic or postsynaptic location (Monti and Monti, 2007). On the other hand, it has been suggested that the activation of D3 receptors (D3Rs) induces somnolence, since pramipexole, which is a D3R-preferring agonist, induces sleep in both rodents and humans (Hauser et al., 2000; Lagos et al., 1998). Taken together, these reports suggest that the wake-promoting effect of dopamine could be mediated by its action on different dopamine receptors. However, little is known about the role of D4 receptors (D4Rs) in the regulation of sleep–wake states. D4Rs are widely expressed in the brain, including prefrontal cortex (PFC), striatum, thalamus, and hypothalamus (Khan et al., 1998), all of which are involved in the regulation of sleep–wake states. It has been reported that the D4R antagonist L-741,741 induces a complex effect on the duration of sleep and wake states in rats (Cavas and Navarro, 2006). On the other hand, the influence of D4R agonists on the sleep architecture has not been studied so far. To clarify the role of the activation of D4R in the regulation of sleep–wake states, in the present study we investigated the effect of two structurally different D4R agonists, Ro 10-5824 and A412997, on the sleep architecture in rats. Ro 10-5824 is a selective partial agonist of D4R (Table 1) that has a high binding affinity (Ki = 5.2nM) and presents a 250-fold selectivity for D4R over D3R, and more than 1,000-fold selectivity over D1, D2, and D5 receptors (Powell et al., 2003). A-412997 is a selective full agonist (Table 1) of D4R that has a high binding affinity (Ki = 7.9nM), with negligible binding affinities for other dopamine receptor subtypes (Moreland et al., 2005). Both compounds have been widely used for the assessment of D4Rs in vivo (Browman et al., 2005; González et al., 2012; Murai et al., 2014a; Powell et al., 2003; Woolley et al., 2008).
P
80 81
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
D
2
Agonist activity was measured in human D4.4R using 35S-GTPγS binding assays. b Agonist activity was measured in rat D4R using Ca2+ flux assays.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
131 132 133 134 135 136 137 138
142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
169 170 171 172 173 174 175 176 177 178 179 180
184 185 186 187 188
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
We first examined the influence of the agonists Ro 10-5824 and A412997 on sleep architecture during the 3 h recoding period. As shown in Fig. 1A, Ro 10-5824 significantly increased the duration of WAKE (F3,18 = 11.38, p = 0.0002) and reduced the duration of NREM (F3,18 = 16.68, p b 0.0001). On the other hand, the duration of REM was not affected (F3,18 = 0.4159, p = 0.7437). The post hoc Dunnett test showed that administration of Ro 10-5824 resulted in a significant increase in the duration of WAKE (p b 0.01) and a decrease in the duration of NREM (p b 0.01), at all doses tested (Fig. 1A). Accordingly, as shown in Fig. 2A, the partial agonist Ro 10-5824 significantly delayed the onset of NREM sleep (F3,18 = 5.816, p = 0.0058), but not the onset of REM sleep (F3,18 = 0.02361, p = 0.9949). Administration of the full agonist A-412997 also resulted in wake promotion, as shown in Fig. 1B and Fig. 2B. This agonist significantly increased the duration of WAKE (F3,18 = 7.413, p = 0.0020) and reduced the duration of NREM (F3,18 = 4.191, p = 0.0205) as well as that of REM (F3,18 = 11.43, p = 0.0002). The increase in the duration of WAKE and the decrease in the duration of both NREM and REM reached statistical significance (post hoc tests, p b 0.01) only at a dose of 10 mg/kg of A412997. Sleep onset for both NREM (F3,18 = 29.33, p b 0.0001) and REM (F3,18 = 53.13, p b 0.0001) were delayed by the administration of A-412997 (Fig. 2B). Sleep latency of NREM was significantly increased
208 209 210 211 212 213 214 215
3.2. Effects of D4R agonists on the spectral power density of EEGs
240
218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239
To characterize further the arousal effects of the agonists Ro 10-5824 241 and A-412997, their influence on the spectral power density of EEG was 242 examined. For easier visualization, in Fig. 4 we show only the 243
C
206 207
E
204 205
R
202 203
R
200 201
N C O
198 199
U
196 197
F
194 195
O
3.1. Effects of Ro 10-5824 and A-412997 on sleep architecture in rats
R O
193
P
3. Results
216 217
D
192
at a dose higher than 3 mg/kg (p b 0.05) of A-412997, while the increase in sleep latency of REM reached statistical significance only at a dose of 10 mg/kg (p b 0.001). Thus, both Ro 10-5824 and A-412997 showed wake-promoting effects in rats, but these two drugs affected REM sleep in a different manner. To assess further the effect of these two drugs, changes in time spent in each of WAKE, NREM, and REM states in every 1 h block were analyzed and are shown in Fig. 3. A significant increase in time spent in WAKE and a decrease in time spent in NREM were clearly seen 1 h after the administration of Ro 10-5824, at all doses tested (p b 0.05), although delayed effects at 3 mg/kg were only found in the 2–3 h period (Fig. 3A). In addition, reduction of the duration of NREM by the administration of Ro 10-5824 at 3 and 10 mg/kg reached a trend, but not significance in the 1–2 h period (p = 0.0609 and p = 0.0698, respectively). No significant effect on time spent in REM was found at any doses or times. The administration of A-412997 at 10 mg/kg caused a significant increase in time spent in WAKE (p b 0.001), and a decrease in time spent in NREM (p b 0.001) within the first hour, although the effects on REM (p b 0.001) were found at 2–3 h (Fig. 3B). Taken together, the administration of both Ro 10-5824 and A412997 resulted in an enhancement of waking states within the first hour. The partial agonist Ro 10-5824 did not influence REM sleep, while the full agonist A-412997 caused a reduction in REM sleep at 2–3 h, and a delay on the onset of REM sleep.
T
191
post hoc Dunnett tests. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., CA, USA). A p value less than 0.05 was considered statistically significant.
E
189 190
3
Fig. 1. Effect of the partial agonist Ro 10-5824 or the full agonist A-412997 on the duration of WAKE, NREM, and REM in a 3 h recording period in rats. Saline, Ro 10-5824 (A), or A-412997 (B) was administered subcutaneously 10 min before the start of recordings (light-on). Each bar represents the mean and SEM of seven rats, expressed as the time (min) spent in each state. *p b 0.05), **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
C
T
E
D
P
R O
O
F
4
244
E
Fig. 2. Effect of the partial agonist Ro 10-5824 or the full agonist A-412997 on the onset of NREM and REM sleep in rats. Saline, Ro 10-5824 (A), or A-412997 (B) was administered subcutaneously 10 min before the start of recordings (light-on). Each bar represents the mean and SEM of seven rats, expressed as the latency (min) to the first occurrence of each state. *p b 0.05, **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
262
4. Discussion
263 264
The present study shows that two structurally distinct dopamine D4 receptor agonists, Ro 10-5824 and A-412997, generally increased waking duration, and conversely, reduced duration of NREM sleep in rats. In addition, the onset of NREM was generally delayed, although the full agonist A-412997, but not the partial agonist Ro 10-5824, influenced the onset and duration of REM sleep. Furthermore, these effects were accompanied with an enhancement of EEG spectral power in the theta
254 255 256 257 258 259
265 266 267 268 269
R
O
252 253
C
250 251
N
248 249
U
246 247
R
260 261
comparison between the saline treatment and the highest dose of the two drugs (i.e., 10 mg/kg). As shown in Fig. 4A, the partial agonist Ro 10-5824 reduced TOTAL EEG spectral power in the delta and increased it in the gamma frequency bands during a 2 h period (F4,24 = 7.946, p = 0.0003). Analysis of EEG profiles in each state revealed that Ro 10-5824 significantly changed EEG power during NREM (F4,24 = 6.647, p = 0.0009), but not during WAKE (F4,24 = 0.5258, p = 0.7178) or REM (F4,24 = 0.9848, p = 0.4346) periods. The partial agonist Ro 10-5824 increased spectral power in the theta, alpha, beta, and gamma frequency bands during the NREM period (Fig. 4A). As shown in Fig. 4B, the full agonist A-412997 produced a greater change in EEG spectral power during a 2 h period (i.e., TOTAL) as well as during each state (i.e., WAKE, NREM, and REM) than Ro 10-5824. In particular, A-412997 enhanced theta and gamma powers during the NREM period (F4,24 = 6.969, p = 0.0007) as well as the WAKE period (F4,24 = 10.46, p b 0.0001). In summary, both Ro 10-5824 and A-412997 generally enhanced theta/gamma powers during the NREM period, but only the full agonist A-412997 induced changes in EEG spectral power during the WAKE and REM periods.
245
and gamma bands. Our results suggest that activation of D4R could in- 270 duce an enhancement of the waking states. This is the first study 271 assessing the effect of D4R agonists on sleep architecture in animals. 272 4.1. Role of D4R in the regulation of sleep–wake states
273
In this study, we found that two chemically distinct dopamine D4R agonists, Ro 10-5824 and A-412997, had a robust effect on arousal. The effect on waking and NREM sleep was clearly observed 1 h after administration of the drugs. This is consistent with the results of other animal studies (Murai et al., 2014a; Powell et al., 2003; Woolley et al., 2008) in which cognitive behavioral experiments were performed 1 h after systemic administration of Ro 10-5824 or A-412997. Both drugs also affected the onset of NREM sleep; however, each compound affected onset and duration of REM sleep differently. This may be due to the different agonist activities of the two compounds, since only A412997, a full agonist of the D4R (Table 1), showed a robust effect on REM sleep, while the partial agonist Ro 10-5824 did not. However, the reported agonist efficacy of Ro 10-5824 was measured using human recombinant systems (Newman-Tancredi et al., 2008; Powell et al., 2003), while efficacy of A-412997 was based on data from rat D4R (Moreland et al., 2005). Therefore, it will be necessary to determine the exact Emax value of the partial agonist Ro 10-5824 in rat D4R, although Ro 10-5824 is widely used as a partial agonist for in vitro and in vivo studies in rodents (González et al., 2012; Powell et al., 2003; Tejas-Juárez et al., 2014). In this study, a significant effect of Ro 10-5824 on sleep parameters was found at all doses tested. On the other hand, the full agonist A-
274
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
5
C
T
E
D
P
R O
O
F
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320
R
N C O
298 299
412997 needed a relatively high dose to produce a statistical significant effect (Figs. 1 and 2). Although we did not compare exposure levels of the study drugs, we assumed that differences in pharmacokinetic parameters such as penetration of the drug in the brain might contribute to the difference in effective doses needed between the two drugs. To verify this, it will be necessary to compare the binding of these two drugs to D4Rs in the brain using specific radiotracers. Unfortunately, there are no agonist radiotracers available for D4Rs, although some promising ligands have been described (Kügler et al., 2011; Leopoldo et al., 2014). Alternatively, we examined the effects of Ro 10-5824 (1, 10 mg/kg) or A-412997 (3, 10 mg/kg) on c-fos mRNA expression levels in the frontal cortex and hippocampus of rats using quantitative RT-PCR as an index of change in neural activity. We found that the two agonists, at the dose that induced arousal, changed c-fos mRNA expression levels in the frontal cortex, suggesting that these agonists at these concentrations activate D4Rs (data not shown). Along with the behavioral parameters mentioned above, we showed the effect of Ro 10-5824 and A-412997 on EEG profiles (Fig. 4). We found an enhancement of the theta/gamma powers during the NREM period with both Ro 10-5824 and A-412997. In addition, only the full agonist A-412997, but not the partial agonist Ro 10-5824, increased the theta/gamma powers during the WAKE period. An enhancement of the theta/gamma powers has been previously reported for wakepromoting agents such as metabotropic glutamate receptor (mGluR2) antagonists or negative allosteric modulators, in rats (Ahnaou et al.,
U
296 297
R
E
Fig. 3. Temporal changes in the duration of WAKE, NREM, and REM in each 1 h block, following subcutaneous administration of saline, Ro 10-5824 (A), or A-412997 (B). Each bar represents the mean and SEM of seven rats, expressed as the time (min) spent in each state. *p b 0.05, **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
2014). Thus, the effect on arousal of D4R agonists that we showed in this study is supported by similar EEG patterns observed for other wake-promoting agents. Interestingly, the full agonist A-412997 produced a broader change in EEG spectral powers than the partial agonist Ro 10-5824. Such a differential effect may account for its different influence on REM sleep. Although this is the first report on the effect of two different D4R agonists on sleep architecture in rats, Cavas and Navarro (2006) have previously reported the effect of a single D4R antagonist on sleep– wake states in rats. In that study, they tested three different i.p. doses of the antagonist (L-741,741), and found a complex effect of this drug on sleep architecture. At a low dose (1.5 mg/kg), they did not find any change in sleep parameters for the entire 3 h recording period following drug injection. A middle dose (3 mg/kg) of L-741,741 significantly reduced total time spent in the active wake state. On the other hand, a higher dose (6 mg/kg) significantly increased total time spent in the active wake state, and reduced total sleep time. REM sleep was not influenced at any of the doses tested. Thus, their findings suggested a biphasic effect of L-741,741 on the duration of sleep and wake states. Our data using D4R agonists showed an opposite action to the intermediate dose of the antagonist L-741,741; therefore, the findings of the two studies have no conflicts in terms of influence on wake time. However, the arousal effect of D4R agonists that we show in our study is inconsistent with the effect of a relatively high dose of the antagonist L-741,741.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
P
R O
O
F
6
357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379
C
E
R
355 356
R
353 354
O
351 352
C
349 350
N
348
We propose three possible explanations for the discrepancy between the effect of the D4R agonists reported here, and those reported by Cavas and Navarro (2006) for a relatively high dose of the antagonist L-741,741. First, it is possible that at higher dose, the antagonist L-741,741 may act as a partial agonist on D4Rs. It is known that some D4R antagonists, for example, the well-known D4R antagonist L745,870, often have weak partial agonist activity in vitro (Gazi et al., 1998, 2000) as well as in vivo (Zawilska et al., 2003). It has been reported that this dual mode of action of D4R antagonists is related to the conditions of the in vitro assay, particularly receptor density (Gazi et al., 1999). Therefore, it is possible that at an intermediate dose, L-741,741 could act as antagonist, but at a higher concentration, it may produce the activation of D4Rs. A second possibility is that the biphasic effect of the D4R antagonist resulted from its action on receptors located pre and postsynaptically, as it has been reported for D2Rs (Monti et al., 1988, 1989). We found a robust dose-dependent effect of two structurally different D4R agonists; however, it is unknown whether D4Rs are located preferentially pre or postsynaptically (Azdad et al., 2003; Gasca-Martinez et al., 2010; Govindaiah et al., 2010). For these reasons, it will be worth testing how the local application of D4R agonists/antagonists in dopaminergic brain areas such as the ventral tegmental area, thalamus, and PFC, could influence sleep architecture in animals. Finally, a third possibility is that methodological differences between the two studies were the most important factor producing the discrepancy. Among these methodological differences, they injected the drug 1 h after lights were on, while we injected the drugs just before lights were on, in a similar manner to the polysomnography studies in humans (Murck et al., 2001; Paterson et al., 2007). In addition, we performed our experiments in a crossover, within-subject comparison manner with 1 week washout periods, which is a similar design to human polysomnography studies, while Cavas and Navarro (2006) used different animals for different doses of L-741,741. All of these methodological differences might have a severe impact on the variability of the sleep parameters to be assessed.
U
346 347
T
E
D
Fig. 4. Changes in spectral power density of EEGs in the delta (0.75–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta (12–30 Hz), and gamma (30–80 Hz) frequency bands over a 2 h period following subcutaneous administration of saline, Ro 10-5824 (A), or A-412997 (B). Spectral power densities in each frequency band in the entire 2 h period (i.e., TOTAL), and during each state (WAKE, NREM, and REM) are expressed as percentage of changes over the saline control (i.e., within-animal comparison). Each bar represents the mean and SEM of seven rats. *p b 0.05, **p b 0.01, and ***p b 0.001 indicate the presence of statistical significant differences between saline- and drug-treated groups.
Further studies (e.g., in D4R-KO mice) will be needed to verify the effect of the blockade of D4Rs in the regulation of sleep. Nevertheless, this is the first study showing a robust arousal effect of D4R agonists on sleep architecture in rats.
380 381 382 383
4.2. Possible mechanisms for the wake-promoting effect of the activation of 384 D4Rs 385 The present study showed the wake-promoting effect of the activation of D4Rs after a systemic administration of two different D4R agonists. The distribution of D4Rs has been studied by immunohistochemistry in the rat brain, and D4R-positive cells were widely found in sleep–regulating regions such as the thalamus, PFC, hypothalamus, and striatum (Khan et al., 1998). All these brain areas could be involved in the findings of the present study. Currently, the exact mechanisms for the wake-promoting effects of the activation of D4Rs are unknown. However, our findings suggest that D4Rs, at least in part, might contribute to the arousal effects of DA-releasing agents such as methylphenidate. It is known that D4Rs are involved in the actions of methylphenidate in vitro (Florán et al., 2004), and in vivo (Erlij et al., 2012; Michaelides et al., 2010). Florán et al. (2004) found that activation of D4Rs with the agonist PD 168,077 modulated the release of GABA induced by depolarization. This is similar to the effect observed for methylphenidate in slices of the thalamic reticular nucleus (TRN), where D4Rs are highly expressed in rodents and monkeys (Khan et al., 1998; Mrzljak et al., 1996). The neurons of the TRN are mainly inhibitory GABAergic interneurons that send their projections to the relay nuclei of the thalamus, playing an important role in the regulation of processes such as attention and sleep (Pinault, 2004; Steriade, 2005). Importantly, the action of methylphenidate in the TRN was blocked by a D4R antagonist, supporting the possible involvement of D4Rs in the arousal effect of methylphenidate in this brain area.
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
445
Conflict of interest
446 447
All authors are employees of Sumitomo Dainippon Pharma, Co., Ltd. This study was funded by Sumitomo Dainippon Pharma.
448
Contributors
449
452
S.N. and K.N. designed the study. S.N., K.N., and H.I. conducted the experiments. S.N. and K.N. analyzed the data. S.N. and J.I. co-wrote the draft of the manuscript, and all authors reviewed and approved the final manuscript.
453
References
454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472
Ahnaou A, Ver Donck L, Drinkenburg WH. Blockade of the metabotropic glutamate (mGluR2) modulates arousal through vigilance states transitions: evidence from sleep–wake EEG in rodents. Behav Brain Res 2014;270:56–67. http://dx.doi.org/10. 1016/j.bbr.2014.05.003. Azdad K, Piet R, Poulain DA, Oliet SH. Dopamine D4 receptor-mediated presynaptic inhibition of GABAergic transmission in the rat supraoptic nucleus. J Neurophysiol 2003; 90(2):559–65. Baskerville TA, Allard J, Wayman C, Douglas AJ. Dopamine-oxytocin interactions in penile erection. Eur J Neurosci 2009;30(11):2151–64. http://dx.doi.org/10.1111/j.14609568.2009.06999.x. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev 2011;63(1):182–217. http://dx.doi.org/10.1124/pr.110. 002642. Berridge CW, Devilbiss DM, Andrzejewski ME, Arnsten AF, Kelley AE, Schmeichel B, et al. Methylphenidate preferentially increases catecholamine neurotransmission within the prefrontal cortex at low doses that enhance cognitive function. Biol Psychiatry 2006;60(10):1111–20. Billiard M. Narcolepsy: current treatment options and future approaches. Neuropsychiatr Dis Treat 2008;4(3):557–66.
434 435
441 442
450 451
T
432 433
C
430 431
E
428 429
R
426 427
R
424 425
N C O
422 423
U
420 421
F
443 444
The results of the present study suggest the involvement of dopamine D4 receptors in the regulation of sleep–wake states. The activation of these receptors could enhance the arousal state as confirmed by the behavioral and electrophysiological patterns shown in this study. Dopamine D4 receptors may contribute to the arousal effect of dopaminereleasing agents such as methylphenidate in humans.
418 419
O
439 440
417
R O
5. Conclusion
415 416
P
438
413 414
Browman KE, Curzon P, Pan JB, Molesky AL, Komater VA, Decker MW, et al. A-412997, a selective dopamine D4 agonist, improves cognitive performance in rats. Pharmacol Biochem Behav 2005;82(1):148–55. Cavas M, Navarro JF. Effects of selective dopamine D4 receptor antagonist, L-741,741, on sleep and wakefulness in the rat. Prog Neuropsychopharmacol Biol Psychiatry 2006; 30(4):668–78. Erlij D, Acosta-García J, Rojas-Márquez M, González-Hernández B, Escartín-Perez E, Aceves J, et al. Dopamine D4 receptor stimulation in GABAergic projections of the globus pallidus to the reticular thalamic nucleus and the substantia nigra reticulata of the rat decreases locomotor activity. Neuropharmacology 2012;62(2):1111–8. http://dx.doi.org/10.1016/j.neuropharm.2011.11.001. Florán B, Florán L, Erlij D, Aceves J. Activation of dopamine D4 receptors modulates [3H]GABA release in slices of the rat thalamic reticular nucleus. Neuropharmacology 2004;46(4):497–503. Gasca-Martinez D, Hernandez A, Sierra A, Valdiosera R, Anaya-Martinez V, Floran B, et al. Dopamine inhibits GABA transmission from the globus pallidus to the thalamic reticular nucleus via presynaptic D4 receptors. Neuroscience 2010;169(4):1672–81. http://dx.doi.org/10.1016/j.neuroscience.2010.05.048. Gazi L, Bobirnac I, Danzeisen M, Schüpbach E, Bruinvels AT, Geisse S, et al. The agonist activities of the putative antipsychotic agents, L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor. Br J Pharmacol 1998;124(5):889–96. Gazi L, Bobirnac I, Danzeisen M, Schüpbach E, Langenegger D, Sommer B, et al. Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L745,870 and U-101958 at human recombinant D4.4 receptors expressed in CHO cells. Br J Pharmacol 1999;128(3):613–20. Gazi L, Schoeffter P, Nunn C, Croskery K, Hoyer D, Feuerbach D. Cloning, expression, functional coupling and pharmacological characterization of the rat dopamine D4 receptor. Naunyn Schmiedebergs Arch Pharmacol 2000;361(5):555–64. González S, Rangel-Barajas C, Peper M, Lorenzo R, Moreno E, Ciruela F, et al. Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain. Mol Psychiatry 2012;17(6):650–62. http://dx.doi.org/10.1038/mp.2011.93. Govindaiah G, Wang T, Gillette MU, Crandall SR, Cox CL. Regulation of inhibitory synapses by presynaptic D₄ dopamine receptors in thalamus. J Neurophysiol 2010;104(5): 2757–65. http://dx.doi.org/10.1152/jn.00361.2010. Gruner JA, Marcy VR, Lin YG, Bozyczko-Coyne D, Marino MJ, Gasior M. The roles of dopamine transport inhibition and dopamine release facilitation in wake enhancement and rebound hypersomnolence induced by dopaminergic agents. Sleep 2009; 32(11):1425–38. Hauser RA, Gauger L, Anderson WM, Zesiewicz TA. Pramipexole-induced somnolence and episodes of daytime sleep. Mov Disord 2000;15(4):658–63. Khan ZU, Gutiérrez A, Martín R, Peñafiel A, Rivera A, De La Calle A. Differential regional and cellular distribution of dopamine D2-like receptors: an immunocytochemical study of subtype-specific antibodies in rat and human brain. J Comp Neurol 1998; 402(3):353–71. Kügler F, Sihver W, Ermert J, Hübner H, Gmeiner P, Prante O, et al. Evaluation of 18 Flabeled benzodioxine piperazine-based dopamine D4 receptor ligands: lipophilicity as a determinate of nonspecific binding. J Med Chem 2011;54(24):8343–52. http:// dx.doi.org/10.1021/jm200762g. Lagos P, Scorza C, Monti JM, Jantos H, Reyes-Parada M, Silveira R, et al. Effects of the D3 preferring dopamine agonist pramipexole on sleep and waking, locomotor activity and striatal dopamine release in rats. Eur Neuropsychopharmacol 1998;8(2): 113–20. Leopoldo M, Selivanova SV, Müller A, Lacivita E, Schetz JA, Ametamey SM. In vitro and in vivo evaluation of N-{2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[(11) C]methoxybenz-amide, a positron emission tomography (PET) radioligand for dopamine D4 receptors, in rodents. Chem Biodivers 2014;11(9):1298–308. http://dx.doi. org/10.1002/cbdv.201400178. Li D, Sham PC, Owen MJ, He L. Meta-analysis shows significant association between dopamine system genes and attention deficit hyperactivity disorder (ADHD). Hum Mol Genet 2006;15(14):2276–84. Michaelides M, Pascau J, Gispert JD, Delis F, Grandy DK, Wang GJ, et al. Dopamine D4 receptors modulate brain metabolic activity in the prefrontal cortex and cerebellum at rest and in response to methylphenidate. Eur J Neurosci 2010;32(4):668–76. http:// dx.doi.org/10.1111/j.1460-9568.2010.07319.x. Monti JM, Jantos H. The roles of dopamine and serotonin, and of their receptors, in regulating sleep and waking. Prog Brain Res 2008;172:625–46. http://dx.doi.org/10.1016/ S0079-6123(08)00929-1. Monti JM, Monti D. The involvement of dopamine in the modulation of sleep and waking. Sleep Med Rev 2007;11(2):113–33. Monti JM, Hawkins M, Jantos H, D’Angelo L, Fernández M. Biphasic effects of dopamine D2 receptor agonists on sleep and wakefulness in the rat. Psychopharmacology (Berl) 1988;95(3):395–400. Monti JM, Jantos H, Fernández M. Effects of the selective dopamine D-2 receptor agonist, quinpirole on sleep and wakefulness in the rat. Eur J Pharmacol 1989;169(1):61–6. Monti JM, Fernández M, Jantos H. Sleep during acute dopamine D1 agonist SKF 38393 or D1 antagonist SCH 23390 administration in rats. Neuropsychopharmacology 1990; 3(3):153–62. Moreland RB, Patel M, Hsieh GC, Wetter JM, Marsh K, Brioni JD. A-412997 is a selective dopamine D4 receptor agonist in rats. Pharmacol Biochem Behav 2005;82(1):140–7. Mrzljak L, Bergson C, Pappy M, Huff R, Levenson R, Goldman-Rakic PS. Localization of dopamine D4 receptors in GABAergic neurons of the primate brain. Nature 1996; 381(6579):245–8. Murai T, Nakako T, Ikeda K, Ikejiri M, Ishiyama T, Taiji M. Lack of dopamine D4 receptor affinity contributes to the procognitive effect of lurasidone. Behav Brain Res 2014a; 261:26–30. http://dx.doi.org/10.1016/j.bbr.2013.11.036.
D
436 437
A study by Michaelides et al. (2010) revealed that in D4R-KO mice there was a modulation of brain metabolic responses induced by methylphenidate in the PFC, as measured with [18 F]2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG PET). In the PFC, D4Rs are highly expressed in both pyramidal and non-pyramidal neurons (Khan et al., 1998; Mrzljak et al., 1996) and are known to differentially modulate glutamatergic and GABAergic neurotransmission in vitro (Wang et al., 2002, 2003; Zhong and Yan, 2014). Therefore, it is possible that the PFC is another region involved in the regulation of sleep–wake states, through the activation of D4Rs. Overall, we could surmise that the promotion of wake produced by the systemic administration of D4R agonists may be mediated by the action of these agonists on multiple areas, such as the PFC and TRN, resulting in the modulation of the neuronal activity of the thalamocortical circuits as a whole. The effect of methylphenidate mentioned above has been studied and discussed in relation to the regulatory roles of DA and D4Rs in hyperactivity and attention (Erlij et al., 2012). This is because methylphenidate has been widely used for the treatment of attention deficit/hyperactive disorder (ADHD), and because of the strong association of polymorphisms in the DRD4 gene with ADHD (Li et al., 2006). However, whether similar mechanisms could be applied to sleep–wake states is currently unknown. Therefore, the role of D4Rs in the regulation of sleep–wake states should be elucidated by further research studies. In fact, in addition to the PFC, TRN, and thalamus, D4Rs are expressed in the hypothalamus and basal ganglia (Baskerville et al., 2009; Khan et al., 1998), which may contribute to the arousal effect of the activation of D4Rs.
E
411 412
7
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
O
F
Tejas-Juárez JG, Cruz-Martínez AM, López-Alonso VE, García-Iglesias B, Mancilla-Díaz JM, Florán-Garduño B, et al. Stimulation of dopamine D4 receptors in the paraventricular nucleus of the hypothalamus of male rats induces hyperphagia: involvement of glutamate. Physiol Behav 2014;133:272–81. http://dx.doi.org/10.1016/j.physbeh.2014. 04.040. Trampus M, Ferri N, Adami M, Ongini E. The dopamine D1 receptor agonists, A68930 and SKF 38393, induce arousal and suppress REM sleep in the rat. Eur J Pharmacol 1993; 235(1):83–7. Wang X, Zhong P, Yan Z. Dopamine D4 receptors modulate GABAergic signaling in pyramidal neurons of prefrontal cortex. J Neurosci 2002;22(21):9185–93. Wang X, Zhong P, Gu Z, Yan Z. Regulation of NMDA receptors by dopamine D4 signaling in prefrontal cortex. J Neurosci 2003;23(30):9852–61. Wisor JP, Nishino S, Sora I, Uhl GH, Mignot E, Edgar DM. Dopaminergic role in stimulantinduced wakefulness. J Neurosci 2001;21(5):1787–94. Woolley ML, Waters KA, Reavill C, Bull S, Lacroix LP, Martyn AJ, et al. Selective dopamine D4 receptor agonist (A-412997) improves cognitive performance and stimulates motor activity without influencing reward-related behaviour in rat. Behav Pharmacol 2008;19(8):765–76. http://dx.doi.org/10.1097/FBP.0b013e32831c3b06. Zawilska JB, Rosiak J, Berezińska M, Nowak JZ. L-745,870 suppresses the nighttime serotonin N-acetyltransferase activity in chick retina: in vivo evidence for agonist activity at D4-dopamine receptors. J Neural Transm 2003;110(3):219–27. Zhong P, Yan Z. Distinct physiological effects of dopamine D4 receptors on prefrontal cortical pyramidal neurons and fast-spiking interneurons. Cereb Cortex 2014. http://dx. doi.org/10.1093/cercor/bhu190.
N
C
O
R
R
E
C
T
E
D
P
Murai T, Nakamichi K, Shimizu I, Ikeda K. Lurasidone suppresses rapid eye movement sleep and improves sleep quality in rats. J Pharmacol Sci 2014b;126(2):164–7. Murck H, Frieboes RM, Antonijevic IA, Steiger A. Distinct temporal pattern of the effects of the combined serotonin-reuptake inhibitor and 5-HT1A agonist EMD 68843 on the sleep EEG in healthy men. Psychopharmacology (Berl) 2001;155(2):187–92. Newman-Tancredi A, Heusler P, Martel JC, Ormière AM, Leduc N, Cussac D. Agonist and antagonist properties of antipsychotics at human dopamine D4.4 receptors: Gprotein activation and K + channel modulation in transfected cells. Int J Neuropsychopharmacol 2008;11(3):293–307. Nieoullon A. Dopamine and the regulation of cognition and attention. Prog Neurobiol 2002;67(1):53–83. Ongini E, Bonizzoni E, Ferri N, Milani S, Trampus M. Differential effects of dopamine D-1 and D-2 receptor antagonist antipsychotics on sleep–wake patterns in the rat. J Pharmacol Exp Ther 1993;266(2):726–31. Paterson LM, Wilson SJ, Nutt DJ, Hutson PH, Ivarsson M. A translational, caffeine-induced model of onset insomnia in rats and healthy volunteers. Psychopharmacology (Berl) 2007;191(4):943–50. Pinault D. The thalamic reticular nucleus: structure, function and concept. Brain Res Brain Res Rev 2004;46(1):1–31. Powell SB, Paulus MP, Hartman DS, Godel T, Geyer MA. RO-10-5824 is a selective dopamine D4 receptor agonist that increases novel object exploration in C57 mice. Neuropharmacology 2003;44(4):473–81. Qu WM, Xu XH, Yan MM, Wang YQ, Urade Y, Huang ZL. Essential role of dopamine D2 receptor in the maintenance of wakefulness, but not in homeostatic regulation of sleep, in mice. J Neurosci 2010;30(12):4382–9. http://dx.doi.org/10.1523/JNEUROSCI.493609.2010. Steriade M. Sleep, epilepsy and thalamic reticular inhibitory neurons. Trends Neurosci 2005;28(6):317–24.
U
559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586
S. Nakazawa et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
R O
8
Please cite this article as: Nakazawa S, et al, Effect of dopamine D4 receptor agonists on sleep architecture in rats, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.006
587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610