Exp Brain Res (2014) 232:935–946 DOI 10.1007/s00221-013-3806-8
RESEARCH ARTICLE
Chronic escitalopram treatment caused dissociative adaptation in serotonin (5‑HT) 2C receptor antagonist‑induced effects in REM sleep, wake and theta wave activity Diána Kostyalik · Zita Kátai · Szilvia Vas · Dorottya Pap · Péter Petschner · Eszter Molnár · István Gyertyán · Lajos Kalmár · László Tóthfalusi · Gyorgy Bagdy
Received: 4 February 2013 / Accepted: 10 December 2013 / Published online: 7 January 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Several multi-target drugs used in treating psychiatric disorders, such as antidepressants (e.g. agomelatine, trazodone, nefazodone, amitriptyline, mirtazapine, mianserin, fluoxetine) or most atypical antipsychotics, have 5-hydroxytryptamine 2C (5-HT2C) receptor-blocking property. Adaptive changes in 5-HT2C receptor-mediated functions are suggested to contribute to therapeutic effects of selective serotonin reuptake inhibitor (SSRI) antidepressants after weeks of treatment, at least in part. Beyond the mediation of anxiety and other functions, 5-HT2C receptors are involved in sleep regulation. Anxiety-related adaptive changes caused by antidepressants have been studied extensively, although sleep- and electroencephalography (EEG)-related functional studies are still lacking. The aim of this study was to investigate the effects of chronic SSRI treatment on 5-HT2C receptor antagonist-induced functions in different vigilance stages and on quantitative EEG (Q-EEG) spectra. Rats were treated with a single dose of the selective 5-HT2C receptor antagonist SB-242084
(1 mg/kg, i.p.) or vehicle at the beginning of passive phase following a 20-day-long SSRI (escitalopram; 10 mg/ kg/day, osmotic minipump) or VEHICLE pretreatment. Fronto-parietal electroencephalogram, electromyogram and motility were recorded during the first 3 h of passive phase. We found that the chronic escitalopram pretreatment attenuated the SB-242084-caused suppression in rapid eye movement sleep (REMS). On the contrary, the 5-HT2C receptor antagonist-induced elevations in passive wake and theta (5–9 Hz) power density during active wake and REMS were not affected by the SSRI. In conclusion, attenuation in certain, but not all vigilance- and Q-EEG-related functions induced by the 5-HT2C receptor antagonist, suggests dissociation in 5-HT2C receptor adaptation. Keywords SB-242084 · REM sleep · Theta waves · Functional adaptation · Anxiety · Depression
Introduction D. Kostyalik · Z. Kátai · S. Vas · D. Pap · P. Petschner · E. Molnár · L. Tóthfalusi · G. Bagdy (*) Department of Pharmacodynamics, Semmelweis University, Budapest, Hungary e-mail:
[email protected] S. Vas · D. Pap · P. Petschner · G. Bagdy MTA-SE, Neuropsychopharmacology and Neurochemistry Research Group, Budapest, Hungary I. Gyertyán Department of Behavioural Pharmacology, Gedeon Richter Plc., Budapest, Hungary L. Kalmár Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
The 5-hydroxytryptamine 2C (5-HT2C) receptors are widely distributed in the central nervous system and involved in the regulation of anxiety, sleep, learning, locomotor activity, hormonal secretion and feeding behaviour (see in reviews: Bagdy 1998; Jensen et al. 2010). The 5-HT2C receptor antagonist property is present in certain antidepressant and atypical antipsychotic drugs, and it seemed to be involved in the therapeutic effects of these drugs. In addition, they could be potential targets in the treatment of sleep disorders, epilepsy, Parkinson’s disease and obesity (Di Giovanni et al. 2006; Bagdy et al. 2007; Jensen et al. 2010). It is well known that many depressed patients do not respond to conventional antidepressants adequately.
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Therefore, multi-target drugs and augmentation strategies have been developed for the management of resistant patients. The 5-HT2C receptors represent promising targets for treating depression or enhancing the activity of serotonin reuptake inhibitor (SSRI) antidepressants. There are some extensively used antidepressants, which inhibit the reuptake of monoamines, and at the same time, they act as antagonists on 5-HT2C receptors (e.g. fluoxetine, mianserine, amitriptyline, agomelatine, nefazodone) (Serretti et al. 2004), although the precise role of these receptors in the therapy of depression has not been determined yet. It is widely assumed that a repeated treatment with SSRIs leads to an agonist-induced desensitization of 5-HT2C receptors occurring secondarily to the elevated extracellular 5-HT release (Leysen 2004); in addition, a decrease in 5-HT2C receptor responses after chronic SSRI treatment has been found to disinhibit the mesolimbic dopamine system, which may be involved in the development of the antidepressant action, at least in part (Prisco and Esposito 1995). Furthermore, the role of 5-HT2C receptors in the acute anxiogenic potential of SSRIs is proven, since the potent and highly subtype-selective 5-HT2C receptor antagonist SB-242084 (Kennett et al. 1997) markedly inhibited the anxiogeniclike effects of acute SSRI treatment in rats (Dekeyne et al. 2000; Bagdy et al. 2001). Anxiety was also reduced after chronic administration of SSRIs (To et al. 1999; To and Bagdy 1999; Bristow et al. 2000). Depression is associated with decreased serotonergic neurotransmission and specific alterations of sleep. The enhanced rapid eye movement sleep (REMS) pressure is typical, and most antidepressant drugs, such as SSRIs, decrease the total amount of REMS and delay the occurrence of the first REMS episode (Steiger and Kimura 2009). In addition, REMS or total sleep deprivation may induce antidepressant effect in depressed patients and a reversal of depressive-like behaviours in animal models of depression (Adrien 2002). So, a pathological link between depression and sleep has been suggested. The 5-HT2C receptors, predominantly expressed by γ-amino-butyric-acid (GABA)-ergic neurons (Serrats et al. 2005), have been detected in numerous brain regions involved in the regulation of sleep and wakefulness, namely the cerebral cortex, hippocampus, hypothalamus, medial pontine reticular formation, locus coeruleus, laterodorsal and pedunculopontine tegmental nuclei and the dorsal raphe nucleus (Pompeiano et al. 1994; Abramowski et al. 1995; Clemett et al. 2000). It is assumed that these receptors may mediate several sleep effects that have been ascribed to 5-HT, since 5-HT2C receptor knockout mice show abnormalities in sleep/wake architecture and an enhanced response to sleep deprivation (Frank et al. 2002). The sleep effects of SSRI antidepressants and several 5-HT2C receptor ligands have been investigated
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extensively, although the alterations in 5-HT2C receptormediated functions in sleep and on the EEG spectra after chronic SSRI treatment have not been studied yet. In this report, to test the hypothesis whether these functions also undergo functional adaptation as a result of chronic treatment with SSRIs, we injected a single dose of the 5-HT2C receptor antagonist SB-242084 at the beginning of passive phase in animals pretreated chronically with escitalopram, the most selective SSRI (Owens et al. 2001). The applied antagonist, the SB-242084, is highly selective for 5-HT2C receptors (pKi = 9.0) and has much lower affinity to 5-HT2A (pKi = 6.8) and 5-HT2B (pKi = 7.0) receptors (Barnes and Sharp 1999).
Materials and methods Animal maintenance All animal experiments and housing conditions were carried out in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC) and the National Institutes of Health “Principles of Laboratory Animal Care” (NIH Publications No. 85–23, revised 1985), as well as specific national laws (the Hungarian Governmental Regulation on animal studies, 31 December Psychopharmacology 1998 Act). Permission was obtained from local ethical committees. Male Wistar rats (n = 25) were purchased from Animal Facility (Semmelweis University, Budapest, Hungary). Rats, weighing 250–280 g at EEG surgery, were kept under controlled environmental conditions (temperature at 21 ± 1 °C, 12-h light/dark cycle starting at 10:00 AM). Food and water were available ad libitum during the whole experiment. Surgery Animals were equipped with electroencephalographic (EEG) and electromyographic (EMG) electrodes as described earlier (Kantor et al. 2004). Briefly, stainless steel screw electrodes were implanted epidurally over the left frontal cortex (L: 2.0 mm and A: 2.0 mm to bregma) and left parietal cortex (L. 2.0 mm and A: 2.0 mm to lambda) for fronto-parietal EEG recordings. The ground electrode was placed over the cerebellum. In addition, EMG electrodes (stainless steel spring electrodes embedded in silicon rubber, Plastics One Inc., Roanoke, VA, USA) were placed in the muscles of the neck. Surgery was performed under halothane (2 %) anaesthesia (Fluotec 3) using a Kopf stereotaxic instrument. After a 7-day recovery period, rats were attached to the polygraph by a flexible recording cable and an electric swivel, fixed above the cages, permitting free movement for the animals. To habituate the animals to the
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Fig. 1 The experimental scheme. VEH, osmotic minipump filled with VEHICLE; SSRI, osmotic minipump filled with escitalopram (10 mg/kg/ day); veh, acute i.p. injection of vehicle; SB, acute i.p. injection of SB-242084 (1 mg/kg)
recording conditions, they were attached to the polygraph and received intraperitoneal (i.p.) injections of physiological saline for 7 days before the experiments. The animals remained connected to the recording cables throughout the study. Study design For the chronic pretreatment, escitalopram oxalate solution (SSRI; provided by Gedeon Richter Plc.; 10 mg/kg/day, dissolved in a solution of 0.3 N HCl in distilled water) or VEHICLE (VEH; solution of 0.3 N HCl in distilled water) was administered by osmotic minipump (2ML4, ALZET, 2.5 μl per hour, 28 days DURECT Corporation, USA) for 20 days. On the 21th day of the chronic pretreatment, rats were injected i.p. with SB-242084 [(SB), 6-chloro-5methyl-1-{[2-(2-methyl-3-pyridyl)oxy]-5-pyridyl carbamoyl}, Tocris, UK, 1 mg/kg, dissolved in a solution of 10 % (2-hydroxypropyl)-β-cyclodextrin] or vehicle [veh, solution of 10 % (2-hydroxypropyl)-β-cyclodextrin] in a volume of 1 ml/kg body weight. Animals used in the EEG studies were randomly divided into four groups: chronic VEHICLE-treated plus acute vehicle or SB-242084 [VEH + veh (n = 6) and VEH + SB (n = 6), respectively], chronic SSRI-treated plus acute vehicle or SB-242084 [SSRI + veh (n = 7) and SSRI + SB (n = 6), respectively]. The experimental scheme is shown on Fig. 1. Sleep recording and scoring EEG, EMG and motor activity were recorded for 3 h after the i.p. injection of SB-242084 or vehicle starting at light onset as described earlier (Filakovszky et al. 1999; Kantor et al. 2002; Graf et al. 2004). Briefly, the signals were amplified (amplification factors approximately 5.000 for EEG and motor activity, 20.000 for EMG), conditioned by analogue filters (filtering, below 0.50 Hz and above 60 Hz at 6 dB/octave), and subjected to analogue to digital conversion with a sampling rate of 128 Hz/channel. The digitized signals were displayed on a monitor and stored on the computer for further analysis. The vigilance states were scored
visually for 4-s periods over 1–3 h as follows: active wakefulness (AW), the EEG is characterized by low-amplitude activity at α (10–13 Hz), β (14–30 Hz) and θ (5–9 Hz) frequencies accompanied by high EMG and motor activity; passive wakefulness (PW), the EEG is characterized by low-amplitude activity at α (10–13 Hz), β (14–30 Hz) and θ (5–9 Hz) frequencies accompanied by low EMG activity and motor activity; light slow-wave sleep (SWS-1), high-voltage slow cortical waves (0.5–4 Hz) interrupted by low-voltage fast EEG activity (spindles 6–15 Hz) accompanied by reduced EMG and motor activity; deep slow-wave sleep (SWS-2), continuous high amplitude slow cortical waves (0.5–4 Hz) with reduced EMG and motor activity; intermediate stage of sleep (IS), a brief stage just prior to REMS and sometimes just after it, characterized by unusual association of high-amplitude spindles (mean 12.5 Hz) and low-frequency (mean 5.4 Hz) theta rhythm; REMS, low-amplitude and high-frequency EEG activity with regular theta waves (5–9 Hz) accompanied by silent EMG and motor activity with occasional twitching. The polygraphic recordings were classified by sleep analysis software (SleepSign for Animal; Kissei Comtec America Inc., USA). After the automatic scoring, recordings were visually revised. The parameters calculated in the present study were the followings: • Time spent in a sleep stage in the summarized first 3 h (1–3 h); • Number of stage episodes in 1–3 h; • Average duration of stage episodes in 1–3 h; A stage episode was defined as a period of a stage (AW, PW, SWS-1, SWS-2 and IS) lasting for ≥4 s. In order to exclude short REMS attempts (sRa), a REMS episode was defined as a period of REMS lasting for ≥16 s and not interrupted by ≥16 s of other vigilance state (Gandolfo et al. 1994); • REMS latency: the time elapsed from the start of sleep until the first consecutive 28 s of REMS (Mendelson 1996);
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• non-REM sleep (NREMS) latency: the time elapsed between light onset and the first consecutive NREMS (SWS-1, SWS-2 and IS) episode lasting at least 3 min and not interrupted by more than 14 consecutive 4-s epochs, or a cumulated total of 240 s not scored as NREMS (Huber et al. 1998); • Sleep fragmentation: the number of awakenings that disconnected any sleep periods (Huber et al. 1998); EEG power spectral analysis EEG power spectra were computed for consecutive 4-s epochs in the frequency range 0.25–60 Hz (fast Fourier transformation routine, Hanning window; frequency resolution, 0.25 Hz). Epochs with artefacts were discarded on the basis of the polygraph records. Adjacent 0.25-Hz bins were summed into 1-Hz bins, and those above 60 Hz were omitted. Bins are marked by their upper limits; thus, 2 Hz refers to 1.25–2.00 Hz (Kantor et al. 2002). The power values of consecutive 4 s EEG epochs in AW, PW, SWS-1, SWS-2, IS and REMS were separately averaged in each of the first 3 h after treatments to obtain the power density values for these vigilance states. Due to the very limited amount of REMS in the 1st hour, the Q-EEG data were analysed only in the 2nd and 3rd hours. Statistical analyses Regarding sleep parameters, to determine whether the chronic escitalopram administration affects the responses of SB-242084 or not, data were evaluated by the interaction effect between the factors pretreatment (VEH or SSRI) and treatment (veh or SB) based on two-way analysis of variance (ANOVA) statistics. In addition, data were also analysed using one-way ANOVA followed by Tukey’s honest significant difference test. The analyses were evaluated for the summarized 3 h. In case of variance inhomogeneity (tested by Levene’s tests for homogeneity), logarithmic data transformation was used regarding the following parameters: time spent in PW, PW number, NREMS latency and AW duration. Regarding the EEG power spectra, the area under the curve (AUC) values of theta frequencies (5–9 Hz) (cumulated power density of theta frequency band divided by the number of theta frequency bins) were log-transformed before analysis in each hours of both AW (1st, 2nd and 3rd hours) and REMS (solely 2nd and 3rd hours). Data were evaluated by the interaction effect of two-way ANOVA [factors: pretreatment (VEH or SSRI) and treatment (veh or SB)] and by one-way ANOVA statistics, followed by Tukey’s honest significant difference test. In addition, to investigate the interrelation between group effects and theta frequencies, theta power values were
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submitted to repeated-measures ANOVA with two main factors: group (non-repeated; VEH + veh, VEH + SB, SSRI + veh or SSRI + SB) and theta frequency bins (repeated; 5–9 Hz), followed by Tukey’s honest significant difference test. P values
