Chronic stress-induced changes in REM sleep on theta oscillations in the rat hippocampus and amygdala

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Chronic stress-induced changes in REM sleep on theta oscillations in the rat hippocampus and amygdala Preethi Hegdea , H.R. Jayakrishnana , Sumantra Chattarji b , Bindu M. Kutty a , T.R. Laxmi a,⁎ a

Department of Neurophysiology, National Institute of Mental Health and Neurosciences (NIMHANS), Hosur Road, P.B. No. 2900, Bangalore 560029 Karnataka, India b National Center for Biological Sciences (NCBS), Tata Institute of Fundamental Research (TIFR), GKVK Campus, Bangalore 560065, India

A R T I C LE I N FO

AB S T R A C T

Article history:

The present study investigated the effect of Chronic Immobilization Stress (CIS) on theta

Accepted 18 January 2011

oscillations in the hippocampus and amygdala during Rapid Eye Movement (REM) sleep. Adult

Available online 27 January 2011

male Wistar rats were subjected to 2 h of CIS daily for 10 days. Polysomnographic recordings with electroencephalogram (EEG) from hippocampus (CA3 and CA1 subregion) and lateral

Keywords:

nucleus of amygdala (LA) were carried out after termination of CIS protocol on the 7th, 14th and

Stress

21st day. The results showed a bimodal distribution on the total REM sleep duration in CIS rats:

Sleep

group of rats exhibited increased REM sleep duration considered as a stress-enhanced REM

Amygdala

(SER) and rats with reduced REM sleep as stress-reduced REM sleep (SRR) group. The bimodal

Hippocampus

distribution in REM sleep was continued to exhibit even after 21 days of termination of stress,

Theta rhythm

showing increased REM sleep in SER and reversible REM sleep in SRR rats. In addition to changes in sleep, increased REM sleep in SER rats was associated with attenuated theta activity in the hippocampus and amygdala, while the SRR rats did not show attenuated theta activities during the stress recovery period. Thus, the study demonstrates the dependence of synchronized amygdalo-hippocampal theta activity with the CIS-induced enhanced REM sleep duration. This raises the possibility that CIS-induced manifestations of the anxiety may be associated with synchronized theta oscillations in the hippocampus and amygdala. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

It is well documented that stress is a biologically powerful contributory factor in the cause and progression of many psychiatric diseases (Breslau et al., 2004; Habukawa et al., 2007; Mellman et al., 2002; Reist et al., 1995; Ross et al., 1994). Recent studies in rodents suggest that repeated stress that

produces degenerative changes in the hippocampus impairs hippocampal-dependent learning (McEwen and Sapolsky, 1995; Sapolsky, 1996), while dendritic hypertrophy in the basolateral amygdala (BLA) facilitates aversive learning and anxiety-like behavior (Liang et al., 1994; Shors and Mathew, 1998; Vyas et al., 2002, 2006). In humans, stress-induced anxiety disorders are of serious concern because augmented anxiety is postulated to

⁎ Corresponding author. Fax: +91 80 26564830. E-mail addresses: [email protected], [email protected] (T.R. Laxmi). Abbreviations: CA1, CA1 subregion of the hippocampus; CA3, CA3 subregion of the hippocampus; CIS, Chronic Immobilization stress; EEG, Electroenecephalogram; EMG, Electromyogram; EOG, Electrooculogram; LA, Lateral amygdala; EEG, Electro encephalogram; PTSD, Post traumatic stress disorder; REM, Rapid eye movement sleep; SER, Stress enhanced REM sleep; SRR, Stress reduced REM sleep; SWS, Slow wave sleep; NREM, Non-REM sleep; TST, Total sleep time; TWT, Total wake time; W1, Active wake; W2, Quiet wake 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.01.055

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play a major role in inducing sleep disturbances (Mellman and Uhde, 1989; Reynolds et al., 1983; Ross et al., 1994). Such stressinduced damage is thought to be related to an increase in cortisol levels and glucocorticoids associated with stress (Bremner, 1999). Evidence from both human and animal studies suggest that sleep facilitates information processing and synaptic remodeling (Hill et al., 2008; Lopez et al., 2008). REM sleep, in particular, has been identified as an important stage of sleep in memory consolidation (Hellman and Abel, 2007; McNamara et al., 2009; Stickgold et al., 2001; Stickgold and Walker, 2007). Further investigations into the behavioral basis of rhythmical slow wave activity (theta) from the hippocampus suggest the presence of two types of theta activity in most animal species (Bland, 1986; Montoya and Sainsbury, 1985; Sainsbury, 1970; Vanderwolf et al., 1988). Type 1 theta has a frequency range of 6–12 Hz, is present during voluntary movements such as running, rearing and swimming. Type 2 theta activity usually has a frequency range at 4–9 Hz and occurs during alert immobility, REM sleep and sensory processing (Bland, 1986; Leblanc and Bland, 1979). The existences of rhythmic theta oscillations in the hippocampus and amygdala are essential for normal physiological functions during sleep (Popa et al., 2010; Wilson and McNaughton, 1994) and abolishing them will result in severe behavioral deficits (Winson, 1978; Winson and Abzug, 1978). We have also reported the changes in REM sleep and theta activity following exposure to acute immobilization stress (Hegde et al., 2008). Furthermore, behavioral and morphological changes induced by stress suggest that stress impairs memory through both hippocampal and nonhippocampal mechanisms (Conrad et al., 1996; Vyas et al., 2002, 2006). Amygdala dependent enhancement in the anxiety-related behavior was reported until 21 days after termination of stress (Vyas et al., 2004), whereas hippocampal dependent behavioral impairment returned to normal when left undisturbed under standard home conditions (Conrad et al., 1999; Luine et al., 1994). The above mentioned studies clearly suggest the deleterious effect of stress on sleep–wake behavior and theta activity. It may be plausible to corroborate that the stress-induced changes in REM sleep may be one of the major factors responsible for the genesis of anxiety leading to changes in theta rhythm in the hippocampus and amygdala. The present experiment was designed to further investigate the relationship between the chronic stress and the amygdalo-hippocampal theta activity during REM sleep.

2.

Results

2.1.

Evaluation of sleep architecture

sleep architecture from Control and CIS rats after 21 days of termination of stress. As compared to control animals, the CIS rats showed significant changes in the sleep architecture. This was mainly due to the changes in REM sleep. The CIS (n=13) rats showed a wide range of variations in REM sleep states; from 4.549±1.09% to 16.14±1.192 % of the total recording time. Accordingly, the rats were further categorized as stress-enhanced REM (SER) group (n=6) with a total of 16.14+1.192% REM sleep and stress-reduced REM group (n=7) with 4.549±1.09% of REM sleep. By means of categorical variable analysis, CIS group of rats showed a very clear dichotomy in the distribution of REM sleep duration (t11 =7.080, p<0.0001), and no such dichotomy was seen in Control group (t9 =1.970, p=0.0843) as shown by Student t-test analysis. Two way ANOVA with Bonferroni post-hoc comparison test revealed (ANOVA: F2,53 =19.56, p<0.0001) that total duration of REM sleep was increased (p<0.05) in SER group and reduced (p<0.05) in SRR group when studied after 7 days of termination of CIS (see Fig. 2A). Though the REM sleep has come back to normal by day 14 (T-test: t=1.140, p>0.05), but showed an increasing trend on day 21 (t=1.527, p>0.05) (Fig. 2A). Additionally, the SER rats did not display any significant changes in the total number of REM episodes as compared to Control group (Fig. 2B). SRR group, on the other hand, showed a decreased number of REM episodes (p < 0.05) as compared to Control rats after 7 and 14 days of termination of stress (ANOVA: F2,23 = 8.806, p < 0.001) (Fig. 2B). While the SER rats showed increased REM sleep, SRR group showed a statistically significant decrease in total REM duration on day 7 (t = 2.956, p < 0.01) (Fig. 2A), day 14 (t = 3.890, p < 0.001) and returned to normal levels on day 21 after CIS termination (t = 1.613, p > 0.05) (Fig. 2A). Similarly, REM episodes in SRR group also showed a significant decrease on day 7 (T-test: p < 0.008, t1,15 = 3.064) (Fig. 2B) and day 14 (T-test: p < 0.006, t1,15 = 3.197). The REM latency did not show any significant differences across recording sessions. In contrast to REM sleep, CIS did not affect the total wakefulness of SER rats. While, the SRR group of rats showed statistically significant increase in TWT (T-test: p < 0.008, t1,15 = 3.057) and significant decrease in TST (t1,15 = 3.508, p < 0.003) on day 14 after CIS in comparison with Control rats (Fig. 3A and C). In both SER and SRR groups, the TSWS remained unaffected at all the three time points of sleep recordings (Fig. 3B). These results indicate that CIS-induced increase in REM sleep did not alter the TST, however, CIS-induced reduction in the REM sleep was accompanied with reduced TST specifically 14 days after termination of CIS.

2.2.

Fig. 1B shows the site locations of the electrodes in the different brain regions such as CA1, CA3 regions of the hippocampus and LA. Polysomnographic recordings from the Control rats (n=11) showed that rats spent predominant time in Slow Wave Sleep (SWS) and Rapid Eye Movement (REM) sleep, that is, 51.53±2.49% and 10.52±1.433% of the total 6 h of recording time respectively. The total wake time (TWT) of the animals was 37.41±2.198% as shown in Fig. 1C. The distribution of the sleep–wake cycle was comparable to that observed in the previous study in the laboratory (Raol and Meti, 1998). Fig. 1D shows representative

Theta activity from hippocampus and amygdala

The impact of chronic stress on theta modulation during stress recovery period was evaluated by FFT analysis. In comparison to rhythmic theta activity in the controls, CIS rats showed profound changes in theta activity of both hippocampal (CA1 and CA3) and LA (Figs. 4, 5) neurons, specifically 21 days after termination of CIS. The change in theta activity during REM sleep was very prominent in SER group when compared to SRR and Control group of rats. Spectral analysis from Control and SRR rats showed a predominant distribution of signal power over theta frequency range (4–8 Hz) during REM sleep (Fig. 4). It was observed

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Fig. 1 – Summary of the experimental design. A, An overview of the experimental protocol for the sleep recording for both Control and CIS group of rats; B, Histology showing the chronic placement of the insulated electrodes for recording EEG from CA1, CA3 regions of the hippocampus and Lateral nucleus of the amygdala (LA). C, Sleep architecture of control rats showing the normal distribution of Total Wake Time (TWT), Slow Wave Sleep (SWS), Rapid Eye Movement (REM) sleep; D, Representative example of Polysomnographic recordings from Control and CIS rats showing different stages of sleep: active wake, SWS and REM sleep from control and CIS rats during light phase of the cycle. EMG, electromyogram; LA, lateral nucleus of amygdala; CA1/CA3, hippocampus. Calibration: time, 1 s; Amplitude, 100 μV.

that changes in REM sleep duration were not accompanied with alterations in theta oscillations of the hippocampus and amygdala of both SER and SRR rats on day 7 after termination of stress (Fig. 5A). The statistical analysis with one way ANOVA indicated that SER group of rats showed reduction in the theta power on day 21 with no statistical significant difference in the CA1, CA3 and LA (Fig. 5B) and normal REM

sleep duration as that of Controls. While theta power in LA of SRR group remained unaffected from day 7 till day 21 after termination of CIS. The Control rats did not show statistical significant differences in theta power across different recording periods. In brief, longitudinal study indicated that CIS induced differential effect on the theta activity of hippocampus and amygdala.

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analysis, Student's t-test revealed that SER rats exhibited a decreased correlation in LA versuss CA1 (p<0.01, t1,8 =3.343) (Fig. 6D), LA versuss CA3 (p<0.05, t1,8 =2.250) (Fig. 6E) and CA3 versuss CA1 (p<0.006, t1,9 =3.527) (Fig. 6F) suggestive of reduced synchronization. While the SRR rats showed no significant difference due to reduction in the correlation during REM sleep duration.

3. Fig. 2 – CIS and REM Sleep. Bimodal distribution on REM sleep duration in CIS rats until 21 days after termination of stress. A, Effect of CIS on REM sleep on 7 days, 14 days and 21 days after termination of CIS; the ordinate depicts the mean percent time in 6 hours of total sleep time and abscissa depicts the sleep recording day after CIS termination. B, Total number of REM episodes showing significant decrease in SRR rats. Note that SER rats continued to show the increased REM sleep even after 21 days of CIS, but not in the SRR rats. Normal control, Control; SER, stress-enhanced REM sleep and SRR, stress-reduced REM sleep group of rats. Mean± SEM, *p< 0.05, **p<0.0006.

Further, autocorrelation analysis showed reduced synchronized oscillations in LA and CA3 in SER group with no obvious changes in theta oscillations in SRR group (data not shown). Intergroup comparisons of changes in coherence of theta activity were made in order to elucidate whether the changes that were observed in the SER and SRR rats were constant throughout the recording period. The unpaired comparisons using Student's ttest showed a decreased coherence level between the LA and CA1 regions with a significance level on day 21 (p<0.05, t1,10 =2.232) (Fig. 6A, G). Statistical analysis using Student's t-test showed that coherence was altered between LA versus CA3 (p < 0.01, t1,10 =2.958) (Fig. 6B, H), CA3 versus CA1 (p<0.003, t1,11 =3.716) (Fig. 6C, I) at theta frequency range in SER group. However, SRR rats did not show any trend towards decrease in coherence level until day 21 (Fig. 6G–I). In addition to the coherence analysis, the study also performed auto- and cross-correlation analysis between hippocampus and amygdala. As seen with coherence

Discussion

The present study demonstrated the chronic immobilization stress (CIS) induced changes in REM sleep in rats. CIS has produced a bimodal distribution of the REM sleep states among the rats; the SER group of rats exhibited enhanced REM sleep states and the SRR group with reduced REM sleep states. Additionally, CIS also induced long term changes in the theta activity in the hippocampus and amygdala. In the present study, CIS appears to induce a prolonged effect on the sleep even after termination of the stress. CIS has showed specific discrepancies in the REM sleep without any alterations in NREM among the SER and SRR groups. The SRR group of rats showed decreased REM sleep, whereas the SER group showed an enhanced REM sleep state following exposure to stress. Additionally, in SER group, the total sleep time was also found to be reduced following exposure to stress suggesting that the changes are specific responses to CIS. Based on the studies in rodents and humans, it is suggested that acute and repeated stress affect the sleep, more specifically on REM sleep for a prolonged period of time (Breslau et al., 2004; Hegde et al., 2008; Shors and Mathew, 1998; Singareddy and Balon, 2002). Rampin et al. (1991) showed a greater reduction in the rebound of sleep when the rats were subjected to IS repeatedly than a single exposure to the IS. Subsequent studies by Palma et al. (2000) and Marinesco et al. (1999) suggest that prolonged stress for more than 4 hours impairs the sleep rebound until 24 hours after the stress. In addition, there are a number of evidences revealing that amygdala-dependent emotional memory is enhanced by the late REM-sleep rich period distinctly more than by the early SWS-rich period of sleep (Gais et al., 2003; Plihal and Born, 1999). Increase in REM sleep, in turn, might facilitate consolidation of

Fig. 3 – CIS and Sleep Architecture. Comparisons of sleep patterns between normal control and CIS (SER and SRR group) rats. A, the percentage of time spent in Total Wake; B, percentage of time spent in total SWS during 6 h of sleep recording period. TWT and TSWS were from after 7 days, 14 days and 21 days of termination of CIS. The data was compared with the age-matched controls and represented as Mean ± SEM. The asterisks indicate significant difference from control rats (**p < 0.001).

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Fig. 4 – Effect of CIS on Theta activities of the hippocampus and amygdala. Averaged relative power of EEG (0–20 Hz) obtained from CA1, CA3 region of the hippocampus and LA during REM sleep. Note the significant reduction in theta power of the CA3 and LA on day21. The averaged data was compared between Control, SER and SRR group.

aversive memories acquired during the wakefulness. Our previous study also suggests that the amygdalo-hippocampal pathway may be involved in the consolidation of emotional experience as shown by decreased synchronized theta activity during REM sleep 10 days after termination of acute immobilization stress (AIS) (Hegde et al., 2008). Indeed, we found that post-CIS changes occur specifically in REM sleep suggesting that stress-induced changes in LA-CA1 pathway during REM sleep could be correlated with subsequent development of anxietylike behavior observed in CIS rats (Vyas et al., 2004). Our results further imply that CIS reduces the coordinated interactions between LA and CA1. The reduction in the synchronized theta activity between these regions was directly correlated with increased REM sleep. Additionally, variation in theta activity was observed only in the rats that exhibited increased REM sleep and not in SRR group. It is generally accepted that aversive information processing occurs during REM sleep (Nishida et al., 2009) which is intimately related to hippocampal theta expression (Vertes, 2004; Vertes et al., 2004). It was suggested that increased synchronization between the amygdala and hippocampal theta activities are strongly related with fear memory retrieval

(Seidenbecher et al., 2003). Due to diverse inputs from various nuclei of the amygdala into the basal forebrain structures (Pare and Smith, 1994; Szymusiak, 1995), its potential involvement in the regulation of REM sleep could not be separated. The changes in theta activity resulting from CIS, thus, can be expected to occur concomitantly during REM sleep. Decreased theta activity between LA and CA1 with increased REM sleep could be attributed to the CIS-induced facilitation of stress hormones on the glucocorticoids (Plihal et al., 1999). We have further observed a decreased theta power in both the hippocampal sub-regions (CA1 and CA3) during REM sleep following termination of the stress in SER rats. However, CIS did not affect the theta frequency power of the lateral amygdala of SER rats. In addition, we have also found that CIS reduced the coherent theta oscillations between CA1 and LA during REM sleep in SER rats, reflecting a lesser temporal co-incidence of synaptic activity (Volgushev et al., 1998). Aligned with this, there are several evidences to suggest that amygdala plays a central role in modulating stress-related changes in the hippocampal synaptic plasticity (Kim and Diamond, 2002; Richter-Levin, 2004) such as amygdalar inactivation blocks stress-induced

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Fig. 5 – CIS and Power Spectral Density. CIS-induced changes in the spectral power during REM sleep after 7 days and 21 days of termination of stress. A. Representative spectrogram from CA1, CA3 and LA showing predominant frequency band at theta range (4–8 Hz) from normal control (left graph), SER (middle graph) and SRR (right graph) group after 7 days of CIS; B, Representative example showing spectrogram from CA1, CA3 and LA after 21 days of CIS; C, Averaged relative spectral power from CA1, CA3 and LA from Control, SER and SRR rats; Control; SER, stress-enhanced REM sleep and SRR, stress-reduced REM sleep rats. day 7, day 14 and day 21 after termination of CIS. Data represented are in Mean+ SEM, *p < 0.05 statistical significant from control group.

impairment in hippocampal long term potentiation (Kim et al., 2005). Subsequent work supporting the role of amygdala in the regulation of REM sleep indicating that inactivation of central amygdala selectively reduced REM sleep and total sleep (Sanford et al., 2002). Activity in the central amygdala depends on the excitatory inputs from the LA (Pare et al., 2004; Pelletier and Pare, 2004; Pitkanen et al., 1995; Royer et al., 1999). Studies on humans suggesting a selective activation of the amygdala following nonhippocampal dependent memory task was benefited particularly from REM sleep (Plihal and Born, 1999; Wagner et al., 2001). Evidence from rodent studies suggest that exposure to a chronic and repeated stress can facilitate fear and anxiety-like behavior in rats immediately after 24 h, as well as 10 days after termination of stress (Conrad et al., 1999; McEwen and Sapolsky,

1995; McGaugh and Roozendaal, 2002; Vyas et al., 2004), which could be mediated by altering the structural plasticity in the hippocampus and amygdala (Vyas et al., 2002). Functional implications of these changes were contrary to the study reported wherein it was suggested that single prolonged exposure to stress caused deficits in both CA1 hippocampus and amygdala LTP (Kohda et al., 2007). But few other reports have shown deficits only in the hippocampal LTP (Foy et al., 1987; Shors et al., 1989), but with enhanced amygdalar LTP following exposure to chronic stress (Kavushansky et al., 2006). The above paradoxical reports on the electrophysiological findings following chronic stress may possibly be due to the bimodal effect of chronic stress on the sleep pattern. Notably, chronic stress-induced decreased theta coherence between hippocampus and amygdala was not

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Fig. 6 – CIS and Theta Synchrony during REM sleep. CIS induced changes in the theta activities between LA and CA1/CA3 during REM sleep. Coherence data averaged at theta frequency range (4–8 Hz) from Control, SER and SRR rats on 21 days after termination of CIS. A, LA versus CA1; B, LA versus CA3; C, CA3 versus CA1; Cross-correlation analysis between D, LA and CA1; E, LA versus CA3; F, CA3 versus CA1; Coherence data at 0–20 Hz G, LA versus CA1; H, LA versus CA3; I, CA3 versus CA1. The data are in comparison with control. Note the CIS has shown to decreased the synchronous activities in SER and not in SRR group during REM sleep after 21 days of termination of CIS; Data are in Mean ± SEM, *p < 0.05.

observed until day 21 after termination of stress. Also, the differences in the neuronal activities were seen only in SER rats, and not in SRR rats. Considering the increase in REM sleep first and delayed changes in the neuronal activity after stressor removal over 21 days indicates that REM sleep alone may be a unique precursor to identify the subsequent behavioral manifestations such as anxiety disorders in the later days of life. The result from SER rats resembles those in Post Traumatic Stress Disorder (PTSD) patients, wherein the sleep disruption is chronic and persistent for decades after the trauma (Harvey et al., 2003). Thus, assessment of the sleep changes due to chronic stress might enable us to identify the individual differences in the

subsequent manifestations of the sleep problems associated with routine stress in humans as well as the development of anxiety disorders in them.

4.

Experimental procedures

4.1.

Subjects

Forty-five to 60 days old adult male Wistar rats were used. They were housed in polypropylene cages, allowed ad libitum access to food and water and maintained on a 12:12 h light/dark cycle. All

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the experiments were carried out in accordance with the guidelines of Central Animal Research Facility (CARF), at National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. Experimental protocol was approved from the institutional animal ethics committee (IAEC) (AEC/31/155(B)/155) and care was taken to minimize pain or discomfort during surgery to the animals.

4.2.

Groups

The rats were divided randomly into two groups. One group of rats (n = 13) were subjected for Chronic Immobilization Stress (CIS) and the second group of rats (n = 11), the Normal Control (Control) was used as the normal age matched control.

4.3.

CIS procedure

Rats were subjected to CIS in a plastic cone, daily for 2 h (10.00 h–12.00 h) for a period of 10 days. During the stress protocol, the rats did not have any access to food and water as per an earlier study (Vyas et al., 2002). Control animals were not subjected to any type of stress and were housed separately in their home cage. However, the rats were handled daily by the experimenter during the same time (10.00 h–12.00 h) while the CIS rats were experiencing the immobilization stress.

4.4.

Implantations procedure

Rats were anesthetized with the combination of ketamine (80 mg/ kg body weight) and xylazine (10 mg/kg body weight). In addition, xylocaine (2%) was injected subcutaneously over the scalp before the surgery. Rats were stereotaxically (David Kopf Stereotaxic instrument, USA) implanted with insulated nichrome wires of 250 μm diameter in CA1 area of hippocampus (CA1) (anteroposterior: −3.3 mm; medio-lateral: 1.5 mm; dorso-ventral: 2.6 mm), CA3 area of hippocampus (CA3) (antero-posterior: −4.16 mm; medio-lateral: 3.6 mm; dorso-ventral: 3.8 mm), and lateral amygdala (LA) (antero-posterior: −3.3 mm; medio-lateral: 5.2 mm; dorso-ventral: 8.0 mm), on the left hemisphere of the rat brain for measuring the EEG during sleep. Paxinos and Watson rat brain atlas was used to locate all the stereotaxic coordinates in relation to bregma and lambda (Paxinos and Watson Atlas 1986). An external screw electrode was implanted in the adjacent parietal lobe subdurally as a reference and ground electrode. In addition, electrodes for Electroculogram (EOG) and electromyogram (EMG) were implanted in external canthus of the eye and posterior nuchal muscle, respectively. This entire electrode ensemble was soldered into a 10-pin socket and fixed on the skull using dental acrylic powder. Healex was applied to all the wound edges and finally placed back into its home cage and care was given to recover from the surgical effects. Following 5 days of post-surgical recovery, the rats were subjected to various experimental protocols.

4.5.

Procedure for sleep recording

After the complete recovery from surgery, the rats were taken to the sleep laboratory of the department and left in its home cage for an hour for habituation. The rats were connected to the swivel commutator and EEG recording was carried out contin-

uously for six hours using 8-channel amplifier (Grasss model 7D). The EEG signals were amplified (Grass model 7D), bandpass filtered (at 0.3 Hz to 3 kHz), digitized (1401 CED as AD converter) and recorded using spike 2 software and stored in a hard disk for the offline analysis. During this period, the spontaneous behavior of the rat was monitored and was recorded online with the spike 2 software. The rats had free access to food and water during the entire recording session. In order to observe the prolonged effect of chronic stress on the sleep and associated changes in the amygdalohippocampal network activities. EEG recordings were carried out on days 7, 14 and 21 after termination of the 10 days of chronic immobilization stress. A summary of the experimental design is depicted in Fig. 1A. Since rats exhibit polyphasic nature of sleep, the sleep architecture was studied during light phase of the cycle. More specifically, the recording was done during 12:00 h–18:00 h. It is known from the literature that rats exhibit maximum time in sleep during this period, 6-h sleep–wake distribution was studied during this period (Raol and Meti, 1998).

4.6.

Recording and quantification of the sleep architecture

The 6 hour sleep–wakefulness cycles were split into 15 s epochs and visually scored on the basis of EEG, EMG and EOG, by a scorer blinded to the study protocol. Each epoch was classified into a particular stage based on predominant sleep stage as per an earlier study (Hegde et al., 2008). The scoring method, described here in detail, is primarily based on five-stage classification of sleep–wake for rats as reported (Bjorvatn and Ursin, 1994; John et al., 1994; Neckelmann and Ursin, 1993; Vetrivelan et al., 2006). The wakeful period was classified into two stages, namely, active wakefulness (W1) based on the presence of Type 1 theta rhythm in CA1 and CA3, tonic EMG level and phasic EOG movements; quiet wakefulness (W2) showing reduced tonic EMG, occasional EOG movements and Type 2 theta rhythm in CA1 and CA3. The sleep period was classified into three stages i.e., light Slow Wave Sleep (SWS) (S1) characterized by the presence of high amplitude and low frequency EEG activity in CA1 and CA3, reduced EMG activity with the absence of locomotor activity and reduced EOG movements; deep SWS (S2) showing synchronized low frequency (delta range) and high amplitude EEG activity in CA1 and CA3, EMG hypotonia and no EOG movements. Rapid Eye Movement (REM) sleep epochs were visually scored showing the presence of synchronized hippocampal EEG at theta frequency (Type 2, 4–9 Hz), EOG with rapid bursts of eye movements and EMG showing muscle atonia. REM onset latency was calculated based on the appearance of first episode of REM sleep from the beginning of sleep recording. The percentage distribution of sleep stages were calculated from the total recording time that includes, SWS percentage (TSWS/Total Recording Time); REM percentage (Total REM sleep/Total Recording Time); Wake Time percentage (Total Waking time/Total Recording time).

4.7.

Quantification of EEG

All the EEG data was sampled at the rate of 1000 Hz and digitized with CED analog-to-digital (A/D) converter (Cambridge, UK) and

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stored on a hard disk for the offline analysis. For the quantification, the longest last episode of REM was selected from which a 30 s duration was considered for further analysis. Each digitized 30 s epoch of EEG signals from REM sleep were submitted to Fast Fourier Transformation (FFT) analysis for the measurement of power spectral density and correlation analysis, to compare synchronized oscillatory activity following CIS. The percentage of absolute power, i.e. relative power from 0 to 20 Hz corresponding to REM sleep was obtained in order to observe the impact of CIS conditions on the dominant frequency range. Comparisons were made between relative power and correlation data obtained for each successive day. Relative power and correlation data were compared between and within Controls and CIS rats. At the end of the experiment, the rats were deeply anaesthetized with halothane and transcardially perfused with 10% formaldehyde. The brains were shelled out and vibratome sections with Cresyl Violet staining method were taken for the histological verification of the electrode placement. The rats with confirmed electrodes in the target were only used for the further evaluation of sleep–wake architecture in both Control and CIS groups.

4.8.

Statistical analysis

Statistical analysis was conducted using SPSS and Prism software. After obtaining significant ANOVAs, post-hoc comparisons of means were carried out either with Bonferroni or Tukey's multiple comparison tests. In addition, T-tests were used to compare the impact of CIS on sleep and EEG details between Control and CIS rats. Statistical significance was set at p < 0.05.

Acknowledgments This work was supported by a research grant from Department of Science and Technology (DST) (Ref: F.NO.SR/SOS/HS-30/2005), Government of India and National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, Karnataka. We are grateful to Professor John Nicholls, via Beirut-2, Trieste, Italy, for the expert critical review of the manuscript.

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