Linghui Yang

Effects of tetrodotoxin (TTX) inactivation of the central nucleus of the amygdala (CNA) on dark period sleep and activity

The amygdala has been implicated in emotional arousal and in the regulation of sleep. Previously, we demonstrated that tetrodotoxin (TTX), a sodium channel blocker that temporarily inactivates neurons and tracts, microinjected into the central nucleus of the amygdala (CNA) during the light period significantly reduced REM, shortened sleep latency, and increased EEG delta power in rats. TTX inactivation of CNA also reduced activity in the open field. These findings suggest that the amygdala modulates arousal in a variety of situations. To test the hypothesis that the amygdala may influence spontaneous arousal, we examined the effects of TTX inactivation of CNA on sleep and activity during the dark period when rats show higher arousal and less sleep. EEG and activity were recorded via telemetry in Wistar rats (n = 8). Bilateral microinjections of TTX (L: 2.5 ng/0.1; H: 5.0 ng/0.2 microl) or SAL (saline, 0.2 microl) were administered before lights off followed by recording throughout the 12-h dark period and following 12-h light period. Microinjections were given at 5-day intervals and were counterbalanced across condition. TTX significantly shortened sleep latency, increased NREM time, decreased REM time, and decreased activity. TTX increased NREM episode duration, whereas the number and duration of REM episodes were decreased. The present results indicate that TTX inactivation of CNA can increase NREM time when spontaneous arousal is high, suggesting a broad role for the amygdala in regulating arousal. The results suggest that understanding the ways in which the amygdala modulates arousal may provide insight into the mechanisms underlying altered sleep in mood and anxiety disorders.

Rat strain differences in sleep after acute mild stressors and short-term sleep loss.

Genetic and physiological diversity amongst rodent strains provide the potential for developing models that may give insight into factors that regulate sleep in response to environmental challenges. We examined home cage activity, behavioral performance in the open field and sleep after a number of mild stressors (cage change [CC], open field [OF]) and after 1 and 4h of sleep deprivation (1hSD and 4hSD) in rat strains (Fischer 344 [F344], Lewis [LEW], Wistar [WST] and Sprague-Dawley [Sp-D], n=16 per strain) that differ in behavior and sleep. F344 and WST rats had greater home cage locomotion than LEW and Sp-D rats, but F344 rats exhibited the least relative locomotion in OF. In 24h baseline recordings of sleep, strain rankings were LEW=WST=Sp-D>F344 in rapid eye movement sleep (REM), and LEW=Sp-D>F344 and LEW>WST in non-REM (NREM). Compared to baseline, total sleep was reduced in all four strains after CC, OF and 1hSD, but not after 4hSD, in the first hour after treatment. Afterwards, increases in REM and NREM were seen after all treatments with the amount and time course varying across treatments and strains. CC induced the weakest and 4hSD the largest effects on sleep, whereas OF and 1hSD had intermediate effects. Among strains, the more anxious F344 rats exhibited the greatest sleep increases during the light period after OF, 1hSD and 4hSD. The results are discussed with respect to the relationship between behavioral and sleep responses to stressors, and to potential mechanisms underlying the strain differences.

Antagonizing Corticotropin-Releasing Factor in the Central Nucleus of the Amygdala Attenuates Fear-induced Reductions in Sleep but not Freezing

STUDY OBJECTIVES Contextual fear is followed by significant reductions in rapid eye movement sleep (REM) that are regulated by the central nucleus of the amygdala (CNA). Corticotropin-releasing factor (CRF) plays a major role in regulating the stress response as well as arousal, and CRF in CNA is implicated in stress-related behavior. To test the hypothesis that CRF regulation of CNA is involved in fear-induced alterations in REM, we determined the effects of microinjections into CNA of the CRF1 antagonist, antalarmin (ANT) on fear-induced reductions in REM. We also evaluated c-Fos activation in the hypothalamic paraventricular nucleus (PVN), locus coeruleus (LC), and dorsal raphe nucleus (DRN) to determine whether activation of these regions was consistent with their roles in regulating stress and in the control of REM. DESIGN On separate days, rats were subjected to baseline and 2 shock training sessions (S1 and S2). Five days later, the rats received bilateral microinjections of ANT (4.8 mM) or vehicle (VEH) prior to exposure to the fearful context. Sleep was recorded for 20 h in each condition. Freezing was assessed during S1, S2, and context. Separate groups of rats received identical training and microinjections or handling control (HC) only, but were sacrificed 2 h after context exposure to assess c-Fos expression. SETTING NA. PATIENTS OR PARTICIPANTS NA. INTERVENTIONS NA. MEASUREMENTS AND RESULTS Compared to baseline, S1 and S2 significantly reduced REM. Exposure to the fearful context reduced REM in VEH treated rats, whereas REM in ANT treated rats did not differ from baseline. ANT did not significantly alter freezing. Fear-induced c-Fos expression was decreased in PVN and LC after ANT compared to VEH. CONCLUSIONS The results demonstrate that CRF receptors in CNA are involved in fear-induced reductions in REM and neural activation (as indicated by c-Fos) in stress and REM regulatory regions, but not in fear-induced freezing.

Individual variation in sleep and motor activity in rats.

We examined individual differences in sleep and motor activity across 2 consecutive days in rats. EEG and motor activity were recorded via telemetry in Wistar rats (n=29) for 48h under well-habituated conditions. Rats were grouped based on sleep amounts and stability across days (short [SS, n=7], intermediate [IS, n=15] and long [LS, n=7] sleep) and comparisons were conducted to determine group differences for measures of sleep and motor activity. We found that correlations across recording days were significant for all selected sleep measures and motor activity counts. Rankings for 24h total sleep time and non-rapid eye movement sleep (NREM) were SS<IS<LS rats whereas amounts of rapid eye movement sleep did not differ among groups. Further analyses of NREM episode parameters found significant differences in mean episode length (SS<IS<LS) but not in the number of episodes. Total and average motor activity counts (per waking min) were greater (32-38%) in SS compared to LS rats on both recording days. The results indicate that individual differences in sleep and motor activity in Wistar rats are stable across days. Differences between SS and LS rats have parallels to those reported for short and long sleep humans.

Mouse strain differences in the effects of corticotropin releasing hormone (CRH) on sleep and wakefulness.

Corticotropin releasing hormone (CRH) plays a major role in central nervous system responses to stressors and has been implicated in stress-induced alterations in sleep. In the absence of stressors, CRH contributes to the regulation of spontaneous waking. We examined the effects of CRH and astressin (AST), a non-specific CRH antagonist, on wakefulness and sleep in two mouse strains with differential responsiveness to stress to determine whether CRH might also differentially affect undisturbed sleep and activity. Less reactive C57BL/6J (n=7) and high reactive BALB/cJ (n=7) male mice were implanted with a transmitter for determining sleep via telemetry and with a guide cannula aimed into a lateral ventricle. After recovery from surgery and habituation to handling, ICV microinjections of CRH (0.04, 0.2, and 0.4 microg), AST (0.1, 0.4, and 1.0 microg) or vehicle alone (pyrogen-free saline, 0.2 microl) were administered during the fourth hour after lights on and sleep was recorded for the subsequent 8 h. Comparisons of wakefulness and sleep were conducted across conditions and across strains. In C57BL/6J mice, REM was significantly decreased after microinjections of CRH (0.2 microg) and CRH (0.4 microg), and NREM and total sleep were decreased after microinjections of CRH (0.4 microg). CRH (0.04 microg) and AST did not significantly change wakefulness or sleep. In BALB/cJ mice, CRH (0.4 microg) increased wakefulness and decreased NREM, REM and total sleep. AST decreased active wakefulness and significantly increased REM at the low and high dosages. These findings demonstrate that CRH produces changes in arousal when given to otherwise undisturbed mice. Strain differences in the effects of CRH and AST may be linked to the relative responsiveness of C57BL/6J and BALB/cJ mice to stressors and to underlying differences in the CRH system.

Interactions between brief restraint, novelty and footshock stress on subsequent sleep and EEG power in rats.

Stress produces significant alterations in sleep that appear to vary with the type, intensity and duration of the stressor. Brief manual restraint may be stressful in rodents but is often required for experimental procedures. We examined the effects of brief manual restraint on sleep and its possible influence on sleep induced after footshock and after the opportunity to explore a neutral enclosure. Sleep was recorded during non-interrupted baseline and during 8-h light and 12-h dark periods after three sessions of 5-min manual restraint (M1-3), after 30 min in neutral enclosure alone (NE) or with previous manual restraint (mNE) and after 20 footshocks presented over the course of 30 min alone (FS) or with previous manual restraint (mFS). Compared to baseline, M1-3 increased total sleep and NREM during both light and dark periods and significantly increased dark period REM. Both NE and mNE increased dark period total sleep, NREM and REM; however, mNE also increased light period total sleep and NREM, but not REM. FS and mFS increased total sleep, NREM and REM during the dark period and total sleep and NREM during light period. FS also significantly decreased light period REM whereas mFS did not. M1, mNE and mFS significantly increased EEG delta power during NREM, but M2-3, NE and FS alone did not. The results revealed that manual restraint can increase sleep and EEG delta power and that increases in sleep may persist across repeated sessions whereas the magnitude of EEG delta power may vary across sessions. In addition, prior manual restraint may significantly alter the changes in sleep and EEG induced by footshock and by the opportunity to explore a neutral enclosure. The results suggest that mild stressors may interact in their effects on sleep.

Basolateral amygdala and the regulation of fear-conditioned changes in sleep: role of corticotropin-releasing factor.

STUDY OBJECTIVE To determine whether corticotropin-releasing factor (CRF) in the basolateral amygdala (BLA) modulated sleep and fear-conditioned alterations in sleep. DESIGN After 2 days of habituation to recording procedures, baseline sleep recordings were obtained. The animals were then habituated to the handling procedure necessary for microinjections over 2 consecutive days. In experiment 1, rats received microinjections of 0.5 μL antalarmin (1.61 or 4.82 mM), a CRF receptor 1 antagonist, or distilled water once a week for 3 wk. In experiment 2, rats received a microinjection of either antalarmin or vehicle prior to inescapable shock training (ST; 20 shocks; 0.8 mA, 0.5 sec; 1 min interstimulus interval). The animals were placed back in the context 7 days later for 30 min without shock (CR; context re-exposure). Sleep was recorded for 8 h after each manipulation. SETTING NA. SUBJECTS Outbred Wistar rats. INTERVENTIONS The rats were surgically implanted with electrodes for recording the electroencephalogram and electromyogram for determining arousal state and with bilateral guide cannulae directed at BLA. MEASUREMENTS AND RESULTS Antalarmin microinjected into BLA did not significantly alter sleep under undisturbed conditions. However, antalarmin microinjected bilaterally into BLA prior to ST blocked reductions in rapid eye movement sleep that ST normally produces. Further, the single microinjection prior to ST blocked the reduction in rapid eye movement typically seen after subsequent CR. Behavioral freezing, an indicator of fear memory, was not altered. CONCLUSIONS CRF in BLA is involved in regulating stress-induced alterations in sleep and it plays a role in modulating how stressful memories influence sleep.

Effects of Stressor Predictability and Controllability on Sleep, Temperature, and Fear Behavior in Mice

STUDY OBJECTIVES Predictability and controllability are important factors in the persisting effects of stress. We trained mice with signaled, escapable shock (SES) and with signaled, inescapable shock (SIS) to determine whether shock predictability can be a significant factor in the effects of stress on sleep. DESIGN Male BALB/cJ mice were implanted with transmitters for recording EEG, activity, and temperature via telemetry. After recovery from surgery, baseline sleep recordings were obtained for 2 days. The mice were then randomly assigned to SES (n = 9) and yoked SIS (n = 9) conditions. The mice were presented cues (90 dB, 2 kHz tones) that started 5.0 sec prior to and co-terminated with footshocks (0.5 mA; 5.0 sec maximum duration). SES mice always received shock but could terminate it by moving to the non-occupied chamber in a shuttlebox. SIS mice received identical tones and shocks, but could not alter shock duration. Twenty cue-shock pairings (1.0-min interstimulus intervals) were presented on 2 days (ST1 and ST2). Seven days after ST2, SES and SIS mice, in their home cages, were presented with cues identical to those presented during ST1 and ST2. SETTING NA. PATIENTS OR PARTICIPANTS NA. INTERVENTIONS NA. MEASUREMENTS AND RESULTS On each training and test day, EEG, activity and temperature were recorded for 20 hours. Freezing was scored in response to the cue alone. Compared to SIS mice, SES mice showed significantly increased REM after ST1 and ST2. Compared to SES mice, SIS mice showed significantly increased NREM after ST1 and ST2. Both groups showed reduced REM in response to cue presentation alone. Both groups showed similar stress-induced increases in temperature and freezing in response to the cue alone. CONCLUSIONS These findings indicate that predictability (modeled by signaled shock) can play a significant role in the effects of stress on sleep.

Corticotropin releasing factor (CRF) modulates fear-induced alterations in sleep in mice

Contextual fear significantly reduces rapid eye movement sleep (REM) during post-exposure sleep in mice and rats. Corticotropin releasing factor (CRF) plays a major role in CNS responses to stressors. We examined the influence of CRF and astressin (AST), a non-specific CRF antagonist, on sleep after contextual fear in BALB/c mice. Male mice were implanted with transmitters for recording sleep via telemetry and with a guide cannula aimed into the lateral ventricle. Recordings for vehicle and handling control were obtained after ICV microinjection of saline (SAL) followed by exposure to a novel chamber. Afterwards, the mice were subjected to shock training (20 trials, 0.5 mA, 0.5 s duration) for 2 sessions. After training, separate groups of mice received ICV microinjections of SAL (0.2 microl, n=9), CRF (0.4 microg, n=8), or AST (1.0 microg, n=8) prior to exposure to the shock context alone. Sleep was then recorded for 20 h (8-hour light and 12-hour dark period). Compared to handling control, contextual fear significantly decreased REM during the 8-h light period in mice receiving SAL and in mice receiving CRF, but not in the mice receiving AST. Mice receiving CRF exhibited reductions in REM during the 12-h dark period after contextual fear, whereas mice receiving SAL or AST did not. CRF also reduced non-REM (NREM) delta (slow wave) amplitude in the EEG. Only mice receiving SAL prior to contextual fear exhibited significant reductions in NREM and total sleep. These findings demonstrate a role for the central CRF system in regulating alterations in sleep induced by contextual fear.

Effects of stressor predictability on escape learning and sleep in mice.

STUDY OBJECTIVES Controllable stress, modeled by escapable shock (ES), can produce significant alterations in post-stress sleep, including increased rapid eye movement (REM) sleep. Recent work has demonstrated that post-stress sleep may be influenced by stressor predictability, modeled by predictive auditory cues. In this study, we trained mice with ES, either signaled (SES) or unsignaled (UES) by auditory cues, and investigated the effects of predictability on escape learning and sleep associated with ES. DESIGN Adult male BALB/cJ mice were implanted for recording electroencephalography and activity via telemetry. After the mice recovered from surgery, baseline sleep recordings were obtained. The mice were then randomly assigned to SES and UES conditions. Both groups had control over the duration of footshocks (0.5 mA; 5.0 sec maximum duration) by moving to the non-occupied chamber in a shuttlebox. SES mice were presented tones (90 dB, 2 kHz, 10 sec maximum duration) that started 5.0 sec prior to and co-terminated with footshocks. UES mice were presented identical tones that were not synchronized to shock presentation. ES training continued for 2 consecutive days (EST1 and EST2) with 20 footshock presentations (1 min inter-stimulus intervals). Seven days after EST2, the animals were re-exposed to the training chamber (context) alone for 30 min. MEASUREMENTS AND RESULTS Escape latency was used to determine successful or unsuccessful escape learning. Sleep was scored for 20 h for baseline and on each treatment day. Freezing in the training context was scored as a behavioral index of fear. Nine of 14 SES mice successfully learned escape (SESl), and 5 failed to learn escape (SESf). Compared with baseline, SESl mice, but not SESf mice, showed significantly increased post-shock REM. All UES mice learned escape and showed enhanced post-shock REM. Freezing and sleep did not differ among groups on the context re-exposure day. CONCLUSIONS The results indicate that information available in a stressful situation can affect an animals ability to learn an appropriate response and post-stress sleep. CITATION Machida M; Yang L; Wellman LL; Sanford LD. Effects of stressor predictability on escape learning and sleep in mice. SLEEP 2013;36(3):421-430.