Laurie L. Wellman

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.

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.

Distinct macrophage subpopulations regulate viral encephalitis but not viral clearance in the CNS.

Intranasal application of vesicular stomatitis virus (VSV) induces acute encephalitis characterized by a pronounced myeloid and T cell infiltrate. The role of distinct phagocytic populations on VSV encephalitis was therefore examined in this study. Ablation of peripheral macrophages did not impair VSV encephalitis or viral clearance from the brain, whereas, depletion of splenic marginal dendritic cells impaired this response and enhanced morbidity/mortality. Selective depletion of brain perivascular macrophages also suppressed this response without altering viral clearance. Thus, two anatomically distinct phagocytic populations regulate VSV encephalitis in a non-redundant fashion although neither population is essential for viral clearance in the CNS.

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.

Effects of microinjections of Group II metabotropic glutamate agents into the amygdala on sleep.

Systemic administration of the Group II metabotropic glutamate (mGlu) receptor agonist, LY379268 (LY37), dose-dependently suppresses rapid eye movement sleep (REM) whereas systemic administration of the mGlu II receptor antagonist, LY341495 (LY34), increases arousal. Group II mGlu receptors are highly expressed in the amygdala, a brain region involved in the regulation of sleep and arousal. To determine whether the amygdala is involved in mediating the effects of Group II mGlu agents on sleep, we microinjected LY37 and LY34 into the basal amygdala (BA) and the central nucleus of the amygdala (CNA) and recorded sleep and wakefulness. Wistar rats were implanted with electrodes for recording sleep and with bilateral cannulae aimed into BA for drug administration. Different groups of rats received bilateral microinjections of LY37 into BA at two dosage ranges (3.2 mM, 5.3 mM or 10.7 mM or 0.1 nM, 2.0 nM or 10.0 nM) or one dosage range of LY34 (1.0 nM, 30.0 nM or 60.0 nM). Microinjections into CNA were conducted at one dosage range for LY37 (0.1 nM, 2.0 nM or 10.0 nM) and for LY34 (1.0 nM, 30.0 nM or 60.0 nM). All drugs or vehicle alone were administered in a counterbalanced order at 5-day intervals. Following microinjection, sleep was recorded for 20 h. Microinjection of LY37 into BA at both nM and mM concentrations significantly decreased REM without significantly altering NREM, total sleep or wakefulness. The high dosage of LY34 in BA significantly suppressed NREM and total sleep. Microinjections of LY37 or LY34 into CNA had no significant impact on sleep. We suggest that Group II mGlu receptors may influence specific cells in BA that control descending output (via the CNA or bed nucleus of the stria terminalis) that in turn regulates pontine REM generator regions.

Effects of corticotropin releasing factor (CRF) on sleep and body temperature following controllable footshock stress in mice

Rapid eye movement sleep (REM) is increased after controllable stress (modeled by escapable footshock, ES) and decreased after uncontrollable stress (modeled by inescapable footshock, IS). Decreases in REM after IS are exacerbated by corticotropin releasing factor (CRF) and attenuated by a CRF antagonist. In this study, we trained mice with ES following injections of CRF, astressin (AST), or saline (SAL) to determine whether CRF would alter REM after ES. Male BALB/cJ mice (n=7) were implanted for recording sleep, activity and body temperature via telemetry and with a guide cannula aimed into a lateral ventricle. After recovery from surgery, sleep following exposure to a novel chamber was recorded as a handling control (HC). The mice received one day of training with ES without injection followed by weekly training sessions in which they received counterbalanced intracerebroventricular (ICV) microinjections of either SAL or CRF (days 7 & 14) or SAL or AST (days 21 & 28) prior to ES. On each experimental day, sleep was recorded for 20 h. Compared to HC, the mice showed significantly increased REM when receiving either SAL or AST prior to ES whereas CRF prior to ES significantly reduced REM. Stress-induced hyperthermia had longer duration after ES compared to HC, and was not significantly altered by CRF or AST compared to SAL. The current results demonstrate that activity in the central CRF system is an important regulator of stress-induced alterations in REM.

Sleep and behavior during vesicular stomatitis virus induced encephalitis in BALB/cJ and C57BL/6J mice.

Intranasal application of vesicular stomatitis virus (VSV) produces a well-characterized model of viral encephalitis in mice. Within one day post-infection (PI), VSV travels to the olfactory bulb and, over the course of 7 days, it infects regions and tracts extending into the brainstem followed by clearance and recovery in most mice by PI day 14 (PI 14). Infectious diseases are commonly accompanied by excessive sleepiness; thus, sleep is considered a component of the acute phase response to infection. In this project, we studied the relationship between sleep and VSV infection using C57BL/6 (B6) and BALB/c mice. Mice were implanted with transmitters for recording EEG, activity and temperature by telemetry. After uninterrupted baseline recordings were collected for 2 days, each animal was infected intranasally with a single low dose of VSV (5×10(4) PFU). Sleep was recorded for 15 consecutive days and analyzed on PI 0, 1, 3, 5, 7, 10, and 14. Compared to baseline, amounts of non-rapid eye movement sleep (NREM) were increased in B6 mice during the dark period of PI 1-5, whereas rapid eye movement sleep (REM) was significantly reduced during the light periods of PI 0-14. In contrast, BALB/c mice showed significantly fewer changes in NREM and REM. These data demonstrate sleep architecture is differentially altered in these mouse strains and suggests that, in B6 mice, VSV can alter sleep before virus progresses into brain regions that control sleep.