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. 2011 Nov 1;34(11):1539–1549. doi: 10.5665/sleep.1394

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

Xianling Liu 1, Laurie L Wellman 1, Linghui Yang 1, Marta A Ambrozewicz 1, Xiangdong Tang 1, Larry D Sanford 1,✉
PMCID: PMC3198209  PMID: 22043125

Abstract

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.

Citation:

Liu X; Wellman LL; Yang L; Ambrozewicz MA; Tang X; Sanford LD. Antagonizing corticotropin-releasing factor in the central nucleus of the amygdala attenuates fear-induced reductions in sleep but not freezing. SLEEP 2011;34(11):1539-1549.

Keywords: Central nucleus of the amygdala, contextual fear, corticotropin releasing factor, dorsal raphe nucleus, locus coeruleus, paraventricular nucleus

INTRODUCTION

Fear conditioning associated with stressful inescapable footshock training produces marked changes in subsequent sleep. Specifically, training with inescapable footshock is followed by a significant decrease in rapid eye movement sleep (REM) that may persist for hours after the stressful experience.1–3 After training, cues (specific stimuli such as light or auditory tones) and contexts (the shock training environment) made fearful through association with inescapable footshock also decrease subsequent REM,1–3 thereby demonstrating that fearful memories can produce significant alterations in sleep similar to those produced by inescapable footshock itself.

The amygdala has been prominently linked to fear behavior that occurs in response to footshock stress4,5 and to the regulation of arousal.6–8 The central nucleus of the amygdala (CNA), in particular, has been linked to the regulation of REM8,9 and microinjection into CNA of the GABAA agonist muscimol selectively decreases REM, whereas microinjection of the GABAA antagonist bicuculline selectively increases REM.9 Recently, we demonstrated that microinjections of bicuculline into CNA attenuated footshock-induced reductions in REM, whereas microinjections of muscimol did not.10 Microinjections of bicuculline, but not muscimol, also reduced footshock-induced c-Fos expression, a marker of neuronal activity,11,12 in the locus coeruleus (LC), a brain region implicated in the regulation of REM13 and stress.14,15 This work indicated that CNA likely has a significant role in regulating the alterations in REM that occur in response to stress.

Corticotropin-releasing factor (CRF) plays a major role in regulating central aspects of the stress response.16,17 CRF also has been implicated in stress-induced alterations in sleep.18 For example, CRF antagonists have been reported to eliminate REM rebound after immobilization stress19 and to decrease REM rebound after sleep deprivation.20 In the absence of stressors, CRF may contribute to the regulation of spontaneous waking21 as evidenced by findings that the ICV administration of CRF increases wakefulness in rats22 and rabbits.23 The critical role of CNA in conditioned fear and its role in regulating REM in response to stress suggest that it may be an important site mediating the effects of CRF on post-stress arousal. CRF in CNA is also implicated in conditioned fear.24

Our previous study10 demonstrated that CNA was involved in regulating REM in the aftermath of footshock stress, but did not determine whether it was also involved in regulating fear-conditioned alterations in REM. In this study, we examined the effects of microinjections of the relatively specific CRF1 antagonist, antalarmin (ANT) into CNA on contextual fear-induced alterations in sleep to test the hypothesis that CRF regulation of CNA was involved in fear-induced alterations in REM. We also examined freezing as a measure of the fear response,25–29 and we measured c-Fos in LC, the hypothalamic paraventricular nucleus (PVN), and the dorsal raphe nucleus (DRN) to determine whether activation of these regions were consistent with their roles in regulating REM and the stress response.

METHODS

Subjects

The subjects were 35 male Wistar rats at approximately 90 days of age at the time of surgery. The animals were given ad libitum access to food and water. The recording room was kept on a 12:12-h light-dark cycle with lights on from 07:00 to 19:00. Ambient temperature was maintained at 24.5 ± 0.5°C.

Surgery

The rats were implanted with skull screw electrodes for recording their electroencephalogram (EEG) and with stainless steel wire electrodes sutured to the dorsal neck musculature for recording their electromyogram (EMG). Leads from the recording electrodes were routed to a 9-pin miniature plug that mated to one attached to a recording cable. Guide cannulae (26 g) for microinjections into CNA were implanted bilaterally with their tips aimed 1.0 mm above CNA (A 6.3, ML ± 4.0, DV 7.0).30 The recording plug and cannulae were affixed to the skull with dental acrylic and stainless steel anchor screws.

All surgical procedures were performed stereotaxically under aseptic conditions. The rats were anesthetized with isoflurane (5% induction; 2% maintenance). Ibuprofen (15 mg/kg) was available in their water supply for relief of postoperative pain. The rats were allowed a minimum of 14 days to recover prior to beginning the experiment. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals and were approved by Eastern Virginia Medical School's Animal Care and Use Committee (Protocol 07-005).

Procedure

After recovery from surgery, a subset of rats (n = 16) was habituated to the handling and recording procedures over 2 consecutive days, after which a single 20-h uninterrupted baseline recording was obtained. Afterwards, all animals received 2 days of footshock training as described below. No microinjections were given during footshock training. Five days later, the rats were randomly assigned to receive bilateral microinjections of either ANT (4.8 mM; n = 8) or vehicle (VEH, n = 8) into CNA prior to exposure to the fearful context alone. After shock training and exposure to the fearful context, the rats were returned to their home cage and their undisturbed wakefulness and sleep was recorded for 20 h.

A separate subset of animals (n = 19) was used to examine c-Fos expression in the PVN, LC, and DRN. These rats also were implanted for recording sleep. Fourteen rats received treatment identical to that described above, except that they were killed 2 h after exposure to the fearful context. Seven animals received microinjections of ANT, and 7 animals received microinjections of VEH into CNA. Five handling control (HC) animals did not experience shock, but received microinjections of VEH to control for handling associated with the injection procedure.

The study was completed utilizing different cohorts of animals. Separate shock training and recording sessions were completed for different cohorts of rats.

Microinjections

ANT (antalarmin hydrochloride, N-Butyl-N-ethyl-2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolol[2,3-d]pyrimidin-4-amine hydrochloride) was obtained from Sigma-Aldrich, St. Louis, MO, USA. ANT was prepared in a VEH of 10% Cremophor EL (Sigma-Aldrich, St. Louis, MO) in pyrogen-free saline.31

For microinjections, injection cannulae (33 g) were secured in place within the guide cannulae. These projected 1.0 mm beyond the tip of the guide cannulae for delivery of drug into CNA. The injection cannulae were connected to one end of lengths of polyethylene tubing that had the other end connected to 5.0 μL Hamilton syringes. The injection cannulae and tubing were prefilled with the solution to be injected. Drugs were infused slowly over 3 min (0.2 μL). Microinjections were administered 15 min prior to introduction of the animal into the fearful context. The timing of the control injections and beginning recording for the HC group was similar to that used for the other groups, but the animals were not placed in the shock chamber.

Footshock Training

After recovery from surgery, the rats were subjected to handling control followed by 2 shock training sessions (S1 and S2), which were conducted at the same time on separate days. For training, the rats were placed in the shock chambers (Coulbourn Habitest cages equipped with grid floors (Model E10-18RF) that were housed in Coulbourn Isolation Cubicles (Model H10-23). The rats were allowed to freely explore for 5 min, after which they were presented with 20 inescapable footshocks (0.5 s, 0.8 mA) at 1.0-min intervals over the course of 20 min. Shock was produced by Coulbourn Precision Regulated Animal Shockers (Model E13-14) and presented via the grid floor of the shock chamber. Five min after the last shock, the rats were returned to their home cages. The entire procedure lasted 30 min.

The shock chamber was thoroughly cleaned with diluted alcohol prior to each training session. All experimental manipulations were conducted prior to the fifth hour after lights on, and sleep recording began at the beginning of the fifth hour. This time was chosen because it is prior to a period in which the animals are sleeping well, and it is far enough into the light period that it should be beyond potential influences of light onset.

Behavioral Freezing

The shock training sessions and the contextual fear session were videotaped for the 16 rats used in the primary sleep study to allow subsequent scoring of freezing, defined as the absence of body movement except for respiration.25,26 Freezing was scored by a trained observer blind to condition in 5-sec intervals during 1.0-min observation periods over the course of the 30 min the rats were in the shock chamber. The percentage time spent freezing was calculated (FT%: freezing time/observed time × 100) for each animal for each observation period.

Sleep Recording and Scoring

For recording, each animal, in its home cage, was placed in a chamber outfitted for electrophysiological recording and a lightweight, shielded cable was connected to the miniature plug on the rat's head. The cable was attached to a swivel that permitted free movement of the rat within its cage. EEG and EMG signals were processed by a Grass, Model 12 polygraph equipped with model 12A5 amplifiers. The amplified signals were routed to an A/D board (Eagle PC30) housed in a Pentium class PC. The signals were digitized at 128 Hz and collected in 10-s epochs using a custom sleep data collection program. The epochs were visually scored as wakefulness, NREM, or REM based on EEG and EMG activity using standard electrographic criteria.8,9,32 Wakefulness was scored based on the presence of low-voltage fast EEG, high-amplitude tonic EMG level, and phasic EMG bursts that could be associated with gross body movements. NREM was scored based on the presence of spindles interspersed with slow waves, lower muscle tone, and no gross body movements or EEG desynchronization. For scoring REM, onset was noted immediately following the last sleep spindle of NREM that occurred in conjunction with decreasing or fully relaxed muscle tone. Afterward, REM was scored continuously during the presence of low voltage, fast EEG, theta rhythm, and muscle atonia.

Histology

The rats were anesthetized with isoflurane (inhalation: 5% induction, 2% maintenance) and then transcardially perfused with 250 mL ice cold saline, followed by 150 mL 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). To localize the microinjection sites, brain slices (50 μm) were made through the amygdala, and the sections were mounted on slides. Sections from the 16 rats in the primary sleep study were stained with cresyl violet to aid in localization of the injection sites. Injection sites in the other 19 rats were visually localized as the sections were cut. In these rats, as the sections containing CNA and PVN overlap, it was not possible to optimize sections for both c-Fos staining and simultaneously cut sections to fully track the cannulae placement in CNA. The sections were examined in conjunction with a stereotaxic atlas30 to confirm cannulae placements.

The rats used to assess c-Fos activation were perfused immediately upon completion of the 2-h sleep recordings to obtain brain tissue for c-Fos immunohistochemistry and to determine the localization of the microinjection site in CNA. The rats were anesthetized with isoflurane (inhalation: 5% induction, 2% maintenance) and then transcardially perfused with 250 mL ice cold saline, followed by 150 mL 4% paraformaldehyde in 0.1 M PB (pH 7.4). Brains were immediately removed and post-fixed in the same fixative at 4°C for 24-48 h and then immersed in 30% sucrose in 0.1 M PB for 48 h at 4°C.

c-Fos Immunohistochemistry Staining

Fifty-μm thick coronal sections were cut from frozen blocks from AP 8.08 to 6.70 mm for PVN and from AP 1.96 to −1.52 mm in the brainstem for DRN and LC. Every fifth section was collected as a sample. One sample was used for c-Fos staining. A second sample was used as a blank control. Free floating methods were used for immunohistochemical staining as described previously.33 Briefly, sections were washed in 0.01 M phosphate buffered saline (PBS, pH 7.4) and incubated in 0.3% hydrogen peroxide (H2O2)-2% normal goat serum in 0.01 M PBS for 30 min at room temperature to eliminate endogenous peroxidase activity and to block nonspecific binding sites. Then sections were washed 3 times for 10 min in PBS and were then incubated for 48 h at 4°C with the c-Fos antibody (1:20,000, Ab-5, Oncogene Research Products, Cat# PC38) in PBS containing 0.3% Triton X-100 and 2% normal goat serum. After washing with PBS, sections were incubated for 2 h at room temperature with biotinylated goat anti-rabbit secondary antibody (1:600, Sigma, Product No.B8895) in PBS containing 2% normal goat serum. Subsequently, the sections were washed and incubated for 1 h at room temperature with horseradish peroxidase avidin-biotin complex (1:100 ABC reagent in PBS-TX, Avidin- Biotin Complex, Vector ABC kit). After washing, sections were reacted with DAB, mounted to slides, and allowed to dry for 48 h. Then sections were dehydrated through graded alcohol, cleared by xylene, and protected by cover slips for visualization.

The primary antibody was absent in control sections, which were otherwise processed identically. Omission of the primary antibody resulted in complete loss of nuclear staining.

c-Fos Quantification

c-Fos expression was visualized in brain sections using a Nikon Eclipse E800 microscope. The sections were standardized as much as possible using a rat brain atlas.30 Digital photographs of the selected regions were taken with a Spot digital camera attached to Nikon Eclipse E800 microscope under 10× magnification. c-Fos positive cells in PVN, LC, and DRN were counted by a researcher blind to experimental condition with the Metamorph Image Analysis program. c-Fos expression in PVN, LC, and DRN was summed over 3 sections and expressed as number of c-Fos positive cells in each anatomical region.

Data Analysis

Statistical analyses were conducted using SigmaStat (SPSS, Inc, Chicago, IL). For the 20-h sleep data, comparisons between groups for each of 4-h time blocks were conducted using 2-way mixed factors (Group (VEH and ANT) × Treatment (Baseline, S1, S2, and context) ANOVAs with repeated measures on Treatment. Across Group and Treatment, we compared total REM and total NREM. Comparisons across 4-h time blocks were not conducted, as these values would be expected to differ simply as a matter of the passage of time. Amount of sleep in each h of the 2-h recordings in the rats used for assessment of c-Fos activation was analyzed across Group and Treatment. The freezing data were analyzed in 3 periods: the 5-min pre-shock period, the following 20-min shock period (during which shock was experienced on S1 and S2, but not during context re-exposure), and the 5-min post-shock period. The data for the pre-shock, shock, and post-shock periods were analyzed with separate 2-way mixed-factors (Group (VEH and ANT) × Treatment (S1, S2, and Context) ANOVAs with repeated measures on Treatment. All reported sleep, c-Fos, and freezing data were examined for and passed the normality test prior to conducting the relevant ANOVA. Significant ANOVAs were followed by post hoc Tukey tests.

RESULTS

Microinjection Sites

Figure 1 shows the location of the microinjections sites in the amygdala for animals used in the sleep study. Locations of microinjection sites for animals used in the sleep and c-Fos expression study were similar and are not plotted. Though there were rostral-caudal variations in the placements among animals, the histology indicated that ANT or VEH would have been infused into CNA and adjacent areas in all the rats, and all animals were used in the data analyses.

Figure 1.

Figure 1

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Drawing showing microinjection sites in 16 animals used for the 20-h primary sleep recordings. CNA, central nucleus of the amygdala; ▪VEH, vehicle control group: ⋆: ANT, antalarmin group.

Effects of ANT on Fear-induced Alterations in Sleep

Rapid eye movement sleep

Total REM amounts in each 4-h Block are plotted in Figure 2. The ANOVAs for total REM revealed significant Group × Treatment interactions for Block 1 (F3,42 = 4.49, P < 0.008) and Block 2 (F3,42 = 4.49, P < 0.008), and a significant Treatment effect for Block 4 (F3,42 = 10.74, P < 0.001). The post hoc analysis of total REM found significantly reduced total REM in Block 1 and Block 2 after S1 and S2 in both the VEH (Figure 2A) and ANT (Figure 2B) groups (note: the treatment of both groups was identical for baseline, S1, and S2). After exposure to the fearful context, the VEH group showed significantly reduced total REM compared to their baseline amounts in both Block 1 and 2. By comparison, the ANT group showed total REM amounts that were not significantly different from their baseline amounts, except in Block 4 where the ANT group showed elevated REM compared to baseline. Direct comparisons of the VEH and ANT groups found significant differences in total REM amounts in Block 1 and Block 2 (Figure 2C).

Figure 2.

Figure 2

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Total REM amounts plotted in 4-h blocks (B1-B5) across the 20-h recording period. (A) Total REM amounts plotted for baseline, S1, S2, and context in the vehicle (VEH) control group. (B) Total REM amounts plotted for baseline, S1, S2, and context in the antalarmin (ANT) group. (C) Direct comparisons of total REM amounts for VEH and ANT in each 4-h Block after re-exposure to the fearful context alone. S1, shock training 1; S2, shock training 2. *P < 0.05; **P < 0.01; and ***P < 0.001 compared to baseline. +P < 0.05; ++P < 0.01; and +++P < 0.001 compared to context.

Non-rapid eye movement sleep

NREM amounts in each 4-h Block are plotted in Figure 3. The ANOVAs for total NREM revealed a significant Treatment effect for Block 1 (F3,42 = 24.15, P < 0.001). Total NREM was reduced compared to baseline on S1 and S2, but did not differ between baseline and context in either the VEH (Figure 3A) or ANT (Figure 3B) groups. Direct comparisons of the VEH and ANT groups (Figure 3C) found significant differences in total NREM amounts only in Block 1 of the context day.

Figure 3.

Figure 3

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Total NREM amounts plotted in 4-h blocks (B1-B5) across the 20-h recording period. (A) Total NREM amounts plotted for baseline, S1, S2, and context in the vehicle (VEH) control group. (B) Total NREM amounts plotted for baseline, S1, S2, and context in the antalarmin (ANT) group. (C) Direct comparisons of total NREM amounts for VEH and ANT in each 4 hour Block after re-exposure to the fearful context alone. S1, shock training 1; S2, shock training 2. *P < 0.05; **P < 0.01; and ***P < 0.001 compared to baseline. +P < 0.05; ++P < 0.01; and +++P < 0.001 compared to context.

Effects of ANT on Fear-induced Alterations in Sleep and c-Fos

Effects on sleep

Comparisons were made for amounts of sleep within each hour of the 2 h of sleep recording that were obtained for the VEH and ANT groups used to assess c-Fos expression. REM sleep in h 1 did not differ across group or treatment condition (P > 0.05). The ANOVA for REM in h 2 revealed a significant Group × Treatment interaction (F3,36 = 4.76, P < 0.007). In h 2 (Figure 4A, B), REM was significantly reduced compared to baseline during S1 and S2 in both the VEH and ANT groups. Compared to baseline, REM was also significantly reduced after fearful context in the VEH group, but not in the ANT group.

Figure 4.

Figure 4

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Total REM amounts and total NREM amounts plotted for 2 h in the vehicle (VEH) and antalarmin (ANT) groups used to assess Fos expression. (A) Total REM amounts in baseline, S1, S2, and context in h 1 and h 2 in the VEH control group. (B) Total REM amounts in baseline, S1, S2, and context in h 1 and h 2 in the ANT group. (C) Total NREM amounts in baseline, S1, S2, and context in h 1 and h 2 in the VEH control group. S1, shock training 1; S2, shock training 2. **P < 0.01 and ***P < 0.001 compared to baseline. ++P < 0.01 compared to context.

NREM did not significantly differ across experimental treatment groups in h 1 (P > 0.05). In h 2 (Figure 4C, D), the ANOVA revealed a significant Treatment effect (F3,36 = 4.91, P < 0.006). The only post hoc comparison that was significant was that NREM was reduced in S2 compared to the fearful context in the ANT group. No other comparisons were significant.

We also made between group comparisons of the VEH and ANT groups and the HC animals that received only microinjections of VEH only prior to sleep recording and sacrifice. These groups did not differ in baseline NREM or REM. On the experimental day, no difference was found during h 1 or 2 for NREM. The analysis of REM found significant differences in h 2 (F2,17 = 7.13, P < 0.006), but not h 1. REM during h 2 was significantly reduced in the VEH group compared to the HC and the ANT groups whereas REM in the HC and ANT groups did not differ.

Effects on c-Fos expression

ANOVAs found significant group effects for c-Fos expression the PVN (F2,17 = 9.28, P < 0.002), LC (F2,17 = 12.56, P < 0.001), and DRN (F2,17 = 3.70, P < 0.05). Post hoc comparisons of c-Fos in the PVN found greater expression in the VEH group than in the ANT or HC groups, which did not significantly differ. In the LC, c-Fos expression in the VEH group was greater than in the ANT and HC groups, and expression in the ANT group was also greater than in the HC group. Although the ANOVA was significant, post hoc Tukey tests did not reveal any significant between groups differences in c-Fos expression in the DRN. Figure 5 presents graphic representation of the findings and sample sections showing c-Fos immunohistochemistry in PVN (Figure 5A-D), LC (Figure 5E-H), and DRN (Figure 5I-L) in each experimental condition.

Figure 5.

Figure 5

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Fos expression in hypothalamic paraventricular nucleus (PVN), locus coeruleus (LC), and dorsal raphe nucleus (DRN) at 2 h after re-exposure to the context alone. (A) Mean Fos counts in PVN in the vehicle (VEH), antalarmin (ANT), and handling control (HC) groups. Representative samples of Fos expression in PVN in the (B) VEH, (C) ANT, and (D) HC groups. (E) Mean Fos counts in LC in the VEH, ANT, and HC groups. Representative samples of Fos expression in LC in the (F) VEH, (G) ANT, and (H) HC groups. (I) Mean Fos counts in DRN in the VEH, ANT, and HC groups. Representative samples of Fos expression in DRN in the (J) VEH, (K) ANT, and (L) HC groups. 3V, 3rd ventricle; 4V, 4th ventricle; Aq, aqueduct. *P < 0.05 ANT compared to VEH; #P < 0.05 HC compared to VEH.

Correlation between REM Amounts and c-Fos Expression

All 19 rats were used in an analysis that examined the correlation between the amount of REM in h 2 of recording and amount of c-Fos expression in PVN, LC, and DRN. c-Fos expression in PVN (r = −0.55, P < 0.05) and LC (r = −0.62, P < 0.01), but not DRN (r = −0.28, NS) was significantly and negatively correlated with the amount of REM the rats exhibited during h 2.

Effects of ANT on Fear-induced Freezing

Significant Treatment effects were found in the ANOVAs for the pre-shock (F2,28 = 163.71, P < 0.001), shock (F2,28 = 42.29, P < 0.001), and post-shock (F2,28 = 22.71, P < 0.001) periods. Shock training resulted in similar significant increases in FT% compared to the pre-shock period in both VEH treated (Figure 6A) and ANT treated (Figure 6B) rats. FT% was also enhanced during context re-exposure (no shock presented) compared to the pre-shock period. FT% was reduced in the shock and post-shock periods of context compared to S1 and S2. However, the rats in the VEH and ANT groups did not show differences in FT% during pre-shock, shock or post-shock in S1 (Figure 6C), S2 (Figure 6D), or context (Figure 6E)

Figure 6.

Figure 6

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Fear-induced freezing in rats pretreated with vehicle (VEH) or antalarmin (ANT). (A) Freezing time % during pre-shock, shock, and post-shock in S1, S2, and context in the VEH control group. (B) Freezing time % during pre-shock, shock, and post-shock in S1, S2, and context in ANT group. (C) Freezing time% during pre-shock, shock, and post-shock in VEH control group and ANT group in S1. (D) Freezing time% during pre-shock, shock, and post-shock in VEH control group and ANT group in S2. (E) Freezing time% during pre-shock, shock, and post-shock in VEH control group and ANT group after re-exposure to the fearful context alone. **P < 0.01; ***P < 0.001.

DISCUSSION

Consistent with previous work in rats1,2,34 and mice,33,35 training with inescapable footshock and re-exposure to the shock context alone reduced REM in the following sleep records. Contextual fear resulted in significant reductions in REM in animals receiving VEH microinjections into CNA. However, the effect of contextual fear on REM was attenuated in animals receiving microinjections of ANT into CNA. The attenuation of the reduction in REM with ANT was associated with reduced c-Fos expression in PVN and LC and occurred even though microinjections of ANT into CNA did not significantly alter freezing in the fearful context compared to VEH controls. These results demonstrate a significant role for CRF receptors in CNA in regulating fear-induced changes in REM and also in regulating activation of brain regions implicated in stress and the control of REM. By comparison, the effects on NREM were less, though there was a significant difference between rats receiving VEH and ANT in the first 4 hours of recording.

Amygdala, CRF, and Fear-Induced Reductions in Sleep

We recently demonstrated that ICV administration into mice of the nonspecific CRF antagonist astressin attenuated contextual fear-induced reductions in REM, whereas ICV administered CRF exacerbated the reduction in REM.36 However, there has been very little work with respect to the effects of local application of CRF compounds into the brain on sleep. Microinjections of a 1.0 ng dosage of CRF into CNA of rats decreased average amounts of REM over 4 h post-injection.37 Recent in vitro work has implicated CRF in the modulation of excitatory glutamatergic synaptic transmission in CNA, with CRF1 and CRF2 receptors apparently playing inhibitory and facilitatory roles, respectively.38 ANT is a relatively specific CRF1 antagonist, suggesting that the primary effect may have been blocking this inhibition. This conclusion is consistent with findings of studies showing that inhibition of CNA reduces REM.8,9

The present results complement a previous study in which we treated rats with microinjections of muscimol, bicuculline, or saline vehicle into CNA prior to training with inescapable footshock.10 In rats treated with saline, training with inescapable footshock selectively reduced electrographically defined REM and increased c-Fos expression in LC compared to rats that received a microinjection of vehicle alone. Rats treated with muscimol, which temporarily inactivates neurons in CNA, also showed reduced REM and increased c-Fos expression in LC. By comparison, microinjection of bicuculline into CNA prior to shock training attenuated the reduction in REM and also attenuated c-Fos expression in LC.

However, even though we found that microinjections into CNA could block fear-induced reductions in REM, we have found no evidence that ANT can impact REM when microinjected into the amygdala of non-stressed rats. For example, microinjections of ANT into CNA (1.6 mM or 4.8 mM in a volume of 0.2 μL), into the basolateral amygdala (1.6 mM or 4.8 mM in a volume of 0.5 μL), or in the other major output of the amygdala, the bed nucleus of the stria terminalis (1.6 mM or 4.8 mM in a volume of 0.2 μL), did not significantly alter REM in non-stressed animals (Unpublished Results).

Amygdala, CRF, and Fear-Induced Alterations of c-Fos Expression in Stress and Arousal Circuitry

Noradrenergic neurons in LC and serotonergic neurons in DRN are central in current understanding of the regulation of REM, and they play important roles in regulating the stress response. Both types of neurons are virtually silent during REM, and this inactivity is thought to be permissive for REM.13 Thus, activation of noradrenergic and serotonergic neurons could be associated with reduced REM. However, only the LC showed a significant correlation between c-Fos expression and REM amounts. The LC in rats is primarily composed of noradrenergic neurons,39 whereas DRN contains a heterogeneous population of cell types,40 many of which do not show state-related changes in firing in association with REM.41,42 Thus, a factor may be that LC allowed a more direct correlation between REM and cell types considered important in its generation.

PVN is the final common pathway for information influencing the HPA axis43,44 and is a key site for integrating neuroendocrine, autonomic, and behavioral responses to stress.45–47 While we did not measure corticosterone as an indicator of peripheral HPA activation, c-Fos activation in PVN has been interpreted as an indicator of the amplitude of the stress response and the degree of activation of the HPA axis.48,49 The reduction of c-Fos in PVN with microinjections of ANT in CNA thus suggests the potential for an alteration in peripheral markers of stress, though this will need empirical verification.

CNA has minimal direct projections to PVN50 and lesions of CNA do not directly influence PVN activation.51 However, CNA can influence PVN via trans-synaptic pathways through the dorsomedial hypothalamic and bed nucleus of the stria terminalis,50,52 which can inhibit PVN and reduce ACTH secretion.44 CNA projections to LC and DRN53–57 potentially could also impact PVN. PVN receives a large noradrenergic projection from brainstem A1 and A2 groups and a smaller projection from LC.58 Lesions of LC reduce ACTH and corticosterone responses to acute stress.59 DRN has serotonergic projections to PVN,60,61 and serotonin agonists enhance PVN activity as indicated by increased plasma corticosterone levels and c-Fos expression.62 Indirect pathways may also play a role in serotonergic and noradrenergic regulation of PVN.63

Amygdala, CRF, and Freezing

Behavioral freezing has been used to evaluate fear and fear memory, with greater FT% being interpreted as indicating stronger fear reactions.25–29 FT% was almost nonexistent and did not differ between groups in the initial pre-shock period. Thus, within-subject increases of FT% after the initial pre-shock period indicates that the shock chamber acquired fear-inducing characteristics after presentation of shock. Shock training resulted in similar significant increases in FT% compared to the pre-shock period in both groups of rats. FT% was also enhanced during context re-exposure (no shock presented) compared to the pre-shock period. However, compared to VEH, microinjections of ANT into CNA did not significantly alter FT% during context re-exposure, even though the post-exposure reduction in REM was significantly attenuated.

We have demonstrated that continued freezing in a fearful context is associated with reduced REM, whereas extinguished fear in a fearful context is associated with normalized post-exposure REM.64 Thus, the apparent disassociation of freezing and later REM amounts may appear surprising. However, freezing and REM are also dissociated in mice trained with escapable and inescapable footshock. We found that BALB/cJ mice trained with escapable footshock showed significant post-training increases in REM, whereas yoked control mice receiving inescapable footshock showed significant reductions in REM. Re-exposure to the shock context was also followed by enhanced REM in mice trained with escapable footshock, whereas those trained with inescapable footshock showed reduced REM. Significant freezing was observed in both instances suggesting that directionally different changes in REM could occur even with substantial behavioral evidence of fear. Indeed, freezing can be greater after escapable footshock than after inescapable footshock.65

Previous work reported that antagonism of CRF receptors in CNA with α-helical CRF9-41 reduced stress-induced freezing that occurred immediately after shock presentation and in response to contextual fear alone.66 High dosages of the selective CRF1 antagonist, NBI27914 microinjected into CNA also reduced shock induced freezing in rats.67 Procedural differences and the considerably more extensive training protocol we used may account for some of the differences between the present experiment and these previous studies. The previous studies presented shock in one session that consisted of three 1.0 to 1.5 mA footshocks administered at 20-sec intervals, whereas we presented twenty 0.8 mA footshocks on each of two training days. Another potentially critical difference was the greater volume (1.0 μL,66 0.5 μL67) microinjected into CNA in the previous studies compared to the volume (0.2 μL) that we microinjected. These greater volumes make it much more likely that diffusion of the drugs went further beyond the boundaries of the relatively small CNA and influenced other regions of the amygdala, such as the adjacent basolateral amygdala, which has greater densities of CRF receptors than does CNA.68,69 However, our data are consistent with a recent report that inactivation of CNA by muscimol or microinjection into CNA of CRF antisense oligonucleotides did not impair contextual freezing during a retention test.24

CONCLUSION

Compared to microinjections of VEH, microinjections of ANT into CNA attenuated the reductions in REM that occur after exposure to a fearful context and reduced fear-induced c-Fos expression in PVN and LC. These effects were independent of fear-induced freezing, suggesting that CRF in CNA is an important regulator of post-stress alterations in REM as well as stress-induced activation in the brain, but not fear memory.

DISCLOSURE STATEMENT

This was not an industry supported study. The authors have indicated no financial conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by NIH research grants MH64827 and MH61716.

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