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GABA and REM Sleep

GABAergic Antagonism of the Central Nucleus of the Amygdala Attenuates Reductions in Rapid Eye Movement Sleep After Inescapable Footshock Stress Xianling Liu, MD, PhD; Linghui Yang, MS; Laurie L. Wellman, PhD; Xiangdong Tang, PhD; Larry D. Sanford, PhD Sleep Research Laboratory, Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk, VA

Study Objectives: Rapid eye movement sleep (REM) appears to be especially susceptible to the effects of stress; inescapable footshock stress (IS) can produce reductions in REM that can occur without recovery sleep. The amygdala has well-established roles in stress and emotion; the central nucleus of the amygdala (CNA) projects to REM regulatory regions in the brainstem and has been found to play a key role in the regulation of REM. The objective of this study was to determine whether the reduction in REM induced by IS could be regulated by CNA and brainstem regions. Design: The GABAergic agonist muscimol (MUS) and GABAergic antagonist bicuculline (BIC) were microinjected into CNA before IS, and sleep was recorded for 20 h. In a second experiment using the same manipulations, sleep was recorded for 2 h, after which the rats were killed to evaluate Fos expression (a marker of neuronal activity) in the locus coeruleus (LC), a brainstem REM regulatory region. Setting: NA.

Patients or Participants: The subjects were male, outbred Wistar rats. Interventions: The rats were surgically implanted with standard electrodes or with telemetry transmitters for determining arousal state. Measurements and Results: IS preceded by control or MUS microinjections selectively reduced REM and increased Fos expression in LC. By comparison, microinjection of BIC into CNA prior to IS attenuated both the reduction in REM and Fos expression in LC to levels seen in non-shocked controls. Conclusions: The results suggest that the effects of IS on REM may involve local GABAergic inhibition in CNA and activation of LC. Keywords: GABA, footshock, stress, REM sleep, central amygdala, locus coeruleus Citation: Liu X; Yang L; Wellman LL; Tang X; Sanford LD. Gabaergic antagonism of the central nucleus of the amygdala attenuates reductions in rapid eye movement sleep after inescapable footshock stress. SLEEP 2009;32(7):888-896.

RAPID EYE MOVEMENT SLEEP (REM) APPEARS TO BE ESPECIALLY SUSCEPTIBLE TO THE EFFECTS OF STRESS.1,2 FOOTSHOCK IS A WIDELY USED STRESSOR in studies involving rodents, and presentation of inescapable footshock stress (IS) to rats and mice can produce subsequent significant decreases in REM after the stressor is removed.2-4 The decreases in REM may be relatively long lasting and can vary with the reactivity and emotionality of rat4 and mouse2,3 strains. By comparison, the effects on NREM have greater variability with decreases, no significant alterations, or increases in NREM amounts, depending on strain.2-4 The pons contains regions critical for the generation and regulation of REM.5 The locus coeruleus (LC) is activated by footshock,6,7 and it is thought to be a major REM regulating region.5 The firing of LC noradrenergic neurons has been linked to the suppression of REM, whereas their silence (along with the silence of serotonergic neurons in the dorsal raphe nucleus) has been hypothesized to permit REM.5,8 Activation of LC neurons also occurs in response to stressful stimuli.9,10 Forebrain regions can also have significant influences on the generation of REM. The central nucleus of the amygdala (CNA), in particular, has been linked to the regulation of REM.11-15 The amygdala has also been prominently linked to fear be-

havior that occurs in response to IS16,17 and to the regulation of arousal.13,15,18 CNA output is tightly regulated by GABA.19 Microinjection into CNA of the GABAA agonist, muscimol (MUS), selectively decreases REM; whereas microinjection of the GABAA antagonist, bicuculline (BIC), selectively increases REM.12 These findings suggest that GABAergic inhibition of CNA suppresses REM, whereas blocking GABAergic inhibition of CNA promotes REM. CNA has direct projections to the LC20,21 that may be important for certain stress responses.22 In a previous study in mice, we found that training with IS enhanced Fos expression in the LC and in some regions of the amygdala.23 In the amygdala, the CNA failed to show enhanced Fos expression after IS,23 a finding that complements other studies reporting that CNA does not show enhanced Fos expression after certain types of stress.24,25 Intense Fos activation in LC and lack of significant Fos expression in CNA after IS are compatible with the hypothesis that activation of the noradrenergic LC and inhibition of CNA may be factors in the IS-induced suppression of REM. Thus, the purpose of this study was to determine whether CNA and LC participate in reducing REM after IS. To determine whether reduced REM following IS could involve regulation by CNA, we microinjected MUS and BIC into CNA prior to IS and compared post-shock sleep to sleep exhibited by non-shocked rats receiving control microinjections alone and to shocked rats receiving control microinjections. Similar IS-induced alterations in REM after microinjections of MUS and vehicle and attenuation of the reduction after microinjections of BIC would suggest that inhibition of CNA is a significant factor in the effects of IS on REM. In a second experiment, we conducted the same manipulations in different groups of rats but killed the animals after 2 h of sleep recording

Submitted for publication October, 2008 Submitted in final revised form March, 2009 Accepted for publication March, 2009 Address correspondence to: Larry Sanford, PhD, Sleep Research Laboratory, Department of Pathology and Anatomy, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23507; Tel: (757) 446-7081; Fax: (757) 446-5719; E-mail: [email protected] SLEEP, Vol. 32, No. 7, 2009

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to examine Fos protein expression as a marker of neuronal activity in the LC. Enhanced Fos expression in the LC associated with reduced REM would suggest a suppressing role for noradrenergic mechanisms in LC. Attenuation of the REM reduction and Fos expression in the LC after BIC microinjected into CNA would suggest that the CNA-LC pathway is important in the regulation of REM in the aftermath of IS.

The study was completed utilizing separate shock training and recording sessions for different cohorts of rats. Microinjections BIC (1[S],9[R]-[−]-bicuculline methiodide) and MUS (muscimol hydrobromide, 5-aminomethyl-3-hydroxyisoxazole) were obtained from Sigma-Aldrich, St. Louis, MO, USA. All solutions were prepared at the desired concentrations in pyrogen-free saline. For microinjections, injection cannulae (33 ga.) 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 injected bilaterally and infused slowly over 2 min (0.1 µL /min). Each animal was recorded under each condition in its respective drug study.

MATERIALS AND METHODS Experiment 1 Subjects The subjects were 32 male Wistar rats 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

Footshock Training

The rats were implanted with skull screw electrodes (A: 2.0 [Bregma], L: 1.5; P: 7.0 [Bregma], L: 1.5 contralateral) for recording the electroencephalogram (EEG); stainless steel wire electrodes were sutured to the dorsal neck musculature for recording the 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 ga.; Plastics One Inc., Roanoke VA, USA) 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).26 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 weight) 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 (Protocols #04-005 and #07005).

Rats were placed in the shock chambers (Coulbourn Habitest cages; Coulbourn Instruments, Whitehall, PA, USA) 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 minutes after which they were presented with 15 footshocks (0.2 mA, 0.5-s duration) at 1.0min intervals over the course of 15 min. Shock was produced by Coulbourn Precision Regulated Animal Shockers (Model E1314) 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 duration of the entire procedure was 25 min. The shock chamber was thoroughly cleaned with diluted alcohol prior to each training session. All experimental manipulations were conducted in the 3rd h after lights on, and sleep recording began at the beginning of the 4th h. Sleep Recording, Scoring, and Data Analysis 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 (West Warwick, RI), Model 12 polygraph equipped with model 12A5 amplifiers. 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 either wakefulness, NREM, or REM based on EEG and gross whole body activity using standard electrographic criteria.12,15,27 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

Procedure After recovery from surgery, the rats were habituated to the handling and recording procedures over 2 consecutive days. The rats were then randomly placed in 1 of 4 microinjection and IS groups: SAL/CON (saline control [0.2 µL] with no footshock, n = 8); SAL/SHK (saline [0.2 µL] plus footshock, n = 8); MUS/SHK (MUS [1.0 uM/0.2 µL] plus footshock, n = 8); BIC/SHK (BIC [333 pM/0.2 µl] plus footshock, n = 8). Following microinjection, all animals received 15 footshocks. The rats were then returned to their home cage, and their undisturbed wakefulness and sleep was recorded for 20 h. One to 2 rats were recorded concurrently in each microinjection treatment group. SLEEP, Vol. 32, No. 7, 2009

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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. Statistical analyses were conducted using SigmaStat (SPSS, Inc, Chicago, IL, USA). Comparisons between groups were conducted using one-way between factors ANOVAs for each 4-h time block. Post hoc tests following significant ANOVAs were conducted with Tukey tests. Comparisons across time were not conducted, as these values would be expected to differ simply as a matter of the passage of time.

Histology

Histology

Fos Immunohistochemistry Staining

To localize the microinjection sites, brain slices (50 µm) were made through the amygdala, and the sections were mounted on slides and stained with cresyl violet. The sections were then examined in conjunction with a stereotaxic atlas26 to confirm cannulae placements.

Fifty-µm thick coronal sections were cut from frozen blocks from AP –0.30 to –1.30 mm of the brain stem. Every fifth section was collected as a sample. One sample was used for Fos staining. A second sample was used as a blank control. Free floating methods were used for immunohistochemical staining as described previously.23 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 Fos antibody (1:20,000, Ab5, 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 avidinbiotin 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 on the slides for 48 h. Then sections were dehydrated through graded alcohol, cleared by xylene, and protected by cover slides 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.

Immediately upon completion of the sleep recording, the rats were perfused to obtain brain tissue for Fos immunohistochemistry and to determine the localization of the microinjection site in CNA as described in Experiment 1. 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). 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.

Experiment 2 Subjects The subjects were 19 male Wistar rats approximately 90 days of age at the time of surgery. Husbandry and housing was as described in Experiment 1. Surgery The rats were implanted with a telemetry transmitter (ETA10F20; DataSciences, St. Paul MN, USA) for recording EEG and activity via telemetry as described previously.28,29 The body of the transmitter was implanted subcutaneously off midline and posterior to the scapula, and it was attached to the skin with 3 sutures for stabilization. Leads from the transmitter were led subcutaneously to the skull and the bare ends placed in contact with the dura through holes in the skull. Guide cannulae (26 ga.) for microinjections into CNA were implanted as described above. Surgical procedures, anesthesia, and recovery were as described in Experiment 1. Procedure

Data Quantification and Analyses

After recovery from surgery, the rats were habituated to the handling and recording procedures over 2 consecutive days. The rats were then randomly placed in one of 4 groups (SAL/ CON, n = 5; SAL/SHK, n = 4; MUS/SHK, n = 5; BIC/SHK, n = 5); microinjections and IS training was performed as described in Experiment 1. The rats were then returned to their home cages, and their undisturbed wakefulness and sleep was recorded for 2 h. They were then killed and the brains processed for Fos immunohistochemistry in LC. Sleep recording and scoring and data analysis were conducted using procedures similar to those described in Experiment 1, except that the analyses were limited to the first and second h of recording. Post hoc comparisons following significant ANOVAs were conducted with Tukey tests. SLEEP, Vol. 32, No. 7, 2009

Fos expression was visualized in brain sections of LC using a Nikon Eclipse E800 microscope. The sections were standardized as much as possible using a rat brain atlas.26 Digital photographs of the selected LC regions were taken with a Spot digital camera attached to Nikon Eclipse E800 microscope under 10 X magnification. Fos positive cells in LC were counted by a researcher blind to experimental condition with MetaMorph Image Analysis program. Fos expression in LC was summarized of 3 sections and was expressed as number of Fos positive cells in LC. Comparisons between groups were conducted using one-way between-factors ANOVAs. Post hoc tests following significant ANOVAs were conducted with Tukey tests. 890

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Figure 2

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Figure 2—Total REM, number of REM episodes, duration of REM episodes, and REM percentage (of total sleep) plotted in 4-h blocks over the 20-h recording period for the saline control without shock (SAL/CON, n = 8) and bicuculline shock (BIC/ SHK, n = 8) conditions. There were no significant differences in REM between groups.

Figure 1—Line drawings illustrating the rostral-caudal extent of microinjection sites in the central nucleus of the amygdala (CNA) for animals studied in Experiment 1. Saline control without shock (●: SAL/CON, n = 8); saline shock (▲: SAL/SHK, n = 8); muscimol shock ( : MUS/SHK, n = 8); bicuculline shock (■: BIC/ SHK, n = 8).

period in any REM parameter we examined (Figure 2). In addition, comparisons among groups revealed virtually identical differences between the SAL/SHK and MUS/SHK groups and the BIC/SHK and SAL/CON groups. Therefore, for clarity only the groups that received footshock are presented in Figure 3, which compares the effects of vehicle, MUS, and BIC on REM after IS. In general, IS produced a significant reduction in total REM (Figure 3A), number of REM episodes (Figure 3B), and REM duration (Figure 3C) in the first 4-h block in the SAL/SHK and MUS/SHK groups compared to the BIC/SHK group and SAL/ CON group (not shown). The only exception to this pattern was that the reduction in the number of REM episodes in the SAL/SHK group did not reach significance compared to either SAL/CON or BIC/SHK, whereas the reduction in the number of REM episodes in the MUS/SHK group was significantly reduced compared to all other treatment groups, including the SAL/SHK group. Significant differences were not found in any analyses of REM in the other 4-h blocks. Post hoc analyses of REM percentage found significant reductions in both SAL/SHK and MUS/SHK groups compared to both BIC/SHK (Figure 3D) and SAL/CON (not shown) groups during the first, second, and third 4-h blocks. In the fourth 4-h block, REM percentage in the MUS/SHK group was significantly reduced compared to the BIC/SHK group. Though there were some slight reductions in NREM amounts that may have contributed to the significant increase in REM percentage, no significant differences among groups were found for any of the analyses for NREM parameters, total sleep (Figure 4), sleep efficiency, or amount of wakefulness (not shown).

RESULTS Experiment 1 Microinjection Sites Figure 1 shows the location of the microinjections sites in the amygdala. Though there were rostral-caudal variations in the placements among animals, the histology indicated that drug or SAL would have been infused into CNA and adjacent areas in all the rats. Therefore, all 32 rats were used in the analysis of sleep. Effects on MUS and BIC Microinjections on Footshock-induced Alterations in Sleep The ANOVAs for the first 4-h block were significant for total REM (F3,28 = 11.73, P < 0.001), number of REM episodes (F3,28 = 12.78, P < 0.001), and REM duration (F3,28 = 8.56, P < 0.001). The ANOVAs for REM percentage were significant for the first (F3,28 = 1.00, P < 0.001), second (F3,28 = 13.41, P < 0.001), third (F3,28 = 5.95, P < 0 .003), and fourth (F3,28 = 3.73, P < 0.022) 4-h blocks. The ANOVA for the fifth 4-h block did not reach significance, (P = 0.073). The post hoc tests found no significant differences between the SAL/CON and BIC/SHK groups during any measurement SLEEP, Vol. 32, No. 7, 2009

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Figure 4

Figure 3 A. Total REM

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Figure 3—Total REM, number of REM episodes, duration of REM episodes, and REM percentage (of total sleep) plotted in 4-h blocks over the 20-h recording period for the saline control without shock (SAL/CON, n = 8), muscimol shock (MUS/SHK, n = 8) and bicuculline shock (BIC/SHK, n = 8) groups. Comparisons of SAL/SHK and BIC/SHK (*, P < 0.05; **, P < 0.01; ***, P < 0.001); comparisons of MUS/SHK and BIC/SHK (+, P < 0.05; ++, P < 0.01; +++, P < 0.001) and comparisons of SAL/SHK and MUS/SHK (#, P < 0.05). Comparisons were conducted with Tukey tests.

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Figure 4—Total NREM, number of NREM episodes, duration of NREM episodes and total sleep plotted in 4-h blocks over the 20-h recording period. Saline control without shock (SAL/CON, n = 8); saline shock (SAL/SHK, n = 8); muscimol shock (MUS/SHK, n = 8); bicuculline shock (BIC/SHK, n = 8). No significant differences were found for any of these measures.

SAL/CON (7.20 ± 1.32; P < 0.001) treatment group (Figure 6B). Fos expression in LC was also greater in the BIC/SHK (19.00 ± 1.81) group than in the SAL/CON (P < 0.01) group, but was significantly less than in the SAL/SHK and MUS/SHK groups (P < 0.001). The difference among groups is also demonstrated in Figure 6 D-G, which present representative samples showing Fos immunohistochemistry in LC in each experimental condition.

Experiment 2 Microinjection Sites Figure 5 shows the location of the microinjections sites for the rats studied in Experiment 2. The histology indicated that drug or SAL would have been infused into CNA and immediately adjacent areas in all the rats and all 19 rats were used in the analysis of sleep and Fos expression in LC.

Correlation between REM Amounts and Fos Expression in LC The reciprocal relationship between amounts of REM in hour 2 of recording and amounts of Fos expression in LC are demonstrated in Figure 6A and B. All 19 rats were used in an analysis that examined the correlation between the amount of REM and amount of Fos expression in LC. This analysis revealed that the amount of Fos expression in LC was significantly and negatively correlated with the amount REM the rats exhibited during hour 2 (r = −0.834, P < 0.001). This relationship is demonstrated graphically in scatterplot form in Figure 6C.

Effects on Sleep Comparisons were made for amounts of sleep within each h of the 2 h of sleep recording that was conducted after IS. REM sleep in hour 1 did not differ across treatment condition (P > 0.05). However, in hour 2 (Figure 6A), REM in the SAL/ SHK (1.02 ± 0.28) and MUS/SHK (0.58 ± 0.41) treatment groups was significantly less than in the SAL/CON (5.05 ± 0.68; P < 0.01) and BIC/SHK (5.18 ± 0.9; P < 0.01) treatment groups. The amount of REM in hour 2 did not significantly differ between the SAL/CON and BIC/SHK (P > 0.05) treatment groups. NREM did not significantly differ across experimental treatment groups in either hour 1 or 2 (P > 0.05).

DISCUSSION Training with IS selectively reduced electrographically defined REM and increased Fos expression in LC. GABAergic inhibition of CNA with microinjections of MUS prior to IS did not block the effects of IS on sleep or on Fos expression in LC. By comparison, microinjection of BIC into CNA prior to IS attenuated the reduction in REM and also attenuated Fos expression in LC. The results suggest that the reduction in REM in the aftermath of IS may involve local GABAergic inhibi-

Fos Expression in LC Fos expression in LC was greater in the SAL/SHK (40.25 ± 1.84) and MUS/SHK (48.20 ± 2.35) treatment groups than the SLEEP, Vol. 32, No. 7, 2009

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Figure 5

Figure 6 A REM in H 2

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Figure 5—Line drawings illustrating the rostral-caudal extent of microinjection sites in the central nucleus of the amygdala (CNA) for animals studied in Experiment 2. Saline control without shock (●: SAL/CON, n = 5); saline shock (▲: SAL/SHK, n = 4); muscimol shock ( : MUS/SHK, n = 5); bicuculline shock (■: BIC/ SHK, n = 5).

Figure 6—Minutes of REM in the second recording hour (H2, A) and number of Fos granules counted in LC examined at the end of H2 (B) plotted for each treatment condition. The correlation between REM in H2 and Fos counts in LC is shown in scatterplot form (C). Example sections showing Fos immunohistochemistry in LC for each experimental condition are shown in panels D-G. Saline control without shock (SAL/CON, n = 5); saline shock (SAL/ SHK, n = 4); muscimol shock (MUS/SHK, n = 5); bicuculline shock (BIC/SHK, n = 5). **, P < 0.01; ***, 0.001 compared to SAL/CON; ++, P < 0.01; +++, 0.001 compared to SAL/SHK, ###, P < 0.001 compared to MUS/SHK. Comparisons were conducted with Tukey tests. 4V: fourth ventricle; me5: motor trigeminal nucleus.

tory mechanisms in CNA and activation of LC. By comparison, the relative lack of effects on NREM amounts suggests that GABAergic regulation of CNA is minimally involved in stressinduced changes in NREM. Amygdala and the Regulation of Sleep and Arousal

Fos Expression in LC After Footshock

The attenuation of the reduction in REM after IS by microinjections of BIC (but not MUS) into CNA suggests that IS produces a GABAergic inhibition of CNA descending output that regulates the stress-induced reduction in REM. Normal sources of GABAergic inhibition of CNA projection could arise from the intercalated cell bodies, which are small GABAergic cell clusters30-32 located between the basolateral complex and CNA33 that have been shown to exert inhibitory control over CNA.34,35 Other potential sources of GABAergic inhibition include the lateral CNA32,34,35 and the caudal portion of the medial CNA.36 The inputs that regulate GABAergic inhibition of CNA and resulting decrease in REM in the aftermath of IS have not been determined; however, the intercalated cell bodies receive excitatory glutamatergic input from regions that have been implicated in mediating the effects of stress, including the basal and lateral nuclei of the amygdala and the infralimbic portion of the ventromedial prefrontal cortex (reviewed by Akirav37). SLEEP, Vol. 32, No. 7, 2009

Fos protein has been widely used to detect neuronal populations that are “activated” during a variety of behavioral and stressful paradigms including footshock.38-41 Fos has low basal expression levels in most neural systems and is rapidly upregulated in response to stimuli. Fos is typically reported to peak around 90 to 120 minutes after stimuli followed by a rapid decline.42 However, Fos expression also may be prolonged or delayed.43 We observed elevated Fos in LC at 2, 4, and 6 h after shock training in BALB/cJ mice, using a virtually identical IS paradigm to the one used in the current study. These mice also had significantly decreased REM for up to 4 h after experiencing IS, suggesting that LC activation (as indicated by Fos) was related to the decrease in REM. The LC in rats is primarily composed of noradrenergic neurons.44 Thus, the hypothesized role of the LC in regulating REM5 and the strong correlation between Fos expression and reduced amounts of REM that we observed suggest that activation of noradrener893

Amygdala Regulation of Sleep After Stress—Liu et al

gic neurons in LC may play a significant role in the inhibition of REM after IS. The excitatory effects of footshock and other noxious stimuli on LC also appear to be mediated by the nucleus paragigantocellularis (PGi) in the rostral medulla6,7,45 via an excitatory amino acid pathway.6,45 Injections of lidocaine, GABA, or a synaptic decoupling solution into the PGi eliminates responses of LC neurons to sciatic nerve stimulation or foot- or tail-pinch46 and activation of the LC by intravenous administration of nicotine.47 Blocking PGi also blocks the response of individual LC neurons to footshock.6,7 There is also an inhibitory projection from PGi to LC48 and recent work has reported REM-on neurons in the dorsal PGi that may be inhibitory to LC and other wake promoting regions.49 The LC has been implicated in alertness and attention as well as in the regulation of arousal,50 and it has been suggested that PGi may integrate a wide range of sensory information in order to foster behaviorally adaptive responses in urgent situations.51 The higher Fos expression in the LC of rats in the BIC/SHK group compared to the SAL/CON group suggests that microinjections of BIC in CNA did not completely block IS activation of LC that may have arisen via the PGi-LC pathway. If so, initial activation of the putative “urgent response” system may not have been significantly affected, and the attenuation of activity in LC, as indicated by reduced Fos expression, may have occurred in the period after IS was completed.

tion, the activation of LC neurons by electrical stimulation in the vicinity of CNA may be mediated by fibers of passage. Implications for the Influence of Stress on Sleep The purpose of the stress response is to restore homeostasis.57 Therefore, recovery sleep after stress may reflect the restoration of homeostasis as the stress response follows its normal course, and thus may play a positive functional role in coping with stress.4 Indeed, stressors such as avoidable footshock,58,59 novel object,15,60 open field,15,61 ether exposure,62 cage change,15,61 and social stress63 induce initial periods of arousal that are often followed by subsequent sleep recovery, including a period of enhanced REM. By comparison, the increased arousal that occurs after IS can occur without subsequent recovery REM,4,64 and we have suggested that the relatively unique lack of REM rebound may play a role in the development of pathologies that have been linked uncontrollable stress.4 In this study, the immediate suppression of REM after IS in the SAL/SHK and MUS/SHK groups was followed by a return to control levels. However, no rebound was observed as significant differences between groups for total REM were found only in the first 4-h block. Indeed, REM percentage in the SAL/SHK and MUS/SHK groups remained reduced in the second and third 4-h block for the SAL/SHK group and all but the last 4-h block of the dark period in the MUS/SHK groups. By comparison, we recently found that training with escapable footshock in a shuttlebox, in a paradigm in which rats always received shock but could terminate it by moving to a safe chamber, led to significant post-stress increases in REM amounts and REM percentage.65 These increases occurred on 2 successive training days that also were characterized by significant decreases in NREM and total sleep during the light period. The changes in REM amounts and REM percentage did not appear to be due simply due to a recovery of lost sleep but were long-lasting increases that were observed in both light and dark periods. Our study with escapable footshock also demonstrated that inactivation of CNA with microinjections of MUS following training with escapable footshock prevented the increase in REM, whereas microinjections of saline did not. Together, these studies suggest that CNA plays a significant role in regulating REM in the aftermath of stress.

CNA Regulation of LC Previous studies found that footshock activates almost all subregions of the amygdala except CNA.23,25 Both IS and pharmacological inhibition of CNA are associated with significant reductions in REM, suggesting that CNA was inhibited following footshock. This hypothesis is consistent with our current finding that inactivation of CNA with muscimol prior to IS produced reductions in REM similar to, or greater than, those produced by footshock preceded by control microinjections. Tract tracing studies have demonstrated that CNA has direct projections to LC.20,21 Fibers from CNA terminate on noradrenergic dendrites in the rostrolateral periceruleus.52 Functional connectivity has been illustrated by studies that have electrically stimulated CNA and recorded in neurons in LC. Short highfrequency trains (200 Hz) delivered at 800 μA in CNA evoked phasic responses in 90% of the neurons recorded in LC.53 Activation of LC after electrical stimulation of LC appears to be at odds with the present results. One possibility is that different populations of neurons could be affected; this seems unlikely, however, as both MUS and TTX produce nonselective inactivation of neurons. However, electrical simulation also stimulates fibers of passage, and fiber bundles surrounding and coursing through CNA54 were most likely affected. This would affect output from CNA as well as the output from the basal amygdala that is mediated by fibers running through CNA toward the stria terminalis.55,56 Target regions for both include the lateral hypothalamus and basal forebrain, though it is currently thought that only CNA output reaches brainstem regions involved in regulating REM.54 Inactivation of both cell bodies and fibers of passage with TTX produce significant reductions in arousal as well as decreases in REM.15 As activity of LC is associated with arousal and attenSLEEP, Vol. 32, No. 7, 2009

Conclusion The results suggest that reductions in REM after IS involve GABAergic regulation of CNA and activation of LC. The attenuation of IS-induced reductions in REM and elevations in Fos expression by prior microinjections of BIC are consistent with the hypothesis that CNA is an important region for modulating the impact of stress on REM. Further studies are needed to determine the role CNA plays in the alterations in REM produced by other stressors and to determine the potential role that REM plays in recovery from stress. Acknowledgments This work was supported by NIH research grants MH64827 and MH61716. 894

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Disclosure Statement

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