Neural circuitry of stress-induced insomnia in rats

Abstract

Sleep architecture is often disturbed after a stressful event; nevertheless, little is known about the brain circuitry responsible for the sleep perturbations induced by stress. We exposed rats to a psychological stressor (cage exchange) that initially causes an acute stress response, but several hours later generates a pattern of sleep disturbances similar to that observed in stress-induced insomnia in humans: increased sleep latency, decreased non-REM (nREM) and REM sleep, increased fragmentation, and high-frequency EEG activity during nREM sleep. We examined the pattern of Fos expression to identify the brain circuitry activated, and found increased Fos in the cerebral cortex, limbic system, and parts of the arousal and autonomic systems. Surprisingly, there was simultaneous activation of the sleep-promoting areas, most likely driven by ongoing circadian and homeostatic pressure. The activity in the cerebral cortex and arousal system while sleeping generates a novel intermediate state characterized by EEG high-frequency activity, distinctive of waking, during nREM sleep. Inactivation of discrete limbic and arousal regions allowed the recovery of specific sleep components and altered the Fos pattern, suggesting a hierarchical organization of limbic areas that in turn activate the arousal system and subsequently the cerebral cortex, generating the high-frequency activity. This high-frequency activity during nREM was eliminated in the stressed rats after inactivating parts of the arousal system. These results suggest that shutting down the residual activity of the limbic-arousal system might be a better approach to treat stress-induced insomnia, rather than potentiation of the sleep system, which remains fully active.

Figure 1.

A, Stress-induced hyperthermia peaks at 10:30 A.M. in rats transferred to a clean cage (0.95 ± 0.14°C) or a dirty cage (1.29 ± 0.09°C) at 10:00 A.M. and returns to basal levels after 1 h. During the sleep disturbance period, temperature is slightly higher in cage exchange (Cage Exch) rats (0.2°C), but not significantly different from controls. For the sake of clarity, error bars have been omitted in this graph that plots temperature every 5 min, but none of the time points is statistically different between the two groups. B–H, Fos counts in several parvicellular subdivisions of the PVH are higher in rats killed 90 min after cage exchange (Cage Exc; D) than in rats killed 5.5 h after cage exchange (F, H) and rats killed 90 min after being transferred to a clean cage (Clean Cg; C), which have similar numbers of Fos counts. Clean cage controls killed after 5.5 h (E) showed little Fos immunoreactivity. Photomicrographs show Fos expression in all these experimental groups at the level of the caudal mpPVH (bregma, −2.12 mm) as shown in the template (G) modified from Figure 26 of the rat brain atlas by Paxinos and Watson (1998) with permission from Elsevier. Rats killed 5.5 h after cage exchange showed high variability in Fos expression, ranging from rats with little Fos expression (F) to animals with extensive Fos (H). In B, data for each parvicellular subdivision were analyzed with one-way ANOVA followed by Fisher's PLSD (horizontal bars at top indicate significant differences, p < 0.05). All values are the mean ± SEM. Scale bar, 100 μm. 3V, Third ventricle.

Figure 2.

Species-specific stress causes initial and late sleep disturbances (acute insomnia). A, C, Rats that are placed in a cage previously occupied by another male rat (cage exchange) sleep significantly less during the first and second hours (initial stress response) and during the fifth and sixth hours (stress-induced acute insomnia) after cage exchange. E, There is loss of REM sleep across the entire period. B, D, F, The number of nREM bouts is not different (D) between controls and stressed rats, but there are fewer transitions from nREM to REM sleep (F), and more to wakefulness (B), indicating a period of sleep fragmentation. The x-axis is marked in hours before or after the cage exchange. Data were analyzed by repeated-measures ANOVA, followed by unpaired t tests for comparisons at each time point. *p < 0.05; ⁽*⁾p between 0.05 and 0.09. All values are the mean ± SEM.

Figure 3.

Rats typically sleep mainly during the light cycle, from 7:00 A.M. to 7:00 P.M. At 10:00 A.M. (peak of sleep), we place a male rat in a dirty cage that has been previously occupied by another male rat for 1 week, which induces a generalized stress response (fight or flight). Control rats are placed in clean cages. Stressed rats are mostly awake during the first 2 h after cage exchange compared with controls. The next 2 h, stressed rats sleep more or less normally as a result of the homeostatic and circadian drives. This is followed by a 2 h period of sleep disturbances in stressed rats in which they sleep ∼20% less than controls. REM sleep is decreased along the whole period after cage exchange. This period of stress-induced acute insomnia 4–6 h after cage exchange (from 2:00 to 4:00 P.M.) is consistently observed in all rats. To assess what brain areas are activated, rats were killed at 3:30 P.M., 90 min after the beginning of this late period of sleep disturbances (optimal time for detection of Fos evoked by a specific stimulus). wk, Wake.

Figure 4.

Previous exposure to a stressor (cage exchange 5–6 h before) induces Fos expression in neurons in both sleep-promoting areas (MnPO, VLPOc, and VLPOex) and arousal systems, including the limbic system (IFC, BST, and CeA) and wake-promoting areas (TMN and LC). Fos expression was substantially lower in the clean cage controls (which were sleeping normally at this time) in all regions except in the sleep-promoting areas, MnPO and VLPOc, in which it was comparable to stressed animals. Rats with amounts of spontaneously occurring sleep (awake 50% of the time during the previous 90 min) similar to the amounts of sleep in stressed rats in hours 5 and 6 had lower Fos counts than stressed animals in all regions except in the TMN, where Fos counts are thought to mirror recent waking behavior. In a first analysis, we compared controls and stressed rats, using an unpaired t test for each area, to determine whether there were differences in Fos expression as a result of the treatment (^@for comparisons between these two groups, p < 0.05). In a second analysis, we compared the three groups (controls, stressed rats, and rats spontaneously awake 50% of the time), using a one-way ANOVA followed by Fisher's PLSD, to determine whether the differences we observed in the first analysis were attributable to the fact that stressed rats were awake 50% of the time (*for pair comparisons among the three groups, p < 0.05). All values are the mean ± SEM.

Figure 5.

Fos activation in the brain during stress-induced insomnia differs from both wake and sleep states. The somatosensory cortex (Ctx; A–D), VLPO (E–H), LC (I–L), and TMN (M–P) are shown in all three states, as well as in rats that have spontaneously naturally slept 50% of the previous 90 min period. Note that cage exchange (Cage Exc) rats show activation of the cortex and TMN that is equivalent to wakefulness, and even higher activation of the LC, but paradoxical simultaneous activation of the VLPO, similar to that observed in sleeping rats. Scale bar, 200 μm. 4V, Fourth ventricle; Cg, cage.

Figure 6.

A–C, Fos activation (black nuclei) in orexin neurons (brown staining) in the lateral hypothalamus–perifornical area during sleep (A), stress-induced acute insomnia (B), and initially after cage exchange (mainly wakefulness) (C). Note that the orexin neurons are normally active during wakefulness but not during normal or disturbed sleep. Scale bar, 200 μm. f, Fornix.

Figure 7.

The high levels of Fos expression in the IFC, BST, and CeA demonstrate that portions of the limbic system are strongly activated during stress-induced insomnia (4–6 h after cage exchange; A, C, E) but not during normal sleep (clean cage controls at the same time; B, D, F). Scale bars: A (for A, B), E (for E, F), 200 μm; C (for C, D), 100 μm. ac, Anterior commissure; opt, optic tract.

Figure 8.

Microarchitecture of sleep during stress-induced insomnia (5–6 h after cage exchange). A, When sleep was scored in 4 s epochs, only the number of REM bouts changed compared with scoring in 12 s epochs. B, There was a trend (p between 0.05 and 0.08) to longer wake bouts and shorter nREM bouts in stressed rats, which accounts for the difference in percentage of wake and nREM sleep with respect to controls. These results ruled out rapid switching between wake and sleep states as the cause of simultaneous Fos expression in wake and sleep structures during stress-induced sleep disturbances. C, D, On the other hand, the ratio of the EEG power spectra during nREM sleep between the experimental day and the baseline showed an increase in high-frequency activity during the sleep-disturbed period from ∼45 to 58 Hz, within the gamma band, which is usually associated with waking cortical activity in rats. Each point represents the average for each frequency at 0.25 Hz intervals (n = 9 and n = 16, for controls and stressed rats, respectively). In D, the average power for each frequency band (delta = 1.5–4 Hz; theta = 4.25–8.75 Hz; sigma = 9–14 Hz; beta = 14.25–30 Hz; and gamma = 30.25–58 Hz) from controls and stressed rats were compared using unpaired t tests (p < 0.05). *p < 0.05 between treatments at each time point; ⁽*⁾p values of 0.05 and 0.08 for mean duration of wake bouts and nREM bouts, respectively, in B. All values are the mean ± SEM.

Figure 9.

The extent of the lesions in the IFC, CeA, and BST are noted by arrowheads in Nissl-stained sections. The extent of the LC lesions was assessed by counting the remaining noradrenergic neurons in sections labeled for dopamine-β-hydroxylase (in the photomicrograph of the LC, there are 3 remaining immunoreactive neurons). Scale bar, 200 μm.

Figure 10.

A, Sleep latency in stressed rats is almost double than in controls. CeA–BST lesions restore sleep latency similar to control levels, whereas IFC lesions have no effect. LC lesions and immepip treatment before cage exchange decrease sleep latency ∼10 min with respect to stressed rats, but this difference is not statistically significant. B, C, During the first hour after cage exchange, which corresponds to the primary stress response, almost all groups have increased wake (B) and decreased nREM (C) compared with clean cage controls. D, REM levels are minimal in all groups, including controls. During the second hour, controls and CeA–BST-lesioned rats fall asleep similarly (C), whereas the other groups have increased wake (B) and decreased nREM (C) compared with controls. REM is decreased in all groups during this second hour. During the acute insomnia period (fifth and sixth hours after cage exchange), all treatments restore more or less the normal amounts of wake (B) and nREM (C), as in controls, but REM sleep is only recovered after CeA–BST lesions and to a lesser extent after IFC lesions (D). Percentages of wake, nREM, and REM at each time point for all treatments are provided in Figure 12. Data were analyzed by one-way ANOVA followed by Fisher's PLSD. ^#Significantly different from controls; *significantly different from cage exchange (Cage Exch) group. ^#,*p < 0.05; ^(#),(*⁾p between 0.05 and 0.09. All values are the mean ± SEM.

Figure 11.

Number of bouts (wake, nREM, and REM) during the fragmentation period (third and fourth hours after cage exchange). The sleep fragmentation induced by stress seems to be caused by activation of the IFC and the arousal system (LC and TMN), but not the limbic system (CeA–BST). The results were analyzed using one-way ANOVA followed by Fisher's PLSD [each treatment compared with clean cage and cage exchange (Cage Exch) rats; for the sake of clarity, comparisons between treatments are not included]. ^#Significantly different from controls; *significantly different from cage exchange group. ^#,*p < 0.05; ⁽*⁾p = 0.08. All values are the mean ± SEM.

Figure 12.

Percentages of wake, nREM and REM sleep at each time point for all treatments. These data are complementary to Figure 10. Comparisons were done with repeated-measures ANOVA followed by planned comparisons using Fisher's PLSD [each treatment compared with controls and cage exchange (Cage Exch)]. ⁺Significantly different from controls; ^@significantly different from cage exchange group. ^+,@p < 0.05; ^(+),(@)p between 0.05 and 0.09. Comparisons between controls and cage exchange rats are not included in these graphs (shown in Fig. 2). The x-axis is marked in hours before or after the cage exchange. All values are the mean ± SEM. les, Lesion.

Figure 13.

Total Fos counts in sleep-promoting areas, limbic regions, and arousal system for each treatment. These data are complementary to Table 2. Comparisons were done using one-way ANOVA followed by Fisher's PLSD for each planned comparison (*p < 0.05). All values are the mean ± SEM. Exch, Exchange.

Figure 14.

Simplified putative circuitry involved in stress-induced acute insomnia based on the results (sleep and Fos) of specific lesions and immepip treatment before stress exposure and on anatomical connections described in previous literature. The olfactory signals of a competing male rat are conveyed to the CeA–BST via the accessory olfactory bulb (AOB) and the medial amygdala (MeA). The CeA–BST, which projects densely to the lateral hypothalamus–perifornical area (LH–PeF), activates mainly nonorexin (non-Orx) neurons in this area. In addition, the LC and the TMN receive moderate and dense projections from the CeA–BST and the LH–PeF, respectively, which most likely causes the activation of these arousal regions during stress-induced insomnia. The cortex, which receives dense projections from the arousal system and the LH–PeF, becomes highly activated; this high cortical activity is associated with gamma activity during nREM in the insomnia period. The reciprocal inhibition between the VLPO (VLPOc and VLPOex) and the arousal system (LC and TMN) would ordinarily prevent coactivation. However, homeostatic and circadian sleep pressure keep the VLPO activated, whereas stress activates the arousal system, resulting in the unique pattern of Fos activation and EEG power spectrum seen during this acute insomnia period.

Figure 15.

The increased high-frequency activity within the last portion of the gamma band (45–58 Hz) during nREM sleep observed in stressed rats during the acute insomnia period seems to be caused by activation of the LC and the TMN. A, LC lesions abolished this high-frequency activity, and the power spectrum of the gamma band was similar to clean cage controls. LC lesions also increased theta (4.5–8.5 Hz) and sigma (9–14 Hz) power. B, Immepip treatment, which inhibits the TMN, decreased the power along the whole gamma band (30–58 Hz) to lower levels than controls, and also increased delta power. In A and B, each point represents the average of the ratio of power spectra during nREM sleep between the experimental day and the baseline for each frequency at 0.25 Hz intervals (n = 9 for controls; n = 16 for cage exchange; n = 8 for LC lesions; n = 6 for immepip treatment). Exc, Exchange. C, The averages for each frequency band (delta = 1.5–4 Hz; theta = 4.25–8.75 Hz; sigma = 9–14 Hz; beta = 14.25–30 Hz; and gamma = 30.25–58 Hz) from controls, cage exchange, LC-lesioned rats, and immepip-treated rats were compared using one-way ANOVA followed by Fisher's PLSD for pair comparisons (*significantly different with respect to clean cage controls, p < 0.05; ^@significantly different with respect to cage exchange rats, p < 0.05). CExc, Cage exchange.