- Email: [email protected]
Induction of FOS expression by acute immobilization stress is reduced in locus coeruleus and medial amygdala of Wistar–Kyoto rats compared to Sprague–Dawley rats
Neuroscience 124 (2004) 963–972
INDUCTION OF FOS EXPRESSION BY ACUTE IMMOBILIZATION STRESS IS REDUCED IN LOCUS COERULEUS AND MEDIAL AMYGDALA OF WISTAR–KYOTO RATS COMPARED TO SPRAGUE–DAWLEY RATS S. MA AND D. A. MORILAK*
sequence. Nonetheless, understanding the mechanisms underlying altered stress reactivity in models such as the WKY rat may contribute to a better understanding of stress-related psychopathologies such as depression, post-traumatic stress disorder or other anxiety disorders. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
Department of Pharmacology and Center for Biomedical Neuroscience, MC 7764, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
Abstract—Activation of the brain noradrenergic system during acute stress is thought to play an important integrative function in coping and stress adaptation by facilitating transmission in many brain regions involved in regulating behavioral and physiologic components of the stress response. Compared with outbred control Sprague–Dawley (SD) rats, inbred Wistar–Kyoto (WKY) rats exhibit an exaggerated hypothalamic–pituitary–adrenal (HPA) response as well as increased susceptibility to certain forms of stress-related pathology. However, we have also shown previously that WKY rats exhibit reduced anxiety-like behavioral reactivity to acute stress, associated with reduced activation of the brain noradrenergic system. Thus, to understand better the possible neurobiological mechanisms underlying dysregulation of the stress response in WKY rats, we investigated potential strain differences in stress-induced neuronal activation in brain regions that are both involved in regulating behavioral and neuroendocrine stress responses, and are related to the noradrenergic system, either as targets of noradrenergic modulation or as sources of afferent innervation of noradrenergic neurons. This was accomplished by visualizing stress-induced expression of Fos immunoreactivity in the paraventricular nucleus of the hypothalamus, lateral bed nucleus of the stria terminalis, central nucleus of the amygdala, and medial nucleus of the amygdala (MeA), as well as the noradrenergic nucleus locus coeruleus (LC). Stress-induced Fos expression was found to be decreased in the LC and MeA of WKY rats compared with similarly stressed SD rats, whereas no strain differences were observed in any of the other brain regions. This suggests that strain-related differences in activation of the MeA may be involved in the abnormal neuroendocrine and behavioral stress responses exhibited by WKY rats. Moreover, as the MeA is both an afferent as well as an efferent target of the brainstem noradrenergic system, reduced MeA activation may either be a source of reduced noradrenergic reactivity seen in WKY rats, or possibly a con-
Key words: anxiety, arousal, HPA axis, norepinephrine, strain differences, stress vulnerability.
Activation of the brain noradrenergic system during acute stress is thought to play an important integrative function in coping and stress adaptation by facilitating transmission in many brain regions involved in regulating behavioral and physiologic components of the stress response. Noradrenergic transmission promotes attention and arousal, biasing the organism toward responding to salient environmental stimuli (Aston-Jones et al., 1999b). Elevated noradrenergic activity in specific limbic forebrain regions also enhances anxiety-like behavioral responses to acute stress, and facilitates activation of the hypothalamic–pituitary–adrenal (HPA) axis, the major neuroendocrine effector of the stress response (Cecchi et al., 2002a,b). An inability to initiate or regulate the stress response appropriately has been proposed to be a critical factor in the pathophysiology of various stress-related disorders, and dysregulation of noradrenergic neurotransmission has been implicated specifically in stress-related psychiatric diseases such as depression, post-traumatic stress disorder (PTSD) and other anxiety disorders (Schatzberg and Schildkraut, 1995; Southwick et al., 1993; Sullivan et al., 1999). The inbred Wistar–Kyoto (WKY) rat strain has been suggested to be a model of genetic stress vulnerability (Pare and Redei, 1993; Redei et al., 1994). Compared with other inbred strains as well as outbred control strains, including Wistar and Sprague–Dawley (SD) rats, WKY rats exhibit an exaggerated HPA response to acute stress, and a greater tendency to develop stress-induced gastric ulcers (Pare and Redei, 1993; Redei et al., 1994). We have shown previously, however, that despite their enhanced HPA stress reactivity, WKY rats show reduced behavioral activity and attenuated anxiety-like behavioral reactivity to acute stress (Pardon et al., 2002). Compared with SD rats, WKY rats showed depressed baseline acoustic startle responding and lower baseline exploratory and social activity. Further, WKY rats showed a lack of fear-potentiation of the startle response, and attenuation of stress-induced reductions in social behavior and open-arm exploration on
*Corresponding author. Tel: ⫹1-210-567-4174; fax: ⫹1-210-567-4303. E-mail address: [email protected] (D. Morilak). Abbreviations: ACTH, adrenocorticotropic hormone; BST, bed nucleus of the stria terminalis; BSTL, lateral bed nucleus of the stria terminalis; BSTLd, lateral bed nucleus of the stria terminalis, dorsal subdivision; BSTLv, lateral bed nucleus of the stria terminalis, ventral subdivision; CeA, central nucleus of the amygdala; CRH, corticotropin-releasing hormone; HPA, hypothalamic–pituitary–adrenal; LC, locus coeruleus; MeA, medial nucleus of the amygdala; NE, norepinephrine; PBS, phosphate-buffered saline; PTSD, post-traumatic stress disorder; PVN, paraventricular nucleus; SD, Sprague–Dawley; WKY, Wistar–Kyoto.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.12.028
963
964
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
the plus maze, indicating reduced anxiety-like behavioral stress reactivity (Pardon et al., 2002). Consistent with this, brain noradrenergic reactivity to stress was also reduced in WKY rats, seen as a blunted elevation in tyrosine hydroxylase mRNA expression in the locus coeruleus (LC), and an attenuation of stress-induced norepinephrine (NE) release in the bed nucleus of the stria terminalis (BST). Further, not only was stress-induced activation of the noradrenergic system reduced, but the facilitation of stressinduced activation of the HPA axis that was shown to be exerted by NE release in the BST of SD rats was found to be absent in WKY rats. Thus, dysregulation of the stress response and increased susceptibility to stress-related pathology in WKY rats may be related to a deficit of brain noradrenergic modulatory activity, though the mechanisms by which this may occur are unknown. Brainstem noradrenergic neurons are found primarily in the LC, as well as in other medullary and pontine cell groups. These cells both project to and receive afferent input from a number of forebrain limbic regions involved directly or indirectly in regulating and modulating behavioral and neuroendocrine responses to stress, including, among others, the lateral BST (BSTL), the central and medial nuclei of the amygdala (CeA and MeA), and the hypothalamic paraventricular nucleus (PVN). The BSTL and CeA are involved in fear and anxiety-like behavioral responses (e.g. Cecchi et al., 2002a; Davis and Shi, 1999), and represent sources of extrahypothalamic modulation of the hormonal HPA stress response mediated in the PVN (Cecchi et al., 2002a; Herman and Cullinan, 1997; Herman et al., 1994). Modulatory effects of NE on behavioral and physiologic responses to various stressors have been described in the BSTL, as well as CeA and PVN (Cecchi et al., 2002a,b; Cole and Sawchenko, 2002; Delfs et al., 2000; Koob, 1999; Plotsky, 1987; Szafarczyk et al., 1987). Although there have been relatively few studies of stress modulatory effects of NE in the MeA, recent observations suggest that descending projections from the MeA, perhaps relaying in the PVN, may be important in the activation of brainstem noradrenergic neurons, specifically by psychogenic stressors (Dayas et al., 1999), thus identifying the MeA as a potentially important upstream afferent regulating the stress reactivity of the brain noradrenergic system. Thus, to understand better the possible neurobiological mechanisms underlying dysregulation of the stress response and increased stress vulnerability of WKY rats, we investigated potential strain differences in stress-induced neuronal activation in brain regions that are involved in behavioral and neuroendocrine stress responses, and are related to the noradrenergic system either as targets of noradrenergic modulation, sources of afferent innervation of noradrenergic neurons, or both, by visualizing stressinduced expression of Fos immunoreactivity in BSTL, CeA, MeA and PVN, as well as the LC. Portions of this work have been presented in abstract form (Ma and Morilak, 2003).
EXPERIMENTAL PROCEDURES Animals Experiments were performed on 13 adult male SD and 13 adult male WKY rats (Charles River, Wilmington, MA, USA), weighing 225–250 g upon arrival. Rats were allowed to acclimate to the animal facility for 7–10 days prior to use, during which they were housed three per cage, all from the same strain, on a 12-h light/dark cycle (lights on at 07:00 h). Food and water were available ad libitum. All effort was made to minimize animal suffering and the number of animals used, and all procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and conformed to NIH guidelines on the ethical use of animals. All experiments were conducted between 09:00 and 12:00 h, during the light portion of the cycle. Rats from each strain were randomly assigned to one of three groups (n⫽4 –5/group). One group from each strain served as unstressed controls, and all other rats were subjected to 1 h immobilization stress. One group of stressed rats from each strain were killed immediately at the termination of 60 min stress, and the remaining groups were killed 1 h after termination of the stress, during which they recovered in their home cages (i.e. 2 h after the onset of 60 min immobilization stress).
Immobilization stress For immobilization stress, rats were held prone on a flat plastic rack large enough to support their entire body (26 cm⫻13 cm). Their limbs were taped to the rack gently but securely using medical adhesive tape. Strips of tape were placed across the back of the neck and head to prevent excessive head movements. At the termination of the 1 h stress period, the animal was held gently in place while the tape was removed.
Fos immunohistochemistry At the designated time point, stressed rats as well as unstressed control rats from each strain were deeply anesthetized (cocktail of ketamine 43 mg/ml, acepromazine 1.4 mg/ml, xylazine 8.6 mg/ml, given in a dose of 0.8 ml/kg, i.m.), and perfused transcardially with 50 ml of 100 mM phosphate-buffered saline (PBS), pH 7.4 containing 1000 IU/ml heparin, followed by 350 ml of 4% paraformaldehyde in PBS, at a flow rate of approximately 50 ml/min. Brains were post-fixed for 1 h in the same fixative, rinsed and then cryoprotected for 48 h in 20% sucrose–PBS at 4 °C. After the brains had sunk, they were frozen in isopentane on dry ice and stored at ⫺70 °C until they could be sectioned and processed (⬍1 month). With the exception of the PVN, six sequential series of 50 m coronal sections were cut on a cryostat through the BSTL, CeA, MeA and LC. Due to the small size of the PVN, only three series of sections were cut through this structure. One series through each region from each brain were used for Fos immunohistochemistry. After rinsing in 20 mM PBS (pH 7.4), free-floating sections were incubated for 30 min at 24 °C in blocking buffer (5% normal sheep serum, 0.3% Triton X-100, 0.05% thimerosal in PBS). They were then incubated in a rabbit anti-cFos antibody (Oncogene Sciences, San Diego, CA, USA), diluted 1/30,000 in blocking buffer for 2 h at 24 °C followed by 40 h at 4 °C. After rinsing, sections were incubated for 4 h at 24 °C in biotinylated sheep anti-rabbit IgG (Sigma) diluted 1/400 in blocking buffer containing 1% normal sheep serum, then for 90 min in avidin– horseradish peroxidase (Sigma) diluted 1/2000 in PBS. Immunoreactivity was then visualized by a peroxidase reaction with 3,3⬘diaminobenzidine as the substrate. Following the reaction, sections were mounted on silanized slides and coverslipped. An adjacent series of sections from each brain were stained with
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
965
Fig. 1. Representative micrographs of Cresyl Violet-stained sections through (A) BSTLd, BSTLv; (B) PVN, parvocellular subdivision (PVNpc); (C) CeA, MeA; and (D) LC, Outlines indicate typical boundaries of regions analyzed for Fos immunoreactivity within each brain area. Scale bar⫽1.00 mm (A, C), 1.25 mm (B) and 1.50 mm (D).
Cresyl Violet to aid in determination and delineation of the cytoarchitectural boundaries of each region of interest. For each region, four to six sections from each brain were analyzed by an investigator blind to the treatment condition, using the NIH-Image software package (v. 1.54; Wayne Rasband, NIH). Sections selected for analysis corresponded to plates 18 –20 (BSTL), 28 –31 (CeA), 29 –32 (MeA), 26 –27 (PVN) and 57–59 (LC) in the atlas of Paxinos and Watson (1998); (see Fig. 1). The dorsal and ventral BSTL (BSTLd and BSTLv, respectively) were analyzed separately. For analysis of Fos expression, digitized images were captured using a SONY XC77 CCD camera mounted on a Nikon microscope coupled to a Scion LG3 capture board in a PowerMac 7100 computer. Images from immunostained sections were aligned with the corresponding image from an adjacent Cresyl Violet-stained section using the Live Paste and capture function. The region of interest was first delineated on the Cresyl Violet-stained section, then the selection outline was transferred to the immunostained section for counting of Fos-positive cell nuclei within the specified region. The mean of all scores within a region was then calculated to generate a single value for each brain, expressed as cells/1000 pixels (corresponding to an area of approximately 6475 m2 at the magnification at which BST, MeA and CeA were analyzed, and approximately 25,900 m2 for PVN and LC). Data were then analyzed by two way ANOVA (Strain⫻Time), followed where appropriate by post hoc analyses using the Newman-Keuls test. Significance was always determined at P⬍0.05.
RESULTS In the PVN (Fig. 2), very few Fos-positive neurons were seen in unstressed control rats of either strain. Significant induction of Fos expression was observed in PVN following 60 min immobilization stress (Main effect for Time: F(2,18)⫽54.324, P⬍0.0001). This induction was significant in both strains relative to their respective unstressed controls (post hoc comparisons, P⬍0.05). Expression of Fos in PVN remained significantly elevated after 60 min of recovery in both strains. Although the number of Fos-
positive cells in PVN was slightly higher at both post-stress time points in WKY rats, there were no significant differences between the strains at any time point (post hoc comparisons, P⬎0.05). In the BSTLv (Fig. 3A, C), very few Fos-positive neurons were seen in unstressed control rats of either strain. Significant induction of Fos expression was observed in BSTLv following 60 min immobilization stress (F(2,18)⫽34.897, P⬍0.0001), and this induction was significant in both strains relative to their respective unstressed controls (P⬍0.05). Expression of Fos in BSTLv remained significantly elevated after 60 min of recovery in both strains. However, there was neither a main effect of Strain nor a significant Strain⫻Time interaction, and post hoc comparisons revealed no significant differences between the strains at any time points (P⬎0.05). By contrast, in the BSTLd (Fig. 3B, C), no significant induction of Fos expression was observed in stressed rats of either strain (F(2,18)⫽0.326). There were no main effects of Time or Strain, and no Strain⫻Time interaction (all P⬎0.05). In CeA (Fig. 4), very few neurons expressed Fos immunoreactivity in unstressed control rats of either strain. ANOVA indicated a significant main effect for Time (F(2,18)⫽4.688, P⬍0.05). The modest increase seen at both 60 and 120 min compared with baseline was, however, significant only when the data were collapsed across strains. When post hoc comparisons were made between each time point and the respective baseline within a strain, none of these comparisons achieved significance. Further, there was no main effect of Strain (F(1,18)⫽2.256), nor a Strain⫻Time interaction (F(2,18)⫽0.207). Likewise, very little Fos expression was seen in MeA of unstressed control rats of either strain (Fig. 5). By contrast with CeA, however, immobilization stress induced an ele-
966
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
Fig. 2. Immobilization stress induced a similar degree of Fos expression in the PVN of both SD and WKY rats. (A) Fos expression induced in PVN of SD and WKY rats by immobilization stress, expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group). * P⬍0.05 compared with same-strain baseline. (B) Representative micrographs of stress-induced Fos immunoreactivity in PVN of SD and WKY rats. Medial is to the right. Scale bar⫽250 m.
vation of Fos expression in MeA (F(2,18)⫽187.643, P⬍0.0001), which was significant in both WKY and SD rats compared with their respective controls (P⬍0.05). Further, there was a significant main effect of Strain (F(1,18)⫽13.979, P⬍0.01), as well as a Strain⫻Time interaction (F(2,18)⫽4.615, P⬍0.05). Post hoc comparisons between strains showed that the induction of Fos expression in MeA immediately after the termination of stress was significantly less in WKY rats compared with SD rats (P⬍0.05). The level of Fos expression in MeA was still elevated after 60 min of post-stress recovery in both strains. However, there was no longer a significant difference between the strains at this time point (see Fig. 5). In the LC of both SD and WKY rats, few Fos positive neurons were seen in unstressed controls (Fig. 6). Following immobilization stress, a significant induction of Fos expression was observed in LC (F(2,18)⫽44.669, P⬍0.0001), which was significant in both strains relative to their respective controls (P⬍0.05). There was also a significant main effect of Strain (F(1,18)⫽9.748, P⬍0.01). The increase in Fos expression in LC, which was still evident after 60 min of post-stress recovery in both strains, was
lower in WKY rats compared with SD rats at this time point (P⬍0.05). Nonetheless, the Strain⫻Time interaction did not reach significance (F(2,18)⫽2.491).
DISCUSSION The brain noradrenergic system is activated by acute stress, and plays an important modulatory role in the response to stress. Acute stress increases NE neurotransmission in limbic forebrain regions such as the CeA and BST (Cecchi et al., 2002a; Pacak et al., 1993, 1995), where it acts to facilitate a number of acute behavioral and neuroendocrine responses to stress, including activation of the HPA axis (Cecchi et al., 2002a,b). In a previous study, we observed reduced activation of the brain noradrenergic system by acute stress in WKY rats, seen as a blunted elevation of tyrosine hydroxylase mRNA expression in the LC and an attenuation of stress-induced NE release in the limbic forebrain compared with SD rats (Pardon et al., 2002). This observation was further confirmed in the present study. Fos induction was moderately lower in LC in WKY rats compared with SD rats immediately after termi-
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
967
Fig. 3. Immobilization stress induced Fos expression to a similar degree in BSTLv of SD and WKY rats. (A) By contrast, Fos expression was not induced in BSTLd of either strain. (B) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group); * P⬍0.05 compared with same-strain baseline. (C) Stress-induced Fos immunoreactivity shown in representative digital photomontages of BST in SD and WKY rats. Medial is to the right. Scale bar⫽250 m.
nation of the 1 h stress, although this difference did not achieve significance, whereas Fos expression was further and significantly reduced in LC of WKY rats compared with SD rats 2 h after the onset of stress (i.e. 1 h after termination of the stressor). A more rapid return toward baseline may also be indicative of a less robust induction of c-fos expression initially. However, because the Fos expression was still significantly elevated in WKY rats at the 120 min time point relative to baseline, and because the level of Fos expression was slightly lower immediately after the 60 min stress as well, we hesitate to conclude that the time course of response was substantively different in the two strains, only that the overall magnitude of response was lower in WKY rats. Consistent with a reduction in noradrenergic activation, we have also reported an attenuation of stress-induced behavioral responses in WKY rats, including some responses that had been shown previously in SD rats to be facilitated by NE (Pardon et al., 2002). In addition, noradrenergic facilitation of the HPA axis, shown to occur in the
BSTL of SD rats, was also lacking in WKY rats, despite their elevated HPA responses (Pardon et al., 2002). Thus, it appears that acute activation of the brain noradrenergic system, and subsequent noradrenergic facilitation of stress-induced activity in target regions such as the BSTL may both be reduced in WKY rats. In the present study, Fos expression in the ventrolateral, but not the dorsolateral subdivision of BST was elevated significantly by acute stress. The BST is a major target of ascending noradrenergic innervation (Moore and Bloom, 1979; Swanson and Hartman, 1975), particularly the ventrolateral subdivision (Phelix et al., 1992a). Noradrenergic innervation of the BST arises primarily from the medullary A2 cell group, with lesser projections from the A1 region and the LC (Aston-Jones et al., 1999a; Delfs et al., 2000). The medullary noradrenergic cell groups were not examined in the present study because, unlike the LC, they are distributed heterogeneously and often sparsely among other cell types in the dorsomedial and ventrolateral medulla (Kalia et al., 1985), making it impossible to
968
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
Fig. 4. Immobilization stress induced only moderate Fos expression in CeA, which was not significant compared with the same-strain baseline in either SD or WKY rats. (A) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group). (B) Fos expression in CeA of SD and WKY rats. Medial is to the left. Scale bar⫽250 m.
identify Fos expression in these neurons in the absence of double-labeling procedures. Nonetheless, the fact that we have previously demonstrated a reduction in stress-activated NE release specifically in the BSTL of WKY rats (Pardon et al., 2002) would suggest that activation of the medullary noradrenergic cell groups was attenuated as well as the LC. In the present study, however, there was no strain difference in stress-induced Fos expression in either BSTLv or BSTLd. This might suggest that other stressresponsive afferents to BSTLv, such as a serotonergic pathway from midbrain raphe nuclei (Phelix et al., 1992b), or an extrahypothalamic input utilizing corticotropin-releasing hormone (CRH; Koob and Heinrichs, 1999; Lee and Davis, 1997), might be sufficient to activate a similar number of cells in this region in WKY and SD rats, despite the difference in noradrenergic activity. Alternatively, it is possible that strain differences in noradrenergic transmission in BSTL may be reflected by differences in the induction of expression of immediate early genes other than c-fos. For instance, both acute swim stress and restraint stress increased the expression of c-fos mRNA in the medial, but not in the lateral part of the BST, whereas the same stimuli increased expression of zif/268 in the lateral but not the medial BST (Cullinan et al., 1995). It is not known whether the deficit of stress-induced noradrenergic activation in WKY rats results from a primary alteration in the activity or excitability of noradrenergic neurons, or from an alteration in excitatory afferent input to the noradrenergic system in this strain. Both the CeA and MeA have been shown to provide excitatory
afferent input to brainstem noradrenergic neurons. CeA has been shown to play a role in behavioral responses to stress (Koob, 1999) and in fear-conditioned behavioral responses (Walker and Davis, 1997). A pathway from CeA to LC utilizing CRH has been shown to be essential to activation of the LC in response to acute hypotensive stress (Curtis et al., 2002). However, the CeA appears not to be involved in activation of noradrenergic neurons by psychogenic stressors such as immobilization (Dayas and Day, 2002), the stimulus used in the present study. Consistent with this, we observed only a modest induction of Fos expression in the CeA following immobilization stress, and there were no strain differences seen in this region. This is also consistent with previous reports describing little or no induction of Fos expression in CeA by acute restraint stress (Arnold et al., 1992; Chen and Herbert, 1995). Similarly, CRH mRNA expression in the CeA has also been reported to be unaffected by immobilization (Pacak et al., 1996). By contrast, the MeA has been shown to be involved in the activation of brainstem noradrenergic neurons in response to psychogenic stress (Dayas and Day, 2002). Further, Fos expression has been shown to be induced in MeA in response to stressful stimuli, particularly to emotional or psychogenic stressors such as restraint (Bhatnagar and Dallman, 1998; Chen and Herbert, 1995; Cullinan et al., 1995), exposure to conditioned aversive stimuli (Pezzone et al., 1992), swim stress (Cullinan et al., 1995) or footshock (Li and Sawchenko, 1998). In the present study, Fos expression was elevated in MeA after immobilization
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
969
Fig. 5. Immobilization stress induced a significant increase in Fos expression in MeA of SD and WKY rats immediately after 60 min stress, and after 1 h post-stress recovery (i.e. 120 min after stress onset). Stress induced significantly higher Fos expression in MeA of SD rats at 60 min, with no strain difference seen after 1 h recovery. (A) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group); * P⬍0.05 compared with same-strain baseline; ⫹ P⬍0.05 between strains at the same time point. (B) Stress-induced Fos expression shown in representative digital photomontages of MeA. Medial is to the right. Scale bar⫽250 m.
Fig. 6. Immobilization stress induced a significant increase in Fos expression in LC of SD and WKY rats immediately after 60 min stress, and after 1 h post-stress recovery (i.e. 120 min after stress onset). The induction of Fos expression was greater in the LC of SD rats, the strain difference being significant at 120 min. (A) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group); * P⬍0.05 compared with same-strain baseline; ⫹ P⬍0.05 between strains at the same time point. (B) Stress-induced Fos expression in LC. Medial is to the right. Scale bar⫽250 m.
970
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
stress, and the magnitude of this induction was significantly attenuated in WKY rats compared with SD rats. Thus, a reduction in stress-induced activation of the MeA could be a potential mechanism to account for the reduction in stress-induced activation of the noradrenergic system in WKY rats. However, in addition to providing a descending excitatory input to the brainstem noradrenergic system, the MeA also receives a substantial noradrenergic innervation (Roder and Ciriello, 1993; Sadikot and Parent, 1990). Thus, it is not possible to determine from the present data whether the attenuated activation of MeA in WKY rats represents an upstream mechanism accounting for reduced noradrenergic activation in this strain, or instead represents a downstream consequence of the reduced noradrenergic activation. A limited amount of data have implicated the MeA in modulation of the neuroendocrine response to stress. Efferent projections from the MeA target many regions of the limbic forebrain and hypothalamus that themselves modulate HPA activation, including the anterior and ventromedial hypothalamus, BST, lateral septum, medial preoptic area and ventral hippocampus (Canteras et al., 1995). Lesions of the MeA attenuated adrenocorticotropic hormone (ACTH) and glucocorticoid secretion induced by acoustic or photic stress (Feldman et al., 1994), and reduced the stress-induced expression of Fos in CRH- and oxytocin-synthesizing neurons in the PVN and supraoptic nuclei (Dayas et al., 1999). Thus, a reduced activation of the MeA by stress could represent one potential substrate for some of the differences in the behavioral and neuroendocrine responses to acute stress seen in WKY rats compared with SD rats. As for a potential role of NE in the vulnerability to stress exhibited by WKY rats, we have hypothesized previously that the observed deficit of noradrenergic facilitatory influence, and the consequent attenuation of acute behavioral responses to stress in WKY rats, may hinder their ability to cope effectively with acute stress, thereby increasing the perceived magnitude or relative “impact” of stress (Pardon et al., 2002). This could account secondarily for the elevated HPA response, as well as the increased vulnerability to stress-related pathology seen in this strain. Thus, whereas stress-induced activation of the brain noradrenergic system and activation of target regions that are facilitated by NE may both be reduced in WKY rats, it is possible that stress-induced activation of other brain regions that respond independently of noradrenergic facilitation might be unaffected or even increased. Consistent with this, in the present study, the number of Fos immunoreactive cells seen in the PVN of WKY rats following stress was slightly higher, by approximately 20 –25%, than the number of cells seen in SD rats, although this was not a statistically significant difference. It is important to note that the measure of Fos induction in this experiment, i.e. the number of immunoreactive cell nuclei, is essentially “digital” in nature. As such, it can indicate only how many cells were affected by the stimulus, not the degree to which these cells were affected. The number of cells expressing Fos in PVN after immobilization stress was very high in
both strains. Thus, it is possible that any strain difference in response magnitude in this region might perhaps be reflected not in the number of cells that were activated, but in the intensity with which they were activated, and the immunohistochemical approach cannot detect such graded differences in response magnitude. Nonetheless, based on the slightly elevated HPA response of WKY relative to SD rats, it was not surprising to see at least a comparable activation of PVN in the two strains, if not a slightly greater induction in WKY rats. Indeed, the ACTH response to restraint stress is greater in WKY rats than in several other strains, including SD and Wistar (Pardon et al., 2002; Pare and Redei, 1993; Redei et al., 1994). In cultured anterior pituitary cells from WKY rats compared with Wistar rats, CRH binding sites and receptor mRNA expression were reduced, activation of ACTH secretion in response to CRH or vasopressin was markedly impaired, and vasopressin failed to potentiate CRH-stimulated ACTH secretion (Hauger et al., 2002). These observations may indicate that elevated release of hypothalamic secretagogues, sufficient to induce receptor down-regulation and desensitization, may contribute to the increased sensitivity of the HPA axis in WKY rats. Others have suggested that glucocorticoid feedback may be impaired in WKY rats (Redei et al., 1994). Such interpretations might be consistent with the very slight elevation of Fos expression seen in the PVN of WKY rats compared with SD rats in the present study. However, this was not a significant difference, and other studies have revealed no difference in CRH mRNA expression in PVN of WKY rats compared with SD rats, either before or after acute or chronic immobilization stress (Gomez et al., 1996; Krukoff et al., 1999), suggesting that the strain difference in the HPA response to stress may not be attributable to differences in activation of the PVN. In conclusion, altered stress responsivity of the brain noradrenergic system and of specific stress-responsive brain regions that are related to it, such as the MeA, may contribute to the abnormal neuroendocrine and behavioral stress responses exhibited by WKY rats compared with the outbred control SD strain. Further, these aberrant responses may contribute to the demonstrated susceptibility of WKY rats to stress-related pathology (Redei et al., 1994), making them a potentially useful model for understanding the neural mechanisms linking stress to certain psychopathological disorders, such as depression, PTSD or other anxiety disorders, in susceptible individuals (Gold and Chrousos, 1999; Schatzberg and Schildkraut, 1995; Southwick et al., 1993; Sullivan et al., 1999). Thus, understanding how changes in these neural systems influence the behavioral and neuroendocrine response characteristics of WKY rats may contribute to the development of better strategies for prevention or treatment of stress-related psychiatric disorders. Acknowledgements—Expert technical assistance was provided by Gabe Barrera, April Garcia, and Angelica Hernandez. Supported by a research grant from the NIMH (MH53851).
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
REFERENCES Arnold FJ, De Lucas Bueno M, Shiers H, Hancock DC, Evan GI, Herbert J (1992) Expression of c-fos in regions of the basal limbic forebrain following intracerebroventricular corticotropin-releasing factor in unstressed or stressed male rats. Neuroscience 51:377– 390. Aston-Jones G, Delfs JM, Druhan J, Zhu Y (1999a) The bed nucleus of the stria terminalis: a target site for noradrenergic actions in opiate withdrawal. Ann NY Acad Sci 877:486 –498. Aston-Jones G, Rajkowski J, Cohen J (1999b) Role of locus coeruleus in attention and behavioral flexibility. Biol Psychiatry 46:1309–1320. Bhatnagar S, Dallman M (1998) Neuroanatomical basis for facilitation of hypothalamic-pituitary-adrenal responses to a novel stressor after chronic stress. Neuroscience 84:1025–1039. Canteras NS, Simerly RB, Swanson LW (1995) Organization of projections from the medial nucleus of the amygdala: a PHAL study in the rat. J Comp Neurol 360:213–245. Cecchi M, Khoshbouei H, Javors M, Morilak DA (2002a) Modulatory effects of norepinephrine in the lateral bed nucleus of the stria terminalis on behavioral and neuroendocrine responses to acute stress. Neuroscience 112:13–21. Cecchi M, Khoshbouei H, Morilak DA (2002b) Modulatory effects of norepinephrine, acting on ␣1-receptors in the central nucleus of the amygdala, on behavioral and neuroendocrine responses to acute immobilization stress. Neuropharmacology 43:1139 –1147. Chen X, Herbert J (1995) Regional changes in c-fos expression in the basal forebrain and brainstem during adaptation to repeated stress: correlations with cardiovascular, hypothermic and endocrine responses. Neuroscience 64:675–685. Cole RL, Sawchenko PE (2002) Neurotransmitter regulation of cellular activation and neuropeptide gene expression in the paraventricular nucleus of the hypothalamus. J Neurosci 22:959 –969. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ (1995) Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64:477–505. Curtis AL, Bello NT, Connolly KR, Valentino RJ (2002) Corticotropinreleasing factor neurones of the central nucleus of the amygdala mediate locus coeruleus activation by cardiovascular stress. J Neuroendocrinol 14:667–682. Davis M, Shi C (1999) The extended amygdala: are the central nucleus of the amygdala and the bed nucleus of the stria terminalis differentially involved in fear versus anxiety? Ann NY Acad Sci 877:281–291. Dayas CV, Buller KM, Day TA (1999) Neuroendocrine responses to an emotional stressor: evidence for involvement of the medial but not the central amygdala. Eur J Neurosci 11:2312–2322. Dayas CV, Day TA (2002) Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor. Eur J Neurosci 15:1712–1718. Delfs J, Zhu Y, Druhan J, Aston-Jones G (2000) Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion. Nature 403:430 –434. Feldman S, Conforti N, Itzak A, Weidenfeld J (1994) Differential effect of amygdaloid lesions on CRF-41, ACTH and corticosterone responses following neural stimuli. Brain Res 658:21–26. Gold PW, Chrousos GP (1999) The endocrinology of melancholic and atypical depression: relation to neurocircuitry and somatic consequences. Proc Assoc Am Physicians 111:22–34. Gomez F, Lahmame A, De Kloet ER, Armario A (1996) Hypothalamicpituitary-adrenal response to chronic stress in five inbred rat strains: differential responses are mainly located at the adrenocortical level. Neuroendocrinology 63:327–337. Hauger RL, Shelat SG, Redei EE (2002) Decreased corticotropinreleasing factor receptor expression and adrenocorticotropic hormone responsiveness in anterior pituitary cells of Wistar-Kyoto rats. J Neuroendocrinol 14:126 –134. Herman JP, Cullinan WE (1997) Neurocircuitry of stress: central con-
971
trol of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 20:78 –84. Herman JP, Cullinan WE, Watson SJ (1994) Involvement of the bed nucleus of the stria terminalis in tonic regulation of paraventricular hypothalamic CRH and AVP mRNA expression. J Neuroendocrinol 6:433–442. Kalia M, Woodward DJ, Smith WK, Fuxe K (1985) Rat medulla oblongata: IV. Topographical distribution of catecholaminergic neurons with quantitative three-dimensional computer reconstruction. J Comp Neurol 233:350 –364. Koob GF (1999) Corticotropin-releasing factor, norepinephrine and stress. Biol Psychiatry 46:1167–1180. Koob GF, Heinrichs SC (1999) A role for corticotropin releasing factor and urocortin in behavioral responses to stressors. Brain Res 848: 141–152. Krukoff TL, MacTavish D, Jhamandas JH (1999) Hypertensive rats exhibit heightened expression of corticotropin-releasing factor in activated central neurons in response to restraint stress. Mol Brain Res 65:70 –79. Lee Y, Davis M (1997) Role of the hippocampus, the bed nucleus of the stria terminalis, and the amygdala in the excitatory effect of corticotropin-releasing hormone on the acoustic startle reflex. J Neurosci 17:6434 –6446. Li HY, Sawchenko PE (1998) Hypothalamic effector neurons and extended circuitries activated in “neurogenic” stress: a comparison of footshock effects exerted acutely, chronically, and in animals with controlled glucocorticoid levels. J Comp Neurol 393:244 –266. Ma S, Morilak DA (2003) Reduced c-Fos induction by immobilization stress in locus coeruleus and medial amygdala of Wistar-Kyoto compared to Sprague-Dawley rats. Soc Neurosci Abstr 29, Online program no. 712.6. Moore RY, Bloom FE (1979) Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu Rev Neurosci 2:113–168. Pacak K, McCarty R, Palkovits M, Kopin IJ, Goldstein DS (1995) Effects of immobilization on in vivo release of norepinephrine in the bed nucleus of the stria terminalis in conscious rats. Brain Res 688:242–246. Pacak K, Palkovits M, Kvetnansky R, Fukuhara K, Kopin IJ, Goldstein DS (1993) Effects of single or repeated immobilization on release of norepinephrine and its metabolites in the central nucleus of the amygdala in conscious rats. Neuroendocrinology 57:623–633. Pacak K, Palkovits M, Makino S, Kopin IJ, Goldstein DS (1996) Brainstem hemisection decreases corticotropin-releasing hormone mRNA in the paraventricular nucleus but not in the central amygdaloid nucleus. J Neuroendocrinol 8:543–551. Pardon M-C, Gould GG, Garcia A, Phillips L, Cook MC, Miller SA, Mason PA, Morilak DA (2002) Stress reactivity of the brain noradrenergic system in three rat strains differing in their neuroendocrine and behavioral responses to stress: implications for susceptibility to stress-related neuropsychiatric disorders. Neuroscience 115:229–242. Pare WP, Redei E (1993) Depressive behavior and stress ulcer in Wistar Kyoto rats. J Physiol 87:229 –238. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, 4th edition. San Diego: Academic Press. Pezzone MA, Lee WS, Hoffman GE, Rabin BS (1992) Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli. Brain Res 597:41–50. Phelix CF, Liposits Z, Paull WK (1992a) Monoamine innervation of bed nucleus nucleus of stria terminalis: an electron microscopic investigation. Brain Res Bull 28:949 –965. Phelix CF, Liposits Z, Paull WK (1992b) Serotonin-CRF interaction in the bed nucleus of the stria terminalis: a light microscopic doublelabel immunocytochemical analysis. Brain Res Bull 28:943–948. Plotsky PM (1987) Facilitation of immunoreactive corticotropin-releasing factor secretion into the hypophysial-portal circulation after activation of catecholaminergic pathways or central norepinephrine injection. Endocrinology 121:924 –930.
972
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
Redei E, Pare WP, Aird F, Kluczynski J (1994) Strain differences in hypothalamic-pituitary-adrenal activity and stress ulcer. Am J Physiol 266:R353–360. Roder S, Ciriello J (1993) Innervation of the amygdaloid complex by catecholaminergic cell groups of the ventrolateral medulla. J Comp Neurol 332:105–122. Sadikot AF, Parent A (1990) The monoaminergic innervation of the amygdala in the squirrel monkey: an immunohistochemical study. Neuroscience 36:431–447. Schatzberg AF, Schildkraut JJ (1995) Recent studies on norepinephrine systems in mood disorders. In: Psychopharmacology: the fourth generation of progress (Bloom FE, Kupfer DJ, eds), pp 911–920. New York: Raven Press, Ltd. Southwick SM, Krystal JH, Morgan CA, Johnson D, Nagy LM, Nicolaou A, Heninger GR, Charney DS (1993) Abnormal noradrenergic function in posttraumatic stress disorder. Arch Gen Psychiatry 50: 266 –274.
Sullivan GM, Coplan JD, Kent JM, Gorman JM (1999) The noradrenergic system in pathological anxiety: a focus on panic with relevance to generalized anxiety and phobias. Biol Psychiatry 46: 1205–1218. Swanson LW, Hartman BK (1975) The central adrenergic system: an immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker. J Comp Neurol 163:467–505. Szafarczyk A, Malaval F, Laurent A, Gibaud R, Assenmacher I (1987) Further evidence for a central stimulatory action of catecholamines on adrenocorticotropin release in the rat. Endocrinology 121:883– 892. Walker DL, Davis M (1997) Double dissociation between the involvement of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in startle increases produced by conditioned versus unconditioned fear. J Neurosci 17:9375– 9383.
(Accepted 18 December 2003)
INDUCTION OF FOS EXPRESSION BY ACUTE IMMOBILIZATION STRESS IS REDUCED IN LOCUS COERULEUS AND MEDIAL AMYGDALA OF WISTAR–KYOTO RATS COMPARED TO SPRAGUE–DAWLEY RATS S. MA AND D. A. MORILAK*
sequence. Nonetheless, understanding the mechanisms underlying altered stress reactivity in models such as the WKY rat may contribute to a better understanding of stress-related psychopathologies such as depression, post-traumatic stress disorder or other anxiety disorders. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
Department of Pharmacology and Center for Biomedical Neuroscience, MC 7764, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
Abstract—Activation of the brain noradrenergic system during acute stress is thought to play an important integrative function in coping and stress adaptation by facilitating transmission in many brain regions involved in regulating behavioral and physiologic components of the stress response. Compared with outbred control Sprague–Dawley (SD) rats, inbred Wistar–Kyoto (WKY) rats exhibit an exaggerated hypothalamic–pituitary–adrenal (HPA) response as well as increased susceptibility to certain forms of stress-related pathology. However, we have also shown previously that WKY rats exhibit reduced anxiety-like behavioral reactivity to acute stress, associated with reduced activation of the brain noradrenergic system. Thus, to understand better the possible neurobiological mechanisms underlying dysregulation of the stress response in WKY rats, we investigated potential strain differences in stress-induced neuronal activation in brain regions that are both involved in regulating behavioral and neuroendocrine stress responses, and are related to the noradrenergic system, either as targets of noradrenergic modulation or as sources of afferent innervation of noradrenergic neurons. This was accomplished by visualizing stress-induced expression of Fos immunoreactivity in the paraventricular nucleus of the hypothalamus, lateral bed nucleus of the stria terminalis, central nucleus of the amygdala, and medial nucleus of the amygdala (MeA), as well as the noradrenergic nucleus locus coeruleus (LC). Stress-induced Fos expression was found to be decreased in the LC and MeA of WKY rats compared with similarly stressed SD rats, whereas no strain differences were observed in any of the other brain regions. This suggests that strain-related differences in activation of the MeA may be involved in the abnormal neuroendocrine and behavioral stress responses exhibited by WKY rats. Moreover, as the MeA is both an afferent as well as an efferent target of the brainstem noradrenergic system, reduced MeA activation may either be a source of reduced noradrenergic reactivity seen in WKY rats, or possibly a con-
Key words: anxiety, arousal, HPA axis, norepinephrine, strain differences, stress vulnerability.
Activation of the brain noradrenergic system during acute stress is thought to play an important integrative function in coping and stress adaptation by facilitating transmission in many brain regions involved in regulating behavioral and physiologic components of the stress response. Noradrenergic transmission promotes attention and arousal, biasing the organism toward responding to salient environmental stimuli (Aston-Jones et al., 1999b). Elevated noradrenergic activity in specific limbic forebrain regions also enhances anxiety-like behavioral responses to acute stress, and facilitates activation of the hypothalamic–pituitary–adrenal (HPA) axis, the major neuroendocrine effector of the stress response (Cecchi et al., 2002a,b). An inability to initiate or regulate the stress response appropriately has been proposed to be a critical factor in the pathophysiology of various stress-related disorders, and dysregulation of noradrenergic neurotransmission has been implicated specifically in stress-related psychiatric diseases such as depression, post-traumatic stress disorder (PTSD) and other anxiety disorders (Schatzberg and Schildkraut, 1995; Southwick et al., 1993; Sullivan et al., 1999). The inbred Wistar–Kyoto (WKY) rat strain has been suggested to be a model of genetic stress vulnerability (Pare and Redei, 1993; Redei et al., 1994). Compared with other inbred strains as well as outbred control strains, including Wistar and Sprague–Dawley (SD) rats, WKY rats exhibit an exaggerated HPA response to acute stress, and a greater tendency to develop stress-induced gastric ulcers (Pare and Redei, 1993; Redei et al., 1994). We have shown previously, however, that despite their enhanced HPA stress reactivity, WKY rats show reduced behavioral activity and attenuated anxiety-like behavioral reactivity to acute stress (Pardon et al., 2002). Compared with SD rats, WKY rats showed depressed baseline acoustic startle responding and lower baseline exploratory and social activity. Further, WKY rats showed a lack of fear-potentiation of the startle response, and attenuation of stress-induced reductions in social behavior and open-arm exploration on
*Corresponding author. Tel: ⫹1-210-567-4174; fax: ⫹1-210-567-4303. E-mail address: [email protected] (D. Morilak). Abbreviations: ACTH, adrenocorticotropic hormone; BST, bed nucleus of the stria terminalis; BSTL, lateral bed nucleus of the stria terminalis; BSTLd, lateral bed nucleus of the stria terminalis, dorsal subdivision; BSTLv, lateral bed nucleus of the stria terminalis, ventral subdivision; CeA, central nucleus of the amygdala; CRH, corticotropin-releasing hormone; HPA, hypothalamic–pituitary–adrenal; LC, locus coeruleus; MeA, medial nucleus of the amygdala; NE, norepinephrine; PBS, phosphate-buffered saline; PTSD, post-traumatic stress disorder; PVN, paraventricular nucleus; SD, Sprague–Dawley; WKY, Wistar–Kyoto.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.12.028
963
964
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
the plus maze, indicating reduced anxiety-like behavioral stress reactivity (Pardon et al., 2002). Consistent with this, brain noradrenergic reactivity to stress was also reduced in WKY rats, seen as a blunted elevation in tyrosine hydroxylase mRNA expression in the locus coeruleus (LC), and an attenuation of stress-induced norepinephrine (NE) release in the bed nucleus of the stria terminalis (BST). Further, not only was stress-induced activation of the noradrenergic system reduced, but the facilitation of stressinduced activation of the HPA axis that was shown to be exerted by NE release in the BST of SD rats was found to be absent in WKY rats. Thus, dysregulation of the stress response and increased susceptibility to stress-related pathology in WKY rats may be related to a deficit of brain noradrenergic modulatory activity, though the mechanisms by which this may occur are unknown. Brainstem noradrenergic neurons are found primarily in the LC, as well as in other medullary and pontine cell groups. These cells both project to and receive afferent input from a number of forebrain limbic regions involved directly or indirectly in regulating and modulating behavioral and neuroendocrine responses to stress, including, among others, the lateral BST (BSTL), the central and medial nuclei of the amygdala (CeA and MeA), and the hypothalamic paraventricular nucleus (PVN). The BSTL and CeA are involved in fear and anxiety-like behavioral responses (e.g. Cecchi et al., 2002a; Davis and Shi, 1999), and represent sources of extrahypothalamic modulation of the hormonal HPA stress response mediated in the PVN (Cecchi et al., 2002a; Herman and Cullinan, 1997; Herman et al., 1994). Modulatory effects of NE on behavioral and physiologic responses to various stressors have been described in the BSTL, as well as CeA and PVN (Cecchi et al., 2002a,b; Cole and Sawchenko, 2002; Delfs et al., 2000; Koob, 1999; Plotsky, 1987; Szafarczyk et al., 1987). Although there have been relatively few studies of stress modulatory effects of NE in the MeA, recent observations suggest that descending projections from the MeA, perhaps relaying in the PVN, may be important in the activation of brainstem noradrenergic neurons, specifically by psychogenic stressors (Dayas et al., 1999), thus identifying the MeA as a potentially important upstream afferent regulating the stress reactivity of the brain noradrenergic system. Thus, to understand better the possible neurobiological mechanisms underlying dysregulation of the stress response and increased stress vulnerability of WKY rats, we investigated potential strain differences in stress-induced neuronal activation in brain regions that are involved in behavioral and neuroendocrine stress responses, and are related to the noradrenergic system either as targets of noradrenergic modulation, sources of afferent innervation of noradrenergic neurons, or both, by visualizing stressinduced expression of Fos immunoreactivity in BSTL, CeA, MeA and PVN, as well as the LC. Portions of this work have been presented in abstract form (Ma and Morilak, 2003).
EXPERIMENTAL PROCEDURES Animals Experiments were performed on 13 adult male SD and 13 adult male WKY rats (Charles River, Wilmington, MA, USA), weighing 225–250 g upon arrival. Rats were allowed to acclimate to the animal facility for 7–10 days prior to use, during which they were housed three per cage, all from the same strain, on a 12-h light/dark cycle (lights on at 07:00 h). Food and water were available ad libitum. All effort was made to minimize animal suffering and the number of animals used, and all procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and conformed to NIH guidelines on the ethical use of animals. All experiments were conducted between 09:00 and 12:00 h, during the light portion of the cycle. Rats from each strain were randomly assigned to one of three groups (n⫽4 –5/group). One group from each strain served as unstressed controls, and all other rats were subjected to 1 h immobilization stress. One group of stressed rats from each strain were killed immediately at the termination of 60 min stress, and the remaining groups were killed 1 h after termination of the stress, during which they recovered in their home cages (i.e. 2 h after the onset of 60 min immobilization stress).
Immobilization stress For immobilization stress, rats were held prone on a flat plastic rack large enough to support their entire body (26 cm⫻13 cm). Their limbs were taped to the rack gently but securely using medical adhesive tape. Strips of tape were placed across the back of the neck and head to prevent excessive head movements. At the termination of the 1 h stress period, the animal was held gently in place while the tape was removed.
Fos immunohistochemistry At the designated time point, stressed rats as well as unstressed control rats from each strain were deeply anesthetized (cocktail of ketamine 43 mg/ml, acepromazine 1.4 mg/ml, xylazine 8.6 mg/ml, given in a dose of 0.8 ml/kg, i.m.), and perfused transcardially with 50 ml of 100 mM phosphate-buffered saline (PBS), pH 7.4 containing 1000 IU/ml heparin, followed by 350 ml of 4% paraformaldehyde in PBS, at a flow rate of approximately 50 ml/min. Brains were post-fixed for 1 h in the same fixative, rinsed and then cryoprotected for 48 h in 20% sucrose–PBS at 4 °C. After the brains had sunk, they were frozen in isopentane on dry ice and stored at ⫺70 °C until they could be sectioned and processed (⬍1 month). With the exception of the PVN, six sequential series of 50 m coronal sections were cut on a cryostat through the BSTL, CeA, MeA and LC. Due to the small size of the PVN, only three series of sections were cut through this structure. One series through each region from each brain were used for Fos immunohistochemistry. After rinsing in 20 mM PBS (pH 7.4), free-floating sections were incubated for 30 min at 24 °C in blocking buffer (5% normal sheep serum, 0.3% Triton X-100, 0.05% thimerosal in PBS). They were then incubated in a rabbit anti-cFos antibody (Oncogene Sciences, San Diego, CA, USA), diluted 1/30,000 in blocking buffer for 2 h at 24 °C followed by 40 h at 4 °C. After rinsing, sections were incubated for 4 h at 24 °C in biotinylated sheep anti-rabbit IgG (Sigma) diluted 1/400 in blocking buffer containing 1% normal sheep serum, then for 90 min in avidin– horseradish peroxidase (Sigma) diluted 1/2000 in PBS. Immunoreactivity was then visualized by a peroxidase reaction with 3,3⬘diaminobenzidine as the substrate. Following the reaction, sections were mounted on silanized slides and coverslipped. An adjacent series of sections from each brain were stained with
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
965
Fig. 1. Representative micrographs of Cresyl Violet-stained sections through (A) BSTLd, BSTLv; (B) PVN, parvocellular subdivision (PVNpc); (C) CeA, MeA; and (D) LC, Outlines indicate typical boundaries of regions analyzed for Fos immunoreactivity within each brain area. Scale bar⫽1.00 mm (A, C), 1.25 mm (B) and 1.50 mm (D).
Cresyl Violet to aid in determination and delineation of the cytoarchitectural boundaries of each region of interest. For each region, four to six sections from each brain were analyzed by an investigator blind to the treatment condition, using the NIH-Image software package (v. 1.54; Wayne Rasband, NIH). Sections selected for analysis corresponded to plates 18 –20 (BSTL), 28 –31 (CeA), 29 –32 (MeA), 26 –27 (PVN) and 57–59 (LC) in the atlas of Paxinos and Watson (1998); (see Fig. 1). The dorsal and ventral BSTL (BSTLd and BSTLv, respectively) were analyzed separately. For analysis of Fos expression, digitized images were captured using a SONY XC77 CCD camera mounted on a Nikon microscope coupled to a Scion LG3 capture board in a PowerMac 7100 computer. Images from immunostained sections were aligned with the corresponding image from an adjacent Cresyl Violet-stained section using the Live Paste and capture function. The region of interest was first delineated on the Cresyl Violet-stained section, then the selection outline was transferred to the immunostained section for counting of Fos-positive cell nuclei within the specified region. The mean of all scores within a region was then calculated to generate a single value for each brain, expressed as cells/1000 pixels (corresponding to an area of approximately 6475 m2 at the magnification at which BST, MeA and CeA were analyzed, and approximately 25,900 m2 for PVN and LC). Data were then analyzed by two way ANOVA (Strain⫻Time), followed where appropriate by post hoc analyses using the Newman-Keuls test. Significance was always determined at P⬍0.05.
RESULTS In the PVN (Fig. 2), very few Fos-positive neurons were seen in unstressed control rats of either strain. Significant induction of Fos expression was observed in PVN following 60 min immobilization stress (Main effect for Time: F(2,18)⫽54.324, P⬍0.0001). This induction was significant in both strains relative to their respective unstressed controls (post hoc comparisons, P⬍0.05). Expression of Fos in PVN remained significantly elevated after 60 min of recovery in both strains. Although the number of Fos-
positive cells in PVN was slightly higher at both post-stress time points in WKY rats, there were no significant differences between the strains at any time point (post hoc comparisons, P⬎0.05). In the BSTLv (Fig. 3A, C), very few Fos-positive neurons were seen in unstressed control rats of either strain. Significant induction of Fos expression was observed in BSTLv following 60 min immobilization stress (F(2,18)⫽34.897, P⬍0.0001), and this induction was significant in both strains relative to their respective unstressed controls (P⬍0.05). Expression of Fos in BSTLv remained significantly elevated after 60 min of recovery in both strains. However, there was neither a main effect of Strain nor a significant Strain⫻Time interaction, and post hoc comparisons revealed no significant differences between the strains at any time points (P⬎0.05). By contrast, in the BSTLd (Fig. 3B, C), no significant induction of Fos expression was observed in stressed rats of either strain (F(2,18)⫽0.326). There were no main effects of Time or Strain, and no Strain⫻Time interaction (all P⬎0.05). In CeA (Fig. 4), very few neurons expressed Fos immunoreactivity in unstressed control rats of either strain. ANOVA indicated a significant main effect for Time (F(2,18)⫽4.688, P⬍0.05). The modest increase seen at both 60 and 120 min compared with baseline was, however, significant only when the data were collapsed across strains. When post hoc comparisons were made between each time point and the respective baseline within a strain, none of these comparisons achieved significance. Further, there was no main effect of Strain (F(1,18)⫽2.256), nor a Strain⫻Time interaction (F(2,18)⫽0.207). Likewise, very little Fos expression was seen in MeA of unstressed control rats of either strain (Fig. 5). By contrast with CeA, however, immobilization stress induced an ele-
966
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
Fig. 2. Immobilization stress induced a similar degree of Fos expression in the PVN of both SD and WKY rats. (A) Fos expression induced in PVN of SD and WKY rats by immobilization stress, expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group). * P⬍0.05 compared with same-strain baseline. (B) Representative micrographs of stress-induced Fos immunoreactivity in PVN of SD and WKY rats. Medial is to the right. Scale bar⫽250 m.
vation of Fos expression in MeA (F(2,18)⫽187.643, P⬍0.0001), which was significant in both WKY and SD rats compared with their respective controls (P⬍0.05). Further, there was a significant main effect of Strain (F(1,18)⫽13.979, P⬍0.01), as well as a Strain⫻Time interaction (F(2,18)⫽4.615, P⬍0.05). Post hoc comparisons between strains showed that the induction of Fos expression in MeA immediately after the termination of stress was significantly less in WKY rats compared with SD rats (P⬍0.05). The level of Fos expression in MeA was still elevated after 60 min of post-stress recovery in both strains. However, there was no longer a significant difference between the strains at this time point (see Fig. 5). In the LC of both SD and WKY rats, few Fos positive neurons were seen in unstressed controls (Fig. 6). Following immobilization stress, a significant induction of Fos expression was observed in LC (F(2,18)⫽44.669, P⬍0.0001), which was significant in both strains relative to their respective controls (P⬍0.05). There was also a significant main effect of Strain (F(1,18)⫽9.748, P⬍0.01). The increase in Fos expression in LC, which was still evident after 60 min of post-stress recovery in both strains, was
lower in WKY rats compared with SD rats at this time point (P⬍0.05). Nonetheless, the Strain⫻Time interaction did not reach significance (F(2,18)⫽2.491).
DISCUSSION The brain noradrenergic system is activated by acute stress, and plays an important modulatory role in the response to stress. Acute stress increases NE neurotransmission in limbic forebrain regions such as the CeA and BST (Cecchi et al., 2002a; Pacak et al., 1993, 1995), where it acts to facilitate a number of acute behavioral and neuroendocrine responses to stress, including activation of the HPA axis (Cecchi et al., 2002a,b). In a previous study, we observed reduced activation of the brain noradrenergic system by acute stress in WKY rats, seen as a blunted elevation of tyrosine hydroxylase mRNA expression in the LC and an attenuation of stress-induced NE release in the limbic forebrain compared with SD rats (Pardon et al., 2002). This observation was further confirmed in the present study. Fos induction was moderately lower in LC in WKY rats compared with SD rats immediately after termi-
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
967
Fig. 3. Immobilization stress induced Fos expression to a similar degree in BSTLv of SD and WKY rats. (A) By contrast, Fos expression was not induced in BSTLd of either strain. (B) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group); * P⬍0.05 compared with same-strain baseline. (C) Stress-induced Fos immunoreactivity shown in representative digital photomontages of BST in SD and WKY rats. Medial is to the right. Scale bar⫽250 m.
nation of the 1 h stress, although this difference did not achieve significance, whereas Fos expression was further and significantly reduced in LC of WKY rats compared with SD rats 2 h after the onset of stress (i.e. 1 h after termination of the stressor). A more rapid return toward baseline may also be indicative of a less robust induction of c-fos expression initially. However, because the Fos expression was still significantly elevated in WKY rats at the 120 min time point relative to baseline, and because the level of Fos expression was slightly lower immediately after the 60 min stress as well, we hesitate to conclude that the time course of response was substantively different in the two strains, only that the overall magnitude of response was lower in WKY rats. Consistent with a reduction in noradrenergic activation, we have also reported an attenuation of stress-induced behavioral responses in WKY rats, including some responses that had been shown previously in SD rats to be facilitated by NE (Pardon et al., 2002). In addition, noradrenergic facilitation of the HPA axis, shown to occur in the
BSTL of SD rats, was also lacking in WKY rats, despite their elevated HPA responses (Pardon et al., 2002). Thus, it appears that acute activation of the brain noradrenergic system, and subsequent noradrenergic facilitation of stress-induced activity in target regions such as the BSTL may both be reduced in WKY rats. In the present study, Fos expression in the ventrolateral, but not the dorsolateral subdivision of BST was elevated significantly by acute stress. The BST is a major target of ascending noradrenergic innervation (Moore and Bloom, 1979; Swanson and Hartman, 1975), particularly the ventrolateral subdivision (Phelix et al., 1992a). Noradrenergic innervation of the BST arises primarily from the medullary A2 cell group, with lesser projections from the A1 region and the LC (Aston-Jones et al., 1999a; Delfs et al., 2000). The medullary noradrenergic cell groups were not examined in the present study because, unlike the LC, they are distributed heterogeneously and often sparsely among other cell types in the dorsomedial and ventrolateral medulla (Kalia et al., 1985), making it impossible to
968
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
Fig. 4. Immobilization stress induced only moderate Fos expression in CeA, which was not significant compared with the same-strain baseline in either SD or WKY rats. (A) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group). (B) Fos expression in CeA of SD and WKY rats. Medial is to the left. Scale bar⫽250 m.
identify Fos expression in these neurons in the absence of double-labeling procedures. Nonetheless, the fact that we have previously demonstrated a reduction in stress-activated NE release specifically in the BSTL of WKY rats (Pardon et al., 2002) would suggest that activation of the medullary noradrenergic cell groups was attenuated as well as the LC. In the present study, however, there was no strain difference in stress-induced Fos expression in either BSTLv or BSTLd. This might suggest that other stressresponsive afferents to BSTLv, such as a serotonergic pathway from midbrain raphe nuclei (Phelix et al., 1992b), or an extrahypothalamic input utilizing corticotropin-releasing hormone (CRH; Koob and Heinrichs, 1999; Lee and Davis, 1997), might be sufficient to activate a similar number of cells in this region in WKY and SD rats, despite the difference in noradrenergic activity. Alternatively, it is possible that strain differences in noradrenergic transmission in BSTL may be reflected by differences in the induction of expression of immediate early genes other than c-fos. For instance, both acute swim stress and restraint stress increased the expression of c-fos mRNA in the medial, but not in the lateral part of the BST, whereas the same stimuli increased expression of zif/268 in the lateral but not the medial BST (Cullinan et al., 1995). It is not known whether the deficit of stress-induced noradrenergic activation in WKY rats results from a primary alteration in the activity or excitability of noradrenergic neurons, or from an alteration in excitatory afferent input to the noradrenergic system in this strain. Both the CeA and MeA have been shown to provide excitatory
afferent input to brainstem noradrenergic neurons. CeA has been shown to play a role in behavioral responses to stress (Koob, 1999) and in fear-conditioned behavioral responses (Walker and Davis, 1997). A pathway from CeA to LC utilizing CRH has been shown to be essential to activation of the LC in response to acute hypotensive stress (Curtis et al., 2002). However, the CeA appears not to be involved in activation of noradrenergic neurons by psychogenic stressors such as immobilization (Dayas and Day, 2002), the stimulus used in the present study. Consistent with this, we observed only a modest induction of Fos expression in the CeA following immobilization stress, and there were no strain differences seen in this region. This is also consistent with previous reports describing little or no induction of Fos expression in CeA by acute restraint stress (Arnold et al., 1992; Chen and Herbert, 1995). Similarly, CRH mRNA expression in the CeA has also been reported to be unaffected by immobilization (Pacak et al., 1996). By contrast, the MeA has been shown to be involved in the activation of brainstem noradrenergic neurons in response to psychogenic stress (Dayas and Day, 2002). Further, Fos expression has been shown to be induced in MeA in response to stressful stimuli, particularly to emotional or psychogenic stressors such as restraint (Bhatnagar and Dallman, 1998; Chen and Herbert, 1995; Cullinan et al., 1995), exposure to conditioned aversive stimuli (Pezzone et al., 1992), swim stress (Cullinan et al., 1995) or footshock (Li and Sawchenko, 1998). In the present study, Fos expression was elevated in MeA after immobilization
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
969
Fig. 5. Immobilization stress induced a significant increase in Fos expression in MeA of SD and WKY rats immediately after 60 min stress, and after 1 h post-stress recovery (i.e. 120 min after stress onset). Stress induced significantly higher Fos expression in MeA of SD rats at 60 min, with no strain difference seen after 1 h recovery. (A) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group); * P⬍0.05 compared with same-strain baseline; ⫹ P⬍0.05 between strains at the same time point. (B) Stress-induced Fos expression shown in representative digital photomontages of MeA. Medial is to the right. Scale bar⫽250 m.
Fig. 6. Immobilization stress induced a significant increase in Fos expression in LC of SD and WKY rats immediately after 60 min stress, and after 1 h post-stress recovery (i.e. 120 min after stress onset). The induction of Fos expression was greater in the LC of SD rats, the strain difference being significant at 120 min. (A) Data expressed as cells/1000 pixels2 (mean⫾S.E.M.; n⫽4 –5/group); * P⬍0.05 compared with same-strain baseline; ⫹ P⬍0.05 between strains at the same time point. (B) Stress-induced Fos expression in LC. Medial is to the right. Scale bar⫽250 m.
970
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
stress, and the magnitude of this induction was significantly attenuated in WKY rats compared with SD rats. Thus, a reduction in stress-induced activation of the MeA could be a potential mechanism to account for the reduction in stress-induced activation of the noradrenergic system in WKY rats. However, in addition to providing a descending excitatory input to the brainstem noradrenergic system, the MeA also receives a substantial noradrenergic innervation (Roder and Ciriello, 1993; Sadikot and Parent, 1990). Thus, it is not possible to determine from the present data whether the attenuated activation of MeA in WKY rats represents an upstream mechanism accounting for reduced noradrenergic activation in this strain, or instead represents a downstream consequence of the reduced noradrenergic activation. A limited amount of data have implicated the MeA in modulation of the neuroendocrine response to stress. Efferent projections from the MeA target many regions of the limbic forebrain and hypothalamus that themselves modulate HPA activation, including the anterior and ventromedial hypothalamus, BST, lateral septum, medial preoptic area and ventral hippocampus (Canteras et al., 1995). Lesions of the MeA attenuated adrenocorticotropic hormone (ACTH) and glucocorticoid secretion induced by acoustic or photic stress (Feldman et al., 1994), and reduced the stress-induced expression of Fos in CRH- and oxytocin-synthesizing neurons in the PVN and supraoptic nuclei (Dayas et al., 1999). Thus, a reduced activation of the MeA by stress could represent one potential substrate for some of the differences in the behavioral and neuroendocrine responses to acute stress seen in WKY rats compared with SD rats. As for a potential role of NE in the vulnerability to stress exhibited by WKY rats, we have hypothesized previously that the observed deficit of noradrenergic facilitatory influence, and the consequent attenuation of acute behavioral responses to stress in WKY rats, may hinder their ability to cope effectively with acute stress, thereby increasing the perceived magnitude or relative “impact” of stress (Pardon et al., 2002). This could account secondarily for the elevated HPA response, as well as the increased vulnerability to stress-related pathology seen in this strain. Thus, whereas stress-induced activation of the brain noradrenergic system and activation of target regions that are facilitated by NE may both be reduced in WKY rats, it is possible that stress-induced activation of other brain regions that respond independently of noradrenergic facilitation might be unaffected or even increased. Consistent with this, in the present study, the number of Fos immunoreactive cells seen in the PVN of WKY rats following stress was slightly higher, by approximately 20 –25%, than the number of cells seen in SD rats, although this was not a statistically significant difference. It is important to note that the measure of Fos induction in this experiment, i.e. the number of immunoreactive cell nuclei, is essentially “digital” in nature. As such, it can indicate only how many cells were affected by the stimulus, not the degree to which these cells were affected. The number of cells expressing Fos in PVN after immobilization stress was very high in
both strains. Thus, it is possible that any strain difference in response magnitude in this region might perhaps be reflected not in the number of cells that were activated, but in the intensity with which they were activated, and the immunohistochemical approach cannot detect such graded differences in response magnitude. Nonetheless, based on the slightly elevated HPA response of WKY relative to SD rats, it was not surprising to see at least a comparable activation of PVN in the two strains, if not a slightly greater induction in WKY rats. Indeed, the ACTH response to restraint stress is greater in WKY rats than in several other strains, including SD and Wistar (Pardon et al., 2002; Pare and Redei, 1993; Redei et al., 1994). In cultured anterior pituitary cells from WKY rats compared with Wistar rats, CRH binding sites and receptor mRNA expression were reduced, activation of ACTH secretion in response to CRH or vasopressin was markedly impaired, and vasopressin failed to potentiate CRH-stimulated ACTH secretion (Hauger et al., 2002). These observations may indicate that elevated release of hypothalamic secretagogues, sufficient to induce receptor down-regulation and desensitization, may contribute to the increased sensitivity of the HPA axis in WKY rats. Others have suggested that glucocorticoid feedback may be impaired in WKY rats (Redei et al., 1994). Such interpretations might be consistent with the very slight elevation of Fos expression seen in the PVN of WKY rats compared with SD rats in the present study. However, this was not a significant difference, and other studies have revealed no difference in CRH mRNA expression in PVN of WKY rats compared with SD rats, either before or after acute or chronic immobilization stress (Gomez et al., 1996; Krukoff et al., 1999), suggesting that the strain difference in the HPA response to stress may not be attributable to differences in activation of the PVN. In conclusion, altered stress responsivity of the brain noradrenergic system and of specific stress-responsive brain regions that are related to it, such as the MeA, may contribute to the abnormal neuroendocrine and behavioral stress responses exhibited by WKY rats compared with the outbred control SD strain. Further, these aberrant responses may contribute to the demonstrated susceptibility of WKY rats to stress-related pathology (Redei et al., 1994), making them a potentially useful model for understanding the neural mechanisms linking stress to certain psychopathological disorders, such as depression, PTSD or other anxiety disorders, in susceptible individuals (Gold and Chrousos, 1999; Schatzberg and Schildkraut, 1995; Southwick et al., 1993; Sullivan et al., 1999). Thus, understanding how changes in these neural systems influence the behavioral and neuroendocrine response characteristics of WKY rats may contribute to the development of better strategies for prevention or treatment of stress-related psychiatric disorders. Acknowledgements—Expert technical assistance was provided by Gabe Barrera, April Garcia, and Angelica Hernandez. Supported by a research grant from the NIMH (MH53851).
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
REFERENCES Arnold FJ, De Lucas Bueno M, Shiers H, Hancock DC, Evan GI, Herbert J (1992) Expression of c-fos in regions of the basal limbic forebrain following intracerebroventricular corticotropin-releasing factor in unstressed or stressed male rats. Neuroscience 51:377– 390. Aston-Jones G, Delfs JM, Druhan J, Zhu Y (1999a) The bed nucleus of the stria terminalis: a target site for noradrenergic actions in opiate withdrawal. Ann NY Acad Sci 877:486 –498. Aston-Jones G, Rajkowski J, Cohen J (1999b) Role of locus coeruleus in attention and behavioral flexibility. Biol Psychiatry 46:1309–1320. Bhatnagar S, Dallman M (1998) Neuroanatomical basis for facilitation of hypothalamic-pituitary-adrenal responses to a novel stressor after chronic stress. Neuroscience 84:1025–1039. Canteras NS, Simerly RB, Swanson LW (1995) Organization of projections from the medial nucleus of the amygdala: a PHAL study in the rat. J Comp Neurol 360:213–245. Cecchi M, Khoshbouei H, Javors M, Morilak DA (2002a) Modulatory effects of norepinephrine in the lateral bed nucleus of the stria terminalis on behavioral and neuroendocrine responses to acute stress. Neuroscience 112:13–21. Cecchi M, Khoshbouei H, Morilak DA (2002b) Modulatory effects of norepinephrine, acting on ␣1-receptors in the central nucleus of the amygdala, on behavioral and neuroendocrine responses to acute immobilization stress. Neuropharmacology 43:1139 –1147. Chen X, Herbert J (1995) Regional changes in c-fos expression in the basal forebrain and brainstem during adaptation to repeated stress: correlations with cardiovascular, hypothermic and endocrine responses. Neuroscience 64:675–685. Cole RL, Sawchenko PE (2002) Neurotransmitter regulation of cellular activation and neuropeptide gene expression in the paraventricular nucleus of the hypothalamus. J Neurosci 22:959 –969. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ (1995) Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64:477–505. Curtis AL, Bello NT, Connolly KR, Valentino RJ (2002) Corticotropinreleasing factor neurones of the central nucleus of the amygdala mediate locus coeruleus activation by cardiovascular stress. J Neuroendocrinol 14:667–682. Davis M, Shi C (1999) The extended amygdala: are the central nucleus of the amygdala and the bed nucleus of the stria terminalis differentially involved in fear versus anxiety? Ann NY Acad Sci 877:281–291. Dayas CV, Buller KM, Day TA (1999) Neuroendocrine responses to an emotional stressor: evidence for involvement of the medial but not the central amygdala. Eur J Neurosci 11:2312–2322. Dayas CV, Day TA (2002) Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor. Eur J Neurosci 15:1712–1718. Delfs J, Zhu Y, Druhan J, Aston-Jones G (2000) Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion. Nature 403:430 –434. Feldman S, Conforti N, Itzak A, Weidenfeld J (1994) Differential effect of amygdaloid lesions on CRF-41, ACTH and corticosterone responses following neural stimuli. Brain Res 658:21–26. Gold PW, Chrousos GP (1999) The endocrinology of melancholic and atypical depression: relation to neurocircuitry and somatic consequences. Proc Assoc Am Physicians 111:22–34. Gomez F, Lahmame A, De Kloet ER, Armario A (1996) Hypothalamicpituitary-adrenal response to chronic stress in five inbred rat strains: differential responses are mainly located at the adrenocortical level. Neuroendocrinology 63:327–337. Hauger RL, Shelat SG, Redei EE (2002) Decreased corticotropinreleasing factor receptor expression and adrenocorticotropic hormone responsiveness in anterior pituitary cells of Wistar-Kyoto rats. J Neuroendocrinol 14:126 –134. Herman JP, Cullinan WE (1997) Neurocircuitry of stress: central con-
971
trol of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 20:78 –84. Herman JP, Cullinan WE, Watson SJ (1994) Involvement of the bed nucleus of the stria terminalis in tonic regulation of paraventricular hypothalamic CRH and AVP mRNA expression. J Neuroendocrinol 6:433–442. Kalia M, Woodward DJ, Smith WK, Fuxe K (1985) Rat medulla oblongata: IV. Topographical distribution of catecholaminergic neurons with quantitative three-dimensional computer reconstruction. J Comp Neurol 233:350 –364. Koob GF (1999) Corticotropin-releasing factor, norepinephrine and stress. Biol Psychiatry 46:1167–1180. Koob GF, Heinrichs SC (1999) A role for corticotropin releasing factor and urocortin in behavioral responses to stressors. Brain Res 848: 141–152. Krukoff TL, MacTavish D, Jhamandas JH (1999) Hypertensive rats exhibit heightened expression of corticotropin-releasing factor in activated central neurons in response to restraint stress. Mol Brain Res 65:70 –79. Lee Y, Davis M (1997) Role of the hippocampus, the bed nucleus of the stria terminalis, and the amygdala in the excitatory effect of corticotropin-releasing hormone on the acoustic startle reflex. J Neurosci 17:6434 –6446. Li HY, Sawchenko PE (1998) Hypothalamic effector neurons and extended circuitries activated in “neurogenic” stress: a comparison of footshock effects exerted acutely, chronically, and in animals with controlled glucocorticoid levels. J Comp Neurol 393:244 –266. Ma S, Morilak DA (2003) Reduced c-Fos induction by immobilization stress in locus coeruleus and medial amygdala of Wistar-Kyoto compared to Sprague-Dawley rats. Soc Neurosci Abstr 29, Online program no. 712.6. Moore RY, Bloom FE (1979) Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu Rev Neurosci 2:113–168. Pacak K, McCarty R, Palkovits M, Kopin IJ, Goldstein DS (1995) Effects of immobilization on in vivo release of norepinephrine in the bed nucleus of the stria terminalis in conscious rats. Brain Res 688:242–246. Pacak K, Palkovits M, Kvetnansky R, Fukuhara K, Kopin IJ, Goldstein DS (1993) Effects of single or repeated immobilization on release of norepinephrine and its metabolites in the central nucleus of the amygdala in conscious rats. Neuroendocrinology 57:623–633. Pacak K, Palkovits M, Makino S, Kopin IJ, Goldstein DS (1996) Brainstem hemisection decreases corticotropin-releasing hormone mRNA in the paraventricular nucleus but not in the central amygdaloid nucleus. J Neuroendocrinol 8:543–551. Pardon M-C, Gould GG, Garcia A, Phillips L, Cook MC, Miller SA, Mason PA, Morilak DA (2002) Stress reactivity of the brain noradrenergic system in three rat strains differing in their neuroendocrine and behavioral responses to stress: implications for susceptibility to stress-related neuropsychiatric disorders. Neuroscience 115:229–242. Pare WP, Redei E (1993) Depressive behavior and stress ulcer in Wistar Kyoto rats. J Physiol 87:229 –238. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, 4th edition. San Diego: Academic Press. Pezzone MA, Lee WS, Hoffman GE, Rabin BS (1992) Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli. Brain Res 597:41–50. Phelix CF, Liposits Z, Paull WK (1992a) Monoamine innervation of bed nucleus nucleus of stria terminalis: an electron microscopic investigation. Brain Res Bull 28:949 –965. Phelix CF, Liposits Z, Paull WK (1992b) Serotonin-CRF interaction in the bed nucleus of the stria terminalis: a light microscopic doublelabel immunocytochemical analysis. Brain Res Bull 28:943–948. Plotsky PM (1987) Facilitation of immunoreactive corticotropin-releasing factor secretion into the hypophysial-portal circulation after activation of catecholaminergic pathways or central norepinephrine injection. Endocrinology 121:924 –930.
972
S. Ma and D. A. Morilak / Neuroscience 124 (2004) 963–972
Redei E, Pare WP, Aird F, Kluczynski J (1994) Strain differences in hypothalamic-pituitary-adrenal activity and stress ulcer. Am J Physiol 266:R353–360. Roder S, Ciriello J (1993) Innervation of the amygdaloid complex by catecholaminergic cell groups of the ventrolateral medulla. J Comp Neurol 332:105–122. Sadikot AF, Parent A (1990) The monoaminergic innervation of the amygdala in the squirrel monkey: an immunohistochemical study. Neuroscience 36:431–447. Schatzberg AF, Schildkraut JJ (1995) Recent studies on norepinephrine systems in mood disorders. In: Psychopharmacology: the fourth generation of progress (Bloom FE, Kupfer DJ, eds), pp 911–920. New York: Raven Press, Ltd. Southwick SM, Krystal JH, Morgan CA, Johnson D, Nagy LM, Nicolaou A, Heninger GR, Charney DS (1993) Abnormal noradrenergic function in posttraumatic stress disorder. Arch Gen Psychiatry 50: 266 –274.
Sullivan GM, Coplan JD, Kent JM, Gorman JM (1999) The noradrenergic system in pathological anxiety: a focus on panic with relevance to generalized anxiety and phobias. Biol Psychiatry 46: 1205–1218. Swanson LW, Hartman BK (1975) The central adrenergic system: an immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker. J Comp Neurol 163:467–505. Szafarczyk A, Malaval F, Laurent A, Gibaud R, Assenmacher I (1987) Further evidence for a central stimulatory action of catecholamines on adrenocorticotropin release in the rat. Endocrinology 121:883– 892. Walker DL, Davis M (1997) Double dissociation between the involvement of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in startle increases produced by conditioned versus unconditioned fear. J Neurosci 17:9375– 9383.
(Accepted 18 December 2003)