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Involvement of central angiotensin receptors in stress adaptation
Neuroscience Vol. 93, No. 3, pp. 877–884, 1999 877 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
Involvement of central angiotensin receptors in stress adaptation
Pergamon PII: S0306-4522(99)00206-7
INVOLVEMENT OF CENTRAL ANGIOTENSIN RECEPTORS IN STRESS ADAPTATION E. C. DUMONT, S. RAFRAFI, S. LAFOREST and G. DROLET* Laval University Medical Research Centre, Neuroscience Unit, and Faculty of Medicine, Universite´ Laval, 2705 boul. Laurier, Sainte-Foy, Que´bec, Canada G1V 4G2
Abstract—The present study examined the effects of acute and chronic neurogenic stressors on the expression of two distinct angiotensin receptors in two stress-related brain nuclei: angiotensin type 1A receptor in the paraventricular nucleus of the hypothalamus and angiotensin type 2 receptor in the nucleus locus coeruleus. Male Wistar rats were divided into four experimental groups. The first two groups were subjected once to an acute 90-min immobilization or air-jet stress session, respectively. The other two groups were subjected to 10 days of daily 90-min immobilization sessions and, on the 11th day, one group was exposed to an additional 90-min immobilization and the other to a single air-jet stress (heterotypic but still neurogenic) session. In each group, rats were perfused before stress (0 min), immediately following stress (90 min) or 150, 180, 270 or 360 min (and 24 h in chronic immobilization) after the beginning of the last stress session. Basal expression of both angiotensin receptor subtype 1A and angiotensin receptor subtype 2 messenger RNA was minimal in non-stressed animals. Acute immobilization as well as air-jet stress induced similar patterns (time-course and maximal values) of angiotensin receptor subtype 1A messenger RNA expression in the paraventricular nucleus. Angiotensin receptor subtype 1A messenger RNA expression increased 90–150 min after the beginning of the stress and returned to basal levels by 360 min. Chronic stress immobilization slightly modified the pattern, but not maximal values of angiotensin receptor subtype 1A messenger RNA expression to further immobilization (homotypic) or air-jet stress (heterotypic). Acute immobilization and air-jet stress sessions induced similar locus coeruleus-specific angiotensin receptor subtype 2 messenger RNA expression. This expression increased 90 min following the onset of the stress session and remained elevated for at least 360 min. Chronic immobilization stress increased angiotensin receptor subtype 2 messenger RNA expression to levels comparable to those observed in acute stress conditions. Novel acute exposure to neurogenic stressors did not further increase these levels in either homotypic (immobilization) or in heterotypic (air-jet stress) conditions. These results suggest that central angiotensin receptors are targets of regulation in stress; therefore, stress may modulate angiotensin function in the paraventricular nucleus and locus coeruleus during chronic exposure to neurogenic stressors. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: paraventricular nucleus of the hypothalamus, locus coeruleus, corticotropin-releasing hormone, autonomic nervous system, hypothalamo-hypophyso-adrenal.
That chronic exposure to stressful events induces compensatory mechanisms that attenuate the deleterious effects of repeated stress is a well-documented fact. The ability to adapt to repetitive or prolonged stress is apparent at the levels of both major stress effectors: the hypothalamo-hypophysoadrenal (HPA) axis and the autonomic nervous system. 40 However, the nature and specificity (or generality) of these adaptational phenomena in the autonomic nervous system and HPA are controversial. Nevertheless, it appears that habituation of the HPA and autonomic responses to chronic stressors are not due to reduced responsiveness of the system per se. Changes in the afferent control of hypothalamic paraventricular corticotropin-releasing hormone (CRH) neurons and/or other systems involved in the release of adrenocorticotropin hormone (ACTH) secretagogues are thought to underline these modifications. 1,30 Indeed, the mechanisms responsible for the potential intact responsiveness of the system are still not known. The central renin–angiotensin system may significantly modulate the effect of the stress response. 7,16 It has been
suggested that angiotensin stimulates ACTH release by augmenting CRH liberation, presumably through an action on CRH neurons in the paraventricular nucleus of the hypothalamus (PVH), and also by potentiating the effects of CRH in the anterior pituitary. 27,28,33 The release of CRH into the hypophyseal portal circulation from the parvocellular neurons of the PVH initiates a cascade of well-defined physiological and behavioral responses. 31 These effects of angiotensin at the PVH level appear to be mediated through angiotensin type 1A receptors (AT1A), which are richly distributed in this nucleus, 32,38 especially on CRHergic neurons of the parvocellular division. 2,3 In contrast, the involvement of the angiotensin type 2 receptor (AT2) in the stress response is less understood. However, both AT2 receptor mRNA and AT2 binding sites have been identified in several regions of the brain, 22 for example in the nucleus locus coeruleus (LC), a well-defined stress-related brain structure. The investigation of chronic adaptation to stress depends very much on the nature of the stressors. Indeed, recent studies have shown that different families of stressors (rather than the stressor itself) use distinct neuronal circuits for the regulation of HPA and autonomic axes. 15,23,30 It follows that, when seeking an accurate description of chronic adaptation to stress, the choice of stressor is of critical importance. The objective of the present study was to investigate the effects of acute and chronic neurogenic stressors on two distinct angiotensin receptors in stress-related brain nuclei, AT1A in
*To whom correspondence should be addressed. E-mail address: [email protected] (G. Drolet) Abbreviations: ACTH, adrenocorticotropin hormone; AT1A, angiotensin receptor subtype 1A; AT2, angiotensin receptor subtype 2; CRH, corticotropin-releasing hormone; EDTA, ethylenediaminetetra-acetate; HPA, hypothalamo-hypophyso-adrenal; LC, locus coeruleus; O.D., optical density; PVH, paraventricular nucleus of the hypothalamus. 877
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the PVH and AT2 in the LC. To investigate the specificity of stress adaptation, we chose to study the effects of two different painless neurogenic stressors; immobilization and air jet, to ascertain whether the psychogenic nature of the stressor or the stressor itself is important for adaptation to chronic exposure to stress. EXPERIMENTAL PROCEDURES
Animals Adult male Wistar rats (Charles River, St Constant, Quebec; 12–14 weeks) weighing 225–250 g were acclimatized for five to seven days to standard laboratory conditions (14-h light/10-h dark cycle: lights on at 06.00 and off at 20.00), and given free access to rat chow and tap water. The investigation was performed in accordance with the guidelines of the Canadian Council on Animal Care, and approved by institutional animal care committee. Stress paradigm design Rats were divided into four experimental groups. The first two groups were subjected once to an acute 90-min immobilization (acute immobilization) or air-jet stress (acute air-jet stress) session, respectively. The other two groups were subjected to 10 days of daily 90-min immobilization sessions and, on the 11th day, one group was exposed to an additional 90-min immobilization (chronic immobilization, homotypic), while the remaining group was subjected to a single air-jet stress (chronic air-jet stress, heterotypic but still neurogenic) session. Adjustable restraining cages (Centrap) were used to immobilize the rats. Air-jet stress was accomplished by coupling immobilization stress with bursts of air at 55 psi aimed at the animals’ snouts. The rats were placed in an immobilization cage and secured to the air-jet delivery device. A computer software randomly controlled both the duration (0.5–10 s) and occurrence interval (10–60 s) of the air-jet bursts. Rats from each group were killed before stress (time 0), or were placed in an individual immobilization cage for 90 min or subjected to a 90-min air-jet session. The rats were then deeply anesthetized with a ketamine (Rogarsetice, 80 mg/kg)/ xylazine (Rompune, 10 mg/kg) solution (i.p.) 90, 150, 180, 270 and 360 min (and 24 h for the chronic immobilization group) after the onset of the stress session, and then perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 48C). The brains were removed from the skull, postfixed for four to seven days and then placed in 20% sucrose/4% paraformaldehyde buffer 48 h at 48C. Frozen brains were mounted on a microtome and cut into 30-mm coronal sections. The slices were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer, 30% ethyleneglycol and 20% glycerol) and stored at 2208C. In situ hybridization histochemistry Histochemical hybridization localization of each transcript was carried out as described previously. 24 AT1A probe (from Dr Deschepper, IRCM, Montre´al, Que´bec, Canada) was synthesized from the Sal I/ Bam H1 fragment of the rat adrenal cDNA library, subcloned into pSPORT 1 and linearized with Sal I (antisense) or Bam H1 (sense). AT2 probe (from Dr Harrison, University of Florida, Gainesville, FL) was produced in pCDM8, subcloned in SKII 1 and linearized with Hind III (antisense) or Bam H1 (sense). Radioisotope-labeled antisense cRNA copies were synthesized by incubating 250 ng of linearized plasmid in 5×transcription buffer (6 mM MgCl2, 30–40 mM Tris, pH 7.9, 10 mM NaCl), 10 mM dithiothreitol, 0.2 mM ATP/GTP/CTP, 200 mCi [a- 35S]UTP, 40 U RNasin and 20 U SP6 (AT1A) or T3 (AT2) RNA polymerase, together for 60 min at 378C. Unincorporated nucleotides were removed using the ammonium acetate method, where 100 ml DNase solution (1 ml DNase, 2.5 ml of 10 mg/ml transfer RNA, 94 ml of 10 mM Tris/ 10 mM MgCl2, 2.5 ml diethylpyrocarbonate water) was added, and followed 10 min later by phenol–chloroform solution extraction. The cRNA was precipitated with 80 ml of 5 M ammonium acetate and 500 ml of 100% ethanol for 20 min on dry ice. The pellet was resuspended in 100 ml of 10 mM Tris/1 mM EDTA (pH 8.0). A concentration of 10 7 c.p.m. probe was mixed into 1 ml hybridization solution (518 ml formamide, 62 ml of 5 M NaCl, 10 ml of 1 M Tris, pH 8.0, 2 ml of 0.5 M EDTA, pH 8.0, 20 ml of 50×Denhardt’s solution, 207 ml of
50% dextran sulfate, 50 ml of 10 mg/ml transfer RNA, 10 ml of 1 M dithiothreitol, 118 ml diethylpyrocarbonate water-volume of probe used). This solution was mixed and heated for 5 min at 658C before applying to slides. Radioisotope-labeled sense (control) cRNA copies were also prepared to verify the specificity of each probe (T7 for both probes). Hybridization with these probes did not reveal any positive signal in the rat brain. Quantitative analysis Blind quantitative analysis of hybridization signal for the mRNA signals was performed on X-ray film over at least two PVHs (bilateral) and two LCs for each animal. Transmittance values (referred to here as optical density, O.D.) of the hybridization signals were measured using a Northern Light Desktop Illuminator (Imaging Research) with a Sony Camera Video System attached to a Micro-Nikkor 55-mm Vivitar extension tube set for a Nikon lens and coupled to a Macintosh computer (Power Macintosh 8600/300), then analysed with NIH Image software, version 1.61 (W. Rasband, National Institutes of Health, Bethesda, MD). The O.D. value for each pixel was calculated using a known standard of intensity and distance measurements from a logarithmic specter adapted from Bioimage Visage 110s (Millipore, Ann Arbor, MI). The wedge was calibrated before correcting for film saturation, which was determined by sampling the darkest PVHs or LCs then adjusting the light source and exposure time. All samples that emitted a clear positive signal were evaluated within a linear range to avoid pixel saturation and underestimation. Sections from experimental and control animals were digitalized and subjected to densitometric analysis that yielded values of mean density per area. The O.D. of each side of the nucleus (LC and PVH) was corrected for the average background signal by subtracting the O.D. of areas without positive signal located immediately outside the digitalized nucleus. Statistical analysis Data from graphical figures are expressed as relative O.D. (arbitrary units) for mRNA in the PVH or the LC of control (time 0), stressed (time 90 min) and post-stressed (times 150, 180, 270, 360 min and 24 h) Wistar rats. The results were analysed using a three-way ANOVA (factorial); post hoc multiple comparisons were made using a Bonferroni test. Factors were identified as follows: time after the beginning of stress (0, 90, 150, 180, 270, 360 min), the type of stressor (immobilization, air-jet stress) and the stress duration (acute, chronic). Levels were deemed significant at P,0.05. RESULTS
Paraventricular nucleus/angiotensin receptor subtype 1A messenger RNA expression Acute immobilization. Basal expression of AT1A receptor mRNA within the PVH was barely detectable in non-stressed animals (time 0, O.D. 8.0^2.4, n7; Fig. 1, upper left, Fig. 2A). Acute exposure to a 90-min immobilization session increased the expression of AT1A receptor mRNA in the parvocellular part of the PVH, specifically (as reported previously). 2,22 This expression was detectable and reached a maximal value 150 min after the onset of the homotypic stress session (O.D. 32.4^4.7, n5, P,0.001; Fig. 1, lower left, Fig. 2A). The positive signal was still present at 180 (O.D. 28.9^4.5, n6, P,0.001; Fig. 2A) and 270 min (O.D. 30.3^3.9, n4, P,0.001; Fig. 2A), but returned to basal levels 360 min (O.D. 16.8^1.8, n2, P.0.05; Fig. 2A) after the onset of the stress session. Chronic immobilization (homotypic stressor). Expression of AT1A receptor mRNA within the PVH was also barely detectable following chronic exposure to immobilization stress (O.D. 5.6^2.5, n5; Fig. 1, upper right, Fig. 2A). Subsequent exposure to immobilization induced a similar maximal increase of AT1A receptor mRNA when compared with the acute condition measured at 150 min (O.D.
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Fig. 1. Distribution of the mRNA encoding the AT1A receptor in the brain of rats exposed to an acute 90-min (left panel) or chronic (10 days × 90 min, right panel) immobilization stress session. Representative examples of coronal brain slices at 0 (upper), 90 (middle) and 150 (lower) min following the onset of the stress session are shown. Acute exposure to a 90-min immobilization session increased the expression of AT1A receptor mRNA specifically in the parvocellular part of the PVH. This expression was detectable and reached a maximal value 150 min following the onset of the stress session. Chronic exposure to immobilization stress sessions modified the time-course of AT1A expression in the PVH to subsequent stress exposure, without altering the maximal values. AT1A expression was already detectable 90 min after the onset of the last stress session as compared to 150 min in acute conditions.
32.4^4.6, n5; Fig. 1, lower right, Fig. 2A). However, chronic exposure to immobilization stress modified the time-course of AT1A receptor mRNA expression in the PVH. Indeed, in chronic conditions, AT1A expression was detectable at as early as 90 min (O.D. 19.1^2.3, n6 and 3.6^1.8, n4, P0.002, chronic vs acute; Fig. 1, middle panels, Fig. 2A). Moreover, the signal returned more rapidly to baseline levels in chronic conditions (O.D. 10.2^2.7, n5 at 270 min, P0.003 compared with acute; Fig. 2A). Acute air-jet stress. The basal expression of AT1A receptor mRNA was barely detectable in the acute air-jet stress group (O.D. 5.3^2.0, n3; Fig. 2B). Acute exposure to a 90-min air-jet stress session also induced the expression of AT1A receptor mRNA in the PVH, which was detectable immedi-
ately after the offset of the stress session (O.D. 27.3^3.3, n5, P0.02; Fig. 2B), reaching a maximal value 150 min after the onset of the stress session (O.D. 32.7^6.1, n5, P0.004; Fig. 2B). The positive signal was still present at 180 (O.D. 28.5^6.3, n6, P0.01; Fig. 2B) and 270 min (O.D. 26.3^5.4, n4, P0.03; Fig. 2B), but returned to basal levels 360 min (O.D. 14.3^0.8, n2; Fig. 2B) after the onset of the stress session. Chronic immobilization (heterotypic stressor). Expression of AT1A receptor mRNA was barely detectable following chronic exposure to immobilization stress sessions in this group (O.D. 5.5^1.2, n4; Fig. 2B). Subsequent exposure to heterotypic stress (air jet) induced a comparable maximal increase in AT1A receptor mRNA when compared with the
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Fig. 2. Effect of acute (open symbols) and chronic (filled symbols) immobilization (A) and air-jet (B) stress on the average relative O.D. for the AT1A receptor mRNA hybridization signal in the PVH. The effects of both neurogenic stress paradigms on AT1A receptor mRNA were compared in acute (C) and chronic (D) conditions. In acute conditions, the time-course of expression and the maximal intensity of the signal were comparable between immobilization and air-jet stress. Chronic exposure to immobilization stress sessions modified the time-course of AT1A expression in the PVH to subsequent stress exposure, without altering the maximal values reached in both immobilization (homotypic stressor) and air-jet stress (heterotypic stressor) groups. These results represent the means^S.E.M. of three to seven rats per time-point. Statistical analysis was performed using a factorial three-way ANOVA followed by a Bonferroni post hoc test. T represents a significant (at least P,0.05) effect of time and G a significant difference between two groups at a particular time-point.
acute condition (O.D. 32.3^1.4, n3; Fig. 2B). However, the time-course of AT1A receptor mRNA expression in the PVH induced by air-jet stress was slightly modified by chronic exposure to immobilization stress. Indeed, under chronic conditions, a smaller increase of AT1A expression was observed at 90 min (O.D. 18.8^1.6, n5, P0.04 compared with acute; Fig. 2B). Moreover, under chronic conditions, the signal tended to fall more rapidly to baseline levels (Fig. 2B). No significant difference was observed between the AT1A receptor expression induced by either neurogenic stressor in acute (Fig. 2C) or chronic conditions (Fig. 2D). Locus coeruleus/angiotensin receptor subtype 2 messenger RNA expression Acute immobilization. Basal expression of AT2 receptor mRNA within the LC was barely detectable in non-stressed animals (time 0, O.D. 4.9^3.3, n8; Fig. 4A). Acute exposure to a 90-min immobilization session increased the mRNA expression of that receptor specifically in the LC. This expression reached a maximal value 90 min after the onset of the stress session (O.D. 42.3^6.9, n3, P,0.001; Fig. 4A). The positive signal was still present at 150 min (O.D. 31.9^7.0, n9, P,0.001; Fig. 4A), 180 min (O.D. 29.0^5.1, n6, P0.004; Fig. 4A), 270 min (O.D. 29.0^4.1, n5, P0.006; Fig. 4A) and 360 min (O.D. 39.3^3.2, n9, P0.01; Fig. 4A) after the onset of the stress session. Chronic immobilization (homotypic). Expression of AT2
receptor mRNA was increased by chronic exposure to immobilization stress sessions (O.D. 20.9^4.6, n5; Fig. 4A). Subsequent exposure to immobilization did not significantly modify AT2 receptor mRNA expression (Fig. 4A). Acute air-jet stress. Basal expression of AT2 receptor mRNA within the LC was barely detectable in non-stressed animals of the air-jet stress group (time 0, O.D. 1.5^0.8, n3; Fig. 3, upper left, Fig. 4B). Exposure to a 90-min airjet session increased the expression of AT2 receptor mRNA specifically in the LC. This expression was detectable 90 min after the onset of the stress session (O.D. 19.8^3.0, n7, P,0.001; Fig. 3, middle left, Fig. 4B). The positive signal was still present 150 min (O.D. 29.1^3.4, n6, P,0.001; Fig. 4B), 180 min (O.D. 26.0^5.8, n6, P0.02; Fig. 4B), 270 min (O.D. 33.1^3.8, n6, P,0.001; Fig. 4B) and 360 min (O.D. 32.8^8.6, n3, P0.001; Fig. 3, lower left, Fig. 4B) after the onset of the stress session. Chronic immobilization (heterotypic stressor). Expression of AT2 receptor mRNA was increased by chronic exposure to immobilization sessions (O.D. 25.6^3.1, n4; Fig. 3, upper right, Fig. 4B). Subsequent exposure to air-jet stress did not modify AT2 receptor mRNA expression (Fig. 3, middle and lower right, Fig. 4B). There was no significant difference between the AT2 mRNA receptor expression induced by either neurogenic stressor in acute (Fig. 4C) or chronic conditions (Fig. 4D).
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Fig. 3. Distribution of the mRNA encoding the AT2 receptor in the brain of rats exposed to an acute 90-min air-jet stress session (left panel) or to a 90-min airjet stress session following chronic exposure to immobilization (10 days × 90 min, right panel). Representative examples of coronal brain slices at 0 (upper), 90 (middle) and 360 (lower) min after the onset of the stress session are shown. Acute exposure to a 90-min air-jet stress session induced the expression of AT2 receptor mRNA specifically in the LC. Moreover, expression of AT2 receptor mRNA was increased by chronic exposure to an immobilization stress session at levels comparable with maximal values obtained with acute exposure to air-jet stress. This increase remained elevated for at least 360 min in both acute and chronic conditions.
DISCUSSION
These results suggest that central angiotensin receptors are important targets of stress regulation; therefore, stress could modulate angiotensin function in the PVH and the LC during chronic exposure to neurogenic stressors. In fact, acute exposure to neurogenic stressors induced the expression of AT1A and AT2 receptor mRNA in the two stress-associated brain nuclei, the PVH and LC, respectively. Moreover, chronic intermittent stress did not attenuate the expression of the AT1A receptor within the PVH, suggesting that the integrity of these receptors may be essential for the maintenance of reactivity to stress of PVH neurons. Furthermore, the present study reports, for the first time, that chronic immobilization stress increases basal expression of AT2 within the LC and
AT2 mRNA increases in the LC to a level that approaches the maximal acute response. This again suggests that the central angiotensin system plays an important role in the adaptive cellular mechanisms that drive the response of the central stress system when assaulted by repeated neurogenic stress. The angiotensin receptor subtype 1A and the paraventricular nucleus of the hypothalamus A physiological role for angiotensin as a central neurotransmitter within the PVH was provided by a study that demonstrated the local release of angiotensin in response to physiological and chemical stimuli. 11 This endogenous release of angiotensin within the PVH mediated its actions
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Fig. 4. Effect of acute (open symbols) and chronic (filled symbols) immobilization (A) and air-jet (B) stress on the average relative O.D. for the AT2 receptor mRNA hybridization signal in the LC. The effects of both neurogenic stress paradigms on AT2 receptor mRNA were compared in acute (C) and chronic (D) conditions. In acute conditions, the time-course of expression and the maximal intensity of the signal were comparable between immobilization and air-jet stress. The expression of AT2 receptor mRNA was increased by chronic exposure to an immobilization stress session. This expression was not further modified by subsequent acute exposure to an immobilization (homotypic stressor) or air-jet stress (heterotypic stressor) session. These results represent the means ^S.E.M. of three to eight rats per time-point. Statistical analysis was performed using a factorial three-way ANOVA followed by a Bonferroni post hoc test. T represents a significant (at least P,0.05) effect of time and G a significant difference between two groups at a particular time-point.
via the AT1A receptor, which is highly expressed in CRHergic neurons of the parvocellular division. 2,3,32,38 Therefore, angiotensin could modulate the activity of CRH neurons directly through AT1A receptors in PVH–CRH perikarya. Effectively, central administration of angiotensin dramatically increases the expression of CRH mRNA within the PVH, suggesting that the release of angiotensin in the PVH during stressful situations may be involved in the elevated CRH mRNA levels seen following acute stress. 25,33 Moreover, it has been reported that repeated immobilization exposure resulted in up-regulation of angiotensin receptors in the PVH. 7 Therefore, the present increase of AT1A expression in the PVH after chronic as well as acute stress is likely to be translated by the synthesis of new AT1A receptors. The increase in angiotensin binding sites 7 and AT1A receptor mRNA, as seen in the present study, could be mediated by high plasma levels of glucocorticoids that occur during stress sessions. Consistent with this are reports that dexamethasone injection increased the expression of AT1A within the PVH, whereas adrenalectomy induced a significant decrease that could be prevented by glucocorticoid treatment. 2 The functional significance of the AT1 receptor in the neurobiology of stress needs to be clarified, but its presence within the parvocellular part of the PVH provides clear anatomical evidence that this receptor could directly modulate both neuroendocrine and autonomic functions. Despite reports of the involvement of the AT1A receptor in HPA regulation, some inconsistencies regarding its exact role persist. Presently, it appears that angiotensin may differentially affect different components of the HPA. For instance, angiotensin modulates the expression of CRF mRNA in the PVH in a
direct and positive manner via activation of AT1 receptors. 3 However, the blockade of central AT1 receptor did not reduce the restraint stress-induced elevated plasma levels of ACTH and corticosterone. 2,3,18 This divergence between the effects of AT1 receptor on different components of the HPA is consistent with the recent finding of functional dissociation within the regulation of different components of the HPA. In a twopaper series, Watts’ group reported that the secretion of ACTH and activation of the CRF gene have distinct and separate stimulus thresholds, suggesting some degree of mechanistic separation. 36,37 These studies raised the point that temporal organization and functional interaction between the components of the HPA could be dissociated. Alternatively, the role of the AT1 receptor within the PVH could be related to autonomic regulation. Indeed, it has been demonstrated that angiotensin is required for the full expression of sympathetic, pressor and neuroendocrine responses during stress. 16,21,29 Moreover, intracerebroventricular administration of an AT1 receptor antagonist (losartan) blunted the normal rise in plasma norepinephrine and epinephrine observed during acute immobilization, which hints at an important contribution of the central AT1 receptor in the mediation of the autonomic response during neurogenic stress. 18 Whether or not the autonomic PVH neurons that express the AT1 receptor are implicated in sympathetic activation remains to be determined. Taken together, these results support the hypothesis that angiotensin and its AT1A receptors are involved in PVH regulation and could represent an important factor in the mediation of the stress response by modulating both the HPA and autonomic axes.
Involvement of central angiotensin receptors in stress adaptation
The angiotensin receptor subtype 2 and the locus coeruleus Noradrenergic neurons of the LC project widely throughout the CNS, thus innervating many forebrain and limbic regions involved in stress and behavior. The LC is thought to be important in processes such as attention, arousal and behavioral activation, 6,10 and may subserve an important integrative function in the response to stress. 17,35,39 The importance of these higher order functions are not restricted to the acute stress response; they also prevail over the subsequent learning and plasticity that mold the processes of habituation and adaptation, as well as the ability to better cope with repeated or prolonged stress. One intriguing finding of the present study is the apparent induction of AT2 expression within the LC after chronic exposure to stress. Unlike the AT1-mediated effects within the PVH, the functional role of AT2 receptors in the LC is not well delineated. The AT2 receptors are abundantly and widely expressed in the fetal and immature brain, which is consistent with the suggestion that AT2 receptors may be involved in tissue growth and differentiation. 22 However, one of the few examples of an AT2 receptor-mediated effect in adult brain was the ability of angiotensin to inhibit glutamate-induced depolarization in LC neurons, 41 an effect abolished selectively by an AT2 antagonist. Therefore, the increase in AT2 mRNA observed in the LC after exposure to neurogenic stressors may be related in some way to one of the processes in neuron injury caused by glutamate. The glutamate afferent to the LC originates from the nucleus paragigantocellularis, which is involved in stress, vigilance and autonomic functions. 5,6 The role of the AT2 receptor in LC neurons in response to stress could be related to a buffering action on LC neurons that reduces their excitability or desensitization of excitatory inputs to the LC, or both. It is noteworthy that LC neurons are considered to be universally activated by all types of stressors and the relative absence of attenuation of responsiveness with chronic stress. 26,34 The presence of AT2 receptors in the LC may contribute to long-lasting changes in synaptic efficacy, thereby contributing to plasticity of neural circuits and adaptation to stress. Adaptation to stress The concept of stress as a general adaptation to any demand made on an individual is being replaced by a more differentiated model. The stress response is a complex, orchestrated response, coded at the sensory, motor, endocrine, autonomic and integrative levels. Chronic exposure to stressful events induces compensatory mechanisms that attenuate the deleterious effects of repetitive stress. These mechanisms play a defensive role that maintains homeostasis. It is generally reported that habituations of the HPA 1,4 and autonomic axes 19,20 are specific to the stressor to which the animals were repeatedly exposed (homotypic stressor). In contrast, the HPA and autonomic axes are still responsive to other types of stressors (heterotypic), suggesting that habituations
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of the HPA and the autonomic nervous system response to chronic stressors are not due to a decrease in the responsiveness of the system per se. It appears that the central organization of neural pathways, which convey stress-related information to the effector systems, may be crucially dependent on stimulus attributes. For instance, the HPA response to stress should be under control of a different set of neuromodulators that would be sensitive to one type of stressor but not another. The central components of the stress response system, especially the pathways involved in HPA regulation, have recently been revisited in view of the fact that various categories of stressors (rather than the stressor itself) are handled, to some extent, in different ways. 12,15,30 Indeed, the results of the present study support the view that the nature of the stressor is a determining factor regarding the direction of the stress responses. The use of two different stressors of the same nature (psychogenic) did not show significant differences between the expression of PVH–AT1A or LC–AT2 responses to immobilization versus air-jet stress. This is consistent with the existence of different stress circuitries participating in the integration of different types of stressors, such as cognitive stressors (processive) or systemic stressors. 12,14,23,30 It could be argued that the air-jet stress paradigm is not really different from immobilization stress, since the rats have to be immobilized during the air-jet stress procedure. However, the absence of controllability and the unpredictability of the air-jet delivery confer a more stressful connotation for the air-jet stress procedure, compared to restraint alone. Indeed, air-jet stress induced a higher increase of plasma ACTH, corticosterone and catecholamine levels (unpublished observations). CONCLUSION
The design of future experiments that test repeated or prolonged stress exposure must include careful consideration of the nature of the stressors to be used if a complete and accurate understanding of the adaptational process (e.g., habituation or sensitization) is to be realized. Our results suggest that the AT1 receptor in the PVH and the AT2 receptor in the LC are integral components of the central stress pathways that could be essential for the maintenance of reactivity of the stress effector, despite the repetition of the activation of these effector systems. 8,9,13,15
Acknowledgements—This research was supported by grants from the Canadian Medical Research Council (MRC), the Heart and Stroke Foundation of Que´bec (HSFQ) and from a Medical School Grant from Merck Frosst. G.D. holds a scholarship from le Fonds de la Recherche en Sante´ du Que´bec (FRSQ). We thank Dr Deschepper (Institut de Recherches Cliniques, Montre´al, Que´bec, Canada) for the rat AT1A cDNA and Dr Harrison (University of Florida, Gainesville, FL, U.S.A.) for the rat AT2 cDNA. We also acknowledge the expert assistance of Ms Susanne Richardson (editorial), Ms France Couture (infographic) and Mr Marc Auger (photographic).
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(1996) Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms. Proc. natn. Acad. Sci. U.S.A. 93, 2359–2364. Mansi J. A., Rivest S. and Drolet G. (1996) Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of the rat hypothalamus by exogenous CRF. Endocrinology 137, 4619–4629. Mansi J. A., Rivest S. and Drolet G. (1998) Effect of immobilization stress on transcriptional activity of inducible immediate-early genes, corticotropinreleasing factor, its type 1 receptor, and enkephalin in the hypothalamus of borderline hypertensive rats. J. Neurochem. 70, 1556–1566. Pacak K., Palkovits M., Kopin I. J. and Goldstein D. S. (1995) Stress-induced norepinephrine release in the hypothalamic paraventricular nucleus and pituitary–adrenocortical and sympathoadrenal activity: in vivo microdialysis studies. Front. Neuroendocr. 16, 89–150. Phillips M. (1987) Functions of angiotensin in the central nervous system. A. Rev. Physiol. 49, 413–435. Rivier C. and Vale W. (1983) Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin. Nature 305, 325–327. Saiki Y., Watanabe T., Tan N., Matsuzaki M. and Nakamura S. (1997) Role of central ANG II receptors in stress-induced cardiovascular and hyperthermic responses in rats. Am. J. Physiol. 272, R26–R33. Sawchenko P. E., Brown E. R., Chan R. K., Ericsson A., Li H. Y., Roland B. L. and Kovacs K. J. (1996) The paraventricular nucleus of the hypothalamus and the functional neuroanatomy of visceromotor responses to stress. Prog. Brain Res. 107, 201–222. Sawchenko P. E. and Swanson L. W. (1990) Organization of CRF immunoreactive cells and fibers in the rat brain: immunohistochemical studies. In Corticotropin-releasing Factor: Basic and Clinical Studies of a Neuropeptide (eds De Souza E. and Nemeroff C.), pp. 29–51. CRC, Boca Raton, FL. Song K., Allen A., Paxinos G. and Mendelsohn F. (1992) Mapping of angiotensin II receptor subtype heterogeneity in rat brain. J. comp. Neurol. 316, 467–484. Spinedi E. and Negro-Vilar A. (1983) Angiotensin II and ACTH release: site of action and potency relative to corticotropin releasing factor and vasopressin. Neuroendocrinology 37, 446–453. Stanford S. C. (1995) Central noradrenergic neurones and stress. Pharmac. Ther. 68, 297–342. Svensson T. H. (1987) Peripheral, autonomic regulation of locus coeruleus noradrenergic neurons in brain: putative implications for psychiatry and psychopharmacology. Psychopharmacology 92, 1–7. Tanimura S. M., Sanchez-Watts G. and Watts A. G. (1998) Peptide gene activation, secretion, and steroid feedback during stimulation of rat neuroendocrine corticotropin-releasing hormone neurons. Endocrinology 139, 3822–3829. Tanimura S. M. and Watts A. G. (1998) Corticosterone can facilitate as well as inhibit corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus. Endocrinology 139, 3830–3836. Tsutsumi K. and Saavedra J. (1991) Angiotensin-II receptor subtypes in median eminence and basal forebrain areas involved in regulation of pituitary function. Endocrinology 129, 3001–3008. Valentino R. J. (1989) Corticotropin-releasing factor: putative neurotransmitter in the noradrenergic nucleus locus ceruleus. Psychopharmac. Bull. 25, 306–311. Whitnall M. H. (1993) Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system. Prog. Neurobiol. 40, 573–629. Xiong H. and Marshall K. C. (1994) Angiotensin II depresses glutamate depolarizations and excitatory postsynaptic potentials in locus coeruleus through angiotensin II subtype 2 receptors. Neuroscience 62, 163–175. (Accepted 29 March 1999)
Involvement of central angiotensin receptors in stress adaptation
Pergamon PII: S0306-4522(99)00206-7
INVOLVEMENT OF CENTRAL ANGIOTENSIN RECEPTORS IN STRESS ADAPTATION E. C. DUMONT, S. RAFRAFI, S. LAFOREST and G. DROLET* Laval University Medical Research Centre, Neuroscience Unit, and Faculty of Medicine, Universite´ Laval, 2705 boul. Laurier, Sainte-Foy, Que´bec, Canada G1V 4G2
Abstract—The present study examined the effects of acute and chronic neurogenic stressors on the expression of two distinct angiotensin receptors in two stress-related brain nuclei: angiotensin type 1A receptor in the paraventricular nucleus of the hypothalamus and angiotensin type 2 receptor in the nucleus locus coeruleus. Male Wistar rats were divided into four experimental groups. The first two groups were subjected once to an acute 90-min immobilization or air-jet stress session, respectively. The other two groups were subjected to 10 days of daily 90-min immobilization sessions and, on the 11th day, one group was exposed to an additional 90-min immobilization and the other to a single air-jet stress (heterotypic but still neurogenic) session. In each group, rats were perfused before stress (0 min), immediately following stress (90 min) or 150, 180, 270 or 360 min (and 24 h in chronic immobilization) after the beginning of the last stress session. Basal expression of both angiotensin receptor subtype 1A and angiotensin receptor subtype 2 messenger RNA was minimal in non-stressed animals. Acute immobilization as well as air-jet stress induced similar patterns (time-course and maximal values) of angiotensin receptor subtype 1A messenger RNA expression in the paraventricular nucleus. Angiotensin receptor subtype 1A messenger RNA expression increased 90–150 min after the beginning of the stress and returned to basal levels by 360 min. Chronic stress immobilization slightly modified the pattern, but not maximal values of angiotensin receptor subtype 1A messenger RNA expression to further immobilization (homotypic) or air-jet stress (heterotypic). Acute immobilization and air-jet stress sessions induced similar locus coeruleus-specific angiotensin receptor subtype 2 messenger RNA expression. This expression increased 90 min following the onset of the stress session and remained elevated for at least 360 min. Chronic immobilization stress increased angiotensin receptor subtype 2 messenger RNA expression to levels comparable to those observed in acute stress conditions. Novel acute exposure to neurogenic stressors did not further increase these levels in either homotypic (immobilization) or in heterotypic (air-jet stress) conditions. These results suggest that central angiotensin receptors are targets of regulation in stress; therefore, stress may modulate angiotensin function in the paraventricular nucleus and locus coeruleus during chronic exposure to neurogenic stressors. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: paraventricular nucleus of the hypothalamus, locus coeruleus, corticotropin-releasing hormone, autonomic nervous system, hypothalamo-hypophyso-adrenal.
That chronic exposure to stressful events induces compensatory mechanisms that attenuate the deleterious effects of repeated stress is a well-documented fact. The ability to adapt to repetitive or prolonged stress is apparent at the levels of both major stress effectors: the hypothalamo-hypophysoadrenal (HPA) axis and the autonomic nervous system. 40 However, the nature and specificity (or generality) of these adaptational phenomena in the autonomic nervous system and HPA are controversial. Nevertheless, it appears that habituation of the HPA and autonomic responses to chronic stressors are not due to reduced responsiveness of the system per se. Changes in the afferent control of hypothalamic paraventricular corticotropin-releasing hormone (CRH) neurons and/or other systems involved in the release of adrenocorticotropin hormone (ACTH) secretagogues are thought to underline these modifications. 1,30 Indeed, the mechanisms responsible for the potential intact responsiveness of the system are still not known. The central renin–angiotensin system may significantly modulate the effect of the stress response. 7,16 It has been
suggested that angiotensin stimulates ACTH release by augmenting CRH liberation, presumably through an action on CRH neurons in the paraventricular nucleus of the hypothalamus (PVH), and also by potentiating the effects of CRH in the anterior pituitary. 27,28,33 The release of CRH into the hypophyseal portal circulation from the parvocellular neurons of the PVH initiates a cascade of well-defined physiological and behavioral responses. 31 These effects of angiotensin at the PVH level appear to be mediated through angiotensin type 1A receptors (AT1A), which are richly distributed in this nucleus, 32,38 especially on CRHergic neurons of the parvocellular division. 2,3 In contrast, the involvement of the angiotensin type 2 receptor (AT2) in the stress response is less understood. However, both AT2 receptor mRNA and AT2 binding sites have been identified in several regions of the brain, 22 for example in the nucleus locus coeruleus (LC), a well-defined stress-related brain structure. The investigation of chronic adaptation to stress depends very much on the nature of the stressors. Indeed, recent studies have shown that different families of stressors (rather than the stressor itself) use distinct neuronal circuits for the regulation of HPA and autonomic axes. 15,23,30 It follows that, when seeking an accurate description of chronic adaptation to stress, the choice of stressor is of critical importance. The objective of the present study was to investigate the effects of acute and chronic neurogenic stressors on two distinct angiotensin receptors in stress-related brain nuclei, AT1A in
*To whom correspondence should be addressed. E-mail address: [email protected] (G. Drolet) Abbreviations: ACTH, adrenocorticotropin hormone; AT1A, angiotensin receptor subtype 1A; AT2, angiotensin receptor subtype 2; CRH, corticotropin-releasing hormone; EDTA, ethylenediaminetetra-acetate; HPA, hypothalamo-hypophyso-adrenal; LC, locus coeruleus; O.D., optical density; PVH, paraventricular nucleus of the hypothalamus. 877
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the PVH and AT2 in the LC. To investigate the specificity of stress adaptation, we chose to study the effects of two different painless neurogenic stressors; immobilization and air jet, to ascertain whether the psychogenic nature of the stressor or the stressor itself is important for adaptation to chronic exposure to stress. EXPERIMENTAL PROCEDURES
Animals Adult male Wistar rats (Charles River, St Constant, Quebec; 12–14 weeks) weighing 225–250 g were acclimatized for five to seven days to standard laboratory conditions (14-h light/10-h dark cycle: lights on at 06.00 and off at 20.00), and given free access to rat chow and tap water. The investigation was performed in accordance with the guidelines of the Canadian Council on Animal Care, and approved by institutional animal care committee. Stress paradigm design Rats were divided into four experimental groups. The first two groups were subjected once to an acute 90-min immobilization (acute immobilization) or air-jet stress (acute air-jet stress) session, respectively. The other two groups were subjected to 10 days of daily 90-min immobilization sessions and, on the 11th day, one group was exposed to an additional 90-min immobilization (chronic immobilization, homotypic), while the remaining group was subjected to a single air-jet stress (chronic air-jet stress, heterotypic but still neurogenic) session. Adjustable restraining cages (Centrap) were used to immobilize the rats. Air-jet stress was accomplished by coupling immobilization stress with bursts of air at 55 psi aimed at the animals’ snouts. The rats were placed in an immobilization cage and secured to the air-jet delivery device. A computer software randomly controlled both the duration (0.5–10 s) and occurrence interval (10–60 s) of the air-jet bursts. Rats from each group were killed before stress (time 0), or were placed in an individual immobilization cage for 90 min or subjected to a 90-min air-jet session. The rats were then deeply anesthetized with a ketamine (Rogarsetice, 80 mg/kg)/ xylazine (Rompune, 10 mg/kg) solution (i.p.) 90, 150, 180, 270 and 360 min (and 24 h for the chronic immobilization group) after the onset of the stress session, and then perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 48C). The brains were removed from the skull, postfixed for four to seven days and then placed in 20% sucrose/4% paraformaldehyde buffer 48 h at 48C. Frozen brains were mounted on a microtome and cut into 30-mm coronal sections. The slices were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer, 30% ethyleneglycol and 20% glycerol) and stored at 2208C. In situ hybridization histochemistry Histochemical hybridization localization of each transcript was carried out as described previously. 24 AT1A probe (from Dr Deschepper, IRCM, Montre´al, Que´bec, Canada) was synthesized from the Sal I/ Bam H1 fragment of the rat adrenal cDNA library, subcloned into pSPORT 1 and linearized with Sal I (antisense) or Bam H1 (sense). AT2 probe (from Dr Harrison, University of Florida, Gainesville, FL) was produced in pCDM8, subcloned in SKII 1 and linearized with Hind III (antisense) or Bam H1 (sense). Radioisotope-labeled antisense cRNA copies were synthesized by incubating 250 ng of linearized plasmid in 5×transcription buffer (6 mM MgCl2, 30–40 mM Tris, pH 7.9, 10 mM NaCl), 10 mM dithiothreitol, 0.2 mM ATP/GTP/CTP, 200 mCi [a- 35S]UTP, 40 U RNasin and 20 U SP6 (AT1A) or T3 (AT2) RNA polymerase, together for 60 min at 378C. Unincorporated nucleotides were removed using the ammonium acetate method, where 100 ml DNase solution (1 ml DNase, 2.5 ml of 10 mg/ml transfer RNA, 94 ml of 10 mM Tris/ 10 mM MgCl2, 2.5 ml diethylpyrocarbonate water) was added, and followed 10 min later by phenol–chloroform solution extraction. The cRNA was precipitated with 80 ml of 5 M ammonium acetate and 500 ml of 100% ethanol for 20 min on dry ice. The pellet was resuspended in 100 ml of 10 mM Tris/1 mM EDTA (pH 8.0). A concentration of 10 7 c.p.m. probe was mixed into 1 ml hybridization solution (518 ml formamide, 62 ml of 5 M NaCl, 10 ml of 1 M Tris, pH 8.0, 2 ml of 0.5 M EDTA, pH 8.0, 20 ml of 50×Denhardt’s solution, 207 ml of
50% dextran sulfate, 50 ml of 10 mg/ml transfer RNA, 10 ml of 1 M dithiothreitol, 118 ml diethylpyrocarbonate water-volume of probe used). This solution was mixed and heated for 5 min at 658C before applying to slides. Radioisotope-labeled sense (control) cRNA copies were also prepared to verify the specificity of each probe (T7 for both probes). Hybridization with these probes did not reveal any positive signal in the rat brain. Quantitative analysis Blind quantitative analysis of hybridization signal for the mRNA signals was performed on X-ray film over at least two PVHs (bilateral) and two LCs for each animal. Transmittance values (referred to here as optical density, O.D.) of the hybridization signals were measured using a Northern Light Desktop Illuminator (Imaging Research) with a Sony Camera Video System attached to a Micro-Nikkor 55-mm Vivitar extension tube set for a Nikon lens and coupled to a Macintosh computer (Power Macintosh 8600/300), then analysed with NIH Image software, version 1.61 (W. Rasband, National Institutes of Health, Bethesda, MD). The O.D. value for each pixel was calculated using a known standard of intensity and distance measurements from a logarithmic specter adapted from Bioimage Visage 110s (Millipore, Ann Arbor, MI). The wedge was calibrated before correcting for film saturation, which was determined by sampling the darkest PVHs or LCs then adjusting the light source and exposure time. All samples that emitted a clear positive signal were evaluated within a linear range to avoid pixel saturation and underestimation. Sections from experimental and control animals were digitalized and subjected to densitometric analysis that yielded values of mean density per area. The O.D. of each side of the nucleus (LC and PVH) was corrected for the average background signal by subtracting the O.D. of areas without positive signal located immediately outside the digitalized nucleus. Statistical analysis Data from graphical figures are expressed as relative O.D. (arbitrary units) for mRNA in the PVH or the LC of control (time 0), stressed (time 90 min) and post-stressed (times 150, 180, 270, 360 min and 24 h) Wistar rats. The results were analysed using a three-way ANOVA (factorial); post hoc multiple comparisons were made using a Bonferroni test. Factors were identified as follows: time after the beginning of stress (0, 90, 150, 180, 270, 360 min), the type of stressor (immobilization, air-jet stress) and the stress duration (acute, chronic). Levels were deemed significant at P,0.05. RESULTS
Paraventricular nucleus/angiotensin receptor subtype 1A messenger RNA expression Acute immobilization. Basal expression of AT1A receptor mRNA within the PVH was barely detectable in non-stressed animals (time 0, O.D. 8.0^2.4, n7; Fig. 1, upper left, Fig. 2A). Acute exposure to a 90-min immobilization session increased the expression of AT1A receptor mRNA in the parvocellular part of the PVH, specifically (as reported previously). 2,22 This expression was detectable and reached a maximal value 150 min after the onset of the homotypic stress session (O.D. 32.4^4.7, n5, P,0.001; Fig. 1, lower left, Fig. 2A). The positive signal was still present at 180 (O.D. 28.9^4.5, n6, P,0.001; Fig. 2A) and 270 min (O.D. 30.3^3.9, n4, P,0.001; Fig. 2A), but returned to basal levels 360 min (O.D. 16.8^1.8, n2, P.0.05; Fig. 2A) after the onset of the stress session. Chronic immobilization (homotypic stressor). Expression of AT1A receptor mRNA within the PVH was also barely detectable following chronic exposure to immobilization stress (O.D. 5.6^2.5, n5; Fig. 1, upper right, Fig. 2A). Subsequent exposure to immobilization induced a similar maximal increase of AT1A receptor mRNA when compared with the acute condition measured at 150 min (O.D.
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Fig. 1. Distribution of the mRNA encoding the AT1A receptor in the brain of rats exposed to an acute 90-min (left panel) or chronic (10 days × 90 min, right panel) immobilization stress session. Representative examples of coronal brain slices at 0 (upper), 90 (middle) and 150 (lower) min following the onset of the stress session are shown. Acute exposure to a 90-min immobilization session increased the expression of AT1A receptor mRNA specifically in the parvocellular part of the PVH. This expression was detectable and reached a maximal value 150 min following the onset of the stress session. Chronic exposure to immobilization stress sessions modified the time-course of AT1A expression in the PVH to subsequent stress exposure, without altering the maximal values. AT1A expression was already detectable 90 min after the onset of the last stress session as compared to 150 min in acute conditions.
32.4^4.6, n5; Fig. 1, lower right, Fig. 2A). However, chronic exposure to immobilization stress modified the time-course of AT1A receptor mRNA expression in the PVH. Indeed, in chronic conditions, AT1A expression was detectable at as early as 90 min (O.D. 19.1^2.3, n6 and 3.6^1.8, n4, P0.002, chronic vs acute; Fig. 1, middle panels, Fig. 2A). Moreover, the signal returned more rapidly to baseline levels in chronic conditions (O.D. 10.2^2.7, n5 at 270 min, P0.003 compared with acute; Fig. 2A). Acute air-jet stress. The basal expression of AT1A receptor mRNA was barely detectable in the acute air-jet stress group (O.D. 5.3^2.0, n3; Fig. 2B). Acute exposure to a 90-min air-jet stress session also induced the expression of AT1A receptor mRNA in the PVH, which was detectable immedi-
ately after the offset of the stress session (O.D. 27.3^3.3, n5, P0.02; Fig. 2B), reaching a maximal value 150 min after the onset of the stress session (O.D. 32.7^6.1, n5, P0.004; Fig. 2B). The positive signal was still present at 180 (O.D. 28.5^6.3, n6, P0.01; Fig. 2B) and 270 min (O.D. 26.3^5.4, n4, P0.03; Fig. 2B), but returned to basal levels 360 min (O.D. 14.3^0.8, n2; Fig. 2B) after the onset of the stress session. Chronic immobilization (heterotypic stressor). Expression of AT1A receptor mRNA was barely detectable following chronic exposure to immobilization stress sessions in this group (O.D. 5.5^1.2, n4; Fig. 2B). Subsequent exposure to heterotypic stress (air jet) induced a comparable maximal increase in AT1A receptor mRNA when compared with the
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Fig. 2. Effect of acute (open symbols) and chronic (filled symbols) immobilization (A) and air-jet (B) stress on the average relative O.D. for the AT1A receptor mRNA hybridization signal in the PVH. The effects of both neurogenic stress paradigms on AT1A receptor mRNA were compared in acute (C) and chronic (D) conditions. In acute conditions, the time-course of expression and the maximal intensity of the signal were comparable between immobilization and air-jet stress. Chronic exposure to immobilization stress sessions modified the time-course of AT1A expression in the PVH to subsequent stress exposure, without altering the maximal values reached in both immobilization (homotypic stressor) and air-jet stress (heterotypic stressor) groups. These results represent the means^S.E.M. of three to seven rats per time-point. Statistical analysis was performed using a factorial three-way ANOVA followed by a Bonferroni post hoc test. T represents a significant (at least P,0.05) effect of time and G a significant difference between two groups at a particular time-point.
acute condition (O.D. 32.3^1.4, n3; Fig. 2B). However, the time-course of AT1A receptor mRNA expression in the PVH induced by air-jet stress was slightly modified by chronic exposure to immobilization stress. Indeed, under chronic conditions, a smaller increase of AT1A expression was observed at 90 min (O.D. 18.8^1.6, n5, P0.04 compared with acute; Fig. 2B). Moreover, under chronic conditions, the signal tended to fall more rapidly to baseline levels (Fig. 2B). No significant difference was observed between the AT1A receptor expression induced by either neurogenic stressor in acute (Fig. 2C) or chronic conditions (Fig. 2D). Locus coeruleus/angiotensin receptor subtype 2 messenger RNA expression Acute immobilization. Basal expression of AT2 receptor mRNA within the LC was barely detectable in non-stressed animals (time 0, O.D. 4.9^3.3, n8; Fig. 4A). Acute exposure to a 90-min immobilization session increased the mRNA expression of that receptor specifically in the LC. This expression reached a maximal value 90 min after the onset of the stress session (O.D. 42.3^6.9, n3, P,0.001; Fig. 4A). The positive signal was still present at 150 min (O.D. 31.9^7.0, n9, P,0.001; Fig. 4A), 180 min (O.D. 29.0^5.1, n6, P0.004; Fig. 4A), 270 min (O.D. 29.0^4.1, n5, P0.006; Fig. 4A) and 360 min (O.D. 39.3^3.2, n9, P0.01; Fig. 4A) after the onset of the stress session. Chronic immobilization (homotypic). Expression of AT2
receptor mRNA was increased by chronic exposure to immobilization stress sessions (O.D. 20.9^4.6, n5; Fig. 4A). Subsequent exposure to immobilization did not significantly modify AT2 receptor mRNA expression (Fig. 4A). Acute air-jet stress. Basal expression of AT2 receptor mRNA within the LC was barely detectable in non-stressed animals of the air-jet stress group (time 0, O.D. 1.5^0.8, n3; Fig. 3, upper left, Fig. 4B). Exposure to a 90-min airjet session increased the expression of AT2 receptor mRNA specifically in the LC. This expression was detectable 90 min after the onset of the stress session (O.D. 19.8^3.0, n7, P,0.001; Fig. 3, middle left, Fig. 4B). The positive signal was still present 150 min (O.D. 29.1^3.4, n6, P,0.001; Fig. 4B), 180 min (O.D. 26.0^5.8, n6, P0.02; Fig. 4B), 270 min (O.D. 33.1^3.8, n6, P,0.001; Fig. 4B) and 360 min (O.D. 32.8^8.6, n3, P0.001; Fig. 3, lower left, Fig. 4B) after the onset of the stress session. Chronic immobilization (heterotypic stressor). Expression of AT2 receptor mRNA was increased by chronic exposure to immobilization sessions (O.D. 25.6^3.1, n4; Fig. 3, upper right, Fig. 4B). Subsequent exposure to air-jet stress did not modify AT2 receptor mRNA expression (Fig. 3, middle and lower right, Fig. 4B). There was no significant difference between the AT2 mRNA receptor expression induced by either neurogenic stressor in acute (Fig. 4C) or chronic conditions (Fig. 4D).
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Fig. 3. Distribution of the mRNA encoding the AT2 receptor in the brain of rats exposed to an acute 90-min air-jet stress session (left panel) or to a 90-min airjet stress session following chronic exposure to immobilization (10 days × 90 min, right panel). Representative examples of coronal brain slices at 0 (upper), 90 (middle) and 360 (lower) min after the onset of the stress session are shown. Acute exposure to a 90-min air-jet stress session induced the expression of AT2 receptor mRNA specifically in the LC. Moreover, expression of AT2 receptor mRNA was increased by chronic exposure to an immobilization stress session at levels comparable with maximal values obtained with acute exposure to air-jet stress. This increase remained elevated for at least 360 min in both acute and chronic conditions.
DISCUSSION
These results suggest that central angiotensin receptors are important targets of stress regulation; therefore, stress could modulate angiotensin function in the PVH and the LC during chronic exposure to neurogenic stressors. In fact, acute exposure to neurogenic stressors induced the expression of AT1A and AT2 receptor mRNA in the two stress-associated brain nuclei, the PVH and LC, respectively. Moreover, chronic intermittent stress did not attenuate the expression of the AT1A receptor within the PVH, suggesting that the integrity of these receptors may be essential for the maintenance of reactivity to stress of PVH neurons. Furthermore, the present study reports, for the first time, that chronic immobilization stress increases basal expression of AT2 within the LC and
AT2 mRNA increases in the LC to a level that approaches the maximal acute response. This again suggests that the central angiotensin system plays an important role in the adaptive cellular mechanisms that drive the response of the central stress system when assaulted by repeated neurogenic stress. The angiotensin receptor subtype 1A and the paraventricular nucleus of the hypothalamus A physiological role for angiotensin as a central neurotransmitter within the PVH was provided by a study that demonstrated the local release of angiotensin in response to physiological and chemical stimuli. 11 This endogenous release of angiotensin within the PVH mediated its actions
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Fig. 4. Effect of acute (open symbols) and chronic (filled symbols) immobilization (A) and air-jet (B) stress on the average relative O.D. for the AT2 receptor mRNA hybridization signal in the LC. The effects of both neurogenic stress paradigms on AT2 receptor mRNA were compared in acute (C) and chronic (D) conditions. In acute conditions, the time-course of expression and the maximal intensity of the signal were comparable between immobilization and air-jet stress. The expression of AT2 receptor mRNA was increased by chronic exposure to an immobilization stress session. This expression was not further modified by subsequent acute exposure to an immobilization (homotypic stressor) or air-jet stress (heterotypic stressor) session. These results represent the means ^S.E.M. of three to eight rats per time-point. Statistical analysis was performed using a factorial three-way ANOVA followed by a Bonferroni post hoc test. T represents a significant (at least P,0.05) effect of time and G a significant difference between two groups at a particular time-point.
via the AT1A receptor, which is highly expressed in CRHergic neurons of the parvocellular division. 2,3,32,38 Therefore, angiotensin could modulate the activity of CRH neurons directly through AT1A receptors in PVH–CRH perikarya. Effectively, central administration of angiotensin dramatically increases the expression of CRH mRNA within the PVH, suggesting that the release of angiotensin in the PVH during stressful situations may be involved in the elevated CRH mRNA levels seen following acute stress. 25,33 Moreover, it has been reported that repeated immobilization exposure resulted in up-regulation of angiotensin receptors in the PVH. 7 Therefore, the present increase of AT1A expression in the PVH after chronic as well as acute stress is likely to be translated by the synthesis of new AT1A receptors. The increase in angiotensin binding sites 7 and AT1A receptor mRNA, as seen in the present study, could be mediated by high plasma levels of glucocorticoids that occur during stress sessions. Consistent with this are reports that dexamethasone injection increased the expression of AT1A within the PVH, whereas adrenalectomy induced a significant decrease that could be prevented by glucocorticoid treatment. 2 The functional significance of the AT1 receptor in the neurobiology of stress needs to be clarified, but its presence within the parvocellular part of the PVH provides clear anatomical evidence that this receptor could directly modulate both neuroendocrine and autonomic functions. Despite reports of the involvement of the AT1A receptor in HPA regulation, some inconsistencies regarding its exact role persist. Presently, it appears that angiotensin may differentially affect different components of the HPA. For instance, angiotensin modulates the expression of CRF mRNA in the PVH in a
direct and positive manner via activation of AT1 receptors. 3 However, the blockade of central AT1 receptor did not reduce the restraint stress-induced elevated plasma levels of ACTH and corticosterone. 2,3,18 This divergence between the effects of AT1 receptor on different components of the HPA is consistent with the recent finding of functional dissociation within the regulation of different components of the HPA. In a twopaper series, Watts’ group reported that the secretion of ACTH and activation of the CRF gene have distinct and separate stimulus thresholds, suggesting some degree of mechanistic separation. 36,37 These studies raised the point that temporal organization and functional interaction between the components of the HPA could be dissociated. Alternatively, the role of the AT1 receptor within the PVH could be related to autonomic regulation. Indeed, it has been demonstrated that angiotensin is required for the full expression of sympathetic, pressor and neuroendocrine responses during stress. 16,21,29 Moreover, intracerebroventricular administration of an AT1 receptor antagonist (losartan) blunted the normal rise in plasma norepinephrine and epinephrine observed during acute immobilization, which hints at an important contribution of the central AT1 receptor in the mediation of the autonomic response during neurogenic stress. 18 Whether or not the autonomic PVH neurons that express the AT1 receptor are implicated in sympathetic activation remains to be determined. Taken together, these results support the hypothesis that angiotensin and its AT1A receptors are involved in PVH regulation and could represent an important factor in the mediation of the stress response by modulating both the HPA and autonomic axes.
Involvement of central angiotensin receptors in stress adaptation
The angiotensin receptor subtype 2 and the locus coeruleus Noradrenergic neurons of the LC project widely throughout the CNS, thus innervating many forebrain and limbic regions involved in stress and behavior. The LC is thought to be important in processes such as attention, arousal and behavioral activation, 6,10 and may subserve an important integrative function in the response to stress. 17,35,39 The importance of these higher order functions are not restricted to the acute stress response; they also prevail over the subsequent learning and plasticity that mold the processes of habituation and adaptation, as well as the ability to better cope with repeated or prolonged stress. One intriguing finding of the present study is the apparent induction of AT2 expression within the LC after chronic exposure to stress. Unlike the AT1-mediated effects within the PVH, the functional role of AT2 receptors in the LC is not well delineated. The AT2 receptors are abundantly and widely expressed in the fetal and immature brain, which is consistent with the suggestion that AT2 receptors may be involved in tissue growth and differentiation. 22 However, one of the few examples of an AT2 receptor-mediated effect in adult brain was the ability of angiotensin to inhibit glutamate-induced depolarization in LC neurons, 41 an effect abolished selectively by an AT2 antagonist. Therefore, the increase in AT2 mRNA observed in the LC after exposure to neurogenic stressors may be related in some way to one of the processes in neuron injury caused by glutamate. The glutamate afferent to the LC originates from the nucleus paragigantocellularis, which is involved in stress, vigilance and autonomic functions. 5,6 The role of the AT2 receptor in LC neurons in response to stress could be related to a buffering action on LC neurons that reduces their excitability or desensitization of excitatory inputs to the LC, or both. It is noteworthy that LC neurons are considered to be universally activated by all types of stressors and the relative absence of attenuation of responsiveness with chronic stress. 26,34 The presence of AT2 receptors in the LC may contribute to long-lasting changes in synaptic efficacy, thereby contributing to plasticity of neural circuits and adaptation to stress. Adaptation to stress The concept of stress as a general adaptation to any demand made on an individual is being replaced by a more differentiated model. The stress response is a complex, orchestrated response, coded at the sensory, motor, endocrine, autonomic and integrative levels. Chronic exposure to stressful events induces compensatory mechanisms that attenuate the deleterious effects of repetitive stress. These mechanisms play a defensive role that maintains homeostasis. It is generally reported that habituations of the HPA 1,4 and autonomic axes 19,20 are specific to the stressor to which the animals were repeatedly exposed (homotypic stressor). In contrast, the HPA and autonomic axes are still responsive to other types of stressors (heterotypic), suggesting that habituations
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of the HPA and the autonomic nervous system response to chronic stressors are not due to a decrease in the responsiveness of the system per se. It appears that the central organization of neural pathways, which convey stress-related information to the effector systems, may be crucially dependent on stimulus attributes. For instance, the HPA response to stress should be under control of a different set of neuromodulators that would be sensitive to one type of stressor but not another. The central components of the stress response system, especially the pathways involved in HPA regulation, have recently been revisited in view of the fact that various categories of stressors (rather than the stressor itself) are handled, to some extent, in different ways. 12,15,30 Indeed, the results of the present study support the view that the nature of the stressor is a determining factor regarding the direction of the stress responses. The use of two different stressors of the same nature (psychogenic) did not show significant differences between the expression of PVH–AT1A or LC–AT2 responses to immobilization versus air-jet stress. This is consistent with the existence of different stress circuitries participating in the integration of different types of stressors, such as cognitive stressors (processive) or systemic stressors. 12,14,23,30 It could be argued that the air-jet stress paradigm is not really different from immobilization stress, since the rats have to be immobilized during the air-jet stress procedure. However, the absence of controllability and the unpredictability of the air-jet delivery confer a more stressful connotation for the air-jet stress procedure, compared to restraint alone. Indeed, air-jet stress induced a higher increase of plasma ACTH, corticosterone and catecholamine levels (unpublished observations). CONCLUSION
The design of future experiments that test repeated or prolonged stress exposure must include careful consideration of the nature of the stressors to be used if a complete and accurate understanding of the adaptational process (e.g., habituation or sensitization) is to be realized. Our results suggest that the AT1 receptor in the PVH and the AT2 receptor in the LC are integral components of the central stress pathways that could be essential for the maintenance of reactivity of the stress effector, despite the repetition of the activation of these effector systems. 8,9,13,15
Acknowledgements—This research was supported by grants from the Canadian Medical Research Council (MRC), the Heart and Stroke Foundation of Que´bec (HSFQ) and from a Medical School Grant from Merck Frosst. G.D. holds a scholarship from le Fonds de la Recherche en Sante´ du Que´bec (FRSQ). We thank Dr Deschepper (Institut de Recherches Cliniques, Montre´al, Que´bec, Canada) for the rat AT1A cDNA and Dr Harrison (University of Florida, Gainesville, FL, U.S.A.) for the rat AT2 cDNA. We also acknowledge the expert assistance of Ms Susanne Richardson (editorial), Ms France Couture (infographic) and Mr Marc Auger (photographic).
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