Impact of acute and chronic stressor experiences on heart atrial and brain natriuretic peptides in response to a subsequent stressor

Hormones and Behavior 58 (2010) 907–916

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Impact of acute and chronic stressor experiences on heart atrial and brain natriuretic peptides in response to a subsequent stressor Boubacar Pasto Wann, Marie-Claude Audet, Hymie Anisman ⁎ Institute of Neuroscience, Carleton University Ottawa, Ontario, Canada K1S 5B6

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Article history: Received 26 May 2010 Revised 24 August 2010 Accepted 1 September 2010 Available online 19 September 2010 Keywords: Atrial and brain natriuretic peptides Norepinephrine Chronic stressor Acute stressor Sensitization Desensitization Locus coeruleus Atrial Ventricle

a b s t r a c t The impact of stressful events on processes related to cardiovascular functioning might vary with previous stressor experiences, just as such sensitization effects have been detected with respect to several neurochemical and hormonal processes. The present investigation assessed the impact of a psychosocial stressor on factors directly or indirectly related to cardiovascular functioning among CD-1 mice that had previously experienced an acute or chronic stressor regimen. These factors included plasma variations of atrial and brain natriuretic peptides (ANP and BNP, respectively), inflammatory cytokines in plasma, mRNA expression of natriuretic peptides and inflammatory cytokines in the ventricles, and norepinephrine (NA) levels and utilization within the locus coeruleus, a brain region implicated in cardiac functioning. A social stressor (exposure to a dominant mouse) increased NE levels and utilization within the locus coeruleus, plasma corticosterone, cytokine and ANP levels. Among mice initially exposed to an acute stressor (restraint), NE utilization, ventricular ANP mRNA expression, and plasma interleukin-6 (IL-6) concentrations were markedly increased by the subsequent social stressor. In chronically stressed mice some of the effects of the social stressor were dampened, including changes of plasma corticosterone, locus coeruleus NE utilization, as well as plasma and ventricular IL-6 mRNA expression. Conversely, plasma ANP was markedly enhanced by the combined stressor events as was ventricular BNP and IL-1β mRNA expression. It seems that stressors may profoundly influence (sensitize or desensitize) on factors that could influence cardiovascular functioning. It remains to be determined whether these actions would be translated as pathophysiological outcomes. © 2010 Elsevier Inc. All rights reserved.

Introduction Stressor experiences have been associated with increased depression and cardiovascular disturbances, and comorbidity between these pathologies has frequently been reported (Lesperance and FrasureSmith, 2000). Numerous processes could be involved in mediating the effects of stressors on the development of cardiovascular disturbances, including peripheral and central nervous system mechanisms. For instance, stressor-provoked effects on norepinephrine (NE) neurons of the locus coeruleus may influence cardiac receptors and increase heart rate, blood pressure and ventricular contraction rate, thereby influencing vulnerability to tachycardia or ventricular fibrillation (Dalack and Roose, 1990). Furthermore, baroreceptors located in the aortic arch and carotid sinus, which control heart rate and blood pressure, can be influenced by stressors and may contribute to cardiac events such as ventricular arrhythmia (Hatton et al., 1997). Indeed, acute stressors were found to reduce the sensitivity of the baroreflex or to reset it to a higher pressure (Hatton et al., 1997; ⁎ Corresponding author. Carleton University, Institute of Neuroscience, Life Science Research Centre, Ottawa, Ontario, Canada K1S 5B6. Fax: + 1 613 520 4052. E-mail address: [email protected] (H. Anisman). 0018-506X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2010.09.001

Porter, 2000) and to increase coronary vasoconstriction, heart rate and blood pressure (Dalack and Roose, 1990). Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones that have been implicated as markers of cardiac diseases, including heart failure, cardiac hypertrophy, arrhythmia and left ventricular dysfunction (Goetze et al., 2004; Mekontso-Dessap and Brochard, 2006). Ordinarily, ANP is synthesized in the atria and ventricular heart, whereas BNP is mainly produced by the ventricular heart. These hormones promote augmented glomerulal filtration and excretion of excessive sodium in urine, normalization of blood pressure, and the reduction of heart NE activity (Piechota et al., 2008). The rise of tachycardia and blood pressure was associated with the release of ANP, and both ANP and BNP were elevated in association with myocardial dysfunction (Grabie et al., 2003; Tavener and Kubes, 2006). Consistent with the view that stressful experiences may be a risk factor for cardiovascular disease, it was reported that emotional stressors and chronic insults increased heart gene expression of natriuretic peptides coupled with increased ANP release (Ueyama et al., 2003; Wann et al., 2010). This does not necessarily suggest that these peptides contribute to the pathology, and indeed, their function may also be one of protecting the heart (Ichihara et al., 2009; McGrath and de Bold, 2005).

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In addition to natriuretic peptides, cardiovascular diseases have been associated with inflammatory factors (Campbell and MacQueen, 2004; Carpeggiani et al., 2004; Lesperance and Frasure-Smith, 2000). For instance, stressors were shown to increase circulating levels of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α (Anisman et al., 2008; Frazure-Smith et al., 2009), which might contribute to altered cardiac functioning (Libetta et al., 2007). These pro-inflammatory cytokines can stimulate BNP synthesis (Campbell and MacQueen, 2004), and it also appears that ANP may be a modulator of immune activity and cytokine functioning (Blumenthal et al., 2005). In addition, both stressors and bacterial endotoxin (lipopolysaccharide) challenges increased ANP concentrations in plasma and in the left ventricle, and increased left atrial and ventricular IL-1β and TNF-α mRNA expression (Wann et al., 2010), and the stressor and endotoxin treatment synergistically increased plasma ANP (Wann et al., 2010). Stressful events have been linked to heart disease, and natriuretic peptides could potentially serve as markers of cardiac dysfunction. However, the data concerning stressor effects on these peptides, as well as on processes that might be tied to them (e.g., cytokines), is limited. Moreover, most studies have been concerned with the immediate effects of stressors on ANP and BNP, and have not focused on the relatively protracted effects that may occur. In this regard, it is known that stressor-induced brain neurochemical changes, and some aspects of hypothalamic-pituitary-adrenal (HPA) functioning, may be subject to a sensitization effect, wherein re-exposure to stressors days or weeks following an initial insult, may promote much greater neurotransmitter or neuroendocrine changes than would ordinarily be elicited by the proximal stressor itself (Anisman and Merali, 2003; Hayley et al., 1999). Given the links between natriuretic peptides, cytokines and locus coeruleus NE activity, in relation to cardiac functioning, it was of interest in the present investigation to assess (a) whether an acute stressor vs. a chronic stressor regimen (comprising variable challenges over 21 days) would have a protracted effect on NE activity within the locus coeruleus, inflammatory factors (cytokines) and ANP and BNP (in plasma and heart), and (b) whether these initial stressor experiences would influence the impact of a subsequent challenge (social disturbance) on these same processes. Methods Subjects Male CD-1 mice (N = 92) were obtained from Charles River Canada (St. Constant, Quebec) at 6 weeks of age. They were housed in groups of four upon arrival and allowed to acclimatize to the laboratory setting for 10–14 days in a temperature-controlled

vivarium with lights on from 0800 h to 2000 h and food and water freely available. All procedures were conducted in accordance with the guidelines set out by the Canadian Council on Animal Care and were approved by the Carleton University Animal Care Committee. Stressor procedures Table 1 provides a description of the sequence of events for each of the treatment conditions. As well, the table indicates the number of mice in each of the groups; however, as will be described shortly, since the number of samples varied in some assays (e.g., plasma cytokines), the df varied in the statistical analyses for the different outcome measures. After the acclimatization period, mice were housed individually and randomly assigned to either a chronic, acute, or no stressor condition. Chronically stressed mice were exposed to a procedure previously shown to elicit depressive-like behaviors and to alter brain monoamine activity (Anisman et al., 2007; Tannenbaum et al., 2002). Specifically, they were exposed to a series of different stressors on each of 21 days (applied twice daily) with a different stressor applied in the morning and afternoon. The stressors comprised the following: physical restraint in semicircular Plexiglas tubes (4 cm diameter× 12 cm long) with tails taped to prevent mice from turning (15 min); exposure to predator odor (rat) by placing the mouse in a cage containing soiled rat bedding (60 min); placing the mouse in a cage containing bedding that was soaked with water (60 min); forced swim in water of 20 °C within a plastic cylinder of 30 cm diameter and 27 cm high (5 min). The animals were returned to their individual home-cages between the two stressor sessions of each day. On the morning of the last day (day 21) of the chronic stressor regimen mice were exposed to a single stressor session that comprised placing them in a tight fitting triangular baggie (with a hole for the nose) resulting in complete restraint (15 min). Mice in the Acute stressor condition experienced the “baggie stressor” (placement in a tight fitting triangular baggie for 15 min) on a single occasion. This permitted direct comparison between those mice that received only the ‘baggie stressor’ on this single occasion and those that had received a series of different challenges prior to the baggie stressor. This particular stressor was selected for the acute condition as it provides complete restraint, and we have found this procedure to be particularly potent given the pronounced neuroendocrine and brain neurochemical changes that are provoked. After the baggie (restraint) stressor session, mice in the chronic and the acute stressor conditions remained in their individual home-cages for the ensuing 21 days without disturbance other than routine changes of bedding. The third group, which comprised mice in the No stressor condition, remained undisturbed in their home-cages over the 42-day period prior to decapitation (i.e., paralleling the 21 days of the initial stressor phase and 21 days of the stressor-free period in the

Table 1 Experimental design and sequence of treatment procedures.

Stressor phase (21 days) Stressor conditions

Resting phase (21 days) Days 1−20

Test day

Day Day21 21 No stressor (n= 15/group)

No stressor

No stressor

No stressor

No stressor Social stressor (n= 16/group)

Acute stressor

No stressor

Restraint

No stressor

No stressor (n= 15/group) Social stressor (n= 15/group)

Chronic stressor

Multiple stressors

Restraint

No stressor

No stressor (n= 15/group) Social stressor (n= 16/group)

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chronic group). On the morning of the day following the 21-day stressor-free period, half of the mice of each condition were exposed to a psychosocial stressor in which they were regrouped with their previous cage mates for a 1-h period, whereas the remaining mice were left undisturbed. This regrouping procedure promotes the reestablishment of the dominance structure and elicits stress responses, such as elevated plasma corticosterone and brain monoamine activity (Anisman et al., 2008). After the 1-h regrouping, mice were returned to their individual home-cages for 90 min after which they were sacrificed by rapid decapitation, and blood, brain and heart tissue were collected. The brain and blood preparations were conducted by one researcher, and preparation of heart samples by a second researcher. Tissue collection and preparation Following decapitation trunk blood was collected in tubes containing 10 μg of EDTA and aprotinin and then placed in separate aliquots. One blood aliquot was centrifuged for 8 min at 3600 rpm and stored at −80 °C until being assayed for levels of corticosterone and cytokines. A second aliquot was centrifuged for 20 min at 2000 ×g and plasma was stored at −80 °C until being assayed for ANP concentrations. Cardiac tissues extraction was conducted as previously described (Tse et al., 2001). Briefly, after sacrifice, the left atria and ventricles were dissected and 1 ml of 1 M acetic acid was added to each atrium sample. Assays were restricted to the left atrium and ventricle as these portions of the heart tend to be more sensitive to pathology than the right (Iyer et al., 2006). The left atria were processed for analysis of ANP concentrations, whereas the ventricles were processed for mRNA expression of cytokines as well as ANP and BNP. Each atrial sample was transferred to a boiling water bath for 10 min, and after cooling on ice, samples were homogenized with a sonicator (Model 100, Sonic Dismembrator, Fisher Scientific), centrifuged at 16,000 ×g for 15 min and the supernatant frozen at −80 °C until assay. The atrial samples were extracted by Sep-Pak C18 cartridges, which had been pre-washed with 10 ml acetonitrile (CH3CN) and then with 10 ml of 0.1% TFA. The acidified samples were then passed 3 times through the cartridges. The sample loaded cartridges were washed with 10 ml of 0.1% TFA. This was followed by a 10 ml wash of 10% CH3CN in 0.1% TFA and finally the samples were eluted slowly with 3 ml of 60% CH3CN in 0.1% TFA and lyophilized. The resulting powder samples were stored at −80 °C until being assayed for ANP levels. Brain tissue was collected immediately after decapitation. Following rapid removal from the skull, a whole brain was placed on a stainless-steel brain matrix (2.5 × 3.75 × 2.0 cm) positioned on a block of ice. The matrix had a series of slots spaced ~500 mm apart that guided razor blades to provide coronal brain sections. Once the brains were sliced, tissue from the locus coeruleus was collected by micropunch using a hollow 20-gauge microdissection needle, following the mouse atlas of Franklin and Paxinos (1997). Tissue punches were placed in 0.3 M monochloroacetic acid containing 10% methanol and internal standards and were stored at − 80 °C for subsequent determination of NE and of its metabolite, 3-methoxy-4hydroxyphenylglycol (MHPG). Plasma analyses Corticosterone Corticosterone levels in plasma were analyzed using a radioimmunoassay kit (ICN Biomedicals CA, USA). Concentrations were determined in duplicate, in a single run to preclude inter-assay variability; the intra-assay variability was less than 10%. Cytokines Owing to the high cost of the assays, only 70 of the plasma samples (N= 11–12/group) were analyzed for the determination of plasma cytokines. Levels of IL-1β and IL-6 in plasma samples were determined

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using a Multiplex cytokine detection kit (Millipore; MPXMCYTO-70K). In order to determine protein concentrations of diluted plasma samples (one part plasma, one part assay buffer), a standard cocktail was used to generate a standard curve with a working range from 3.2 to 10,000 pg/mg. A volume of 25 ml of prepared standards and controls was added, in duplicate, to a pre-wet 96-well filter plate. For the unknown wells, 25 μl of assay buffer and 25 μl of plasma sample were added to each well. The pre-mixed anti-mouse cytokine beads were vortexed, sonicated and 25 μl of the bead solution was added to each well. Following brief vortexing, plates were incubated on a plated shaker overnight at 4 °C. Thereafter, samples were washed twice with 200 ml of wash buffer and removed by vacuum filtration, and 25 μl of detection antibodies beads were added to each well and incubated on a plate shaker for 60 min at room temperature. Streptavidin-PE (25 ml) was then added to each well containing the detection antibodies and incubated with agitation on a plate shaker for 30 min. The excess liquid was removed by vacuum filtration and washed twice using wash buffer and 150 ml of sheath fluid, and the plate was then placed on a plate shaker for 5 min in order to re-suspend the beads. The filter plate was analyzed using a Luminex 100 instrument, fitted with a five-parameter logistic regression curve using Analyst software (Millipore). ANP The levels of ANP in plasma and in cardiac tissue were determined by radioimmunoassay according to the manufacturer's protocol (Phoenix Pharmaceuticals, Inc). After reconstitution with the RIA buffer (150 ml), the standard peptide (1 ml of RIA buffer), the rabbit anti-peptide serum (13 ml of RIA buffer), and the positive control (1 ml of RIA buffer), samples were prepared as indicated in the experimental protocol (Phoenix Pharmaceuticals) and radioactivity was determined using a gamma counter. Locus coeruleus norepinephrine and metabolite determination Levels of NE and MHPG within the locus coeruleus were determined using high-performance liquid chromatography (HPLC) as previously described (Hayley et al., 1999). Briefly, tissue punches were sonicated in a solution obtained from a stock solution containing 500 ml HPLC grade water, 5.0 ml methanol, 0.0186 g EDTA, and 14.17 g monochloroacetic acid. The locus coeruleus was sonicated in 300 ml of this solution. After centrifugation, 20 ml of the supernatant was passed at a flow rate of 1.5 ml/min (1400–1600 p.s.i.) through a system equipped with a M-600 pump (Milford, USA), a guard column, a radial compression column (55 m, c18 reverse phase, 8 mm × 10 cm), and a 3-cell coulometric electrochemical detector (ESA model 5100A). The mobile phase used for separation comprised 1.3 g heptane sulfonic acid, 0.1 g disodium EDTA, 6.5 ml triethylamine, and 35 ml acetonitrile that had been filtered using 0.22-mm filter paper, degassed, and the pH levels adjusted to 2.5 using phosphoric acid. A Hewlett-Packard integrator determined the height and area of the peaks. The protein content of each sample was measured using bicinchoninic acid with a protein analysis kit (Pierce Scientific, Canada), and a spectrophotometer (Brinkman, PC800 colorimeter). The NE and MHPG concentrations were based on protein levels. The lower limit of detection for the NE and its metabolite was 5.0 pg/ml. Determination of ANP, BNP, IL-1β, IL-6 and TNF-α mRNA expression For the QPCR analyses of ventricular ANP, BNP, IL-1β, IL-6, and TNF-α mRNA expression only 90 samples were available (14–16/ group). Total RNA was extracted from frozen samples with Trizol according to the manufacturer's protocol (Invitrogen; Burlington, Ontario, Canada). The total RNA was then reverse transcribed using Superscript II reverse transcriptase (Invitrogen; Burlington, Ontario, Canada). Aliquots of this reaction were then used in simultaneous QPCR reactions.

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cytokines, as well as NE levels and utilization within locus coeruleus were assessed using a 3 (Initial stressor condition: No stressor, Acute stressor, Chronic Stressor) × 2 (Social stressor on the test day: No stressor vs. Social stressor) between-groups analysis of variance (ANOVA). For the mRNA analyses, the Ct values were converted to fold changes using the 2(− Delta Delta Ct) method (Livak and Schmittgen, 2001) and then subjected to a two factor between-groups ANOVA. Follow-up comparisons were conducted using the least significant difference (LSD) to maintain the alpha at 0.05. Results Plasma corticosterone levels

Fig. 1. Plasma corticosterone levels (means ± SEM) among mice that were initially either not stressed or exposed to either an acute or a chronic stressor regimen, and then 21 days later exposed to a social stressor or no treatment. *p b 0.05 relative to mice that had not been stressed on the test day. ●p b 0.05 relative to mice that had only been stressed on the test day.

For QPCR, SYBR green detection was used according to the manufacturer's protocol (Stratagene Brillant QPCR kit). A Bio Rad iQ5 real time thermocycler was used to collect the data. All PCR primer pairs generated amplicons between 100 and 200 bp. Amplicon identity was checked by restriction analysis. Primer efficiency was measured from the slope relation between absolute copy number or RNA quantity and the cycle threshold (Ct) using iQ5 software. All primer pairs had a minimum of 90% efficiency. GAPDH was used for normalization as it is a stably expressed housekeeping gene (Gutkowska et al., 2007). Primer sequences used for QPCR were as follows: GAPDH, forward: AAA TGG TGA AGG TCG GTG TG, reverse: GAA TTT GCC GTG AGT GGA GT. ANP, forward: CCA GAG TGG ACT AGG CTG CAA C, reverse: AAT GTG ACC AAG CTG CG TGA C. BNP, forward: GCA TGG ATC TCC TGA AGG TGC, reverse: GCT GTC TCT GGG CCA TTT CC. IL-1β, forward: TGT CTG AAG CAG CTA TGG CAA C, reverse: CTG CCT GAA GCT CTT GTT GAT G. TNF-α, forward: CTC AGC CTC TTC TCA TTC CTG C, reverse: GGC CAT AGA ACT GAT GAG AGG G. IL-6, forward: TTC TTG GGA CTG ATG CTG GTG, reverse: CAG AAT TGC CAT TGC ACA ACT C.

Statistical analyses Plasma corticosterone and cytokines levels, plasma and atrial ANP concentrations, ventricular mRNA expression of ANP, BNP, and

Plasma corticosterone levels varied as a function of the Initial stressor condition, F(2,86) = 13.33, p b 0.001, the Social stressor on the test day, F(1,86) = 48.06, p b 0.001, and the Initial stressor condition × Social stressor on the test day interaction, F(2,86) = 9.44, p b 0.001. The follow-up tests of the simple effects comprising the interaction indicated that the social stressor applied on the test day provoked a significant rise of plasma corticosterone levels (p b 0.01). The magnitude of the rise, as depicted in Fig. 1, was considerably smaller in mice that had previously been exposed to the chronic stressor than among mice that had either not been stressed or had been acutely stressed (p b 0.01). Norepinephrine and MHPG determination Levels of NE in the locus coeruleus, depicted in Fig. 2A, were increased in mice that had been socially stressed on the test day, F (1,86) = 4.85, p b 0.05, irrespective of the Initial stressor condition. The levels of MHPG in the locus coeruleus were likewise affected by the social stressor on the test day, F(1,86) = 9.69, p b 0.01, as well as by the initial stressor treatment, F(2,86) = 6.50, p b 0.01. The interaction between these variables did not reach statistical significance. However, as specific a priori predictions had been made concerning the influence of previous stressor experiences on the response to a subsequent stressor, follow-up comparisons were made of the simple effects comprising this interaction. As shown in Fig. 2B, among mice that had initially been exposed to an acute or chronic stressor, but not stressed on the test day, the accumulation of MHPG exceeded that of mice that had not been stressed on either occasion (p's b 0.05). Furthermore, the increased MHPG accumulation elicited by the social stressor on the test day was more pronounced among mice that had previously been exposed to an acute stressor (p b 0.01); however, this

Fig. 2. Norepinephrine (A) and MHPG (B) levels (means ± SEM) within the locus coeruleus among mice as a function of the initial stressor condition (no stressor, acute stressor, or chronic stressor) and the stressor treatment applied 21 days later (no stressor vs. social stressor). *p b 0.05 relative to mice that had not been stressed on the test day. ●p b 0.05 relative to mice that had only been stressed on the test day. ○p b 0.05 relative to mice that had never been stressed.

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Fig. 3. Plasma ANP (A) and left atrial ANP (B) concentrations (means ± SEM) as a function of the initial stressor treatment (no stress, acute or chronic stressor) and the stressor condition applied 3 weeks later (social stressor vs. no stressor). *p b 0.05 relative to mice that had not been stressed on the test day.

effect was not apparent in mice that had been chronically stressed previously. Plasma and atrial ANP levels Plasma ANP concentrations varied as a function of Initial stressor condition × Social stressor on the test day interaction, F(2,78) = 3.70, p b 0.05. The follow-up tests of the simple effects comprising this interaction indicated that the social stressor applied on the test day increased plasma ANP levels only among mice that had been initially exposed to the chronic stressor (p b 0.05) and not among mice that had initially been exposed to the acute stressor or that had not been stressed (see Fig. 3A). Atrial ANP levels in each condition are shown in Fig. 3B. The levels of ANP were increased by the social stressor on the test day, F(1,78) = 14.73, p b 0.01, and by the initial stressor treatment, F(2,78) = 4.51, p b 0.05. The interaction between the Initial stressor condition and the Social stressor on the test day approached statistical significance, F(2,78) = 2.59, p = 0.08. Follow-up tests of the simple effects comprising this interaction, based on our hypothesized outcomes, indicated that the social stressor on the test day increased ANP levels in mice that had not previously been stressed and in mice that had previously been exposed to a chronic stressor (p b 0.05), whereas this outcome was not apparent in mice that had previously experienced the acute stressor.

Ventricular ANP and BNP mRNA expression Ventricular ANP mRNA expression varied as a function of the Initial stressor condition × Social stressor on the test day interaction, F(1,84) = 7.35, p b 0.01. It is clear from Fig. 4A, and confirmed by the follow-up statistical tests, that the effects of the social stressor on the test day were limited in mice that had not previously been stressed as well as in mice in the chronic stressor condition (i.e., the increase was less than 0.2 fold changes in both instances). In contrast, among mice that had previously been acutely stressed, the elevation of ANP mRNA was approximately 0.7 fold after the social stressor on the test day and significantly exceeded that of mice that received either of these treatments alone (p's b 0.01). The mRNA expression of BNP within the left ventricle was very different from that of ANP (see Fig. 4B). In particular, both the social stressor applied on the test day, F(1,84) = 4.99, p b 0.05, and the initial stressor regimen, F(2,84) = 5.36, p b 0.01, influenced BNP mRNA expression. As well, mRNA expression of BNP varied as a function of the Initial stressor condition × Social stressor on the test day interaction, F(2,84) = 3.09, p b 0.05. The follow-up comparisons of the simple effects comprising this interaction indicated that the social stressor applied on the test day elicited a greater increase of BNP expression among chronically stressed mice (p b 0.01) relative to nonstressed mice, and the increase was significantly greater than that evident in mice that received only one of the stressor treatments

Fig. 4. Fold changes (means ± SEM) of ANP (A) and BNP (B) mRNA expression within the left ventricle among mice that had initially been either nonstressed or had been exposed to an acute or chronic stressor, followed 3 weeks later by no stressor or a social stressor. *p b 0.05 relative to mice that had not been stressed on the test day. ●p b 0.05 relative to mice that had only been stressed on the test day.

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(p b 0.05). In contrast, among both previously nonstressed and acutely stressed mice, subsequent exposure to the social stressor did not influence BNP mRNA expression. Plasma cytokine concentrations The plasma concentrations of IL-1β were increased by the social stressor on the test day, F(1,64) = 11.85, p b 0.001, whereas neither the initial stressor treatment nor the interaction between the Initial stressor condition and the Social stressor on the test day reached significance (see Fig. 5A). The concentration of IL-6 similarly varied as a function of the social stressor administered on the test day, F(1,64) = 125.00, p b 0.001, but this outcome interacted with the initial stressor treatment mice received, F(2,64) = 25.81, p b 0.001. Comparisons of the simple effects comprising this interaction indicated that subsequent administration of the social stressor on the test day increased IL-6 levels in all groups (p's b 0.01; see Fig. 5B). These elevations were significantly enhanced among those mice that had previously encountered an acute stressor (p b 0.05), whereas among mice that had been chronically stressed the effects of the social stressor treatment were markedly attenuated (p b 0.001). The levels of plasma TNF-α were exceedingly low, often not reaching detection level, and hence are not reported here. Ventricular IL-1β, IL-6 and TNF-α mRNA expression The IL-1β mRNA expression in the left ventricle varied as a function of the initial stressor condition, F(2,84) = 3.40, p b 0.01, but not as a function of the social stressor applied on the test day. The follow-up tests indicated that IL-1β mRNA expression was elevated in chronically stressed mice relative to nonstressed mice or those that had previously been exposed to an acute stressor (p's b 0.05; see Fig. 6A), irrespective of whether they had experienced the social stressor on the test day. The initial stressor experience also influenced TNF-α mRNA expression, F(2,84) = 3.69, p b 0.05, being elevated 21 days after either the acute or the chronic stressor relative to mice that had never been stressed (p's b 0.05 and 0.01, respectively). In contrast to the changes of IL-1β, the social stressor on the test day provoked a marked reduction of TNF-α expression, F(1,84) = 38.69, p b 0.001, irrespective of the previous treatment administered (see Fig. 6B). Unlike the other cytokines, IL-6 mRNA expression varied as a function of the Initial stressor condition × Social stressor on the test day interaction, F(2,84) = 8.79, p b 0.01. As depicted in Fig. 6C, among mice that had not initially been stressed, the social stressor administered on

the test day increased IL-6 expression (p b 0.01). A moderate, nonsignificant increase of IL-6 was also evident among mice that had initially been acutely stressed and then exposed to a stressor on the test day relative to animals that had not subsequently been exposed to the social stressor on the test day (p b 0.10). Interestingly, 21 days after the chronic stressor regimen and in the absence of a social challenge on the test day, the IL-6 mRNA expression was elevated relative to mice that have never been stressed (p b 0.05). However, if these chronically stressed mice were subjected to a social stressor on the test day, then the elevated cytokine expression associated with the chronic stressor treatment was no longer apparent (p b 0.01). Intercorrelations between outcome variables A series of Pearson correlations were conducted to assess the interrelations that existed among the plasma corticosterone and cytokine levels, ANP concentrations in plasma and atria, mRNA expression of ANP, BNP, and cytokines, as well as brain NE and MHPG indices. This was done within each group as well as in the full data set. In general, there were only a few significant correlations between variables, but there did not appear to be any systematic pattern in their occurrence. The mRNA expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α was unrelated to any of the cardiac indices in either the blood or the heart itself. In the full data set, plasma corticosterone was directly correlated with locus coeruleus MHPG (r = 0.38, p b 0.05), atrial ANP (r = 0.25, p b 0.05), and ventricular ANP mRNA expression (r = 0.28, p b 0.05), and inversely related to ventricular TNF-α mRNA expression (r = 0.50, p b 0.01). Summary The initial acute and chronic stressor conditions and the social stressor administered on the test day elicited different sets of outcomes, which varied as a function of the specific dependent measure being considered (plasma corticosterone, ANP, IL-1β, IL-6; locus coeruleus MHPG; atrial ANP; ventricular ANP, BNP, IL-1β, IL-6, and TNF-α mRNA expression). To facilitate appraisal of the data, a summary of the findings are presented in Table 2 in which symbols designate the magnitude and direction of change relative to mice that had not been stressed at any time. As depicted in this table the social stressor applied on the test day (in the absence of a stressor applied earlier) had wide ranging effects, including elevations of plasma corticosterone and locus coeruleus NE activity, plasma cytokines and atrial ANP, as well as ventricular IL-6 mRNA expression. An acute or

Fig. 5. Plasma concentrations (means ± SEM) of IL-1β (A) and IL-6 (B) as a function of the initial stressor treatment (no stress, acute or chronic stressor) and the stressor condition applied 3 weeks later (social stressor vs. no stressor). *p b 0.05 relative to mice that had not been stressed on the test day. ●P b 0.05 relative to mice that had only been stressed on the test day.

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later exposed to the social challenge, sensitized responses were apparent with respect to locus coeruleus MHPG, plasma IL-6 and ventricular mRNA expression of ANP. Among chronically stressed mice a sensitized change was also apparent with respect to plasma ANP and ventricular BNP and IL-1β expression. However, desensitized responses were also evident as some of the effects ordinarily elicited by an acute stressor on the test day (e.g., increased plasma corticosterone and IL-6, locus coeruleus NE utilization, and ventricular IL-6 mRNA expression) were less pronounced or entirely absent among mice that had been chronically stressed and 21 days later and then challenged with the social stressor. Discussion

Fig. 6. Fold changes (means ± SEM) of ventricular IL-1β (A), TNF-α (B) and IL-6 (C) mRNA expression as a function of the initial stressor treatment (no stress, acute or chronic stressor) and the stressor condition applied 3 weeks later (social stressor vs. no stressor). *p b 0.05 relative to mice that had not been stressed on the test day. ●P b 0.05 relative to mice that had only been stressed during the test day. ○P b 0.05 relative to mice that had never been stressed.

chronic stressor applied 21 days earlier had limited protracted effects, but NE utilization was increased and ventricular TNF-α mRNA was increased in mice, and in those mice that had been chronically stressed IL-6 mRNA was also elevated relative to nonstressed mice. It was of particular interest given the purpose of the present investigation that the initial stressor treatment mice received influenced the responses elicited by subsequent exposure to the social stressor. In those mice that had been acutely stressed and then

The effects of acute stressors are relatively transient, such that neurochemical and neuroendocrine consequences elicited by adverse events ordinarily normalize within a few minutes or hours (Anisman and Merali, 2003; Sapolsky et al, 2000). However, as recently reported (Audet and Anisman, 2010), when a social stressor was acutely administered, it had wide ranging effects on biological processes measured 90 min later, including relatively persistent elevations of plasma corticosterone and NE utilization within the locus coeruleus. Moreover, in line with findings that emotional stressors may provoke the up-regulation of the expression of natriuretic peptide genes in the myocardium (Ueyama et al., 2003), the social stressor was associated with relatively persistent increases of factors that might directly be related to cardiac functioning or that might predict adverse cardiac outcomes (e.g., atrial ANP levels and circulating levels of IL-1β and IL6, as well as ventricular mRNA expression of IL-6). It has been reported that heart disease was accompanied by elevations of plasma cytokines and natriuretic peptides (Emdin et al., 2004), as well as inflammatory factors, such as IL-1β and TNF-α, that potentially stimulated BNP release (Campbell and MacQueen, 2004). Moreover, given that heart disease and major depressive disorder are frequently comorbid (Lesperance and Frasure-Smith, 2000) and that both illnesses are accompanied by plasma cytokine variations (Anisman et al., 2008; Brown et al., 2009; Frasure-Smith et al., 2009; Lesperance and Frasure-Smith, 2000), it might be considered that stressor-provoked activation of inflammatory processes contributes to this comorbidity. However, as the social stressor in the present investigation elicited multiple physiological changes, it is uncertain whether they simply occurred in concert with one another or were causally related in some fashion. It was shown, for instance, that cardiomyopathy was associated with progressive elevations of natriuretic peptides and several neurohormones, such as glucocorticoids (Emdin et al, 2004), and that the synthetic glucocorticoid, dexamethasone, increased atrial ANP mRNA expression (Roy et al., 2009). It is thus possible that the elevated atrial ANP elicited by the social stressor in the present investigation might have been related to the elevated corticosterone elicited by this treatment. Indeed, plasma corticosterone levels were found to be significantly correlated with atrial ANP and with ventricular mRNA expression of ANP, supporting the connection between these factors. Yet, both corticosterone and the ANP variations may reflect parallel actions of the social stressor, and it is premature to assume that corticosterone and natriuretic peptides were causally connected to one another. It will be recalled that stressful experiences may proactively influence neurochemical responses to stressors that are subsequently encountered, and might thus promote the emergence of psychopathology (Anisman et al., 2003). These sensitized responses have most often been assessed with regard to neuroendocrine and brain neurochemical processes, where it was shown that an initial stressor experience enhanced the hypothalamic and extrahypothalamic neurochemical responses (e.g., monoamine activity) (Anisman and Merali, 2003), as well as stress-related hormones, such as CRH and corticosterone (Buwalda et al., 1999). In this regard, it was reported

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Table 2 Summary of stressor effects on diverse biological outcome measures.

that the glucocorticoid response became more pronounced with the passage of time following an initial insult, possibly owing to increased co-expression of CRH and AVP within median eminence CRH terminals, leading to synergistic enhancement of pituitary ACTH and adrenal corticosterone upon later stressor experiences (Lynn et al., 2010; Tilders and Schmidt, 1999; Tilders et al., 1993). However, there have also been reports indicating that preliminary exposure to a stressor resulted in the diminution (desensitization) of the corticosterone response upon subsequent stressor experience (Armario et al., 2008). This outcome generally was more notable with relatively strong stressors, although the specific factors that promoted sensitized responses on the one hand, and desensitized responses on the other, have not been identified. Nevertheless, the present investigation indicated that stressful experiences were associated with the sensitization or desensitization of specific physiological processes and it seems that the nature of the initial stressor experience (acute vs. chronic) modulated the responses elicited upon subsequent exposure to a novel, ethologically relevant, social stressor. Specifically, among mice that had initially been exposed to acute restraint, a social challenge encountered 21 days later resulted in exaggerated MHPG accumulation within the locus coeruleus relative to mice that received only one of these treatments. This sensitized response was accompanied by increased levels of circulating IL-6 and ventricular ANP mRNA expression that were greater than those elicited by the initial acute restraint or the social stressor applied individually. It is uncertain which, if any, of these biological responses were linked to one another in the present study. However, locus coeruleus activity has been associated with cardiac functioning (Guo et al., 2002) as well as heart failure (Patel and Zhang, 1996), and thus

it is possible that the sensitized locus coeruleus increase of NE utilization might have been linked to the ventricular ANP changes. As indicated earlier, although the effects of stressors are typically fairly transient, like mice that had initially encountered an acute stressor, those mice that had been chronically exposed to different stressors exhibited exaggerated response to the subsequent social challenge. This sensitization effect was apparent with respect to heart-related factors, such as plasma ANP, as well as ventricular BNP and IL-1β mRNA expression. In contrast to these outcomes, the chronic stressor regimen also elicited desensitized responses characterized by the diminution of the typical effects associated with the social stressor. This included a limited increase of levels of plasma corticosterone and IL-6, of locus coeruleus MHPG accumulation, as well as mRNA expression of ventricular ANP and IL-6. The processes responsible for the sensitized responses on the one hand, and those responsible for a desensitized response on the other, are uncertain, but it did appear that the desensitized response was more common in mice previously treated with the chronic stressor. Although ANP and BNP have both been implicated as markers for cardiovascular illness (Goetze et al., 2004; Mekontso-Dessap and Brochard, 2006), they behaved differently in response to various stressor conditions. Specifically, in response to a social stressor, sensitized ANP mRNA expression was evident in mice that had been exposed to an acute stressor 21 days earlier, whereas a sensitized BNP response was evident among mice in the chronic stressor condition. As discussed earlier, interrelations might exist between natriuretic peptides and cytokine functioning, and IL-1β and TNF-α are known to impair heart contractility and chemo-mechanical energy transduction (Hofmann et al., 2007), and to provoke increased dilatation of the left

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ventricle (Anderson et al., 2003; Aoshima et al., 1997; Bozkurt et al., 1998). Thus, it might have been suspected that the natriuretic changes elicited by the stressor conditions might be associated with the cytokine alterations. However, there were no apparent correlations between the cytokine variations and the heart markers (ANP and BNP), irrespective of the stressor conditions. Of course, these data should not be taken to suggest that cytokines are unrelated to cardiovascular disturbances, but only that under the conditions examined, the ventricular cytokine mRNA expression and the plasma cytokine levels were not tied to ANP and BNP which have been taken as potential markers of cardiac disturbances. Conclusion Consistent with the perspective linking stress, inflammatory factors and heart disease, a potent social stressor markedly influenced atrial ANP levels as well as plasma and ventricular pro-inflammatory cytokines. Furthermore, acute and chronic stressors proactively influenced the response to this later social challenge. In this regard, some of the natriuretic peptide markers and cytokines changes were subject to a sensitization-like effect so that subsequent stress responses were augmented, whereas other effects of the social stressor resulted either in a limited sensitization or an actual desensitization. This was particularly apparent among mice that had been chronically stressed previously. However, direct relations between cytokine and ANP/BNP levels in blood or ventricular expression were not evident. As alluded to earlier, it has been suggested that acute stressors promote a cascade of physiological alterations that are of adaptive significance (increased amine activity, corticosterone release and immune activation) that function to facilitate defensive responses or prevent pathology (Anisman et al., 2008). As the stressor continues, a degree of “adaptation” or tolerance may occur (e.g., compensatory variations of neurotransmitter synthesis and various receptor changes) so that the stressor effects are somewhat attenuated. Ultimately, however, if the stressor persists, then resources may become overly taxed (allostatic overload) leading to pathological outcomes (McEwen, 2006). In the present investigation the chronic stressor was limited to 21 days of treatment, which may allow for adaptation to the effects ordinarily associated with acute stressors. It remains to be established whether cardiac and inflammatory indices would be more profoundly disturbed with still more extended stressor experiences. Acknowledgments This work was supported by Canadian Institute of Health Research (CIHR). HA holds a Canada Research Chair in Neuroscience, and M-C.A is supported by the Fonds de la Recherche en Santé du Québec (FRSQ). References Anderson, J.L., Krause-Steinrauf, H., Goldman, S., Clemson, B.S., Domanski, M.J., Hager, W.D., Murray, D.R., Mann, D.L., Massie, B.M., McNamara, D.M., Oren, R., Rogers, W.J., 2003. Failure of benefit and early hazard of bucindolol for Class IV heart failure. J. Card. Fail. 9, 266–277. Anisman, H., Merali, Z., 2003. Cytokines, stress and depressive illness: brain-immune interactions. Ann. Med. 35, 2–11. Anisman, H., Hayley, S., Merali, Z., 2003. Cytokines and stress: sensitization and crosssensitization. Brain Behav. Immun. 17, 86–93. Anisman, H., Prakash, P., Merali, Z., Poulter, M.O., 2007. Corticotropin releasing hormone receptor alterations elicited by acute and chronic unpredictable stressor challenges in stressor-susceptible and resilient strains of mice. Behav. Brain Res. 181, 180–190. Anisman, H., Merali, Z., Hayley, S., 2008. Neurotransmitter, peptide and cytokine processes in relation to depressive disorder: comorbidity between depression and neurodegenerative disorders. Prog. Neurobiol. 85, 1–74. Aoshima, H., Yokoyama, T., Tanizaki, J., Izu, H., Yamada, M., 1997. The sugar specificity of Na+/glucose cotransporter from rat jejunum. Biosci. Biotechnol. Biochem. 61, 979–983.

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