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Research report
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CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress
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José S. de Andrade a , Milena B. Viana a , Renata O. Abrão a , Jackson C. Bittencourt b,c , Isabel C. Céspedes a,∗ a
Department of Biosciences, Federal University of São Paulo, Av. Ana Costa 95, UNIFESP, 11060-001 Santos, SP, Brazil Laboratory of Chemical Neuroanatomy, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, USP, 05508-000 São Paulo, SP, Brazil c Center of Neuroscience and Behavior, Institute of Psychology, University of São Paulo, Av. Prof. Mello Moraes, 1721, Bloco C, 05508-030, São Paulo, SP, Brazil b
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h i g h l i g h t s • • • •
PVN cells respond to both acute and chronic predictable stressors. EW cells are only activated in response to acute stressors. CRF/Ucn1 neuronal circuits respond coordinately to acute stressors. CRF/Ucn1 neuronal circuits are differently affected by chronic stress.
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a r t i c l e
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a b s t r a c t
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Article history: Received 11 May 2014 Received in revised form 6 June 2014 Accepted 8 June 2014 Available online xxx
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Keywords: CRF Urocortin 1 Paraventricular hypothalamic nucleus Edinger–Westphal nucleus Stress
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1. Introduction
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Corticotropin-releasing factor (CRF) acts to promote stress-like physiological and behavioral responses and is mainly expressed in the paraventricular hypothalamic nucleus (PVN). Urocortin 1 (Ucn1) is also a ligand to CRF type 1 and 2 receptors that has been associated with the stress response. Ucn1 neurons are primarily found in the Edinger–Westphal (EW) nucleus. It has been previously proposed that CRF and Ucn1 differently modulate stress responses to distinct types of stressors. The present study used male Wistar rats to compare the effects of acute restraint stress and unpredictable chronic stress (UCS) through Fos-immunoreactivity (Fos-ir) on CRF-containing neurons of PVN and Ucn1-containing EW centrally projecting neurons. Results showed that PVN neurons responded to both acute restraint and UCS. Also for the PVN, unspecific variables, dependent on the time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute restraint stress. Also, for this nucleus a significant difference was found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remain in the laboratory, interfere with the nucleus activation. These results suggest that CRF/Ucn1 neuronal circuits encompass two interconnected systems, which are coordinated to respond to acute stressors, but are differentially activated during chronic unpredictable stress. © 2014 Published by Elsevier B.V.
Physiological responses to stress encompass an efficient and conserved set of interconnecting systems, aimed to the maintenance of the organism’s integrity under challenging circumstances.
∗ Corresponding author. Tel.: +55 13 3878 3700; fax: +55 13 3223 2592. E-mail addresses: [email protected], [email protected] (I.C. Céspedes).
The autonomic nervous system provides an immediate reaction to stressor exposure – through its sympathetic and parasympathetic divisions – triggering rapid physiological changes through the innervation of end organs [1]. Activation of the hypothalamic–pituitary–adrenocortical (HPA) axis occurs in response to corticotropin-releasing factor (CRF) release from the paraventricular nucleus of the hypothalamus (PVN). Adrenocorticotropic hormone (ACTH) released from the anterior pituitary stimulates the adrenal cortex, resulting in the elevation of plasma circulating glucocorticoids levels [2,3].
http://dx.doi.org/10.1016/j.bbr.2014.06.014 0166-4328/© 2014 Published by Elsevier B.V.
Please cite this article in press as: de Andrade JS, et al. CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.06.014
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CRF, whose characterization and synthesis was performed by Vale et al. [4], seems to be an essential ingredient for the maintenance of an organism’s well being or homeostasis. Nowadays, this peptide, originally implicated in the activation of the HPA axis and known as the “stress hormone”, has also been considered for its role within the central nervous system [5,6] outside the HPA axis [7]. Since CRF was first characterized, a growing family of ligands and receptors has been identified. Aside from CRF, other family members include urocortin1 (Ucn1), Ucn2, and Ucn3, along with two receptors, CRF type 1 (CRFR1) and CRF type 2 (CRFR2), and a CRF binding protein. These family members differ in their tissue distribution and pharmacology. Although, the main sources of CRF are the parvocellular neurons of the PVN, the most abundant expression of urocortin 1 (Ucn1) in mammals has been reported in neurons located in the midbrain in the Edinger–Westphal nucleus (EW) [8–12]. The EW is a small, cholinergic, preganglionic parasympathetic structure, critically involved in oculomotor adaptation [13,14]. It has been proposed that the structure and functions of the EW are more complex than what was originally thought [11,13,15–18]. Thus, to distinguish EW cells that exhibit choline acetyltransferase immunopositivity and project to the ciliary ganglion from those that are positive for Ucn1, but do not innervate the ciliary ganglion, the terminology of non-preganglionic or centrally projecting EW has been introduced [8,11,12,15,19–21]. The later will be the subject of investigation of the present work. Although CRF appears to play a stimulatory role in stress responsivity through activation of CRFR1, the involvement of CRFR2 with stress and anxiety is still a matter of debate [22–25]. Taking this last observation into account and since Ucn1 is the only ligand with high affinity for both CRFR1 and 2, its role in the mediation of stressrelated responses may differ, depending on the type and intensity of the stressor. In a previous study, we employed Fos immunoreactivity (Fos-ir) and in situ hybridization of mRNA of CRF and Ucn1 to investigate how two different acute psychogenic stressors, restraint and footshock, activated the PVN and the EW [26]. The results of this study showed that restraint increased both Fos-ir and CRF mRNA expression in the PVN, while in the EW Fos-ir was higher in the footshock group and Ucn1 mRNA expression was higher in the restraint group. These results suggest that these two nuclei respond differently and in a complex manner to distinct types of acute stressors. Different roles for the PVN and EW have also been proposed when acute and chronic stressors are compared. Thus, it has been suggested that while the PVN has a fast response to acute stimuli, with Fos peaking within an hour [27,28], the EW responds slower to these two different kind of stressors, with Fos-ir peaking only 4 h after stress initiation [29,30]. In line with this notion it was hypothesized that CRF in the PVN, acting via CRFR1, plays a major role in the stress response initiation, whereas Ucn1 in the EW would be activated later [31–33]. Nevertheless, this proposition needs to be better investigated through controlled experimental studies that simultaneously compare both types of stressors. Taking the above into account, this study aims to compare the response to acute restraint stress and unpredictable chronic stress (UCS) of two nuclei that represent the major sources of CRF (PVN) and Ucn1 (EW), thus contributing to a better understanding of the controversial role of both peptides on stress regulation. Fos-ir was used to map the activation of the PVN and EW in animals submitted to either acute restraint or UCS. As formerly noted, the product of the immediate-early gene c-fos is expressed throughout the brain in response to a variety of tasks, thus making it a powerful instrument to study intracellular responses of neurons to different stimuli [34]. The UCS model presents good face validity and has been broadly used to investigate some of the physiological and behavioral
consequences of chronic stress [35–39]. Briefly, in this test, rodents are exposed to a variety of stressors (i.e., restriction, inversion of the light–dark cycles, water/food deprivation, damp sawdust) intermittently, usually for two to three weeks [35–39]. Immediately after the stress procedure, animals were weighted. Independent groups of animals were subjected to measurements of plasma corticosterone after acute restraint or UCS.
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2. Materials and methods
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2.1. Animals
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Male Wistar rats, weighing 250–300 g (CEDEME, Universidade Federal de São Paulo, Campus Santos, Brazil) were housed in groups of 4–6 per cage (50 cm × 60 cm × 22 cm). Room temperature was maintained at 22 ± 1 ◦ C with lights on from 0700 to 1900 h. Food and water were freely available throughout the experiments. UCS animals were housed under the same conditions except during the periods they were exposed to some of the stressors (i.e. food and water restriction/deprivation, inversion of the light–dark cycle). The study was approved by the Ethical Committee for Animal Research of the Federal University of São Paulo and was performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior, which are based on the conditions stated by the “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996). 2.2. Procedure The animals were organized in four groups with 6 individuals each: control (group 1) versus acute restraint stress and control (group 2) versus UCS. Acute restraint: two days after their arrival to the laboratory, animals were individually placed for a period of 30 min in acrylic restraining cylinders (6.3 cm × 6.5 cm × 23.2 cm) adjustable to the size of the animal and with holes on the sides to provide ventilation. The restraining procedure took place during the morning period (from 7:00–9:00 a.m.) in an experimental room dimly lit. Control animals (group 1) were left undisturbed in Plexiglas cages, in the same experimental room and for the same amount of time (30 min). At 1 h after the stress procedure animals were sacrificed. This is the time required to achieve the peak of Fos-ir in activated neurons [26]. UCS: the protocol was performed as described previously [40, see Table 1]. Rats were subjected to different kinds of stressors, which varied from day to day, for 14 consecutive days. There were a total of seven stressors: (1) periods of 1 h of restraint in a acrylic restraining cylinders (7.0 cm × 7.5 cm × 16 cm); (2) lights on overnight; (3) food deprivation overnight, followed by 2 h access to restricted food (resulting from the scattering of pellets of 45 mg in the cage); (4) water deprivation overnight, followed by 1 h contact with an empty water bottle; (5) food restriction for 2 h; (6) damp sawdust overnight; (7) inversion of the light–dark cycle from Friday night to Monday morning. Unstressed animals (control group 2) remained for 14 days in the laboratory, under standard housing conditions. Animals were sacrificed 2 h after the last stimulus (see Table 1, Monday morning, around 9:00 a.m.), so that the peak of Fos-ir corresponding to the last stimulus would not hide the effect of the chronic treatment [40]. 2.3. Fos protein immunohistochemistry (Fos-ir) After each treatment and in the period described above, the animals were anesthetized with ketamine/xylazine 2:1 (1 ml/kg) and
Please cite this article in press as: de Andrade JS, et al. CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.06.014
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Table 1 Unpredictable chronic stress (UCS) protocol. Period
Monday
Tuesday
Wednesday
Thursday
Friday
Morning
Restraint (30 min)
Restraint (30 min)
Food restricition for 2 h
Restraint (30 min)
Afternoon Night
Restraint (30 min) Lights on during the night
Restraint (30 min) Water/fooddeprivation overnight
Restraint (30 min) Water deprivation overnight
Empty water bottle (60 min); Restraint (30 min) Restraint (30 min) Damp sawdust overnight
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perfused with ≈100 ml of 0.9% saline for approximately 1 min, followed by 500–700 ml of 4% formaldehyde (from paraformaldehyde heated to 60–65 ◦ C) and H2 O at 4 ◦ C, pH 9.5, for approximately 25 min. The brains were post-fixed for 1 h in the same fixative solution, and then stored in a solution containing 20% sucrose for cryoprotection at 4 ◦ C. Regularly spaced series (5 × 1-in-5) of 30 m-thick frozen sections were cut in the coronal plane, collected in ethylene glycol-based cryoprotectant solution and stored at −20 ◦ C for later determination of Fos-ir. Fos-ir cells were identified by using a polyclonal anti-serum raised in rabbit against synthetic human Fos (anti-Fos – 1:20,000; Oncogene, Cambridge, MA, USA) in a solution containing KPBS loaded and goat serum. Immunohistochemistry was performed using a conventional avidin–biotin immunoperoxidase protocol [41] and Vectastain Elite reagents (Vector Laboratories, Burlingame, CA, USA). Tissue was pretreated with hydrogen peroxide (0.3%; Sigma, St. Louis, MO, USA) diluted in KPBS before addition of the primary antibody to quench endogenous peroxidase activity in the tissue. The reaction with diaminobenzidine (DAB) (0.05%; Sigma) was amplified using nickel ammoniumsulfate, both diluted in acetate buffer solution. The sections were then mounted on gelatin-coated slides, allowed to dry for approximately 18 h and counterstained with 0.25% thionin in order to visualize the labeled cells within the borders of each nucleus. We quantified Fos-ir cells in sections, having as reference the following AP coordinates [42] bregma: PVN: −1.80 mm and EW: −5.88 mm, under bright-field illumination using the ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA). One slice per brain region was analyzed.
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2.4. Corticosterone measurements
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The animals submitted to acute stress (N = 5) were euthanized by decapitation immediately after the stressor. The animals submitted to UCS (N = 5) were euthanized by decapitation 2 h later the last stimulus. Trunk blood was collected (from 08:00–11:00 a.m.) and centrifuged for 20 min at 4000 rpm, at 4 ◦ C. Aliquots of serum were then removed and stored at −20 ◦ C until assayed. Serum corticosterone concentrations were determined by enzyme immunoassay (ELISA) using a commercial kit (Assay Designs, Inc., Ann Arbor, USA). Unstressed animals (N = 5) were left undisturbed under standard laboratory conditions, for 3 (acute control) or 14 (chronic control) days and also euthanized by decaptiation and their trunk blood collected (from 09:00–11:00 a.m.) and analyzed as described for stressed animals.
2.5. Statistical analysis A two-factor ANOVA was used to analyze Fos-ir neurons (factor 1: stress protocol, restraint or UCS; factor 2: absence or presence of stress). In case of significant results, groups were analyzed by one-way ANOVA followed by the Tukey post-hoc test. Weight and corticosterone measurements were analyzed by independent Ttest (control versus acute restraint; control versus UCS). Values of P ≤ 0.05 were considered significant.
Inversionofthe light–dark cycle over the weekend
Table 2 Weight (mean ± SEM) in control and stressed animals, measured at the end of the experiments. Experimental group
Weight
Acute control Restraint Chronic control UCS
296.67 292.50 306.67 310.00
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3. Results
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3.1. Animals’s weight
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All animals arrived at the laboratory weighting between 260 and 280 g. Table 2 shows the weight of the different groups at the end of the stress procedure. There were no significant changes in body weight between control and acute restraint animals (T(10) = 1.0; P = 0.341), or between control and UCS animals (T(10) = −0.791; P = 0.448). 3.2. Fos-immunoreativity (Fos-ir) The results obtained with the analyses of Fos-ir in the studied brain regions, PVN and EW, in animals submitted to acute or UCS are summarized in Table 3. 3.3. Paraventricular nucleus of the hypothalamus (PVN) For the PVN, two-factor ANOVA showed a significant effect of factor 1 (restraint or UCS) F(1,20) = 56.36; P < 0.001), of factor 2 (absence or presence of stress) F(1, 20) = 260.18; P < 0.001) and of the interaction between factors (F(1,20) = 102.92; P < 0.001). One-way ANOVA showed a significant difference between groups (F(3,23) = 139.82; P < 0.001) (Fig. 1). The Tukey post-hoc test showed significant differences between all the groups tested (P < 0.05), but no significant differences between the two groups of control animals, acute and chronic (P > 0.05), suggesting that a short or a prolonged period of time spent in the laboratory does not seem to be an important factor involved with the activation of this nucleus. 3.4. Edinger–Westphal nucleus (EW) For the EW, two-factor ANOVA showed a significant effect of factor 1 (restraint or UCS) F(1,20) = 158.90; P < 0.001), of factor 2 (absence or presence of stress) F(1, 20) = 57.93; P < 0.001) and of the interaction between factors (F(1,20) = 26.56; P < 0.001). One-way ANOVA also showed a significant difference between groups for the EW (F(3,23) = 81.13; P < 0.001) (Fig. 2). The Tukey post-hoc test showed that the two groups of control animals, acute and chronic, differ significantly (P < 0.001) – an effect possibly related to habituation to the laboratory conditions –, that both group of control animals were also significantly different from acute restraint animals (P < 0.001), and that acute control animals were significantly different from UCS animals (P = 0.010). Nevertheless, there were no
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Fig. 1. Photomicrographs of Fos immunoreactive cells (dark spots) in coronal sections of the paraventricular hypothalamic nucleus (PVN) of animals submitted to acute restraint (upper panel) or unpredictable chronic stress-UCS (lower panel). Scale bar: 300 m; Magnification, ×200. (*) third ventricle.
Fig. 2. Photomicrographs of Fos immunoreactive cells (dark spots) in coronal sections of the Edinger–Westphal (EW) nucleus of animals submitted to acute restraint (upper panel) or unpredictable chronic stress-UCS (lower panel). Scale bar: 300 m; Magnification, ×200. (*) medial longitudinal fasciculus.
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Table 3 Mean ± SEM of Fos immunoreactive cells in the paraventricular hypothalamic nucleus (PVN) and Edinger–Westphal nucleus of unstressed animals (control) and animals submitted to acute restrain to run predictable chronic stress (UCS). Structure
Control (group 1)
Acute restraint
Control (group 2)
UCS
PVN EW
9.00 ± 0.93 25.33 ± 2.86
138.50 ± 7.26* 52.17 ± 2.85*
12.00 ± 3.71 9.67 ± 0.84#
51.50 ± 5.47* , + 14.83 ± 0.83#
* + #
P < 0.05, with respect to all the other groups (group 1). P < 0.05, with respect to control (group 2). P < 0.05, with respect to control (group 1) and acute restraint (ANOVA, followed by the Tukey test).
Table 4 Corticosterone measurements (mean ± SEM) in control and stressed animals.
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Experimental group
Corticosterone measurements (ng/ml)
Acute control Restraint Chronic control UCS
226.60 513.80 221.60 669.61
± ± ± ±
38.10 45.50 24.82 99.04
significant differences between chronic control animals and UCS animais (P > 0.05), suggesting that the EW is not crucial for the organization of responses to chronic stress. 3.5. Corticosterone measurements
Measurements (mean ± SEM) of serum corticosterone levels are shown in Table 3. Independent Student t-test indicated a sig266 267 nificant difference between acute control and restraint animals 268 (T(6.15) = −4.53; P < 0.001). Also, there was a significant difference 269 Q2 between chronic control and UCS animals (T(7) = −4.90; P < 0.01] 270 (Table 4). 265
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4. Discussion The results of the present study showed that PVN neurons responded to both acute and chronic stress. Also, for the PVN unespecific variables, such as the period of time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute stress. Nevertheless, for this nucleus a significant difference was also found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remained in the laboratory (i.e., a short versus a prolonged period of handling), may interfere with the nucleus activation. Measurements of corticosterone levels showed that both stress protocols induced significant increases in serum corticosterone. The central role of the PVN in HPA activation has been well documented by several previous studies performed with exposure to acute stressors [for a review see 43]. For instance, Viau and Sawchenko [28] have demonstrated increased Fos-ir in CRF cells in the PVN after a single 30 min episode of acute restraint that is slightly decreased in animals killed 2 h after the stress protocol. Similar results were also found by Lund et al. [44]. Corroborating these observations, it was also previously shown by our research group that PVN neurons are significantly activated by both acute restraint and acute footshock [26]. The conclusions obtained with Fos-ir are also supported by studies that show rapid phosphorylation of cyclic AMP response element binding protein (CREB) and mitogen activated protein (MAP) kinase in the PVN following acute stressors [45,46]. Acute stress has been shown to induce transcription of CRF and vasopressin genes in the parvocellular PVN, and subsequently increase CRF and vasopressin mRNA [46–48]. Moreover, acute stress exposure also results in increases in portal blood levels of CRF, vasopressin and oxytocin [49–51].
Previous investigations have also shown that the PVN responds effectively to chronic stress exposure. A variety of chronic stress paradigms, such as chronic footshock, immobilization, social subordination, social defeat and also UCS have been shown to cause increases in CRF mRNA expression within the PVN [47,52–54]. In fact, across multiple rat strains, CRF mRNA increases induced by repeated immobilization is observed [55], thus showing that PVN activation is a consistent result of chronic stress. Furthermore, it has been previously shown that chronic stress increases PVN Fos-ir as well as the number of glutamatergic and noradrenergic immunoreactive terminals within the parvocellular PVN [56]. The number of GABAergic-immunoreactive boutons in the PVN, in this same study, was unchanged. Taken together, these data are consistent with a role for both glutamate and norepinephrine in chronic stress enhancement of HPA axis excitability [56]. Nevertheless, it is also important to point out that although the PVN was significantly activated by chronic stress, a greater increase in Fos-ir within the nucleus was observed after acute stress. This observation seems to corroborate the idea that Fos protein expression in CRF cells in the PVN habituate over a prolonged period of time [28], what might also explain why some studies with different chronic stress paradigms have not reported increases in PVN activation [43]. Interestingly, there were no significant differences in PVN Fos-ir in control animals that remained for two or fourteen days in the laboratory. This suggests that unspecific variables (i.e., a short versus a prolonged period of handling) do not seem to be critical factors involved in the activation of this nucleus. In other words, that the important stimuli that activated the nucleus were in fact the ones related to the stressful procedure. While the PVN was activated by both acute and chronic stress, the EW showed a significant activation only after acute restraint stress. In a previous study, we have shown that two different acute stress protocols, restraint and footshock, induced Fos-ir in the EW, although the results obtained with footshock were more pronounced [26]. On the other hand, UCN1 mRNA expression was higher after acute restraint [26]. Several other studies also pointed to the same direction. For instance, it was previously shown that acute pain stress resulted in the activation of urocortinimmunoreactive neurons in the EW, peaking at 4 h after acute pain stress [29]. Also, Weninger et al. [30] showed that acute restraint stress and lipopolysaccharide administration upregulate Ucn1 mRNA in a mouse model. The finding that acute restraint stress stimulated the production of Ucn1 in EW neurons, suggests a role for these neurons in the stress response in the mouse as well, a notion strengthened by the fact that in addition to Ucn1, two other peptides within this same neuronal group, cocaine- and amphetamine-regulated transcript peptide (CART) and nesfatin-1, were upregulated [57]. Contrarily to our results, however, it was also previously shown that chronic predictable/homotypic stress (3 weeks of exposure to ether vapor) activates mouse EW Ucn1 neurons [58]. It is interesting to observe that, in this particular study [58], Fos-ir in EW cells was also more robust after exposure to acute ether stress, an observation that goes in the same direction of our results. On the other hand, regarding the response of the EW to chronic stress,
Please cite this article in press as: de Andrade JS, et al. CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.06.014
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two hypotheses may be raised: (1) the nucleus plays different roles depending on the species investigated (i.e., if mouse or rat); (2) the nucleus plays different roles depending on whether the stressors are predictable or unpredictable. Nevertheless, both propositions warrent further investigation. Also differing from what was observed for the PVN, there were significant differences in EW Fos-ir between control animals that remained for two or fourteen days in the laboratory. This observation suggests that the nucleus suffers habituation overtime. It is intesting to point out that these results do not agree with the proposal of Korosi et al. [58], who suggest that the EW Ucn1 system does not habituate [58], in contrast with the habituating response of the HPA axis. Nevertheless, again here it is important to emphasize that the authors based their suggestion on a study performed with mice and with predictable chronic stimuli. 5. Technical considerations
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The present study used c-Fos immunoreativity to investigate the responses of the PVN and EW to acute and chronic stress. Although c-Fos has also been used as a marker of neuronal activation in response to both types of stressors [26,34,40,59–63], some evidence suggests that Fos-B may be a better marker of neuronal activity, in particular in response to chronic stressors [64], forcFos-immunoreactity desensitizes overtime. If such is the case, one possibility that can be raised is that the induction of Fos immunoreactivity in the EW was desensitized in response to chronic stress. Here again, this proposition needs to be further investigated.
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Altogether, our results point to the possibility that CRF/Ucn1 neuronal circuits in the rat encompass two interconnected systems, which seem to coordinately respond to acute stressors, but are differently activated during chronic unpredictable stress. Our data suggest that before unpredictable chronic stress stimuli, which could (possibly) be interpreted as different acute stimuli, the CRF system remains crucially responsible for the coordination of the stress response. Nevertheless, further studies with different animals models and different types of acute and chronic stressors are still necessary to better investigate this proposition.
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Acknowledgments
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This study was financed by Fundac¸ão de Amparo à Pesquisa do Estado de Alagoas, Brazil, and by Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil.
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Contents lists available at ScienceDirect
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Research report
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CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress
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José S. de Andrade a , Milena B. Viana a , Renata O. Abrão a , Jackson C. Bittencourt b,c , Isabel C. Céspedes a,∗ a
Department of Biosciences, Federal University of São Paulo, Av. Ana Costa 95, UNIFESP, 11060-001 Santos, SP, Brazil Laboratory of Chemical Neuroanatomy, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, USP, 05508-000 São Paulo, SP, Brazil c Center of Neuroscience and Behavior, Institute of Psychology, University of São Paulo, Av. Prof. Mello Moraes, 1721, Bloco C, 05508-030, São Paulo, SP, Brazil b
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h i g h l i g h t s • • • •
PVN cells respond to both acute and chronic predictable stressors. EW cells are only activated in response to acute stressors. CRF/Ucn1 neuronal circuits respond coordinately to acute stressors. CRF/Ucn1 neuronal circuits are differently affected by chronic stress.
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a r t i c l e
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a b s t r a c t
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Article history: Received 11 May 2014 Received in revised form 6 June 2014 Accepted 8 June 2014 Available online xxx
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Keywords: CRF Urocortin 1 Paraventricular hypothalamic nucleus Edinger–Westphal nucleus Stress
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1. Introduction
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Corticotropin-releasing factor (CRF) acts to promote stress-like physiological and behavioral responses and is mainly expressed in the paraventricular hypothalamic nucleus (PVN). Urocortin 1 (Ucn1) is also a ligand to CRF type 1 and 2 receptors that has been associated with the stress response. Ucn1 neurons are primarily found in the Edinger–Westphal (EW) nucleus. It has been previously proposed that CRF and Ucn1 differently modulate stress responses to distinct types of stressors. The present study used male Wistar rats to compare the effects of acute restraint stress and unpredictable chronic stress (UCS) through Fos-immunoreactivity (Fos-ir) on CRF-containing neurons of PVN and Ucn1-containing EW centrally projecting neurons. Results showed that PVN neurons responded to both acute restraint and UCS. Also for the PVN, unspecific variables, dependent on the time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute restraint stress. Also, for this nucleus a significant difference was found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remain in the laboratory, interfere with the nucleus activation. These results suggest that CRF/Ucn1 neuronal circuits encompass two interconnected systems, which are coordinated to respond to acute stressors, but are differentially activated during chronic unpredictable stress. © 2014 Published by Elsevier B.V.
Physiological responses to stress encompass an efficient and conserved set of interconnecting systems, aimed to the maintenance of the organism’s integrity under challenging circumstances.
∗ Corresponding author. Tel.: +55 13 3878 3700; fax: +55 13 3223 2592. E-mail addresses: [email protected], [email protected] (I.C. Céspedes).
The autonomic nervous system provides an immediate reaction to stressor exposure – through its sympathetic and parasympathetic divisions – triggering rapid physiological changes through the innervation of end organs [1]. Activation of the hypothalamic–pituitary–adrenocortical (HPA) axis occurs in response to corticotropin-releasing factor (CRF) release from the paraventricular nucleus of the hypothalamus (PVN). Adrenocorticotropic hormone (ACTH) released from the anterior pituitary stimulates the adrenal cortex, resulting in the elevation of plasma circulating glucocorticoids levels [2,3].
http://dx.doi.org/10.1016/j.bbr.2014.06.014 0166-4328/© 2014 Published by Elsevier B.V.
Please cite this article in press as: de Andrade JS, et al. CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.06.014
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CRF, whose characterization and synthesis was performed by Vale et al. [4], seems to be an essential ingredient for the maintenance of an organism’s well being or homeostasis. Nowadays, this peptide, originally implicated in the activation of the HPA axis and known as the “stress hormone”, has also been considered for its role within the central nervous system [5,6] outside the HPA axis [7]. Since CRF was first characterized, a growing family of ligands and receptors has been identified. Aside from CRF, other family members include urocortin1 (Ucn1), Ucn2, and Ucn3, along with two receptors, CRF type 1 (CRFR1) and CRF type 2 (CRFR2), and a CRF binding protein. These family members differ in their tissue distribution and pharmacology. Although, the main sources of CRF are the parvocellular neurons of the PVN, the most abundant expression of urocortin 1 (Ucn1) in mammals has been reported in neurons located in the midbrain in the Edinger–Westphal nucleus (EW) [8–12]. The EW is a small, cholinergic, preganglionic parasympathetic structure, critically involved in oculomotor adaptation [13,14]. It has been proposed that the structure and functions of the EW are more complex than what was originally thought [11,13,15–18]. Thus, to distinguish EW cells that exhibit choline acetyltransferase immunopositivity and project to the ciliary ganglion from those that are positive for Ucn1, but do not innervate the ciliary ganglion, the terminology of non-preganglionic or centrally projecting EW has been introduced [8,11,12,15,19–21]. The later will be the subject of investigation of the present work. Although CRF appears to play a stimulatory role in stress responsivity through activation of CRFR1, the involvement of CRFR2 with stress and anxiety is still a matter of debate [22–25]. Taking this last observation into account and since Ucn1 is the only ligand with high affinity for both CRFR1 and 2, its role in the mediation of stressrelated responses may differ, depending on the type and intensity of the stressor. In a previous study, we employed Fos immunoreactivity (Fos-ir) and in situ hybridization of mRNA of CRF and Ucn1 to investigate how two different acute psychogenic stressors, restraint and footshock, activated the PVN and the EW [26]. The results of this study showed that restraint increased both Fos-ir and CRF mRNA expression in the PVN, while in the EW Fos-ir was higher in the footshock group and Ucn1 mRNA expression was higher in the restraint group. These results suggest that these two nuclei respond differently and in a complex manner to distinct types of acute stressors. Different roles for the PVN and EW have also been proposed when acute and chronic stressors are compared. Thus, it has been suggested that while the PVN has a fast response to acute stimuli, with Fos peaking within an hour [27,28], the EW responds slower to these two different kind of stressors, with Fos-ir peaking only 4 h after stress initiation [29,30]. In line with this notion it was hypothesized that CRF in the PVN, acting via CRFR1, plays a major role in the stress response initiation, whereas Ucn1 in the EW would be activated later [31–33]. Nevertheless, this proposition needs to be better investigated through controlled experimental studies that simultaneously compare both types of stressors. Taking the above into account, this study aims to compare the response to acute restraint stress and unpredictable chronic stress (UCS) of two nuclei that represent the major sources of CRF (PVN) and Ucn1 (EW), thus contributing to a better understanding of the controversial role of both peptides on stress regulation. Fos-ir was used to map the activation of the PVN and EW in animals submitted to either acute restraint or UCS. As formerly noted, the product of the immediate-early gene c-fos is expressed throughout the brain in response to a variety of tasks, thus making it a powerful instrument to study intracellular responses of neurons to different stimuli [34]. The UCS model presents good face validity and has been broadly used to investigate some of the physiological and behavioral
consequences of chronic stress [35–39]. Briefly, in this test, rodents are exposed to a variety of stressors (i.e., restriction, inversion of the light–dark cycles, water/food deprivation, damp sawdust) intermittently, usually for two to three weeks [35–39]. Immediately after the stress procedure, animals were weighted. Independent groups of animals were subjected to measurements of plasma corticosterone after acute restraint or UCS.
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2. Materials and methods
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2.1. Animals
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Male Wistar rats, weighing 250–300 g (CEDEME, Universidade Federal de São Paulo, Campus Santos, Brazil) were housed in groups of 4–6 per cage (50 cm × 60 cm × 22 cm). Room temperature was maintained at 22 ± 1 ◦ C with lights on from 0700 to 1900 h. Food and water were freely available throughout the experiments. UCS animals were housed under the same conditions except during the periods they were exposed to some of the stressors (i.e. food and water restriction/deprivation, inversion of the light–dark cycle). The study was approved by the Ethical Committee for Animal Research of the Federal University of São Paulo and was performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior, which are based on the conditions stated by the “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996). 2.2. Procedure The animals were organized in four groups with 6 individuals each: control (group 1) versus acute restraint stress and control (group 2) versus UCS. Acute restraint: two days after their arrival to the laboratory, animals were individually placed for a period of 30 min in acrylic restraining cylinders (6.3 cm × 6.5 cm × 23.2 cm) adjustable to the size of the animal and with holes on the sides to provide ventilation. The restraining procedure took place during the morning period (from 7:00–9:00 a.m.) in an experimental room dimly lit. Control animals (group 1) were left undisturbed in Plexiglas cages, in the same experimental room and for the same amount of time (30 min). At 1 h after the stress procedure animals were sacrificed. This is the time required to achieve the peak of Fos-ir in activated neurons [26]. UCS: the protocol was performed as described previously [40, see Table 1]. Rats were subjected to different kinds of stressors, which varied from day to day, for 14 consecutive days. There were a total of seven stressors: (1) periods of 1 h of restraint in a acrylic restraining cylinders (7.0 cm × 7.5 cm × 16 cm); (2) lights on overnight; (3) food deprivation overnight, followed by 2 h access to restricted food (resulting from the scattering of pellets of 45 mg in the cage); (4) water deprivation overnight, followed by 1 h contact with an empty water bottle; (5) food restriction for 2 h; (6) damp sawdust overnight; (7) inversion of the light–dark cycle from Friday night to Monday morning. Unstressed animals (control group 2) remained for 14 days in the laboratory, under standard housing conditions. Animals were sacrificed 2 h after the last stimulus (see Table 1, Monday morning, around 9:00 a.m.), so that the peak of Fos-ir corresponding to the last stimulus would not hide the effect of the chronic treatment [40]. 2.3. Fos protein immunohistochemistry (Fos-ir) After each treatment and in the period described above, the animals were anesthetized with ketamine/xylazine 2:1 (1 ml/kg) and
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Table 1 Unpredictable chronic stress (UCS) protocol. Period
Monday
Tuesday
Wednesday
Thursday
Friday
Morning
Restraint (30 min)
Restraint (30 min)
Food restricition for 2 h
Restraint (30 min)
Afternoon Night
Restraint (30 min) Lights on during the night
Restraint (30 min) Water/fooddeprivation overnight
Restraint (30 min) Water deprivation overnight
Empty water bottle (60 min); Restraint (30 min) Restraint (30 min) Damp sawdust overnight
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perfused with ≈100 ml of 0.9% saline for approximately 1 min, followed by 500–700 ml of 4% formaldehyde (from paraformaldehyde heated to 60–65 ◦ C) and H2 O at 4 ◦ C, pH 9.5, for approximately 25 min. The brains were post-fixed for 1 h in the same fixative solution, and then stored in a solution containing 20% sucrose for cryoprotection at 4 ◦ C. Regularly spaced series (5 × 1-in-5) of 30 m-thick frozen sections were cut in the coronal plane, collected in ethylene glycol-based cryoprotectant solution and stored at −20 ◦ C for later determination of Fos-ir. Fos-ir cells were identified by using a polyclonal anti-serum raised in rabbit against synthetic human Fos (anti-Fos – 1:20,000; Oncogene, Cambridge, MA, USA) in a solution containing KPBS loaded and goat serum. Immunohistochemistry was performed using a conventional avidin–biotin immunoperoxidase protocol [41] and Vectastain Elite reagents (Vector Laboratories, Burlingame, CA, USA). Tissue was pretreated with hydrogen peroxide (0.3%; Sigma, St. Louis, MO, USA) diluted in KPBS before addition of the primary antibody to quench endogenous peroxidase activity in the tissue. The reaction with diaminobenzidine (DAB) (0.05%; Sigma) was amplified using nickel ammoniumsulfate, both diluted in acetate buffer solution. The sections were then mounted on gelatin-coated slides, allowed to dry for approximately 18 h and counterstained with 0.25% thionin in order to visualize the labeled cells within the borders of each nucleus. We quantified Fos-ir cells in sections, having as reference the following AP coordinates [42] bregma: PVN: −1.80 mm and EW: −5.88 mm, under bright-field illumination using the ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA). One slice per brain region was analyzed.
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2.4. Corticosterone measurements
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The animals submitted to acute stress (N = 5) were euthanized by decapitation immediately after the stressor. The animals submitted to UCS (N = 5) were euthanized by decapitation 2 h later the last stimulus. Trunk blood was collected (from 08:00–11:00 a.m.) and centrifuged for 20 min at 4000 rpm, at 4 ◦ C. Aliquots of serum were then removed and stored at −20 ◦ C until assayed. Serum corticosterone concentrations were determined by enzyme immunoassay (ELISA) using a commercial kit (Assay Designs, Inc., Ann Arbor, USA). Unstressed animals (N = 5) were left undisturbed under standard laboratory conditions, for 3 (acute control) or 14 (chronic control) days and also euthanized by decaptiation and their trunk blood collected (from 09:00–11:00 a.m.) and analyzed as described for stressed animals.
2.5. Statistical analysis A two-factor ANOVA was used to analyze Fos-ir neurons (factor 1: stress protocol, restraint or UCS; factor 2: absence or presence of stress). In case of significant results, groups were analyzed by one-way ANOVA followed by the Tukey post-hoc test. Weight and corticosterone measurements were analyzed by independent Ttest (control versus acute restraint; control versus UCS). Values of P ≤ 0.05 were considered significant.
Inversionofthe light–dark cycle over the weekend
Table 2 Weight (mean ± SEM) in control and stressed animals, measured at the end of the experiments. Experimental group
Weight
Acute control Restraint Chronic control UCS
296.67 292.50 306.67 310.00
± ± ± ±
2.79 3.09 2.11 3.65
3. Results
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All animals arrived at the laboratory weighting between 260 and 280 g. Table 2 shows the weight of the different groups at the end of the stress procedure. There were no significant changes in body weight between control and acute restraint animals (T(10) = 1.0; P = 0.341), or between control and UCS animals (T(10) = −0.791; P = 0.448). 3.2. Fos-immunoreativity (Fos-ir) The results obtained with the analyses of Fos-ir in the studied brain regions, PVN and EW, in animals submitted to acute or UCS are summarized in Table 3. 3.3. Paraventricular nucleus of the hypothalamus (PVN) For the PVN, two-factor ANOVA showed a significant effect of factor 1 (restraint or UCS) F(1,20) = 56.36; P < 0.001), of factor 2 (absence or presence of stress) F(1, 20) = 260.18; P < 0.001) and of the interaction between factors (F(1,20) = 102.92; P < 0.001). One-way ANOVA showed a significant difference between groups (F(3,23) = 139.82; P < 0.001) (Fig. 1). The Tukey post-hoc test showed significant differences between all the groups tested (P < 0.05), but no significant differences between the two groups of control animals, acute and chronic (P > 0.05), suggesting that a short or a prolonged period of time spent in the laboratory does not seem to be an important factor involved with the activation of this nucleus. 3.4. Edinger–Westphal nucleus (EW) For the EW, two-factor ANOVA showed a significant effect of factor 1 (restraint or UCS) F(1,20) = 158.90; P < 0.001), of factor 2 (absence or presence of stress) F(1, 20) = 57.93; P < 0.001) and of the interaction between factors (F(1,20) = 26.56; P < 0.001). One-way ANOVA also showed a significant difference between groups for the EW (F(3,23) = 81.13; P < 0.001) (Fig. 2). The Tukey post-hoc test showed that the two groups of control animals, acute and chronic, differ significantly (P < 0.001) – an effect possibly related to habituation to the laboratory conditions –, that both group of control animals were also significantly different from acute restraint animals (P < 0.001), and that acute control animals were significantly different from UCS animals (P = 0.010). Nevertheless, there were no
Please cite this article in press as: de Andrade JS, et al. CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.06.014
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Fig. 1. Photomicrographs of Fos immunoreactive cells (dark spots) in coronal sections of the paraventricular hypothalamic nucleus (PVN) of animals submitted to acute restraint (upper panel) or unpredictable chronic stress-UCS (lower panel). Scale bar: 300 m; Magnification, ×200. (*) third ventricle.
Fig. 2. Photomicrographs of Fos immunoreactive cells (dark spots) in coronal sections of the Edinger–Westphal (EW) nucleus of animals submitted to acute restraint (upper panel) or unpredictable chronic stress-UCS (lower panel). Scale bar: 300 m; Magnification, ×200. (*) medial longitudinal fasciculus.
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Table 3 Mean ± SEM of Fos immunoreactive cells in the paraventricular hypothalamic nucleus (PVN) and Edinger–Westphal nucleus of unstressed animals (control) and animals submitted to acute restrain to run predictable chronic stress (UCS). Structure
Control (group 1)
Acute restraint
Control (group 2)
UCS
PVN EW
9.00 ± 0.93 25.33 ± 2.86
138.50 ± 7.26* 52.17 ± 2.85*
12.00 ± 3.71 9.67 ± 0.84#
51.50 ± 5.47* , + 14.83 ± 0.83#
* + #
P < 0.05, with respect to all the other groups (group 1). P < 0.05, with respect to control (group 2). P < 0.05, with respect to control (group 1) and acute restraint (ANOVA, followed by the Tukey test).
Table 4 Corticosterone measurements (mean ± SEM) in control and stressed animals.
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Experimental group
Corticosterone measurements (ng/ml)
Acute control Restraint Chronic control UCS
226.60 513.80 221.60 669.61
± ± ± ±
38.10 45.50 24.82 99.04
significant differences between chronic control animals and UCS animais (P > 0.05), suggesting that the EW is not crucial for the organization of responses to chronic stress. 3.5. Corticosterone measurements
Measurements (mean ± SEM) of serum corticosterone levels are shown in Table 3. Independent Student t-test indicated a sig266 267 nificant difference between acute control and restraint animals 268 (T(6.15) = −4.53; P < 0.001). Also, there was a significant difference 269 Q2 between chronic control and UCS animals (T(7) = −4.90; P < 0.01] 270 (Table 4). 265
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4. Discussion The results of the present study showed that PVN neurons responded to both acute and chronic stress. Also, for the PVN unespecific variables, such as the period of time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute stress. Nevertheless, for this nucleus a significant difference was also found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remained in the laboratory (i.e., a short versus a prolonged period of handling), may interfere with the nucleus activation. Measurements of corticosterone levels showed that both stress protocols induced significant increases in serum corticosterone. The central role of the PVN in HPA activation has been well documented by several previous studies performed with exposure to acute stressors [for a review see 43]. For instance, Viau and Sawchenko [28] have demonstrated increased Fos-ir in CRF cells in the PVN after a single 30 min episode of acute restraint that is slightly decreased in animals killed 2 h after the stress protocol. Similar results were also found by Lund et al. [44]. Corroborating these observations, it was also previously shown by our research group that PVN neurons are significantly activated by both acute restraint and acute footshock [26]. The conclusions obtained with Fos-ir are also supported by studies that show rapid phosphorylation of cyclic AMP response element binding protein (CREB) and mitogen activated protein (MAP) kinase in the PVN following acute stressors [45,46]. Acute stress has been shown to induce transcription of CRF and vasopressin genes in the parvocellular PVN, and subsequently increase CRF and vasopressin mRNA [46–48]. Moreover, acute stress exposure also results in increases in portal blood levels of CRF, vasopressin and oxytocin [49–51].
Previous investigations have also shown that the PVN responds effectively to chronic stress exposure. A variety of chronic stress paradigms, such as chronic footshock, immobilization, social subordination, social defeat and also UCS have been shown to cause increases in CRF mRNA expression within the PVN [47,52–54]. In fact, across multiple rat strains, CRF mRNA increases induced by repeated immobilization is observed [55], thus showing that PVN activation is a consistent result of chronic stress. Furthermore, it has been previously shown that chronic stress increases PVN Fos-ir as well as the number of glutamatergic and noradrenergic immunoreactive terminals within the parvocellular PVN [56]. The number of GABAergic-immunoreactive boutons in the PVN, in this same study, was unchanged. Taken together, these data are consistent with a role for both glutamate and norepinephrine in chronic stress enhancement of HPA axis excitability [56]. Nevertheless, it is also important to point out that although the PVN was significantly activated by chronic stress, a greater increase in Fos-ir within the nucleus was observed after acute stress. This observation seems to corroborate the idea that Fos protein expression in CRF cells in the PVN habituate over a prolonged period of time [28], what might also explain why some studies with different chronic stress paradigms have not reported increases in PVN activation [43]. Interestingly, there were no significant differences in PVN Fos-ir in control animals that remained for two or fourteen days in the laboratory. This suggests that unspecific variables (i.e., a short versus a prolonged period of handling) do not seem to be critical factors involved in the activation of this nucleus. In other words, that the important stimuli that activated the nucleus were in fact the ones related to the stressful procedure. While the PVN was activated by both acute and chronic stress, the EW showed a significant activation only after acute restraint stress. In a previous study, we have shown that two different acute stress protocols, restraint and footshock, induced Fos-ir in the EW, although the results obtained with footshock were more pronounced [26]. On the other hand, UCN1 mRNA expression was higher after acute restraint [26]. Several other studies also pointed to the same direction. For instance, it was previously shown that acute pain stress resulted in the activation of urocortinimmunoreactive neurons in the EW, peaking at 4 h after acute pain stress [29]. Also, Weninger et al. [30] showed that acute restraint stress and lipopolysaccharide administration upregulate Ucn1 mRNA in a mouse model. The finding that acute restraint stress stimulated the production of Ucn1 in EW neurons, suggests a role for these neurons in the stress response in the mouse as well, a notion strengthened by the fact that in addition to Ucn1, two other peptides within this same neuronal group, cocaine- and amphetamine-regulated transcript peptide (CART) and nesfatin-1, were upregulated [57]. Contrarily to our results, however, it was also previously shown that chronic predictable/homotypic stress (3 weeks of exposure to ether vapor) activates mouse EW Ucn1 neurons [58]. It is interesting to observe that, in this particular study [58], Fos-ir in EW cells was also more robust after exposure to acute ether stress, an observation that goes in the same direction of our results. On the other hand, regarding the response of the EW to chronic stress,
Please cite this article in press as: de Andrade JS, et al. CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.06.014
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two hypotheses may be raised: (1) the nucleus plays different roles depending on the species investigated (i.e., if mouse or rat); (2) the nucleus plays different roles depending on whether the stressors are predictable or unpredictable. Nevertheless, both propositions warrent further investigation. Also differing from what was observed for the PVN, there were significant differences in EW Fos-ir between control animals that remained for two or fourteen days in the laboratory. This observation suggests that the nucleus suffers habituation overtime. It is intesting to point out that these results do not agree with the proposal of Korosi et al. [58], who suggest that the EW Ucn1 system does not habituate [58], in contrast with the habituating response of the HPA axis. Nevertheless, again here it is important to emphasize that the authors based their suggestion on a study performed with mice and with predictable chronic stimuli. 5. Technical considerations
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The present study used c-Fos immunoreativity to investigate the responses of the PVN and EW to acute and chronic stress. Although c-Fos has also been used as a marker of neuronal activation in response to both types of stressors [26,34,40,59–63], some evidence suggests that Fos-B may be a better marker of neuronal activity, in particular in response to chronic stressors [64], forcFos-immunoreactity desensitizes overtime. If such is the case, one possibility that can be raised is that the induction of Fos immunoreactivity in the EW was desensitized in response to chronic stress. Here again, this proposition needs to be further investigated.
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6. Conclusions
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Altogether, our results point to the possibility that CRF/Ucn1 neuronal circuits in the rat encompass two interconnected systems, which seem to coordinately respond to acute stressors, but are differently activated during chronic unpredictable stress. Our data suggest that before unpredictable chronic stress stimuli, which could (possibly) be interpreted as different acute stimuli, the CRF system remains crucially responsible for the coordination of the stress response. Nevertheless, further studies with different animals models and different types of acute and chronic stressors are still necessary to better investigate this proposition.
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Acknowledgments
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This study was financed by Fundac¸ão de Amparo à Pesquisa do Estado de Alagoas, Brazil, and by Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil.
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