Behavioural Brain Research 267 (2014) 46–54

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Chronic corticosterone administration facilitates aversive memory retrieval and increases GR/NOS immunoreactivity Thays B. Santos, Isabel C. Céspedes, Milena B. Viana ∗ Departamento de Biociências, Universidade Federal de São Paulo, 11060-001 Santos, Brazil

h i g h l i g h t s • • • •

Chronic corticosterone improves emotional aversive learning. Chronic corticosterone increases GR immunoreactivity. Chronic corticosterone increases nNOS immunoreactivity. Chronic corticosterone increases GR/nNOS double immunostaining.

a r t i c l e

i n f o

Article history: Received 7 February 2014 Received in revised form 7 March 2014 Accepted 13 March 2014 Available online 21 March 2014 Keywords: Corticosterone Emotional aversive conditioning Inhibitory avoidance Glucocorticoid receptors Nitric oxide

a b s t r a c t Glucocorticoids are stress hormones that mediate the organism’s reaction to stress. It has been previously proposed that the facilitation of emotional aversive conditioning induced by these hormones may involve nitric oxide-pathways. The purpose of the present study was to address this question. For that, male Wistar rats were surgically implanted with slow-release corticosterone (CORT) pellets (21 days) and tested in a step-down inhibitory avoidance task. Additional groups of animals were also submitted to the same treatment conditions and on the 21st day of treatment assayed for GR (glucocorticoid receptors)nNOS (neuronal nitric oxide synthase) immunoreactivity (GRi-nNOSi) or measurements of plasma CORT. Results showed that CORT treatment induced facilitation of step-down inhibitory avoidance. This same treatment also significantly increased CORT plasma levels and GRi in the medial, basolateral and basomedial amygdala, in the paraventricular hypothalamic nucleus (PVN), in the ventral and dorsal dentate gyrus, in the ventral CA1 region and in the dorsal CA1 and CA3 regions. Furthermore, nNOSi and GRi-nNOSi were significantly increased by CORT treatment in the medial amygdala and basolateral amygdaloid complex, in the PVN, subiculum, in the dorsal CA3 region and in the ventral CA1 and CA3 regions. These results indicate that the facilitation of aversive conditioning induced by CORT involves GR-nNOS pathways activation, what may be of relevance for a better understanding of stress-related psychiatric conditions. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Stress can be described as any challenge to the homeostatic balance that demands an adaptive reaction of the subject [1]. One of the main physiological alterations that follows the stress response, is the activation of the hypothalamic–pituitary–adrenal (HPA) axis [2]. Activation of the HPA axis is controlled by the release of corticotropin-releasing factor (CRF) and arginine–vasopressin (AVP) from the paraventricular hypothalamic nucleus (PVN). CRF

∗ Corresponding author at: Av. Ana Costa, 95, 11060-001 Santos, SP, Brazil. Tel.: +55 13 33851535; fax: +55 13 33851535. E-mail address: [email protected] (M.B. Viana). http://dx.doi.org/10.1016/j.bbr.2014.03.020 0166-4328/© 2014 Elsevier B.V. All rights reserved.

and AVP stimulate adrenocorticotropic hormone (ACTH) release, which in turn triggers the release of glucocorticoids (corticosterone and cortisol) from the adrenal cortex. Glucocorticoids are, therefore, hormones involved in the adaptation of the organism to stressors. After being released, these so-called stress hormones can enter the brain and interact with receptors expressed in regions that mediate HPA axis activity and the organism’s responses to stress. There are two types of glucocorticoid receptors: mineralocorticoid (MRs) and glucocorticoid receptors (GRs). Glucocorticoids possess 6-10 times more affinity to MRs [3]. Thus, while MRs seem to mediate the effects of circadian levels of glucocorticoids, GRs are the main receptors activated by glucocorticoids in response to stress or to severe variations in glucocorticoids levels [3].

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GRs are highly concentrated in the pre-frontal cortex, hippocampus and amygdala [3], brain regions involved with emotional conditioning. In fact, it has previously been shown that treatment with glucocorticoids, aside from exerting anxiogenic effects, improves learning and memory associated with aversive stimuli [4–9]. In the last years, a role for nitric oxide (NO) in the modulation, in particular, of emotional aversive conditioning, has also been proposed [10–12]. NO is produced by neuronal nitric oxide synthase (nNOS), in response to the activation of NMDA receptors by glutamate [13,14] and functions as a retrograde neuronal messenger, facilitating synaptic plasticity, long-term potentiation (LTP) and the formation of long-term memories. It has been shown that the pharmacological blockade of NO signaling in rats impairs contextual [11] and cued [10] fear learning. Also, knockout mice with targeted mutation of the nNOS gene showed severe impairment of contextual fear learning [12]. Interestingly, in this same study, an association between increases in plasma corticosterone levels and in the magnitude of contextual freezing responses was also observed. In fact, previous evidences indicate that glucocorticoid hormones may increase glutamate-mediated neurotransmission and alter NO synthesis/release in the Central Nervous System [15,16]. Also, moderate to high levels of nNOS have been found in regions related to the modulation of fear/anxiety [17], such as the hippocampus and the amygdala. Furthermore, stress-exposure seems to induce the activation of NO neurons in the amygdala, hypothalamus, and periaqueductal grey matter [18], structures that integrate the so-called “Brain Aversive System” [19]. There is also evidence that NO affects the activity of the HPA axis [20,21], exerting both stimulatory and inhibitory effects depending on the brain area involved and the type of stress [for a review, see 21]. Nevertheless, to the best of our knowledge the relationship between NO-pathways activation in different brain regions and glucocorticoid-induced improvement of aversive conditioning has not been thoroughly investigated. The purpose of the present study was to investigate the effect of chronic corticosterone (CORT) treatment (21 days, slow-release pellets) on memory retrieval and GR-nNOS immunoreactivity (GRi-nNOSi) in the amygdala, hippocampus and PVN. In a previous study we have shown that this same treatment procedure induces facilitation of the avoidance task of the elevated T-maze model of anxiety, an anxiogenic effect [9]. For behavioral measurements, a step-down inhibitory avoidance task was chosen. It has been previously shown that with this task, aversive conditioning is rapidly acquired [22,23]. Also, the model has been well studied, in terms of its behavioral and biochemical basis [24–26]. In addition, an independent group of animals was subjected to measurements of plasma CORT on the 21st day of treatment.

2. Materials and methods 2.1. Subjects Male Wistar rats (CEDEME, Universidade Federal de São Paulo, Brazil), weighing 280–320 g at the beginning of the experiment, were housed in groups of 5–6 per cage. After surgery, animals were housed in pairs in Plexiglas-walled cages until testing. Room temperature was controlled (22 ± 1 ◦ C) and a light–dark cycle was maintained on a 12-h on–off cycle (07:00–19:00 h lights on). Food and water were available all throughout the experiments. The study was approved by the Ethical Committee for Animal Research of the Federal University of São Paulo (number 0064/12) and was performed in compliance with the recommendations of the

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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. Apparatus – step-down inhibitory avoidance The apparatus was a 40 cm × 25 cm × 20 cm box with a 5-cm high, 8-cm wide and a 25-cm long platform on the left end of a grid composed of a series of parallel 0.1 cm caliber stainless steel bars spaced 1.0 cm apart. Luminosity at the center of box was 60 lux. After each experimental session, the apparatus was cleaned with a 10% ethanol solution. 2.3. Surgery One day after their arrival, rats were anaesthetized with an IP injection of ketamine hydrochloride (80 mg/kg; Agribrands, Brazil) and xylazine (10 mg/kg; Agribrands, Brazil). A 1-cm incision through the skin of the upper back of the animal was made and a 2-cm subcutaneous pocket was open horizontally with a pair of forceps to allow the implant of a slow-release CORT pellet (200 mg, 21-day release, Innovative Research of America, USA – this procedure induces the daily release of approximately 10 mg/kg of CORT). The pellet was inserted into the pocket and the incision sutured. Sham rats were subjected to the same surgical procedure except that a pellet was not implanted. To prevent infections, at the end of the surgery, all animals were injected (IM) with 0.2 ml of a pentabiotic preparation (Pentabiotico Veterinário Pequeno Porte; Forte Dodge, Brazil). 2.4. Procedure Training was performed on the 20th day of treatment. For that, animals (N = 8–9) were placed on the platform of the step-down inhibitory avoidance apparatus and the latency to step down placing the four paws on the grid was measured. In training sessions, after stepping down onto the grid, animals received a 2-s, 0.8-mA footshock. This procedure induced an escape response back to the platform. Animals that did not remain for at least 50 s on the platform after shock were excluded. Twenty-four hours later animals were tested. In test sessions, no footshock was given and the stepdown latency was cut off at 300 s. 2.4.1. Double staining immunohistochemistry: GR and nNOS On the 21st day of treatment, independent groups of CORT and sham animals (N = 6) were anesthetized with ketamine/xylazine as described above and perfused with 100 ml of 0.9% saline for 1 min, followed by 500–700 ml of 4% paraformaldehyde 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 at 4 ◦ C and stored in phosphate buffered saline (PBS) plus 20% sucrose, at 4 ◦ C. Regularly spaced series (5 × 1-in-5) of 30 ␮m-thick frozen sections were cut in the frontal plane, collected in ethylene glycol-based cryoprotectant solution and stored at −20 ◦ C for later determination of double staining. For identification of neurons with immunoreactivity for the GR receptor (GR-ir) polyclonal antibody raised in rabbits against GR (anti-GR, 1:1000; Abcam, Cambridge, United Kingdom) was used. Immunohistochemistry was performed using a conventional avidin–biotin immunoperoxidase protocol [27] and Vectastain Elite reagents (Vector Laboratories, USA). Tissue was pretreated with hydrogen peroxide (0.3%; Sigma, USA) before addition of the primary antibody to squelch endogenous peroxidase activity. The reaction with diaminobenzidine-DAB (0.05%; Sigma, USA) was performed for 40 s and was amplified using nickel ammonium sulfate,

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resulting in black coloring localized in the nucleus. In the same sections and after GR-ir revelation, for identification of neurons with immunoreactivity for nNOS enzyme (nNOS-ir) polyclonal antibody raised in rabbits against the nNOS enzyme (anti-nNOS, 1:200; Abcam, Cambridge, United Kingdom) was used. The immunohistochemistry was performed using the same avidin–biotin immunoperoxidase protocol, Vectastain Elite reagents and the pretreatment with hydrogen peroxide before addition of the primary antibody. Again, the reaction with diaminobenzidine-DAB (0.05%; Sigma, USA) was performed for 40 s but without nickel ammonium sulfate, resulting in a brown coloring localized in the cell’s cytoplasm. The co-localization of the two stainings in the same neuron was considered a double staining. The sections were then mounted on gelatin-coated slides with DPX mounting medium. The PVN, amygdala and the hippocampus were analyzed. As mentioned, these three brain regions have been shown to possess both GR and nNOS-producing neurons [3,17,18]. We quantified GR-ir, NOS-ir and double staining neurons under bright-field illumination using the Image-Pro Plus software (Media Cybernetics, USA). The following bregmas were used for quantification purposes, having as reference the AP coordinates from the Paxinos and Watson Rat Brain Atlas [28]: −1.44 to −1.72 (PVN); −3.14 to −3.2 (amygdala, dorsal CA 1 and 3 and dorsal dentate gyrus); −5.4 to −5.76 (ventral dentate gyrus, ventral CA1 and 3 and subiculum). Sections were analyzed by an observer blind to the treatment conditions. Cells considered GR, nNOS or double staining positive were counted using constant minimum and maximum OD and object size criteria, which were validated with visual counts. 2.5. CORT measurements An independent group of animals (N = 7–8) was euthanized by decapitation on the 21st day of treatment. Trunk blood was collected (from 8:00 to 10:00 a.m.) in ice cold EDTA-containing tubes and centrifuged for 20 min at 4000 rpm, at 4 ◦ C. Aliquots of plasma were then removed and stored at −20 ◦ C until assayed. Plasma corticosterone concentrations were determined by enzyme immunoassay (ELISA, IB79112) using a commercial kit (Assay Designs, Inc., Ann Arbor, USA). Detection lower and upper limits ranged from 0 to 240 nmol/l; within assay variability 79.17 nmol (mean) (%CV 2.44); inter assay variability 74.83 nmol/l (mean) (%CV 6.35).

Fig. 1. Facilitation of step-down inhibitory avoidance induced by chronic treatment with corticosterone. N = 8 (sham) and 9 (corticosterone). P < 0.05, unpaired Student’s t-test.

2.6. Statistical analysis Unpaired Student’s t-test was used to analyze behavioral, immunohistochemistry data and CORT measurements. A value of P ≤ 0.05 was considered significant. 3. Results 3.1. Behavioral results Fig. 1 shows the effects of CORT treatment on step-down inhibitory avoidance measurements. Unpaired Student’s t-test showed that CORT-treated animals presented longer step-down latencies on the test day when compared to sham animals: (T(15) = −2.13; P = 0.05). 3.2. Immunohistochemistry results Table 1 shows the effects of CORT treatment on GRi, nNOSi and GRi/nNOSi. With respect to GRi, unpaired Student’s ttest showed significant differences in the medial (T(10) = −4.78; P = 0.001) (Fig. 2A), basolateral (T(11) = −3.35; P = 0.006) (Fig. 2B) and basomedial amygdala (T(6.01) = −3.70; P = 0.010) (Fig. 2C), PVN (T(5.23) = −4.36; P = 0.007) (Fig. 3A), ventral dentate gyrus (T(12) = −3.62; P = 0.003) (Fig. 4A), ventral CA1 (T(13) = −2.93; P = 0.012) (Fig. 4B), dorsal dentate gyrus (T(11) = −6.87; P < 0.001)

Table 1 Glucocorticoid receptors and neuronal nitric oxide synthase immunoreactivity (mean ± SEM) in brain regions of sham animals and of animals chronically treated with corticosterone. Brain regions

GRi

nNOSi

Sham Medial amygdala Central amygdala Lateral amygdala Basolateral amygdala Basomedial amygdala PVN Dorsal DG Dorsal CA1 Dorsal CA3 Subiculum Ventral DG Ventral CA1 Ventral CA3

29.2 176.0 19.7 8.5 0.2 50.7 17.8 31.8 1.8 7.1 17.0 5.2 10.4

± ± ± ± ± ± ± ± ± ± ± ± ±

16.2 30.0 14.5 4.2 0.2 5.5 3.5 4.0 0.5 4.9 9.1 2.0 2.3

CORT

Sham

125.7 ± 12.1* 244.9 ± 61.5 27.6 ± 8.9 33.7 ± 5.9* 21.6 ± 5.8* 115.4 ± 13.8* 70.4 ± 5.6* 77.0 ± 16.0* 19.9 ± 2.2* 16.2 ± 4.1 69.3 ± 10.4* 15.6 ± 2.6* 17.0 ± 3.5

2.8 ± 1.3 ± 2.8 ± 5.8 ± 0 25.2 ± 22.6 ± 21.3 ± 5.2 ± 6.3 ± 13.7 ± 7.3 ± 8.8 ±

GR/nNOSi CORT 1.8 0.6 0.9 1.8 3.7 3.0 2.9 1.4 3.1 3.6 1.9 1.9

75.2 4.3 15.9 24.1 19.0 82.8 26.4 23.4 14.4 39.5 16.4 16.7 21.6

± ± ± ± ± ± ± ± ± ± ± ± ±

Sham 19.3* 1.4 3.7* 4.8* 2.5* 1.4* 4.0 2.1 2.8* 8.5* 1.4 3.1* 1.8*

1.2 ± 1.0 ± 1.8 ± 2.5 ± 0 10.2 ± 12.8 ± 13.8 ± 1.0 ± 0.6 ± 6.3 ± 2.7 ± 4.6 ±

CORT 1.0 0.5 0.8 1.7 1.3 1.4 2.4 0.4 0.6 2.1 0.9 1.1

46.7 1.9 10.5 17.29 11.6 46.4 13.4 17.1 9.4 13.0 9.9 9.9 11.1

± ± ± ± ± ± ± ± ± ± ± ± ±

13.8* 1.0 2.3* 4.4* 2.3* 2.1* 1.6 2.0 2.1* 4.4* 1.2 2.4* 1.4*

PVN, paraventricular hypothalamic nucleus; DG, dentate gyrus; CA, cornus Ammon; GRi, glucocorticoid immunoreactivity; NOSi, neuronal nitric oxide immunoreactivity; CORT, corticosterone. * P < 0.05, with respect to sham animals in the same group (GRi, nNOSi or GR/nNOSi; Student’s t-test).

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Fig. 2. Photomicrographs of GRi/nNOS immunostaining in coronal sections of different brain regions of sham and corticosterone treated animals (N = 6). (A) Medial amygdala, (B) basolateral amygdala, (C) basomedial amygdala. GR-ir neurons: black coloring localized in the nucleus (arrowhead); nNOS-ir neurons: brown coloring localized in the cytoplasm (small arrow); double staining (large arrow). *Optic tract, **external capsule. Scale bars: 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Fig. 5A), dorsal CA1 (T(11) = −2.54; P = 0.027) (Fig. 5B) and dorsal CA3 (T(7.65) = −7.98; P < 0.001) (Fig. 6A). With respect to nNOSi, unpaired Student’s t-test showed significant differences in the medial (T(5.06) = −3.75; P = 0.013) (Fig. 2A), lateral (T(7.85) = −3.46; P = 0.009) (Fig. 3B), basolateral (T(11) = −3.32; P = 0.007) (Fig. 2B) and basomedial amygdala (T(11) = −7.02; P < 0.001) (Fig. 2 C), PVN (T(6.34) = −14.51; P < 0.001) (Fig. 3A), ventral CA1 (T(13) = −2.27; P = 0.041) (Fig. 4B), ventral CA3 (T(11) = −4.67; P = 0.001) (Fig. 4C), dorsal CA3 (T(12) = −2.70; P = 0.019) (Fig. 6A) and subiculum (T(6.31) = −3.66; P = 0.010) (Fig. 6B). Unpaired Student’s t-test also showed significant differences between sham and CORT animals with respect to GRi/nNOSi in the medial (T(6.06) = −3.30; P = 0.016) (Fig. 2A), lateral (T(8.39) = −3.53; P = 0.007) (Fig. 3B), basolateral (T(11) = −2.91; P = 0.014) (Fig. 2B) and basomedial amygdala (T(11) = −4.71; P = 0.001) (Fig. 2C), PVN (T(6.73) = −14.68; P < 0.001) (Fig. 3A), ventral CA1 (T(8,72) = −2,77; P = 0.023) (Fig. 4B), ventral CA3 (T(11) = −3.33; P = 0.007) (Fig. 4C), dorsal CA3 (T(12) = −3.41;

P = 0.005) (Fig. 6A) and subiculum (T(5.17) = −2.82; P = 0.036) (Fig. 6B). 3.3. CORT measurements Levels of plasma CORT (mean ± SEM) on the 21st day of treatment were 13.65 ± 2.71 ng/ml for the sham group and 428.41 ± 108.31 ng/ml for the CORT group. Unpaired Student’s t-test showed that the group treated with CORT presented significantly higher plasma levels of this glucocorticoid: (T(6.01) = −3.83; P = 0.009). 4. Discussion The purpose of the present study was to investigate behavioral and biochemical alterations induced by chronic treatment with CORT. Results showed that the subcutaneous implant of 200 mg slow-release (21 days) pellets of CORT induced facilitation of stepdown inhibitory avoidance, a task related to emotional aversive

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Fig. 3. Photomicrographs of GRi/nNOS immunostaining in coronal sections of different brain regions of sham and corticosterone treated animals (N = 6). (A) Paraventricular hypothalamic nucleus, (B) lateral amygdala. GR-ir neurons: black coloring localized in the nucleus (arrowhead); nNOS-ir neurons: brown coloring localized in the cytoplasm (small arrow); double staining (large arrow). *Third ventricle, **external capsule. Scale bars: 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

conditioning. Treatment with CORT was capable of inducing significant increases in plasma concentrations of this glucocorticoid, while at the same time increasing GRi in the medial, basolateral and basomedial amygdala, in the PVN, in the ventral and dorsal dentate gyrus, in the ventral CA1 region and in the dorsal CA1 and CA3 regions. Furthermore, nNOSi and GRi-nNOSi were significantly increased by CORT treatment in the medial amygdala and basolateral amygdaloid complex, in the PVN, subiculum, in the dorsal CA3 region and in the ventral CA1 and CA3 regions. The effects induced by CORT on emotional aversive conditioning have been shown to be biphasic. Although some studies point to facilitatory effects [4–8], others suggest that CORT administration impairs memory retrieval [5,29,30]. These differential effects seem to depend not only on the dose of CORT administered, but also on the time of administration, and if administration was performed pre-training, during training or post-training. The procedure used to administer CORT is also an important factor, since with the implant of pellets the circadian rhythm of secretion is lost. Furthermore, it is interesting to mention that most of the studies that point to a facilitatory effect of CORT used aversive conditioning paradigms in which emotional arousal was a critical element of the learning experience and memory improvement. Thus, it has been previously postulated that this enhanced memory for emotional events is due to an interaction between stress hormones and regions involved with emotional conditioning [see 5]. This relationship seems to apply for the present case. Although in our study high doses of CORT were administered for 21 days, we did not show learning/memory impairments. These results corroborate previous data [31,32]. For instance, increases in contextual freezing have been observed when 20 mg/kg of CORT was daily administered for 25 days [31]. Also, acquisition of lithium chloride-conditioned taste aversion was potentiated after 50 mg/kg of CORT was repeatedly administered for 14 days [32]. Furthermore, in a previous study

performed in our laboratory [9], also using the implant of 200 mg slow-release pellets, we found facilitation of inhibitory avoidance responses in the elevated T-maze animal model of anxiety [33], a task that involves emotional aversive conditioning. The anxiogenic effects and the facilitation of aversive conditioning observed with the administration of chronic CORT are consistent with the idea that increases in glucocorticoid levels play an important role in the development of anxiety and mood disorders [34]. It is interesting to point out that the flattening of CORT circadian rhythm induced by the implant of pellets has been shown to be a crucial factor related to the activation of GR. Previous evidence suggests that GR activation (but not MR) varies widely over the normal diurnal variation in CORT levels [35]. In fact, apart from improving aversive learning, chronic treatment with CORT also induced GRi in the basolateral and basomedial amygdala (two nuclei that together with the lateral amygdala form the basolateral complex) [36], medial amygdala, hippocampus and PVN. Previous studies have consistently shown that activation of GR in the PVN occurs in response to stress and glucocorticoids release [37–39]. Stressful stimuli and danger signals activate the HPA axis via neurocircuits that converge to the region and induces the stimulation of CRF-producing neurons [39]. Aside from the PVN, limbic regions, such as the amygdala and the hippocampus, also possess high concentrations of GR [3,40]. It has been previously shown, for instance, that after being released glucocorticoids readily enter the brain and bind to receptors in the basolateral amygdaloid complex [35], thus enhancing memory consolidation of aversive experiences [41]. On the other hand, lesions of the basolateral amygdala or the administration of GR antagonists into the structure block memory facilitation induced by systemic treatment with glucocorticoids [41–43]. The increases in GRi in the medial amygdala corroborate previous data obtained in our laboratory [44], showing that treatment

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Fig. 4. Photomicrographs of GRi/nNOS immunostaining in coronal sections of different brain regions of sham and corticosterone treated animals (N = 6). (A) Ventral dentate gyrus, (B) ventral CA1, (C) ventral CA3. GR-ir neurons: black coloring localized in the nucleus (arrowhead); nNOS-ir neurons: brown coloring localized in the cytoplasm (small arrow); double staining (large arrow). Scale bars: 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

with CORT (same dose-regimen used in the present study) activates Fos immunoreactivity, particularly in this amygdaloid nucleus. Different emotional stressors also stimulate the structure [45,46]. It is known that the region sends projections to the PVN, modulating ACTH and CORT release [47]. According to some authors, the projections from the medial amygdala to the PVN are even more pronounced than the ones from the central nucleus [48,49]. Our results also showed that the lateral amygdala did not present higher GRi in response to treatment. These results corroborate previous observations showing that the lateral amygdala, in comparison to other amygdala nuclei, presents lower concentrations of GRs [3,40,50]. On the other hand, it is interesting to point out that there were no significant differences in GRi between sham and CORT-treated animals in the central amygdala, a region that has been described as possessing high concentrations of GRs [3,40]. However, our data also indicate that the central amygdala of sham animals showed high GRi. Thus, it is possible that a ceiling effect was obtained, masking significant differences between sham and

CORT-treated animals for this particular structure. Furthermore, although increases with respect to treatment were also detected in the subiculum and ventral CA3, these results were not statistically significant. Regarding the hippocampus, it is interesting to mention that some studies performed with stress exposure [51] or repeated injections of CORT [52] have shown, contrarily to our results, downregulation of GR. One more time a possible explanation for these contradictory results might be related to the flattening of CORT circadian rhythm acquired with the implant of pellets [35]. In fact, other studies performed using this same procedure have not shown down-regulation of hippocampal GR [53]. As previously mentioned, both stress and glucocorticoids increase glutamate release [54]. Activation of NMDA receptors by glutamate evokes calcium influx and the activation of a number of enzymes, including NOS, which converts amino-acid l-arginine to N␻-hydroxy-l-arginine, which is posteriorly converted to NO [13,14]. As a retrograde messenger, NO influences the formation of LTP and memory consolidation.

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Fig. 5. Photomicrographs of GRi/nNOS immunostaining in coronal sections of different brain regions of sham and corticosterone treated animals (N = 6). (A) Dorsal dentate gyrus, (B) dorsal CA1. GR-ir neurons: black coloring localized in the nucleus (arrowhead); nNOS-ir neurons: brown coloring localized in the cytoplasm (small arrow); double staining (large arrow). Scale Bars: 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Photomicrographs of GRi/nNOS immunostaining in coronal sections of different brain regions of sham and corticosterone treated animals (N = 6). (A) Dorsal CA3, (B) subiculum. GR-ir neurons: black coloring localized in the nucleus (arrowhead); nNOS-ir neurons: brown coloring localized in the cytoplasm (small arrow); double staining (large arrow). Scale bars: 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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With the exception of the dorsal CA1 region of the hippocampus, all regions that presented significant increases in GRi in response to CORT treatment also presented significant increases in nNOSi. Furthermore, when compared to sham animals, the lateral amygdala, the subiculum and the ventral CA3 region of the hippocampus of CORT-treated animals also showed increases in nNOSi. In fact, nNOS has been previously detected in moderate to high concentrations in all the regions presently studied [17]. In the hypothalamus, the highest density of NO-producing neurons, under basal conditions, is within the PVN [17,55]. Actually, in the PVN both parvo and magnocellular neurons contain nNOS [56], and in these last neurons, nNOS coexists with both vasopressin and oxytocin [57,58]. Also, nNOS activity is increased in the PVN in response to several stress paradigms [59–62]. In the hippocampus, moderate nNOS staining was found in the subiculum and in the CA1 and 3 regions, under basal conditions [17]. As for the PVN, increases in nNOSi in the hippocampus after exposure to stress and to glucocorticoid release have also been previously shown [63,64]. In the amygdala, high concentrations of nNOS were particularly found, under basal conditions, in the basolateral complex [17]. On the other hand, moderate concentrations were found in the medial nucleus [17], while only a weak immunostaining was found in the central amygdala [17]. This last observation could also explain why neither nNOS, nor GRi/nNOSi, were significantly altered by CORT treatment in this nucleus in the present study. In fact, in a previous study that verified activation of NO-neurons in response to novelty and restraint stress [18], it was shown that animals submitted to a new environment presented activation of NO-producing neurons in the medial septum, medial amygdala, hypothalamus, colliculi, raphe and several other hindbrain nuclei. Restraint stress also caused the activation of NO-producing neurons in all of these areas and the activation of additional NO-producing neurons in the diagonal band of Broca, lateral and medial preoptic areas, basomedial and basolateral amygdalar nuclei, among others. The central amygdala, on the other hand, was not activated [18]. At last, one of the main findings of the present study was that all regions that presented significant increases in nNOSi also presented significant increases in GRi/nNOSi. To the best of our knowledge, this is the first study to demonstrate the relationship between GR and nNOS activation in different brain regions related to aversive conditioning. That GR activation influences endogenous NO had been previously observed in the adrenal cortex [65]. In this particular study, treatment with L-NAME, a competitive inhibitor of all isoforms of NOS, significantly decreased GRi [65]. Previous studies have also demonstrated that exogenous NO is capable of activating endothelial GR in cell culture [66] and increase GRi in the lung, liver and kidney in porcine endotoxin sepsis [67]. Since all regions that presented significant nNOSi also presented significant increases in GRi/nNOSi it is possible to affirm that nNOSi was mainly detected in neurons that showed activation of GR by CORT treatment, the same treatment that increased the retrieval of an aversive memory and induced the facilitation of inhibitory avoidance performance. In other words, our data allow the conclusion that the facilitation of aversive conditioning induced by chronic CORT, and which often accompanies anxiety and mood disorders, involves GR-nNOS pathways activation. This observation may be of relevance for the understanding and treatment of these pathological conditions. In this last regard, it has been proposed [64,68] that stress-induced increases in NOS, resulting in NO overproduction and in the consequent inhibition of reduced glutathione (GSH), the non-enzymatic component of anti-oxidant defense, by nitration or nitrosylation may induce oxidative damage and neurobiological changes in stress-related neurocircuitries.

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Acknowledgements This study was financed by Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, FAPESP, Brazil. Thays Brenner dos Santos was the recipient of a fellowship grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil. The authors thank José Simões de Andrade for helpful technical support.

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