Differential immunoreactivity of glucocorticoid receptors and vasopressin in neurons of the anterior and medial parvocellular subdvisions of the hypothalamic paraventricular nucleus

Brain Research Bulletin 82 (2010) 271–278

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Differential immunoreactivity of glucocorticoid receptors and vasopressin in neurons of the anterior and medial parvocellular subdvisions of the hypothalamic paraventricular nucleus Leandro Marques de Souza, Celso Rodrigues Franci ∗ Departamento de Fisiologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brazil

a r t i c l e

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Article history: Received 15 December 2009 Received in revised form 12 May 2010 Accepted 13 May 2010 Available online 26 May 2010 Keywords: Vasopressin Paraventricular nucleus Corticosterone Glucocorticoid receptors Stress Hypothalamus–pituitary–adrenal axis

a b s t r a c t arginine-vasopressin in the parvocellular neurons of the hypothalamic paraventricular nucleus is known to play an important role in the control of the hypothalamo-pituitary–adrenal axis. In the present study, we verify plasma corticosterone levels, the distribution of glucocorticoid receptor- and arginine-vasopressin-positive neurons, and the co-localization of both glucocorticoid receptors and arginine-vasopressin in neurons in the anterior and medial parvocellular subdivisions of the paraventricular nucleus after manipulations of the hypothalamus–pituitary–adrenal axis. Normal, sham surgery, and adrenalectomized male rats were subjected to intraperitoneal injections of saline or dexamethasone to measure plasma corticosterone levels by a radioimmunoassay. We also examined arginine-vasopressin and glucocorticoid receptor immunofluorescence in sections from the paraventricular nucleus. Our results showed that the immunoreactivity of arginine-vasopressin neurons increased in the anterior parvocellular subdivision and decreased in the medial parvocellular subdivision from adrenalectomized rats treated with dexamethasone. On the other hand, we showed that the immunoreactivity of glucocorticoid receptors increased in the anterior and medial parvocellular subdivisions of these same animals. However, the immunoreactivity of glucocorticoid receptors is higher in the medial parvocellular than anterior parvocellular subdivision. The co-localization of arginine-vasopressin and glucocorticoid receptors was found only in the medial parvocellular subdivision. These findings indicate that glucocorticoids have direct actions on arginine-vasopressin-positive neurons in the medial parvocellular but not anterior parvocellular subdivision. There is a differentiated pattern of arginine-vasopressin-positive neuron expression between the anterior and medial parvocellular subdivisions. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Physical or psychological stressors are known to activate the hypothalamus–pituitary–adrenal (HPA) axis, which allows organisms to adapt to environmental changes [2,3,48]. Corticotropinreleasing hormone (CRH) and arginine-vasopressin (AVP) are the major secretagogues of corticotropin (ACTH) in the parvocellular neurons of the paraventricular nucleus (PVH) [7,19,43]. The PVH is known to be highly responsive to stressors, and its outputs (including those to the brainstem, median eminence, and pituitary) are important pathways for the response to stress [6,47]. Levels of CRH and AVP have both been shown to be sensitive to stressful stimuli [49,53]. The expression of CRH mRNA in the parvocellular neurons of the PVH increases sharply following a variety of acute stressors [13,25,36,37]. PVH presents two differ-

∗ Corresponding author. Tel.: +55 16 3602 3022; fax: +55 16 3633 0017. E-mail address: [email protected] (C.R. Franci). 0361-9230/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2010.05.009

ent populations of AVP-positive neurons: magnocellular neurons contain high levels of AVP and are responsive to specific demands (e.g., hypovolaemia or osmolality changes in the extracellular liquid [22]); parvocellular neurons [20] respond to most stressors with increased AVP expression [23,38,46]. AVP itself is a weak ACTH secretagogue, but it potentiates the stimulatory effects of CRH on ACTH release from the corticotrophs [1,19,43]. AVP and CRH in the parvocellular neurons of the PVH are negatively regulated by glucocorticoids [16,44]. The withdrawal of the negative feedback action of glucocorticoids by adrenalectomy results in the up-regulation of the CRH and AVP gene expression in the parvocellular neurons [16,30,31,32,35,44,45]. Glucocorticoid microinjections in the PVH inhibit medial parvocellular neurons (PVHmp) of the PVH, resulting in both the down-regulation of mRNA for CRH and a decrease in ACTH secretion by negative feedback [54]. This direct action on PVN neurons is obvious by the expression of glucocorticoid receptors (GR) in these neurons [52]. GR are expressed in the parvocellular neurons of the PVH, especially in the PVHmp [18,52]. They are part of a group of steroid

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hormone receptors (SHR) that include mineralocorticoids (MR) as well as the progesterone (PR), androgen (AR), and estrogen (ER) receptors. This group comprises a small part of a large family of proteins known as the nuclear receptors [10]. To verify a possible differential participation of parvocellular AVP neurons subpopulations in different conditions of activity of HPA axis, we did investigate the distribution of GR, AVP, GR/AVP neurons in both the anterior (PVHap) and medial (PVHmp) parvocellular subdivisions of the PVH. 2. Materials and methods 2.1. Animals Male Wistar rats weighing 250 ± 10 g were housed one per cage and acclimated for 5 days to a 12 h:12 h light-dark cycle (from 07:00 to 19:00 h). Animals were maintained at an ambient temperature of 23 ± 1 ◦ C with free access to food and water. All protocols were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (USA) and were previously approved by the Ethics Committee for Experiments on Animals of the Medical School of Ribeirão Preto, University of Sao Paulo. 2.2. Experimental design and surgical procedures To reach the objective of this study we work with different conditions of activity of HPA axis: intact condition, effect of absence of adrenal (adrenalectomy) associated or not with hormonal treatment (dexamethasone), effect of surgical stress (sham surgery) associated or not with hormonal treatment (dexamethasone), hormonal treatment (dexamethasone) in intact animal The animals were divided into three groups and six subgroups: • Intact animals – At the end of the fifth day of acclimation (19.00 h), intact animals were divided in two subgroups that received an intraperitoneal injection of saline (0.15 M NaCl; 0.1 mL/100 g b.w, intact-saline group) or 2.5% dexamethasone (0.1 mL/100 g, Sigma, USA, intact-Dexa group). • Groups for surgeries – After five days of habituation to housing conditions, one animals were subjected to sham surgery and another one was subjected to bilateral adrenalectomy (ADX) under tribromoethanol 2.5% (1 mL/100 g, Aldrich, USA) anesthesia. This surgical procedure was performed via the dorsal approach. Control animals underwent sham surgery, in which the adrenal glands were exposed but not excised. After the surgeries, the animals received a veterinary pentabiotic (0.1 mL/100 g, Fort Dodge-BRA) and were housed individually. Adrenalectomized animals (groups IV and VI) also received access to 0.9% saline as drinking fluid. • At the end of the fifth day of recovery (19.00 h), each group subjected to surgery (sham or ADX) was divided in two subgroups that received intraperitoneal injections of saline (0.15 M NaCl; 0.1 mL/100 g b.w., sham-saline group/ADX-saline group) or 2.5% dexamethasone (0.1 mL/100 g, sham-Dexa group/ADX-Dexa group) • Twelve hours after intraperitoneal injection with saline or dexamethasone on the sixth day (7.00 h), animals from all groups were anesthetized to permit withdrawal of a blood sample through an aortic puncture. The sample was stored at ±20 ◦ C until assayed to measure corticosterone levels. Then, approximately 30 ml of 0.01 M phosphate-buffered saline (PBS, pH 7.4) was rapidly perfused through the ascending aorta; this was followed by perfusion of 300 ml of 4% paraformaldehyde in a 0.1 M sodium phosphate buffer (pH 7.4). Brains were removed immediately after perfusion and immersed in 30% sucrose in PBS (48 h at 4 ◦ C), frozen in cooled isopentane, and stored at −70 ◦ C until sectioning for immunohistochemistry. 2.3. Immunohistochemistry 2.3.1. Tissue preparation Serial coronal sections (20 ␮m) (0.9–2.1 mm posterior to bregma) through the anterior (PVHap) and medial (PVHmp) portions of the paraventricular nucleus (PVH) were cut on a cryostat at −20 ◦ C. The 20 ␮m-thick frozen sections were divided into six 1-in-8 series (a–h): section 1a was serial to section 1b, which was serial to section 1c, still 1 h which was then in turn serial to section 2a, and so on. Thus, in a given series (a–h) two sequentially numbered sections (e.g., 1a, 2a) are separated by seven 20 ␮m-thick sections (a spam of 140 ␮m is missing between them). The slices were thawed, and mounted on slides covered with gelatin. Brain sections were rinsed in PBS and then placed into 0.1 M glycine in PBS (20 min) to remove excess aldehydes. After washes in PBS, sections were blocked (1 h) in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) and then incubated with primary antibodies. 2.3.2. Immunofluorescence AVP- and GR-sections of from the PVN (0.9–2.1 mm posterior to bregma) were incubated with a mouse monoclonal anti-vasopressin antibody (1:1000, a kind gift of Dr Harold Gainer, National Institutes of Health, Bethesda, MD, USA) and goat antiglucocorticoid receptor antibody (1:100, sc-1004, Santa Cruz Biotechnology, Santa

Cruz, CA, USA). All antibodies were diluted in PBS containing 0.1% Triton X-100 and 1% BSA, and the sections were incubated overnight (20 h) at 28 ◦ C. Following PBS washes, sections were incubated (1 h) in a cocktail of fluorescent secondary antibodies. To visualize AVP neurons and GR, a donkey anti-mouse IgG labeled with Alexa Fluor 488 (Molecular Probes, USA) and a donkey anti-rabbit IgG labeled with Alexa Fluor 594 (Molecular Probes, USA) were used. The slides were washed with PBS and coverslipped. 2.3.3. Tissue analysis Tissue sections were examined using an Axioskope 2 Plus microscope (Carl Zeiss, Göttingen, Germany), and the images were captured with a Zeiss AxioCam digital camera (Carl Zeiss, Göttingen, Germany). In the double-label immunofluorescence studies for AVP/GR as well as single-label immunoreactivity analyses of AVP or GR, one series of tissue sections through the rostro-caudal extent of the PVH (six sections, with a 140 ␮m interval between sections) was used per animal (n = 5–9 per group). Sections were displayed on the microscope using a 20× objective. The boundaries of different PVH subdivisions were defined according of anatomical parameters of brain maps [51]. The series of sections from each brain were compared with the reference atlas to define the boundaries of different PVH subdivisions. Finally, we used the same size of the optical field for all of the photomicrographs. The criteria for identification of parvocellular neurons was the localization of GR since previous studies showed that GR immunoreactivity is found in parvocellular region but not in magnocellular region in PVH [21,40]. Furthermore, for identifying the parvocellular and the magnocellular neurons of the PVH in the hypothalamus, we referred to crucial characteristics. The magnocellular region has characterized by the compact clustering of the large cells (mean short diameter >12 mm). Between the third ventricle and the compact magnocellular division lies the prominent mass of small neurons forming the parvocellular region (diameter of parvocellular cells <10 mm) [28]. So, we could distinguish the two divisions according to the cell size in high magnification. We made the measures of all neurons using Image J software (NIH, Bethesda, MD, USA) and only those immunereactive neurons with less than 10 mm were quantified. The antibodies used in this study recognize only AVP or GR. All AVP neurons or GR within the section were examined, quantified and designated as immunoreactive to mouse monoclonal anti-vasopressin antibody (1:1000, a kind gift of Dr Harold Gainer, National Institutes of Health, Bethesda, MD, USA) or goat anti-glucocorticoid receptor antibody (1:100, sc-1004, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunopositivity of the cells analyzed is given by the specificity of the antibodies used in this study. All AVP neurons and GR within the section were examined bilaterally. Next, sections scored for the co-staining of AVP and GR were re-examined using Confocal microscopy (TCS SP2 AOBS, Leica Microsystems, Manheim, Germany). All AVP neurons or GR within the section were quantified manually and automatically. The quantitative assessment of AVP neurons, GRs, and double-labeled cells was obtained from captured microscope images using Image J software (NIH, Bethesda, MD, USA). 2.3.4. Immunohistochemistry controls In both single- and double-label experiments, omission of the primary antibody resulted in no labeling (data shown in Fig. 6, G and H). For each double-label combination, primary antibodies were titrated to establish the maximum dilutions that provided robust signals and a minimum of nonspecific staining (data not shown). 2.3.5. Hormone measurement Plasma corticosterone (CORT) was measured by a radioimmunoassay (RIA) that required extraction using ethanol. The following reagents were used for RIA: corticosterone H3 (NEN Life Sciences Products, Boston, USA), specific antibody and standard reference (Sigma, USA). Tritiated corticosterone was used to measure recovery. To avoid variation, all samples from the same experiment were measured in the same assay. The intra-assay error was 5% and the minimum detectable dose was 0.8 ng/ml. 2.3.6. Statistical analysis The mean ± SEM of AVP neurons, GRs, and percent of dual-labeled cells was calculated. Two-way ANOVAs followed by the Bonferroni post hoc analyses for multiple comparisons were used to evaluate the number of AVP neurons, GR and percent of dual-labeled cells calculated per region in the six animal groups. A P-value <0.05 was considered statistically significant.

3. Results 3.1. Effects of adrenalectomy and dexamethasone treatment on plasma corticosterone levels Fig. 1 shows the corticosterone plasma concentrations in normal, sham, and adrenalectomized male rats submitted to intraperitoneal injections of saline or dexamethasone. The present data showed a significant decrease of plasma corticosterone levels after treatment and surgery. Bonferroni post hoc analysis revealed

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Fig. 1. Effects of adrenalectomy (ADX) and dexamethasone (DEXA) treatment on plasma corticosterone concentrations. Each bar represents the mean ± S.E.M. Significant main effects for treatment (F1,33 = 609.1; P < 0.0001) and surgery (F2,33 = 191.4; P < 0.0001). The symbols  for treatment and  for surgery show the difference between groups and the level of significance was set at P < 0.001. A two-way ANOVA indicated a significant interaction between the treatment and surgery factors (F2,33 = 232.6; P < 0.0001).

that plasma corticosterone levels were higher in intact-saline (62.2 ± 3.5 ng/mL) and sham-saline (68.9 ± 2.9 ng/mL) groups than intact-Dexa (3.3 ± 0.5 ng/mL) and sham-Dexa (4.8 ± 0.9 ng/mL) groups. Furthermore, the plasma corticosterone levels (ng/mL) were lower in ADX-saline group (2.6 ± 2.5 ng/mL) than intact-saline group (62.2 ± 8.7 ng/mL). Similar data were obtained between the ADX groups treated with saline (2.6 ± 2.5 ng/mL) or dexamethasone (6.7 ± 3.0 ng/mL). 3.2. AVP and GR immunoreactivity in neurons of the PVHap and PVHmp Fig. 2 shows illustrative photomicrographs (20 ␮m thick section) of the PVHap (A–C) and PVHmp (D–F) with immunofluorescent labels for AVP (green). Photomicrographs from saline + Dexa,

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sham + saline and sham + Dexa groups (no shown) are very similar to the intact group. Fig. 3 presents the number of Arginine-vasopressin (AVP)positive neurons in the PVHap (A) and PVHmp (B). For the treatment factor, a Bonferroni post hoc analysis revealed that the number of AVP-positive neurons in the PVHap was higher in ADX-Dexa group (6.2 ± 1.1/section) than ADX-saline group (1.9 ± 0.5/section) while in the PVHmp was higher in ADX-saline group (34.1 ± 7.4/section) than ADX-Dexa group (17.5 ± 1.6/section). For the surgery factor, a Bonferroni post hoc analysis revealed that the number of AVP-positive neurons was higher in ADX-Dexa group (6.2 ± 2.6/section) than intact-Dexa group (0.8 ± 0.9/section) while in the PVHmp was higher in ADX-saline (34.1 ± 7.4/section) and ADX-Dexa (17.5 ± 1.6/section) groups than intact-saline (4.7 ± 1.1/section) and intact-Dexa (4.2 ± 0.6/section) groups. Dexametasone treatment induced different effects on number of AVP-positive neurons in the PVHap (increased) and PVHmp (decreased). Fig. 4 shows illustrative photomicrographs (20 ␮m thick section) of the PVHap (A–C) and PVHmp (D–F) with immunofluorescent labels for GR (red). Photomicrographs from saline + Dexa, sham + saline and sham + Dexa groups (no shown) are very similar to the intact group. Fig. 5 presents the number of activated glucocorticoid receptors (GR) in the PVHap (A) and PVHmp (B). For the treatment factor, a Bonferroni post hoc analysis revealed that the number of neurons with activated GR immunoreactivity was higher in ADX-Dexa group (PVHap, 59.9 ± 6.7/section; PVHmp, 90.4 ± 4.7/section) than ADX-saline group (PVHap, 1.2 ± 0.4/section; PVHmp, 1.1 ± 0.4/section). For the surgery factor, a Bonferroni post hoc analysis revealed that the number of neurons with activated GR immunoreactivity was higher in ADX-Dexa group (PVHap, 59.9 ± 0.4/section; PVHmp, 90.4 ± 4.7/section) than intactDexa group (PVHap,1.1 ± 1.0/section; PVHmp, 62.8 ± 6.8/section). The number of neurons with activated GR immunoreactivity was higher in the PVHmp than PVHap for all groups but treatment with dexamethasone increased this immunoreactivity for both regions mainly in ADX groups.

Fig. 2. Photomicrographs (20 ␮m thick section) of anterior (PVHap, in panels A–C) and medial (PVHmp, in panels D–F) parvocellular region in the paraventricular nucleus (PVH) from intact rats treated with saline (panels A and D), adrenalectomized (ADX) rats treated with saline (panels B and E), ADX rats treated with dexamethasone (DEXA) (panels C and F). Immunofluorescence label for arginine-vasopressin (AVP, green, Alexa Fluor 488). Scale bars = 50 ␮m. 3 V, third ventricle.

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Fig. 3. Effects of adrenalectomy and dexamethasone treatment on the number of arginine-vasopressin (AVP)-positive neurons in the anterior (PVHap, in (A) panel) and medial (PVHmp, in (B) panel) parvocellular subdivision of the paraventricular nucleus. Each bar represents the mean ± S.E.M. A two-way ANOVA indicated a significant interaction between treatment and surgery factors (F2,30 = 10.5; P = 0.0003 in (A)/F2,30 = 4.5; P = 0.0187 in (B)). Significant main effects were detected for treatment (F1,30 = 9.0; P = 0.0052 in (A)/F1,30 = 7.8; P = 0.0088 in (B)) and for surgery (F2,30 = 21.0; P < 0.0001 in (A)/F2,30 = 31.9; P < 0.0001 in (B)). Bonferroni post hoc analysis was carried out. The symbols  for treatment and  for surgery show the difference between groups and the level of significance was set at P < 0.001.

Fig. 4. Photomicrographs (20 ␮m thick section) of anterior (PVHap, in panels A–C) and medial (PVHmp, in panels D–F) parvocellular region in the paraventricular nucleus (PVH) from intact rats treated with saline (panels A and D), adrenalectomized (ADX) rats treated with saline (panels B and E), ADX rats treated with dexamethasone (DEXA) (panels C and F). Immunofluorescence label for glucocorticoid receptor (GR, red, Alexa Fluor 594). Scale bars = 50 ␮m. 3 V, third ventricle.

Fig. 5. Effects of adrenalectomy and dexamethasone treatment on the number of neurons with activated glucocorticoid receptors (GR) in the anterior (PVHap, in panel A) and medial (PVHmp, in panel B) parvocellular subdivision of the paraventricular nucleus. Each bar represents the mean ± S.E.M. A two-way ANOVA indicated a significant interaction between treatment and surgery factors (F2,24 = 122.1; P < 0.0001 in (A)); (F2,31 = 52.6; P < 0.0001 in (B)). Significant main effects were detected for treatment (F1,24 = 49.9; P < 0.0001) (A); (F1,31 = 84.5; P < 0.0001 in (B)) and surgery (F2,24 = 70.5; P < 0.0001) in (A); (F2,31 = 17.9; P < 0.0001 in (B)). Bonferroni post hoc analysis was carried out. The symbols  for treatment and  for surgery show the difference between groups and the level of significance was set at P < 0.001.

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Fig. 6. Photomicrographs (20 ␮m thick section) of the anterior (PVHap; panels A–C) and medial (PVHmp; panels D–F) parvocellular region in the paraventricular nucleus (PVH) from adrenalectomized (ADX) animals treated with dexamethasone (DEXA). Immunofluorescence labels for arginine-vasopressin (AVP, green, Alexa Fluor 488; panels A and D) and glucocorticoid receptors (GR, red, Alexa Fluor 594; panels B and E) are present. AVP neurons with activated GR were observed in the PVHmp, but no AVP/GR double labeling was detected in the PVHap, as shown in the composite images (panels C and F). Controls in which the primary antibody was omitted from PVHmp sections did not detect AVP (panel G)- or GR (panel H)-positive neurons. Confocal photomicrographs (20 ␮m thick section) of immunofluorescence for AVP (panel I) and GR (panel J) in the PVHmp. The composite image of AVP-GR double labeling in panel K and high magnification of image from panel K in panel L confirm the nuclear GR immunoreactivity in the AVP-positive neurons. Scale bars = 50 ␮m (panels A–H), 8 ␮m (panels I–L). 3 V, third ventricle.

3.3. Neuronal co-localization of GR and AVP Fig. 6 shows photomicrographs (20 ␮m thick section) of the PVHap (A–C) and PVHmp (D–F) in animals from ADX-Dexa

group with immunofluorescent labels for AVP (green; A and D) and GR (red; B and E). Composite images show AVP-positive neurons with activated GR in the PVHmp (F) but not in the PVHap (C). Confocal microscopy analysis confirms the acti-

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Fig. 7. Percent of arginine-vasopressin (AVP)-positive neurons with glucocorticoid receptors (GR) immunoreactivity in the medial (PVHmp) parvocellular subdivision of the paraventricular nucleus of the hypothalamus. Each bar represents the percent of AVP-positive neurons with co-staining GR and we observed the absence of AVPpositive neurons co-staining GR in ADX-saline group. Statistical analysis using a twoway ANOVA followed by the Bonferroni post-test indicated a significant interaction between the treatment and surgery factors (F2,44 = 200.1; P < 0.0001) and significant effects of treatment (F1,44 = 247.1; P < 0.0001) and surgery (F2,44 = 164.6; P < 0.0001). The symbol  for surgery shows the difference between groups and the level of significance was set at P < 0.001.

vated GR in the AVP-positive neurons from the PVHmp (Fig. 6; I–L). Fig. 7 shows the percent of AVP-positive neurons with activated GR in the PVHmp that were detected in intact-saline (0.5 ± 0.1%/section), intact-Dexa (0.6 ± 0.1%/section), sham-saline (0.7 ± 0.1%/section), sham-Dexa (1.7 ± 0.1%/section) and ADX-Dexa (14.2 ± 1.1%/section) groups but not in ADX-saline group. Dexametasone treatment increased the percent of AVP-positive neurons with GR in the PVHmp from ADX animals. 4. Discussion Our results showing a significant decrease of plasma corticosterone levels after treatment and surgery are in agreement with classical knowledge from the literature regarding control of secretion of the hypothalamus–pituitary–adrenal axis [54]. We did not find a difference in the plasma corticosterone levels between the ADX groups treated with saline or dexamethasone. These results demonstrate the effectiveness of the adrenalectomy and the absence of the feedback mechanism response in the animals of these groups. In the present study, the immunoreactivity of AVP did not differ in either the PVHap or PVHmp in response to treatments with saline and dexamethasone in intact and sham animals. These data are supported by previous studies [4], in which the presence of endogenous corticosterone desensitizes the responses of AVP to exogenous glucocorticoids. However, the expression of AVP increased in neurons from the PVHap and decreased in those from the PVHmp from ADX animals treated with dexamethasone in comparison to ADX animals treated with saline. The literature widely shows that adrenalectomy causes a marked increase of AVP expression or immunoreactivity in the parvocellular portion of the PVN [29,32,33,50,55–57]. Previous studies have also shown that corticosterone replacement after adrenalectomy reverses this increase of AVP expression or immunoreactivity, leading to a decrease of AVP expression or immunoreactivity in the parvocellular portion of the PVH [5,26,32,44]. Several researchers verified the effect of glucor-

ticoids administration on mRNA or hnRNA and protein levels [31,35,36,37,44,45]. We examined only protein levels and our results for the PVHmp corroborate these findings described in the literature. In fact, in all of the studies previously mentioned, corticosterone replacement after adrenalectomy reverses the increase of AVP expression or immunoreactivity and leads to a decrease of AVP expression or immunoreactivity in the parvocellular portion of the PVH [5,26,32,44]. However, these studies were performed to analyze the entire parvocellular portion of the PVN. None of these authors conducted an individual analysis of the parvocellular subdivisions. Hence, our results provide information concerning the immunoreactivity of two different subpopulations of AVP neurons in the PVHap and PVHmp in response to adrenalectomy and dexamethasone treatment. The former did not change after adrenalectomy but increased after dexamethasone treatment while the latter increased after adrenalectomy and decreased after dexamethasone treatment. The subpopulation of AVP neurons in the PVHmp is involved clearly in the feedback mechanism to control the HPA axis. The subpopulation of AVP neurons in the PVHap may be involved in the mechanisms to control other functions of the organism but we cannot ruled out its participation also to control of the HPA axis in other different conditions of study, for instance, stress conditions. We did not find any difference of activated GR immunoreactivity between the PVHap and PVHmp in response to treatment with saline or dexamethasone in intact or sham animals. One possible explanation for this lack of difference suggests that the presence of endogenous corticosterone desensitizes responses of the GR to dexamethasone treatment. In agreement with this hypothesis, our study found an increase of activated GR immunoreactivity in both the PVHap and PVHmp from ADX animals treated with saline or dexamethasone. According to previous studies, corticosterone treatment induces GR re-accumulation in the nuclei of parvocellular neurons [18,21,34]. These studies showed that the re-accumulation processes occur more strongly in ADX animals submitted to corticosterone replacement than they do in sham animals. Furthermore, we observed that activated GR immunoreactivity was higher in PVHmp than PVHap for all groups. These data are consistent with previous reports mapping GR in the rat brain that found a higher quantity of GR in the PVHmp [8,14,18]. Previous studies showed that a chaperone complex in the cytoplasm binds to the GR and maintains these receptors as inactive form. After dissociation from chaperone complex, GR translocate to the nucleus to regulate the target gene expression [12]. Nuclear GR immunoreactivity decreased one week after adrenalectomy and increased after corticosteroid replacement [36]. We used antiserum against N-terminus of the GR molecule that labels GR dissociated from chaperone complex, in process of translocation or translocated to the nucleus as show results of this study. Moreover, we did verify also accumulation of GR immunoreactivity in the nucleus after corticosteroid replacement The analysis of GR co-localization in AVP-positive neurons revealed double labeling only in the PVHmp from normal and sham animals treated with saline or dexamethasone as well as ADX animals treated with dexamethasone. Furthermore, statistical analysis showed a large increase in the percentage of neurons co-staining AVP and GR in ADX animals treated with dexamethasone. Although previous studies suggest that low plasma corticosterone levels sensitize the response of AVP-positive neurons to dexamethasone by decreasing of the AVP expression [4], an increase in nuclear GRs re-accumulation also occurs simultaneously [18,21,34]. Thus, this increase of activated GR immunoreactivity can also occur in AVP-positive neurons. On the other hand, the absence of this coexpression in ADX animals treated with saline can be explained by low plasma corticosterone levels along with absence of corti-

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costerone treatment that prevents the nuclear GR re-accumulation processes. Therefore our results taken together indicate that direct and indirect effects of glucocorticoids may occur on parvocellular AVP neurons in PVH. Since AVP neurons in PVHmp co-staining with GR, it is possible a direct effect of glucocorticoids in this region. However, this direct effect would be not possible in the PVHap because we did not find co-staining of GR in AVP neurons. An indirect effect of glucocorticoids on AVP expression was suggested in a previous paper, in which adrenalectomy-induced AVP expression in parvocellular neurons was modulated by synaptic inputs [9]. One candidate for the inhibitory neurotransmitter is GABA, which is shown to exist throughout the hypothalamus [17,27,39]. GABA inhibits the release of CRH and/or AVP in vivo and in vitro [11,24,39,41,42], whereas GABA-A antagonists increase CRH and AVP expression in parvocellular PVN neurons [15]. Thus, we cannot ruled out that the dexamethasone treatment in the present study also decreased AVP immunoractivity in the PVHmp of ADX animals by indirect action through stimulating GABA release from adjacent neurons. Other studies will be necessary to corroborate this hypothesis. On the other hand, in the same studied condition we did find an increase of AVP immunoractivity in the PVHap but not GR co-localization. This indicates that the action of glucocorticoids in PVHap was probably indirect through synaptic inputs. In conclusion, our results show differential responses of parvocellular AVP neurons subpopulations from anterior (PVHap) and medial (PVHmp) regions in the PVH to the adrenalectomy and treatment by dexamethazone. Taken together, the data demonstrate that AVP neurons subpopulation in PVHmp is involved in the negative feedback mechanism to control of HPA axis. On the other hand, the AVP neurons subpopulation in PVHap is not involved in this negative feedback mechanism at least in the studied conditions. We cannot to rule out a possible participation of these neurons to control of the HPA axis in other different conditions, for instance, stress. Acknowledgements The authors would like to thank Rogério Rosário Azevedo and Rubens Fernando de Mello for technical support. This work was supported by FAPESP, CAPES and CNPq financial grants from Brazil. References [1] G. Aguilera, Regulation of pituitary ACTH secretion during chronic stress, Front. Neuroendocrinol. 15 (1994) 321–350. [2] G. Aguilera, S.L. Lightman, A. Kiss, Regulation of the hypothalamic– pituitary–adrenal axis during water deprivation, Endocrinology 132 (1993) 241–248. [3] G. Aguilera, Q. Pham, C. Rabadan-Diehl, Regulation of pituitary vasopressin receptors during chronic stress: relationship to corticotroph responsiveness, J. Neuroendocrinol. 6 (1994) 299–304. [4] G. Aguilera, C. Rabadan-Diehl, Vasopressinergic regulation of the hypothalamic–pituitary–adrenal axis: implications for stress adaptation, Regul. Pept. 96 (2000) 23–29. [5] S.F. Akana, M.F. Dallman, Chronic cold in adrenalectomized, corticosterone (B)treated rats: facilitated corticotropin responses to acute restraint emerge as B increases, Endocrinology 138 (1997) 3249–3258. [6] S.F. Akana, M.F. Dallman, M.J. Bradbury, K.A. Scribner, A.M. Strack, C.D. Walker, Feedback and facilitation in the adrenocortical system: unmasking facilitation by partial inhibition of the glucocorticoid response to prior stress, Endocrinology 131 (1992) 57–68. [7] F.A. Antoni, Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor, Endocr. Rev. 7 (1986) 351–378. [8] M. Aronsson, K. Fuxe, Y. Dong, L.F. Agnati, S. Okret, J.A. Gustafsson, Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 9331–9335. [9] F. Baldino Jr., T.M. O’Kane, S. Fitzpatrick-McElligott, B. Wolfson, Coordinate hormonal and synaptic regulation of vasopressin messenger RNA, Science 241 (1988) 978–981.

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