Anteroventral third ventricle (AV3V) lesion affects hypothalamic neuronal nitric oxide synthase (nNOS) expression following water deprivation

Brain Research Bulletin 86 (2011) 239–245

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Anteroventral third ventricle (AV3V) lesion affects hypothalamic neuronal nitric oxide synthase (nNOS) expression following water deprivation Fábio Alves Aguila a , Gabriela Ravanelli Oliveira-Pelegrin a , Song Tieng Yao b,1 , David Murphy b , Maria José Alves Rocha a,∗ a b

Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, United Kingdom

a r t i c l e

i n f o

Article history: Received 20 April 2011 Received in revised form 21 July 2011 Accepted 27 July 2011 Available online 4 August 2011 Keywords: Supraoptic nucleus Dehydration c-fos Vasopressin Nitric oxide

a b s t r a c t Neuronal nitric oxide synthase (nNOS) has been reported to be up-regulated in the hypothalamic supraoptic nucleus (SON) during dehydration which in turn could increase nitric oxide (NO) production and consequently affect arginine vasopressin (AVP) secretion. The anteroventral third ventricle (AV3V) region has strong afferent connections with the SON. Herein we describe our analysis of the effects of an AV3V lesion on AVP secretion, and c-fos and nNOS expression in the SON following dehydration. Male Wistar rats had their AV3V region electrolytically lesioned or were sham operated. After 21 days they were submitted to dehydration or left as controls (euhydrated). Two days later, one group was anaesthetized, perfused and the brains were processed for Fos protein and nNOS immunohistochemistry (IHC). Another group was decapitated, the blood collected for hematocrit, osmolality, serum sodium and AVP plasma level analysis. The brains were removed for measurement of neurohypophyseal AVP content, and the SON was punched out and processed for nNOS detection by western blotting. The AV3V lesion reduced AVP plasma levels and c-fos expression in the SON following dehydration (P < 0.05). Western blotting revealed an up-regulation of nNOS in the SON of control animals following dehydration, whereas such up-regulation was not observed in AV3V-lesioned rats (P < 0.05). We conclude that the AV3V region plays a role in regulating the expression of nNOS in the SON of rats submitted to dehydration, and thus may affect the local nitric oxide production and the secretion of vasopressin. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Nitric oxide (NO) is a lipophilic gas with free passage through biological membranes under physiological conditions. This gaseous mediator is synthesized by three isoforms of nitric oxide synthases (NOS), inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) which catalyze the conversion of the amino acid l-arginine to lcitruline [5,10,24]. Several studies using histochemistry and imunocytochemistry techniques revealed NO-producing neurons co-expressed with the neuropeptides angiotensin II (Ang II), arginine vasopressin (AVP) and oxytocin (OT), especially in the hypothalamic–neurohypophysial system (HNS) [6,8,48]. These

∗ Corresponding author at: Dept. MEF – Faculdade de Odontologia de Ribeirão Preto, USP., Avenida do Café s/n, CEP 14040-904 Ribeirão Preto-SP, Brazil. Tel.: +55 16 3602 3974; fax: +55 16 3633 0999. E-mail addresses: song.yao@florey.edu.au (S.T. Yao), [email protected] (M.J.A. Rocha). 1 Florey Neuroscience Institutes, University of Melbourne, Royal Parade, Melbourne, Victoria 3010, Australia. 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.07.020

studies lead to the suggestion that NO may act as a neurotransmitter or neuromodulator within the HNS. The role of NO in the release of AVP has been extensively studied, but results have been apparently contradictory, showing either stimulation or inhibition. Studies in vitro using hypothalamus or neural lobe isolated from rats, showed that NO donors reduced the KCl-induced AVP release [52] and inhibition of NOS by N-nitrol-arginine methyl ester (L-NAME) caused an increase in AVP and OT release [20]. In rats, intracerebroventricular (icv) injections of NO donors such as S-nitroso-N-acetylpenicillamine (SNAP) and larginine increased the release of AVP [29,49]. On the other hand the central inhibition of NOS by L-NAME also increased the AVP and OT release [17,18]. Furthermore, in vitro study using punches of rat SON showed that either NO scavenger or NOS inhibitor (L-NMMA) reduced osmotically stimulated AVP release [11]. It seems that these contradictory effects of NO on AVP secretion depend on the production levels of this gaseous mediator, as we suggested in a previous work [7,27]. It has been suggested that NO can regulate locally but also indirectly the release of AVP by modulating the activity of the main afferents to magnocellular neurons [32,41]. In vitro studies showed

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that the iNOS and nNOS mRNAs are increased by noradrenaline, particularly in the SON suggesting that noradrenergic input arising from A1/A2 and A6 cell groups of the brainstem can increase NO production and affect the vasopressin release [12]. While the presence of the three isoforms of NOS have been detected in neurons, nNOS is the major form found in magnocellular neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the rat [45]. Moreover, this isoform is also present in several brain structures with strong connections with the SON and PVN, such as the organum vasculosum of lamina terminalis (OVLT), subfornical organ (SFO), area postrema (AP), the median pre-optic nucleus (MnPO) and also nucleus tractus solitarius (NTS) [4,9,34,37,42]. The presence of nNOS in these structures indicates a central production of NO, which may also affect AVP release [1,17,22]. The anteroventral region of the third ventricle (AV3V) that includes the OVLT, preoptic periventricular nucleus, MnPO, is well known to have an important role in the release of AVP [23,25,28,33]. The AV3V region has strong afferent connections with the SON and PVN, as well as being extremely important in body fluid homeostasis and cardiovascular control [14,15,44,46]. When these connections are disrupted, e.g. by electrolytic lesion, the animals showed a diminished or null response to experimental osmotic challenges (dehydration or hypertonic saline ingestion), and they exhibited decreased fluid intake, c-fos expression and AVP and OT secretion [15,23,25,28,33]. It has also been reported that animals subjected to an osmotic stimulus, such as water deprivation for 48 h, present an up-regulation of nNOS gene expression and NO production in the SON [11,26,40,51]. As the importance of the AV3V region in this context has not been addressed in any study so far, our aim was to determine the role of the AV3V region in the regulation of nNOS of the SON in rats submitted to dehydration. 2. Materials and methods 2.1. Animals Male Wistar rats (250–280 g) provided by the Animal Facility of the Campus of Ribeirão Preto, University of São Paulo, were housed in controlled temperature (25 ± 1 ◦ C) and photoperiodic (12:12 h light: day cycle) conditions, with food (Nuvilab CR-1, NUVITAL) and tap water available ad libitum. All experimental protocols were approved and performed according to the guidelines of the Ethics Committee of the University of São Paulo – Campus Ribeirão Preto. 2.2. Surgery Rats were anesthetized with ketamine–xylazine (100 and 14 mg/kg, respectively) and fixed in a Kopf stereotaxic frame. The animals were subjected to anodal electrolytic lesions (2.0 mA for 20 s) in the AV3V region or to sham-lesions with a 0.6 mm nichrome wire electrode. The stereotaxic coordinates of the midline lesion were: 0.0–0.01 mm posterior to bregma and 7.5–8.0 mm ventral to the midsagital sinus. At the end of the experiment, the extent of the lesions was evaluated using standard histological methods. 2.3. Experimental protocol Following surgery the animals were housed in individual cages on a 12 h light: 12 h dark cycle and provided with ad libitum access to food and water. Animals that drank less than 10 ml over a 24-h period after the lesion were functionally regarded as successfully lesioned. They received a 30% drinking sucrose solution to provide adequate hydration, and were gradually weaned from the sucrose to water. At the start of the experiment, 3 weeks post-surgery, the AV3V-lesioned and sham groups consumed comparable amounts of water and food. There were four groups in the experiment: (1) water replete, sham; (2) 48 h dehydrated, sham; (3) water replete, lesioned; (4) 48 h dehydrated, lesioned. From all these groups, a first set of rats was anesthetized with chloral hydrate (400 mg/Kg, i.p.) and perfused transcardially with 150 ml of phosphate-buffered saline (PBS, pH 7.4) and 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, post-fixed in the same fixative for 4 h, placed in PBS containing 30% sucrose and stored at 4 ◦ C. Coronal brain sections were cut at 40 ␮m thickness and divided into three series for immunohistochemistry analyses. One section was processed for c-fos, another for nNOS, and the last section was cryoprotected and set apart as reserve.

A second set of rats was decapitated by means of a guillotine and blood was collected for measurement of hematocrit, serum sodium, plasma osmolality and vasopressin levels. The neurohypophysis was removed and stored at −80◦ C for measurements of AVP content by radioimmunoassay and of total protein content by the Bradford method. Brains were rapidly excised from the cranium, frozen on dry ice and stored at −80 ◦ C until use. 2.4. Determination of hematocrit, plasma osmolality and serum sodium Hematocrit was measured by centrifugation, plasma osmolality by freezing point depression (Precision System, Inc., Natick, MA, USA) and serum sodium by flame photometry (Micronal, São Paulo, Brazil). 2.5. Radioimmunoassay (RIA) for vasopressin After extraction from plasma, the AVP levels were measured by double antibodyspecific radioimmunoassay. Briefly, plasma samples (1.0 ml) were extracted using the acetone/petroleum ether method. Extracts were lyophilized and stored at −80 ◦ C until analysis. Neurohypophyses were homogenized in 0.1 N HCl. A 50 ␮l aliquot of the homogenate was used to measure protein content by the Bradford method and the remainder was for RIA. At the moment of the assay, the homogenates were diluted 1:4000 in assay buffer. Standard reagents and incubation protocols were used for the peptide assays. AVP measurements were carried out with a commercial antiserum (Peninsula Laboratories, Inc., San Carlos, CA, USA) at a final dilution of 1:40,000. The antiserum is specific and shows essentially no cross-reactivity with other known peptides. The buffer used contained Na2 HPO4 (0.062 M), Na2 EDTA (0.013 M) and 0.5% bovine serum albumin (BSA), pH 7.5. For peptide labeling, 125 I was purchased from a commercial supplier (GE Healthcare). A non-equilibrium assay was used with an incubation volume of 500 ␮l and an incubation time of 4 d at 4 ◦ C. Bound hormone was separated from unbound by a secondary antibody produced in the laboratory of José Antunes-Rodrigues and Lucila L.K. Elias (Faculty of Medicine of Ribeirao Preto, USP, São Paulo, Brazil) where the RIA was performed. The assay sensitivity was 0.9 pg/mL. 2.6. Immunohistochemistry for protein Fos and nNOS Free floating brain sections were washed in PBS (pH 7.4) and incubated in PBS containing 3% hydrogen peroxide for 10 min. After several rinses in PBS for 30 min, they were placed in 5% normal goat serum for 45 min and then incubated for 24–48 h at room temperature in antibodies produced against rabbit Fos protein (SC-52, Santa Cruz) or mouse nNOS (SC-5302, Santa Cruz). After rinsing, the sections were incubated for 1.5 h at room temperature with secondary biotinylated goat-anti-rabbit or mouse IgG antibodies (Vector), one for Fos and the other for nNOS detection, both diluted 1:2000 in PBS. After washing in PBS, the sections were placed for 30 min in avidin–biotin peroxidase complex (ABC, Vectastain). Fos or nNOS immunoreactivity was visualized by incubation with 0.05% 3,3 -diaminobenzidine tetrachloride and 0.001% hydrogen peroxide. The sections were mounted on gelatin-coated slides, dehydrated and coverslipped with Entellan (Merck). 2.7. Immunohistochemistry analysis The sections were analyzed by light microscopy and the labeled neurons were detected using 10× objective of Zeiss KS300 microscope by investigator blind to the treatment. The absence or presence and quantification of labeled neurons were registered using ImageJ software, National Institutes of Health (NIH). For a cell to be considered as expressing Fos-IR, the nucleus of the neurons had to be of appropriate size (cell nucleus diameter ranging approximately from 8 to 15 ␮m) and shape (oval or round) and be distinct from background at 10× magnification. For quantification the number of Fos-IR cells, the coronal sections of the mid level of SON were selected from similar rostro-caudal positions. The anatomical description of brain regions follows that of Swanson [38]. The total number of marked neurons in the whole nucleus of a section was counted unilaterally. Coronal sections containing nNOS-IR neurons in the SON were selected in a similar manner to mentioned above. The quantification was performed by the optical density ratio between the stain average and background of three randomly selected regions. 2.8. Microdissection of the SON nuclei Brains were placed in a cryostat and based on rat brain atlas coordinates [38] and using the optic chiasm as anatomical landmark, two coronal sections of approximately 500 ␮m thickness were removed from the hypothalamic region. The supraoptic nucleus was carefully punched out by means of a stainless steel needle of 1200 ␮m diameter. 2.9. Western blotting for the expression analysis of NOS protein The tissue punches were placed in sample buffer (0.25 M sucrose, 0.1 mM EDTA in 50 mM phosphate buffer, pH 7.2) and homogenized with a sonicator. The

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Fig. 1. Coronal section of rat brain showing a typical lesion of the AV3V region. Superimposed is a diagram adapted from the stereotaxic rat brain atlas [30] delineating structures affected by the injury. 3V, 3rd ventricle; AC, anterior commissure; LV, ventrolateral thalamic nucleus; MnPO, median preoptic nucleus; Och, optic chiasm; OVLT, organum vasculosum of the lamina terminalis.

homogenates were centrifuged at 10,000 × g for 30 min at 4 ◦ C. Protein concentrations of the supernatants were measured by the bicinchoninic acid assay [35] in a 1 ␮l volume at 592 nm in a fluorospectrophotometer (Nanodrop Products, Wilmington, DE, USA). Equal amounts of protein (25 ␮g) of the hypothalamic extracts were separated by SDS-PAGE in a 7.5% polyacrylamide gel. Molecular mass markers of 10–250 kDa (Full-Range Rainbow, Amersham Biosciences) were applied in a lateral lane of the gel for visualization of separation and transfer quality. Following electrophoresis, the proteins were blotted onto a nitrocellulose membrane (0.45 ␮m; Amersham Biosciences) in a tank blotting system. Western blotting was performed under a current of 350 mA for 1.5 h in transfer buffer containing 0.22% NaHCO3 . After blocking for 1 h (5% skimmed milk powder in 0.1 M PBS with 0.2% Tween), membranes were rinsed in PBS and then incubated for 16 h at 4 ◦ C with nNOS antibody (SC-5302, Santa Cruz) diluted 1:1000 in PBS containing 0.05% Tween, 1% normal goat serum and 5% skimmed milk powder, followed by incubation with an HRPconjugated goat-anti-mouse (Amersham Biosciences) secondary antibody diluted 1:4000 in PBS containing 1% skimmed milk powder. A chemiluminescence reaction kit (enhanced chemiluminescence [ECL], Amersham Biosciences,) and X-ray film was used for detection of nNOS protein. The X-ray films were scanned and the ECLdetected protein bands quantified by ImageJ (NIH, public domain program). The results were transformed into arbitrary units. 2.10. Statistical analysis The data are presented as means ±S.E.M. Statistical analysis was performed by two-way ANOVA and a post hoc Student Newman–Keuls (SNK) test. A positive ratio means up-regulation and a negative one down-regulation. A P value ≤0.05 was used as criterion for significance.

3. Results 3.1. Structural damage caused by AV3V lesion All animals with an AV3V lesion exhibited damage to the periventricular tissue between the anterior commissure and the floor of the third ventricle, including the bilateral periventricular tissue from the lamina terminalis through the preoptic area and anterior hypothalamus. The lesion included the anterior wall of the third ventricle with the associated organum vasculosum of the lamina terminalis (OVLT), the ventral part of the median preoptic nucleus (MnPO), and parts of the preoptic and anteroventral periventricular nuclei (Fig. 1). As is typical for rats with an AV3V lesion, the experimental group displayed a significant acute and temporary adipsia in the 24 h period immediately following surgery. The animals regained the initiative to ingest water with

the phased withdrawal of offered sucrose solution. By the time of the experiments the animals had recovered ad libitum intake of water comparable to control animals. 3.2. Effect of AV3V lesions on hematocrit, osmolality and serum sodium levels following dehydration For hematocrit, two-way ANOVA revealed effects in dehydration (F(1,35) = 45.451; P < 0.001) and lesion (F(1,35) = 7.270; P = 0.011). With respect to plasma osmolality, two-way ANOVA revealed effects in dehydration (F(1,29) = 37.410; P < 0.001), lesion (F(1,29) = 41.013; P < 0.001) and interaction between the two (F(1,29) = 7.732; P = 0.009). Finally for serum sodium, two-way ANOVA also showed effects in dehydration (F(1,16) = 27.896; P < 0.001), lesion (F(1,16) = 34.439; P < 0.001) and interaction between the two (F(1,16) = 5.510; P = 0.032). The analysis by post hoc SNK revealed that sham dehydrated animals showed increases in hematocrit levels and plasma osmolality. Under water-replete conditions, AV3V lesioned-rats, though not showing any alteration in hematocrit, presented an increase in plasma osmolality and serum sodium levels. After dehydration, plasma osmolality and serum sodium were further increased in lesioned animals, accompanied by a blunted effect on hematocrit (P ≤ 0.05, Table 1). 3.3. Effect of AV3V lesions on SON Fos-IR and nNOS-IR following dehydration For Fos-IR, two-way ANOVA revealed effects in dehydration (F(1,32) = 66.557; P < 0.001), lesion (F(1,32) = 64.260; P < 0.001) and interaction between the two (F(1,32) = 97.570; P < 0.001). With respect to nNOS-IR, two-way ANOVA revealed an effect in lesion (F(1,28) = 19.770; P < 0.001). The analysis by post hoc SNK revealed that sham dehydrated animals showed increased c-fos expression (P ≤ 0.05) and a tendency to increase nNOS-IR (P = 0.06). In contrast, the AV3V lesion prevented this increase in c-fos expression and resulted in diminished nNOS-IR. While the AV3V lesion itself did not cause any change in the number of Fos-like

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Table 1 Hematocrit, plasma osmolality and serum sodium levels in AV3V-lesioned and sham-operated rats in water-replete condition and under dehydration. Measurements are expressed as mean ± S.E.M. for each group. Number of animals (n) is given in parentheses. Statistical analysis was performed using Two-Way ANOVA followed by SNK tests. Group

Treatment

Hematocrit (%)

Osmolality (mosmol/kg)

Serum sodium (mEq/L)

Sham Sham Lesioned Lesioned

Water-replete Dehydrated Water-replete Dehydrated

43 ± 2 (8) 56 ± 2 (8)* 39 ± 2 (11) 50 ± 2 (12)* , #

278 ± 7 (5) 298 ± 7 (6)* 299 ± 5 (11)# 352 ± 5 (11)* , #

140 ± 2 (4) 145 ± 2 (4) 146 ± 2 (6)# 159 ± 2 (6)* , #

* #

P ≤ 0.05 compared to water-replete group. P ≤ 0.05 compared to sham group.

Fig. 2. Fos immunoreactivity in coronal sections through the SON of rats kept under the following conditions: water replete, sham (A); water-replete, lesioned (B); 48 h dehydration, sham (C); 48 h dehydration, lesioned (D).

Table 2 Immunohistochemistry for Fos protein and nNOS in AV3V-lesioned and sham-operated rats in water-replete condition and under dehydration. Measurements are expressed as mean ± S.E.M. for each group. Number of animals (n) is given in parentheses. Statistical analysis was performed using Two-Way ANOVA followed by SNK tests. Group

Treatment

Number of Fos-IR neurons

Optic density ratio NNOS-IR/background

Sham Sham Lesioned Lesioned

Water-replete Dehydrated Water-replete Dehydrated

5 ± 7 (8) 125 ± 7 (8)* 17 ± 6 (10) 6 ± 6 (10)#

6 ± 1 (7) 8 ± 1 (6) 4 ± 1 (10)# 4 ± 1 (9)#

* #

P ≤ 0.05 compared to water-replete group. P ≤ 0.05 compared to sham group.

immunoreactive neurons (Fos-IR), it promoted a decrease in nNOSIR (P ≤ 0.05) (Figs. 2 and 3 and Table 2). 3.4. Effect of AV3V lesions on AVP plasma level and neurohypophysial content following dehydration For AVP plasma level, two-way ANOVA revealed an effect in the interaction between dehydration and lesion (F(1,13) = 6.599; P < 0.023). For the AVP neurohypophysial content, two-way ANOVA revealed effects in both dehydration (F(1,14) = 33.876; P < 0.001) and lesion (F(1,14) = 18.568; P < 0.001). The analysis by post hoc SNK revealed that sham dehydrated animals showed increased AVP plasma levels and decreased hor-

mone content in the neurohypophysis (P ≤ 0.05). The AV3V lesion prevented both the increase in AVP plasma levels and the decrease in neurohypophysial content in dehydrated animals (P ≤ 0.05). The AV3V lesion alone, however, did not affect AVP plasma levels, but we observed an increased hormone content in the neurohypophysis (P ≤ 0.05) (Fig. 4). 3.5. Effect of AV3V lesions on SON nNOS content following dehydration For nNOS content, two-way ANOVA revealed effects in dehydration (F(1,10) = = 7.819; P = 0.019), lesion (F(1,10) = 20.991; P = 0.001) and interaction between the two (F(1,10) = 12.007; P = = 0.006).

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Fig. 3. nNOS immunoreactivity in coronal sections through the SON of rats kept under the following conditions: water replete, sham (A); water-replete, lesioned (B); 48 h dehydration, sham (C); 48 h dehydration, lesioned (D).

Fig. 4. Effect of AV3V lesioning on plasma vasopressin (A) and neurohypophysial vasopressin content (B) in rats kept for 48 h under water-replete (white bar) or dehydration conditions (black bar). The data are represented as means ± S.E.M. (n = 3–5 animals per group). *P ≤ 0.05 compared to water-replete group, # P ≤ 0.05 compared to sham group.

The analysis by post hoc SNK revealed that dehydration in sham animals caused an up-regulation in the expression of nNOS as observed by western blotting quantification (P ≤ 0.05). The AV3V lesion was able to prevent this up-regulation (P ≤ 0.05). The lesion itself did not alter the content of nNOS in the SON (Fig. 5).

4. Discussion Our results showed, for the first time, that lesioning the AV3V region prevents the up-regulation of nNOS in the SON of rats following dehydration. An increase in NADPH-diaphorase activity, which is a histochemical marker for the presence of NOS, in dehydrated animals was first demonstrated by Pow [31]. Although we could not satisfactorily evidence increased NOS content by means of immunohistochemistry, quantitative western blot analysis clearly showed that the dehydration stimulus caused a robust increase in the content of the nNOS enzyme in the SON. These results corrobo-

Fig. 5. Effect of AV3V lesioning on the content of nNOS enzyme in the supraoptic nucleus of rats submitted to 48 h of dehydration (black bar) or water-replete conditions (white bar). The data are represented as means ± S.E.M. (n = 3–5 animals per group). *P ≤ 0.05 compared to water-replete group, # P ≤ 0.05 compared to sham group.

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rate those of previous investigations using different methodologies [26,40,51]. We tribute the apparently discrepant nNOS results between immunohistochemistry and western blotting observed in this study to the peculiarities of each method. Whereas immunohistochemical analysis is best suited for revealing the spatial distribution of a given target protein in a tissue sample, it is less suited for quantification due to the target’s spread in the tissue, increasing the possibility of background and unspecific noise. In contrast, western blotting is better suited for quantitative analysis, at the cost of a complete lack of spatial information. Moreover, in our immunohistochemistry analysis we chose the most representative region for quantifying nNOS-IR and therefore we do not quantify the entire SON. In western blotting, nNOS-IR was quantified from tissue homogenate of punches that contained the entire SON. Concurrent with the nNOS up-regulation in the SON we observed an increase in AVP plasma levels and a decrease in the neurohypophysial hormone content, suggesting that nNOS may be involved in the neuroendocrine alterations elicited by dehydration. Increase in NO production in SON punches and increase in AVP release was also seen in hyperosmotic rats [11]. Using both methodologies we could observe that lesion of the AV3V region plays a role in the expression of nNOS in the SON of rats subjected to dehydration, which confers significance to our results. Besides preventing nNOS up-regulation, the AV3V lesion, as expected, also prevented the increase in AVP plasma levels and a decrease in the neurohypophysial stocks of this hormone. This leads us to conclude that nNOS up-regulation should be of importance in the modulation of AVP secretion under dehydration. Furthermore, nNOS up-regulation may be a compensatory mechanism for the hormonal increase. As to now, the mechanism by which NO can alter AVP release is not well established and deserves further investigation. A recent study suggests that enhanced nNOS activity in the PVN and SON contributes to the elevation of peripheral AVP concentration, likely through a mechanism that involves prolactin [43]. Clearly, there is a complex interplay of multiple transmitter systems and unraveling such a complex system constitutes a significant challenge and is beyond the scope of the present study. At the cellular level, NO may act as a conventional neurotransmitter, thereby inhibiting the neuronal stimulation involved in hormone release [53]. This is supported by reports showing that NO can inhibit the firing activity of AVP and OT magnocellular neurons [13,19,36]. Alternatively, NO could act indirectly on these hypothalamic nuclei through local activation of a GABAergic inhibitory system [50,54] that may also be activated by N-methyl-d-aspartate (NMDA)-type glutamate receptors that can be found together with NO at certain synaptic sites [16]. Another possibility would be that NO inhibits presynaptic neurons in the circumventricular organs, and thus decrease the release of neurotransmitters such as Ang II that simulate the vasopressin secretion [2]. NO can diffuse into neurons and glial cells and bind to soluble guanylyl cyclase (sGC), this leading to an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). Since cGMP can inhibit or stimulate cellular functions by means of several mechanisms, such as protein kinase or phosphodiesterase activities or ion channel regulation, NO might also act on vasopressinergic neurons through this mediator, as we recently observed in experimental sepsis [27]. Nevertheless there is also evidence that the response of vasopressinergic neurons to an osmotic stimulus can occur independent of the cGMP pathway, or NO may react directly with thiol groups in cell membrane proteins and thus exert its biological effects [16,39]. Electrolytic lesion of the AV3V region disrupts its connections with the hypothalamus leading to impaired neuronal activity and decreased c-fos expression [23,33], reduction in AVP plasma levels, and an increase in the neurohypophysial stocks of this hormone

[25,28]. In the present study, we observed a decrease in AVP secretion in AV3V lesioned rats, and this was accompanied by a reduction in c-fos expression. Nevertheless, although the electrolytic lesion of the AV3V region dramatically prevented the increase in c-fos expression observed following dehydration, it did not completely abolish c-fos expression, as already shown in a previous study from our laboratory [33]. This could be due to the existence of afferents from the SFO, and/or the dorsal MnPO to the SON and PVN that usually remain intact after AV3V lesion and which could sustain the basal c-fos expression observed in hypothalamic magnocellular neurons [33,47]. The increase in osmolality and hypernatremia could be another reason for the basal c-fos expression after dehydration, as magnocellulars are osmosensitive [3,21]. Moreover, the hypovolemia, maybe reflected by the increased hematocrit could also be another stimulus for c-fos expression. Hypovolemia may sensitize volume receptors that, through NTS afferents, may also contribute to the activation of hypothalamic nuclei. Additionally, noradrenergic input into magnocellular neurons from brainstem structures, such as NTS, was seen to alter the enzymatic activity and the expression of the nNOS in the SON magnocellular neurons [12]. These findings indicate that the central NO production may be increased after noradrenergic stimulation occurring during hypotension, what could affect the neurohypophyseal release of AVP. We conclude that the AV3V region clearly plays a role in the expression of nNOS in the SON of rats submitted to dehydration suggesting that NO produced as a consequence of this up-regulation may modulate AVP secretion. Disclosure statement The authors have nothing to disclose. Acknowledgments The authors thank Nadir M. Fernandes, Milene M. Lopes for technical assistance. We also thank Drs. José Antunes-Rodrigues and Lucila L.K. Elias, Faculty of Medicine of Ribeirão Preto, University of São Paulo, for providing the infrastructure for the RIA analyses. Financial support from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) is gratefully acknowledged. References [1] J. Antunes-Rodrigues, M. de Castro, L.L. Elias, M.M. Valenca, S.M. McCann, Neuroendocrine control of body fluid metabolism, Physiol. Rev. 84 (2004) 169–208. [2] J.S. Bains, A.V. Ferguson, Angiotensin II neurotransmitter actions in paraventricular nucleus are potentiated by a nitric oxide synthase inhibitor, Regul. Pept. 50 (1994) 53–59. [3] C.W. Bourque, L.P. Renaud, Activity patterns and osmosensitivity of rat supraoptic neurones in perfused hypothalamic explants, J. Physiol. 349 (1984) 631–642. [4] D.S. Bredt, P.M. Hwang, S.H. Snyder, Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature 347 (1990) 768–770. [5] D.S. Bredt, S.H. Snyder, Nitric oxide: a physiologic messenger molecule, Annu. Rev. Biochem. 63 (1994) 175–195. [6] J. Calka, C.H. Block, Relationship of vasopressin with NADPH-diaphorase in the hypothalamo-neurohypophysial system, Brain Res. Bull. 32 (1993) 207–210. [7] P.B. Correa, J.A. Pancoto, G.R. de Oliveira-Pelegrin, E.C. Carnio, M.J. Rocha, Participation of iNOS-derived NO in hypothalamic activation and vasopressin release during polymicrobial sepsis, J. Neuroimmunol. 183 (2007) 17–25. [8] C.A. Dawson, J.H. Jhamandas, T.L. Krukoff, Activation by systemic angiotensin II of neurochemically identified neurons in rat hypothalamic paraventricular nucleus, J. Neuroendocrinol. 10 (1998) 453–459. [9] T.M. Dawson, D.S. Bredt, M. Fotuhi, P.M. Hwang, S.H. Snyder, Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 7797–7801. [10] F. Feihl, B. Waeber, L. Liaudet, Is nitric oxide overproduction the target of choice for the management of septic shock? Pharmacol. Ther. 91 (2001) 179–213. [11] E.R. Gillard, C.G. Coburn, A. de Leon, E.P. Snissarenko, L.G. Bauce, Q.J. Pittman, B. Hou, M.C. Curras-Collazo, Vasopressin autoreceptors and nitric oxidedependent glutamate release are required for somatodendritic vasopressin

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