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Serotonergic system involvement in the inhibitory action of estrogen on induced sodium appetite in female rats
Physiology & Behavior 104 (2011) 398–407
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Serotonergic system involvement in the inhibitory action of estrogen on induced sodium appetite in female rats Carolina Dalmasso a, José Luis Amigone b, Laura Vivas a,⁎ a b
Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC-CONICET, Córdoba, Argentina Sección de Bioquímica Clínica, Hospital Privado, Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 20 April 2011 Accepted 22 April 2011 Keywords: Serotonergic system Sodium appetite Estrous cycle Vasopressinergic neurons OVLT DRN
a b s t r a c t This study of the participation of the serotonergic system in the inhibitory effect of estrogen on induced sodium appetite in female rats explores sodium appetite induced by Furosemide and low sodium diet treatment (DEP) in normally cycling rats and in ovariectomized rats with and without estradiol replacement (OVX, OVX + E2) . We also analyzed the neural activity of serotonergic neurons of the dorsal raphe nucleus (DRN) as well as the activity of other brain nuclei previously found to be involved in sodium and water balance in sodium depleted animals without access to the intake test. For this purpose, we examined the brain Fos, Fos-serotonin and Fos-vasopressin immunoreactivity patterns in diestrus (D), estrus (E), OVX and OVX + E2 rats subjected to DEP. Female rats in E and OVX + E2 exhibited a significant decrease in induced sodium intake compared with females in D and OVX. This estrogen-dependent inhibition on induced sodium appetite (approximately 50% reduction) can be correlated with changes in Fos activation observed in the organum vasculosum of the lamina terminalis (OVLT) and DRN, in response to sodium depletion. Given our previous observations in males, the expected sodium depletion-induced activity of the OVLT was found to be absent in OVX + E2 females, while the usual inhibitory tonic activity of serotonergic neurons of the DRN, instead of decreasing after sodium depletion, increases or remains unchanged in OVX + E2-DEP and E-DEP females, respectively. Regarding urinary water and sodium excretion 3 h after furosemide treatment, E-DEP and OVX + E2-DEP animals excreted smaller volumes of more highly concentrated urine than depleted D and OVX rats. Twenty hours after sodium depletion, the same groups of animals also showed a significant increase in the number of Fos-AVP immunoreactive neurons within the supraoptic nucleus, compared with D-DEP. In summary, our results demonstrate an estrogen-dependent inhibition of induced sodium appetite in normally cycling rats and ovariectomized animals with estradiol replacement, which may involve an interaction between excitatory neurons of the OVLT and inhibitory serotonergic cells of the DRN. The main finding is thus serotonergic system involvement as a possible mechanism in the inhibitory action of estrogen on induced sodium appetite. © 2011 Elsevier Inc. All rights reserved.
1. Introduction A review of the literature indicates that, after body water and sodium loss, the excitatory effects of angiotensin II (ANG II) and the brain osmo-sodium receptors that stimulate sodium appetite involve interactions with inhibitory hindbrain serotonin (5-HT) circuits in males rats [3,5,6,8,17,20,21,31–34,59,62]. Specifically, for a normal sodium appetite sensation, and consequently appropriate salt drinking after electrolyte and fluid depletion, circulating ANG II should act centrally, both to activate brain angiotensinergic mechanisms that stimulate salt appetite, and also to inhibit brain 5-HT mechanisms that tonically inhibit sodium ingestion in males. ⁎ Corresponding author at: Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC-CONICET, Córdoba, Argentina. Tel.: +54 351 4681465/66x108, 120; fax: +54 351 4695163. E-mail addresses: [email protected], [email protected] (L. Vivas). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.04.029
The central 5-HT circuits underlying this interaction mainly include bi-directional connections between the lamina terminalis and 5-HT neurons of the dorsal raphe nucleus (DRN) [4,24,33,59]. The lamina terminalis (LT) plays a major role in many aspects of body fluid homeostasis [11,15,18,29,36,42]. Anatomically, contains three forebrain structures: the subfornical organ (SFO), the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT). The SFO and OVLT lack a blood-brain barrier and contain cells which are sensitive to humoral signals such as changes in plasma and cerebrospinal fluid sodium concentration, osmolality and ANG II levels [29,36,42]. These neural groups project, directly or indirectly via the MnPO, to the magnocellular portion of the supraoptic nucleus (SON) and the paraventricular hypothalamic nucleus (PVN). Vasopressin and oxytocin synthesis and release, and subsequent hydroelectrolytic balance regulation, are modulated by means of these projections [11,29]. Our previous studies have shown that the activity of neurons of the LT as well as of 5-HT cells within the DRN is affected by body sodium
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status in male rats [3,17,18,20,21]. Fos expression, used as a marker of neuronal activity, increases after sodium depletion and also after sodium consumption in cells of the SFO and OVLT. However, in serotonergic DRN cells, c-fos expression decreases when animals are sodium depleted and increases when animals are either in sodium balance or in the process of restoring sodium balance through ingestion [3,17,20,21]. These results are consistent with the idea that serotonergic cells originating in the DRN tonically inhibit sodium appetite, maybe via the LT. Such an inhibitory influence would likely be reduced in states of fluid deficiency and be increased as animals ingest water and NaCl to restore hydromineral balance. Data obtained by Reis and his colleagues [6] have also implicated the DRN as an inhibitory structure for the control of water and sodium intake in males. In these works, male rats with lesions of the DRN had enhanced sodium consumption under both ad libitum (i.e., need-free) conditions and after different experimental procedures that stimulate sodium appetite. These also suggest that the DRN tonically inhibits sodium intake. On the other hand, studies analyzing estrogen modulation of body fluid balance have been conducted both during the estrous cycle as well as in ovariectomized animals with hormone replacement and, although sometimes contradictory, all together suggest an inhibitory effect of estrogen on sodium consumption [1,7,25,43]. For example, Antunes-Rodrigues et al. (1963) [1] reported that spontaneous sodium intake decreases during estrus, a stage with high estrogen concentrations [35], and increases during diestrus. Furthermore, Scheidler et al. (1994) [43] showed that adult male rats and ovariectomized females ingested 50 to 150% more saline than intact females, when pronounced sodium appetite was induced by a prolonged sodium-free diet or mineralocorticoid administration. In addition, estrogen replacement in ovariectomized animals also decreased sodium intake levels. A series of studies also support an estrogen modulation upon the serotonergic system; it has been shown that estrogen: i) modulates 5-HT synthesis and release [39,40], ii) increases the basal firing rate of serotonergic neurons in female rats [38], iii) augments 5-HT transporter mRNA in the DRN of ovariectomized animals [30,52], and iv) chronically administered to ovariectomized rats, increases tryptophan hydroxylase-2 (TPH2) mRNA in the DRN [13,17]. In summary, as a number of studies have postulated the involvement of estrogen and the serotonergic systems in the inhibition of sodium appetite and the existence of physiological interactions between these, the following question arises: Is this inhibitory effect of estrogen on induced sodium intake in females mediated by the serotonergic system? We hypothesize that female rats in estrus and ovariectomized animals treated with estrogen replacement (OVX + E2) may drink less saline solution after fluid depletion than ovariectomized (OVX) and intact rats during diestrus, in part because the inhibitory serotonergic systems of the DRN are more active in animals with elevated plasma estrogen concentrations. To test this idea, we quantified the number of cells that are double-immunolabeled with Fos-5-HT immunoreactivity within the DRN in diestrus, estrus and ovariectomized animals (with and without estradiol replacement) subjected to sodium depletion by a Furosemide and low sodium diet treatment (DEP). Estrogen also modulates AVP synthesis and release [4,9,10,14,16,22, 23,38,46–50,55,61]. An increase has been reported in plasma AVP concentrations as well as in osmotically stimulated AVP mRNA, in female rats in estrus and ovariectomized animals treated with estrogen replacement [10,16,23,46]. However, the effects of estrogen on AVP synthesis and release are not conclusive; it seems to depend on the methodological approach utilized, since other authors have shown that estrogen has no effect on AVP release and osmotically stimulated AVP mRNA expression in rats [4,9,38]. Taking into account the above mentioned evidences we aim to analyzed the pattern of Fos immunoreactivity within vasopressinergic hypothalamic nuclei and along the
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neuronal groups of the Lamina terminalis nuclei. Additionally, a different set of females were subjected to sodium depletion by DEP, and water and sodium ingestion and excretion were analyzed. 2. Materials and methods 2.1. Animals Sixty-day-old Wistar-derived female rats, born and reared in the vivarium of the Instituto Ferreyra (INIMEC, Córdoba, Argentina) were used in the experiments. Animals weighing 190–230 g were individually housed in metabolic cages, in a temperature-controlled environment, with a 12:12 h light/dark cycle, beginning at 08:00 h. Food, distilled water and 2% sodium solution were available ad libitum during the training period. All experimental protocols were approved by the appropriate animal care and use committee of our institute, following the guidelines of the Public Health Service Guide for the Care and Use of Laboratory Animals. 2.2. Experimental design General considerations: Animals were randomly assigned to different groups defined by the combination of furosemide treatment (animals were administered either furosemide combined with low sodium diet [DEP] or saline with normal sodium diet [CON] and hormonal status (females either in diestrus [D], estrus [E], ovariectomized [OVX] or ovariectomized + estradiol replacement [OVX + E2]). The final groups were arranged as follows: (i) diestrus females with saline and normal sodium diet (D-CON); (ii) diestrus females with furosemide and low sodium diet (D-DEP); (iii) estrus females with saline and normal sodium diet (E-CON); (iv) estrus females with furosemide and low sodium diet (E-DEP); (v) ovariectomized with saline and normal sodium diet (OVX-CON); (vi) ovariectomized with furosemide and low sodium diet (OVX-DEP); (vii) ovariectomized + estradiol replacement with saline and normal sodium diet (OVX + E2-CON); (viii) ovariectomized + estradiol replacement with furosemide and low sodium diet (OVX + E2-DEP). Sodium and water intake tests were performed with a minimum of ten subjects. Additionally, for immunohistochemical analysis, a different set of animals with at least five subjects of each group, were sacrificed before the intake test. 2.3. Surgical procedure and estradiol (E2) replacement Bilateral ovariectomy was performed under aseptic conditions in sixty-day-old rats. Anesthesia was induced by ketamine (60 mg/ kg, i.p.) in combination with xylazine (7.5 mg/kg, i.p.). Estrogen replacement began two weeks after ovariectomy. Specifically, estradiol (E2) (20 μg 17-β-estradiol-3-benzoate/0.2 ml/animal, s.c., Schering Laboratories) or vehicle (neutral corn oil, 0.2 ml/animal, s.c., Gador Laboratories) was injected in OVX + E2 and OVX groups, respectively, twice a day during five consecutive days. Intact female rats were sham-operated and injected with neutral corn oil under the same administration schedule as OVX + E2 and OVX groups. The reproductive status of each animal was determined by microscopic analysis of vaginal smears; only females having two or more regular estrous cycles were used for these experiments. 2.4. Sodium depletion Sodium appetite was stimulated by acute treatment with furosemide (Lasix, Aventis Pharma, Brazil) in combination with a low sodium diet (DEP). Animals were injected subcutaneously with furosemide (20 mg/ kg in isotonic saline vehicle). Immediately after treatment, the animals were placed into clean individual metabolic cages, and they were allowed access to distilled water and low sodium diet (corn flour: 0%
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NaCl) for a 24 h period. Control rats were subjected to a similar procedure, receiving isotonic saline injections and a standard diet (0.2% NaCl), instead. After 24 h of sodium depletion, a two-bottle preference test was performed. Food was removed and two bottles containing distilled water or 0.34 M NaCl solution were offered. Ingested fluid volumes were recorded repeatedly during the intake test, (at 15, 30, 60, 90 and 120 min time points). For statistical analysis, intake scores were expressed as ml/100 g of body weight. Intake measures are corrected for body weight taking into account that there are differences based on treatment (depleted vs. non-depleted) and also due to ovariectomy and hormone replacement.
avidin–biotin peroxidase complex (Vector, 1:200 dilution in 1% NHS-PB 0.1 M) for 2 h at room temperature. The peroxidase label was detected using diaminobenzidine hydrochloride (DAB; Sigma). This method produces a brown cytoplasmatic reaction product, clearly distinguishable from the typical black nuclear stain. Finally, the free-floating sections were mounted on gelatinized slides, air-dried overnight, cleared in xylene, and placed under a coverslip with DePeX. Controls for Fos-ir, Fos-AVP-ir and Fos-5HT-ir were conducted by processing sections without the primary antiserum. No Fos-ir, Fos-5HT-ir or FosAVP-ir neurons were observed after this control procedure. 2.7. Cytoarchitectural and quantitative analysis
2.5. Analysis of urine and plasma osmolarity and electrolytes Urine volume of each rat was recorded 3 h after saline or furosemide injection. Urine osmolarity was measured using an Advanced ® MicroOsmometer Model 3300. This osmometer uses the technique of freezing-point depression to measure osmolarity, expressed as mOsm/ kg H2O. Urine electrolytes were measured using an Ion Selective Electrode (Hitachi Modular P + ISE. Roche Diagnostic). The amount of sodium excreted in urine was calculated by multiplying the sodium of urine by its volume. Urine volumes are corrected for body weight taking into account that there are differences based on treatment (depleted vs. non-depleted) and also due to ovariectomy and hormone replacement. 2.6. Staining procedure for Fos, Fos-5-HT and Fos-AVP immunoreactivity (ir) As mentioned previously, for immunohistochemical analysis, a different set of depleted and non-depleted animals were used. It is important to note that this set of animals was not allowed access to the intake test; we analyzed the brain neural activity during the appetitive phase. The rats were subjected to furosemide or control injection and 22 h later were anesthetized with chloral hydrate (0.6 ml/ 100 g bw) and perfused transcardially with normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2). The brains were removed, fixed in the perfusion solution overnight, and stored at 4 °C in PB 0.1 M containing 30% sucrose. Two equivalent sets of coronal sections (40 μm) were cut with a freezing microtome. One of the sets was processed for Fos-5HT immunodetection. The remaining set was processed for Fos-AVP immunostaining. The staining procedures followed the previously described doublelabeling technique [17,18]. Briefly, sections were placed in a mixture of 10% H2O2 and 10% methanol until oxygen bubbles ceased appearing. They were then incubated in 10% normal horse serum (NHS, Gibco, Auckland, NZ)) in PBS for 2 h to block non-specific binding. After blocking, sections were incubated overnight at room temperature in a rabbit anti-Fos antibody (against a synthetic 14-amino acid sequence corresponding to residues 4–17 of human Fos, Oncogene Science, Manhasset, NY, USA) diluted 1:10,000 in PBS 0.1 M containing 2% NHS and 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO,USA). Twenty-four hours later, sections were incubated in biotin-labeled anti-rabbit immunoglobulin (1:200) for 2 h at room temperature. Afterwards, sections were incubated with avidin–biotin peroxidase complex (Vector, 1:200 dilution in 1% NHS-PB 0.1 M) for 2 h at room temperature. The peroxidase label was detected using diaminobenzidine hydrochloride (DAB; Sigma Chemical Co., St. Louis, MO, USA) intensified with 1% cobalt chloride and 1% nickel ammonium sulfate. This method produces a blue-black nuclear reaction product. After washing in PBS 0.01 M, sections were blocked in 10% NHS in PBS, for 2 h, and then one of the sets was incubated in rabbit anti-5-HT polyclonal antibody (ImmunoStar) diluted 1:5000 and the other in rabbit anti-vasopressin polyclonal antibody (Chemicon) diluted 1:10,000, in a solution containing PBS 0.1 M, 2% NHS and 0.3% Triton X-100. After 48 h of incubation, all sections were washed in PBS 0.01 M, and then incubated in biotin-labeled anti-rabbit immunoglobulin (1:200) for 2 h at room temperature. Then they were incubated with
The brain nuclei exhibiting Fos-ir were identified and delimited according to the rat brain atlas [37]. The Fos-ir neurons of all nuclei were counted at one level. The distance from the bregma of the corresponding plates is as follows: for OVLT= −0.20 mm; SFO =−0.92 mm; MnPO = −0.30 mm; SON=−.3 mm; PaLM=−1.80 mm; DRN =−8.00 mm. Immunostaining cells were quantified with a computerized system that included a Zeiss microscope equipped with a DC 200 Leica digital camera attached to a contrast enhancement device. Images were digitalized and analyzed by Scion Image PC, based on the National Institutes of Health Image 1997 version. Representative sections in each group were acquired at exactly the same level, with the aid of the Adobe Photoshop image analysis program (version 5.5). The counting was done in at least five animals of each condition and was repeated at least twice on each section analyzed, to ensure that the number of profiles obtained was similar. The investigator who conducted the cell counting was blind to the experimental condition. 2.8. Statistical analysis All data are expressed as means ± SEM. Data were analyzed, using Statistica (StatSoft, Tulsa, OK), by means of a 2-way ANOVA, with hormonal status (D, E, OVX or OVX + E2) and furosemide treatment (saline or furosemide) as independent factors. Whenever the analysis showed either main effects or statistically significant interactions, posthoc comparisons were performed (Least Significant Difference -LSDtest, with significance levels set at p b 0.05). In experiment 1, the dependent variables under analysis were urine osmolarity as well as electrolyte concentration. Urine osmolarity and electrolytes obtained 3 h after sodium depletion were analyzed by means of a 1-way ANOVA with hormonal status as the independent factor. Given that no urine samples were obtained from non-depleted animals 3 h after treatment because they did not urinate, only furosemide-treated animals were included in the analysis. In experiment 2, consumption levels of 0.34 M NaCl solution and distilled water were included as repeated measures. In experiment 3, immunohistochemical data derived from Fos and double labeling Fos-5-HT and Fos-AVP immunohistochemistry were also analyzed. 3. Results 3.1. Experiment 1: Effects of hormonal status during estrous cycle and estradiol replacement in OVX rats on water and sodium excretion induced by DEP treatment Urine volume, osmolarity and electrolyte concentration 3 h after DEP treatment: Five different one-way ANOVAs were performed in order to analyze urine volume, osmolarity, and urinary sodium, chloride and potassium concentrations of animals subjected to DEP treatment. The significant main effect of hormonal status was recorded in all cases. [Urine volume: F(3,79) = 6.60; osmolarity: F(3,75) = 26.31; urinary sodium concentration: F(3,75) = 21.90; urinary chloride concentration: F(3,75) = 19.56; urinary potassium concentration: F(3,77) = 12.53. All p values b .005].
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Post-hoc analysis of urinary volume (Fig. 1) showed that E, OVX and OVX + E2 excreted significantly lower volumes than D animals. Moreover OVX + E2 females excreted less urine when compared with the OVX group. Post-hoc tests of urine osmolarity (table 1) indicated that E, OVX and OVX + E2 groups exhibited higher osmolarity than D rats. Interestingly, E and OVX + E2 groups registered greater osmolarity than that observed in OVX animals. Similarly, urine sodium concentration in E, OVX and OVX + E2 females was higher than in D rats. Furthermore, urine sodium concentration in OVX was significantly lower than in E and OVX + E2 groups. Besides, OVX + E2 females excreted the highest concentration of this electrolyte (Table 1). To sum up, these results indicate that, after sodium depletion, sodium concentration varies considerably across hormonal status, even when all animals have excreted the same total amount of this electrolyte. Specifically, E and OVX + E2 rats excreted the lowest urine volumes compared with the remaining groups and showed the highest sodium concentration scores.
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Table 1 Urine osmolarity (mOsm/kg) and urine Na+ concentration (mEq/l) of D, E, OVX and OVX + E2 animals accumulated 3 h after furosemide/low sodium diet (DEP) treatment. DEP
D E OVX OVX + E2
Osm (mOsmol/kg)
Na+ (mEq/l)
245.22 ± 17.17 356.79 ± 8.11 277.33 ± 9.11 341.76 ± 5.48
69.38 ± 6.77 98.66 ± 2.59 86.51 ± 2.69 109.06 ± 1.44
Results are expressed as mean ± SE. DEP: animals subjected to furosemide along with low sodium diet. D: female rats in diestrus; E: female rats in estrus; OVX: ovariectomized female rats; OVX + E2 ovariectomized female rats + estradiol replacement.
Water consumption was also affected by sodium depletion (Fig. 2). Furosemide combined with a low sodium diet induced a significant increase in water intake (F(1,82) = 133.75; p b .0001) in E-DEP, OVXDEP and OVX + E2-DEP. However, when considering hormonal status, there were no differences between the groups. 3.3. Experiment 3
3.2. Experiment 2: Effects of hormonal status during estrous cycle and estradiol replacement to OVX rats on sodium and water intake induced by DEP treatment The statistical analysis showed a main effect of furosemide factor (F(1.82) = 133.75; p b .001), hormonal status (F(3,82) = 11.93; p b .0001) and solution (F(1,82) = 49.17; p b .0001). Additionally, a significant interaction between the three factors was observed (F(3,82) = 5.35; p b .005). Post-hoc comparisons of sodium intake (Fig. 2) indicated that this was increased by ovariectomy in non-depleted animals: OVX rats showed a significant increase in sodium intake compared with E and OVX + E2 groups. It is important to note that these groups (E-CON and OVX + E2-CON) seemed to consume smaller amounts of sodium than D-CON subjects, although this difference did not reach significant levels. As expected, it was observed that furosemide depletion induced an increase in sodium intake in comparison to non-depletion treatment. After 120 min intake test, E-DEP animals ingested significantly lower sodium volumes compared with D-DEP and OVX-DEP groups. In addition, estradiol replacement significantly decreased sodium intake in comparison to D-DEP and OVX-DEP. Ovariectomized-depleted animals ingested the highest sodium volumes registered. These results are consistent with an inhibitory effect of estrogen on sodium consumption.
Fig. 1. Urine volume obtained 3 h after furosemide administration in the different groups: diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.05 compared with D-DEP. : p b 0.05 compared with OVX-DEP.
3.3.1. Effects of hormonal status during estrous cycle and estradiol replacement to OVX rats on Fos-ir along the lamina terminalis induced by DEP treatment Organum vasculosum of the lamina terminalis (OVLT): The inferential analysis showed a main effect of furosemide treatment (F(1,40) = 85.97; p b .0001) as well as a main effect of hormonal status (F(3,40) = 12.52; p b .0001). A significant interaction was also observed between the included factors (F(3,40) = 22.29; p b .0001) (Fig. 3).
Fig. 2. Sodium intake (panel A) and water intake (panel B) (ml/100 g bw; 2 h) of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON; white bars) or furosemide/low sodium diet (DEP; black bars). Vertical lines represent SEM. +: p b 0.001 compared with CON. : p b 0.05 compared with E-CON and OVX + E2-CON. : p b 0.05 compared with D-DEP, OVX-DEP. *: p b 0.005 compared with D-DEP.
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Fig. 3. Average number of Fos immunoreactive (Fos-ir) neurons in the OVLT, SFO and MnPO of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.05 compared with CON. : p b 0.05 compared with D-DEP. : p b 0.01 compared with E-DEP and OVX-DEP. *: p b 0.05 compared with D-CON and E-CON.
A posteriori comparisons showed that in D-DEP, E-DEP, and OVXDEP groups, sodium depletion induced a significant increase in Fos immunoreactivity compared with their respective controls without depletion. In contrast, furosemide treatment did not alter the OVLT basal activity of the OVX + E2 group (Figs. 3, 4). Regardless of the hormonal status exhibited, non-depleted animals did not differ in terms of Fos expression. After sodium depletion, it was observed that the D-DEP group exhibited the highest number of Fos immunostained neurons and the OVX + E2-DEP group the lowest. Subfornical Organ (SFO): The analysis of variance showed a main effect of furosemide factor (F(1,35) = 638.02; p b .0001), and a main effect of hormonal status (F(3,35) = 5.09; p b .005). Furthermore, a significant interaction between the factors was observed (F(3,35) = 4.20; p b .05). Post-hoc comparisons indicated that non-depleted OVX + E2-CON animals exhibited a higher number of Fos positive cells when compared with D-CON and E-CON groups (Fig. 3). The analysis also showed that sodium depletion induced a significant increase in Fos immunoreactivity in all depleted groups in relation to their respective controls without depletion. Additionally, furosemide treatment significantly increased the number of Fos immunostaining cells in E-DEP and OVX-DEP rats, compared with the D-DEP group.
Median Preoptic nucleus (MnPO): In this nucleus, the analysis of the data showed a main effect of furosemide (F(1,42) = 41.32; p b .0001) and a main effect of hormonal status (F(3,42) = 3.51; p b .05). The interaction between both factors did not reach significant levels. Post-hoc comparisons indicated that sodium depletion induced a decrease in the activity of this nucleus, as evidenced by diminished Fos immunostaining (Fig. 3). Considering hormonal status, the analysis showed that OVX group exhibited the highest activation, while E animals presented the lowest Fos-ir. 3.3.2. Effects of hormonal status on DEP-induced activity in serotonergic neurons of the dorsal raphe nucleus (DRN) Fig. 5 shows a schematic representation of the DRN detailing the subnuclei that were evaluated in the analysis of Fos-5-HT immunoreactive pattern. The ANOVA showed a main effect of furosemide factor (F(3,28) = 32.83; p b .0001) as well as a main effect of hormonal status (F(1,28) = 7.50; p b .01). An interaction between the factors (F(3,28) = 13.01; p b .0001) was also observed. A posteriori comparisons (Fig. 6) indicated that OVX-CON presented significantly lower number of Fos positive cells than the remaining nondepleted groups. In D-DEP and OVX-DEP groups, furosemide depletion induced a significant decrease in Fos-5-HT immunoexpression
Fig. 4. Pattern of Fos immunoreactivity in the OVLT of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Plates A, B, C and D (10x) show D, E, OVX and OVX + E2 groups treated with CON. Plates E, F, G and H (10x) show D, E, OVX and OVX + E2 groups treated with DEP. Scale bar = 100 μm.
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3.3.3. Effects of hormonal status on DEP-induced activity in vasopressinergic neurons of the supraoptic (SON) and magnocellular subdivision of the paraventricular nucleus (PaML) It is important to note that we analyzed the neural activity of vasopressinergic neurons of non-depleted and depleted animals before any fluid reestablishment. As expected during this stage, we found a relatively low neural activation in terms of Fos-AVP-ir. Within the SON, the ANOVA showed a main effect of furosemide treatment (F(1,38) = 70.15; p b .0001) as well as a main effect of hormonal status (F(3,38) = 150.08; p b .0001), and also an interaction between the factors considered (F(3,38) = 13.89; p b .0001). Post-hoc comparisons (Fig. 8) indicated that, under non-depleted condition, E and OVX + E2 animals exhibited higher double-labeled cells than D and OVX. It should be noted that OVX + E2 showed the highest number of Fos-AVP-ir cells. Sodium depletion caused an increase in Fos-AVP immunostaining only in E, OVX and OVX + E2 females (Fig. 9). With regard to the neural activity observed in vasopressinergic neurons of the PaLM subdivision, inferential analysis showed a main effect of furosemide treatment (F(1,37) = 7.27; p b .01) as well as a main effect of hormonal status (F(3,37) = 8.15; p b .0005). Regarding furosemide treatment, as expected, depleted animals exhibited a significant increase in Fos-AVP neurons compared with their respective controls (Fig. 8). Post-hoc comparisons showed significantly higher activation (operationalized as an increase in Fos-AVP double-labeled immunoreactivity) in E and OVX + E2 females than in D and OVX rats. In summary, the vasopressinergic activity changes observed between the different groups may reflect hormonal status influences, and can be correlated with the renal excretory response observed in those groups of animals 3 h after furosemide treatment, since E and OVX + E2 rats excreted the lowest urine volumes compared with the remaining groups and showed the highest sodium concentration scores. Fig. 5. Schematic representation of the dorsal raphe nucleus showing the specific subnuclei that were evaluated in the analysis of Fos-5-HT immunoreactive pattern (upper). Microphotography (5x) of the DRN that illustrates the corresponding schematic representation (lower).
compared with their respective non-depleted controls, while provoking a significant increase in the number of double-labeled cells in OVX + E2-DEP in comparison with OVX + E2-CON. In addition E-DEP and OVX + E2-DEP females exhibited significantly higher activation than D-DEP and OVX-DEP groups (Fig. 6, 7).
Fig. 6. Average number of double-immunolabeled Fos-serotonin (Fos-5-HT) neurons in the DRN of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.01 compared with CON. *: p b 0.01 compared with D-CON, E-CON and OVX + E2-CON. : p b 0.01 compared with D-DEP and OVX-DEP.
4. Discussion The main result of this work is consistent with involvement of the serotonergic system as a possible mechanism in the inhibitory action of estrogen on induced sodium intake in female rats. This behavioral inhibition is related to changes in Fos activation observed in serotonergic cells of the DRN and with reduced Fos activity found within OVLT neurons. In other words, hormonal status during the estrous cycle and estradiol replacement after ovariectomy change the neural activity induced by sodium depletion in cells of the OVLT and serotonergic cells of the DRN. In particular, there is an interesting correlation between the reduced Na intake (approximately a 50% reduction) in response to sodium depletion observed in estrus and ovariectomized rats treated with estradiol and the neural activity found in the stimulatory neurons of the OVLT and the inhibitory serotonergic neurons of the DRN. Thus, taking into account our previous observations in males, our results in females showed that the expected sodium depletion-induced activity of the OVLT is absent in ovariectomized rats treated with estradiol, while the usual inhibitory tonic activity of serotonergic neurons of the DRN increases or remains unchanged, rather than decreases, after sodium depletion. Our results also demonstrate a hormonal status modulation of stimulated sodium appetite, suggesting, like prior studies, an inhibitory effect of estrogen on sodium consumption [1,43] and providing new evidence about a possible mechanism involved in such inhibition. It is important to note that one of the lamina terminalis nuclei, the OVLT, often associated with sodium appetite regulation [15,42] showed a correlated neural activity with the level of stimulated sodium ingestion in the present study. The intact females in estrus and the OVX + E2 rats, with relatively lower basal and induced sodium intake, also showed a relatively lower or absent
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Fig. 7. Pattern of double immunoreactivity Fos-5-HT in the DRN of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Plates A, B, C and D (20x) show D, E, OVX and OVX + E2 groups treated with CON. Plates E, F, G and H (20x) show D, E, OVX and OVX + E2 groups treated with DEP. Plates in the upper right corner show higher magnifications (40x) of the neurons pointed by arrows. Scale bar = 50 μm.
activation of the OVLT. On the other hand, diestrus and ovariectomized animals showed higher basal and induced sodium consumption and also had greater OVLT activation induced by sodium depletion. Along the other nuclei of the lamina terminalis, the SFO and the MnPO, we found a similar pattern of activity to that observed in male rats after sodium depletion [18]: specifically, an increased number of Fos-ir cells along the SFO and a diminished number within the MnPO, which we believe signify, respectively, a stimulatory and inhibitory role of these nuclei on sodium intake behavior. The furosemide-low sodium diet treatment is a well-known animal model in which the renin–angiotensin system is stimulated. Numerous previous reports propose an interaction between the estrogen and angiotensinergic receptors along the LT nuclei as an explanation of the estrogen-mediated effect on drinking behavior [19,26,27,31,57,58] (Fig. 10). Lind (1986) has anatomically demonstrated a neural angiotensinergic connection originating in the LT nuclei projecting to the DRN. In addition, ANG II injected via the carotid artery or into the SFO enhances the electrical activity of SFO neurons that project to the DRN [58–60]. A recent microdialysis study [58] indicates that ANG II activation of SFO neurons projecting to the
DRN results in inhibition of neurons in this nucleus to effect a reduction in local 5-HT release. These results suggest that SFO neurons projecting to the DRN may be involved in monitoring circulating levels of ANG II and in carrying such information to the DRN. A comparable projection originating in the OVLT and terminating in the DRN may play a similar role. Our recent connectional studies using retrograde tracers in sodium-depleted male rats ingesting salt suggest that structures of the LT inform the DRN of sodium status or sodium consumption, by a descending neural pathway [4]. Thus, the LT structures may act to inform the DRN of body sodium and hormonal status and to regulate sodium consumption and volume expansion through a serotonergic mechanism (Fig. 10). Taking into account our present results and that: i) serotonin and the DRN have been implicated in the inhibitory control of salt intake in males, ii) estrogen has an inhibitory effect on sodium consumption in females, iii) signals arriving from the LT evoked by fluid depletioninduced sodium ingestion interact with this inhibitory serotonergic system, and iv) estrogen modulates 5-HT synthesis and release, it is possible to postulate serotonergic system involvement in the inhibitory action of estrogen on induced sodium appetite in female rats. Estrogenic modulation of serotonergic system activity may be
Fig. 8. Average number of double-immunolabeled Fos-vasopressin (Fos-AVP) neurons in the SON and PaML of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.01 compared with CON and D-DEP. : p b 0.01 compared with D-CON and OVX-CON. : p b 0.01 compared with D, E and OVX.
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Fig. 9. Pattern of double immunoreactivity Fos-AVP in the SON of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Plates A, B, C and D (10x) show D, E, OVX and OVX + E2 groups treated with CON. Plates E, F, G and H (10x) show D, E, OVX and OVX + E2 groups treated with DEP. Plates in the upper right corner show higher magnifications (40x) of the neurons pointed by arrows. Scale bar = 100 μm.
involved in both tonic and phasic inhibition of sodium appetite [3,17,20,21,32–34], since several studies have shown that estrogen modulates tryptophan–hydroxylase enzyme activity and expression [13,24,41]. Another possibility, also considered in the schematic representation (Fig. 10), is that changes in estrogen levels may modulate ANGsensitive DRN projecting neurons in the OVLT, to suppress their response to circulating ANG II and consequently inhibit sodium intake. Our results also demonstrate that estradiol influences vasopressinergic neural activity and the associated diuresis after fluid depletion, before drinking. The increased basal vasopressinergic-neural activity observed in the SON of E and OVX + E2 animals may explain the different renal response to furosemide treatment in these two groups, since both excreted significantly lower urine volumes than diestrus rats, exhibiting higher sodium concentration in urine
compared with the remaining groups. After sodium depletion, E, OVX + E2 and ovariectomized rats without estradiol replacement (OVX) showed increased activity; however, it should be noted that the furosemide-induced overnight water drinking of OVX animals was significantly decreased compared with all groups (data not shown). Thus, this evidence suggests the existence in ovariectomized animals of differences in underlying osmoregulatory mechanisms, such as the threshold sensitivity to induce thirst and AVP release. The increased vasopressinergic activity observed in the hypothalamic cells of E and OVX + E2 animals is consistent with previous studies showing an increase in plasma AVP concentration as well as AVP-mRNA in female rats in estrus and in OVX rats after estradiol replacement [9,10,38]. Similarly, our data also support reports of an estrogen-mediated increase of AVP in normally menstruating women and post-menopausal women receiving estradiol [54]. Several studies claim that estrogens favour fluid retention by activating the renin–
Fig. 10. Schematic representation showing the possible mechanisms that may contribute to the inhibitory action of estrogen on induced sodium intake in female rats.
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angiotensin–aldosterone and vasopressinergic systems. During the luteal phase, estrogen elevation is associated with fluid redistribution from vascular to the extravascular space and changes in the osmotic threshold for AVP release [48,51,53]. Estrogen may be acting directly on vasopressinergic neurons, modulating estrogen receptor beta density expressed in these neurons [28,48,49,55,56]. Likewise, estrogen may be modulating estrogen alpha receptor density localized in the SFO and OVLT, and, by means of these projections, be acting on vasopressinergic neurons [22,25,32,50,57–59]. On the other hand, taking into account our data regarding the vasopressinergic neural activity and the inhibitory action of estrogen on induced sodium intake, it is possible to speculate that vasopressinmediated fluid retention during high estrogen states may be another physiological mechanism limiting sodium ingestion. Finally, recent evidence demonstrates that estrogen can impair brain cell volume regulation to hyponatremia through the inhibition of Na–K– ATPase activity [2]. In addition, previous reports indicate that estrogen may regulate the activity of the Na–K–ATPase pump in different systems [12,44]. Thus, it is possible to postulate a similar mechanism of estrogen modulation of the Na–K–ATPase activity in another system such as the present in sodium-sensitive cells of the OVLT and SFO, where a Nachannel coupled to the α-subunit of Na–K−ATPase may initiate the signal transduction to regulate salt-intake behavior [45]. In summary, our results demonstrate an estrogen-dependent inhibition of induced sodium appetite in normally cycling rats and ovariectomized animals with estradiol replacement that may involve an interaction between excitatory neurons of the OVLT and inhibitory serotoninergic cells of the DRN key brain cells underlying the responses to hyponatremia and hypovolemia. Acknowledgments This work was, in part, supported by grants from CONICET, ANPCyT and SECyT. Carolina Dalmasso is the recipient of a fellowship from CONICET. References [1] Antunes Rodrigues J, Covian MR. Hypothalamic control of sodium chloride and water intake. Acta Physiol Lat Am 1963;13:94–100. [2] Ayus JC, Achinger SG, Arieff A. Brain cell volume regulation in hyponatremia: role of sex, age, vasopressin, and hypoxia. Am J Physiol Renal Physiol 2008;295(3):F619–24. [3] Badauê-Passos Jr D, Godino A, Johnson AK, Vivas L, Antunes-Rodrigues J. Dorsal raphe nuclei integrate allostatic information evoked by depletion-induced sodium ingestion. Exp Neurol 2007;206(1):86–94. [4] Barron WM, Schreiber J, Lindheimer MD. Effect of ovarian sex steroids on osmoregulation and vasopressin secretion in the rat. Am J Physiol 1986;250 (4Pt1):E352–361. [5] Castro L, Athanazio R, Barbetta M, Ramos AC, Angelo AL, Campos I, Varjao B, Ferreira H, Fregoneze J, De Castro-e-Silva E. Central 5-HT2B/2 C and 5-HT3 receptor stimulation decreases salt intake in sodium-depleted rats. Brain Res 2003;981:151–9. [6] Cavalcante-Lima HR, Lima HR, Costa-e-Sousa RH, Olivares EL, Cedraz-Mercez PL, Reis RO, Badaue-Passos Jr D. de-Lucca Jr W, de Medeiros MA, Cortes WS, Reis LC. Dipsogenic stimulation in ibotenic DRN-lesioned rats induces concomitant sodium appetite. Neurosci Lett 2005;374:5–10. [7] Chow SY, Sakai RR, Witcher JA, Adler NT, Epstein AN. Sex and sodium intake in the rat. Behav Neurosci 1992;106(1):172–80. [8] Colombari DS, Menani JV, Johnson AK. Forebrain angiotensin type 1 receptors and parabrachial serotonin in the control of NaCl and water intake. Am J Physiol 1996;271:R1470–6. [9] Crofton J, Baer P, Share L, Brooks D. Vasopressin release in male and female rats: Effects of gonadectomy and treatment with gonadal steroid hormones. Endocrinology 1985;117: 1195–200. [10] Crowley WR, O'Donohue TL, George JM, Jacobowitz DM. Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones, evidence for mediation by norepinephrine. Life Sciences 1978;23:2579–86. [11] De Luca Jr L, Vivas L, Menani JV. Controle neuroendócrino da ingestao de agua e sal. Cap. 10. Neuroendocrinologia básica e aplicada. Eds. José Antunes-Rodrigues Ayrton Custodio Moreira Lucila Leico Kagohara Margaret De Castro, Rio de Janeiro, Brasil. 2005. [12] Del Castillo AR, Battaner E, Guerra M, Alonso T, Mas M. Regional changes of brain Na+, K+-transporting adenosine triphosphate related to ovarian function. Brain Res 1987;416:113–8.
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Serotonergic system involvement in the inhibitory action of estrogen on induced sodium appetite in female rats Carolina Dalmasso a, José Luis Amigone b, Laura Vivas a,⁎ a b
Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC-CONICET, Córdoba, Argentina Sección de Bioquímica Clínica, Hospital Privado, Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 20 April 2011 Accepted 22 April 2011 Keywords: Serotonergic system Sodium appetite Estrous cycle Vasopressinergic neurons OVLT DRN
a b s t r a c t This study of the participation of the serotonergic system in the inhibitory effect of estrogen on induced sodium appetite in female rats explores sodium appetite induced by Furosemide and low sodium diet treatment (DEP) in normally cycling rats and in ovariectomized rats with and without estradiol replacement (OVX, OVX + E2) . We also analyzed the neural activity of serotonergic neurons of the dorsal raphe nucleus (DRN) as well as the activity of other brain nuclei previously found to be involved in sodium and water balance in sodium depleted animals without access to the intake test. For this purpose, we examined the brain Fos, Fos-serotonin and Fos-vasopressin immunoreactivity patterns in diestrus (D), estrus (E), OVX and OVX + E2 rats subjected to DEP. Female rats in E and OVX + E2 exhibited a significant decrease in induced sodium intake compared with females in D and OVX. This estrogen-dependent inhibition on induced sodium appetite (approximately 50% reduction) can be correlated with changes in Fos activation observed in the organum vasculosum of the lamina terminalis (OVLT) and DRN, in response to sodium depletion. Given our previous observations in males, the expected sodium depletion-induced activity of the OVLT was found to be absent in OVX + E2 females, while the usual inhibitory tonic activity of serotonergic neurons of the DRN, instead of decreasing after sodium depletion, increases or remains unchanged in OVX + E2-DEP and E-DEP females, respectively. Regarding urinary water and sodium excretion 3 h after furosemide treatment, E-DEP and OVX + E2-DEP animals excreted smaller volumes of more highly concentrated urine than depleted D and OVX rats. Twenty hours after sodium depletion, the same groups of animals also showed a significant increase in the number of Fos-AVP immunoreactive neurons within the supraoptic nucleus, compared with D-DEP. In summary, our results demonstrate an estrogen-dependent inhibition of induced sodium appetite in normally cycling rats and ovariectomized animals with estradiol replacement, which may involve an interaction between excitatory neurons of the OVLT and inhibitory serotonergic cells of the DRN. The main finding is thus serotonergic system involvement as a possible mechanism in the inhibitory action of estrogen on induced sodium appetite. © 2011 Elsevier Inc. All rights reserved.
1. Introduction A review of the literature indicates that, after body water and sodium loss, the excitatory effects of angiotensin II (ANG II) and the brain osmo-sodium receptors that stimulate sodium appetite involve interactions with inhibitory hindbrain serotonin (5-HT) circuits in males rats [3,5,6,8,17,20,21,31–34,59,62]. Specifically, for a normal sodium appetite sensation, and consequently appropriate salt drinking after electrolyte and fluid depletion, circulating ANG II should act centrally, both to activate brain angiotensinergic mechanisms that stimulate salt appetite, and also to inhibit brain 5-HT mechanisms that tonically inhibit sodium ingestion in males. ⁎ Corresponding author at: Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC-CONICET, Córdoba, Argentina. Tel.: +54 351 4681465/66x108, 120; fax: +54 351 4695163. E-mail addresses: [email protected], [email protected] (L. Vivas). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.04.029
The central 5-HT circuits underlying this interaction mainly include bi-directional connections between the lamina terminalis and 5-HT neurons of the dorsal raphe nucleus (DRN) [4,24,33,59]. The lamina terminalis (LT) plays a major role in many aspects of body fluid homeostasis [11,15,18,29,36,42]. Anatomically, contains three forebrain structures: the subfornical organ (SFO), the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT). The SFO and OVLT lack a blood-brain barrier and contain cells which are sensitive to humoral signals such as changes in plasma and cerebrospinal fluid sodium concentration, osmolality and ANG II levels [29,36,42]. These neural groups project, directly or indirectly via the MnPO, to the magnocellular portion of the supraoptic nucleus (SON) and the paraventricular hypothalamic nucleus (PVN). Vasopressin and oxytocin synthesis and release, and subsequent hydroelectrolytic balance regulation, are modulated by means of these projections [11,29]. Our previous studies have shown that the activity of neurons of the LT as well as of 5-HT cells within the DRN is affected by body sodium
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status in male rats [3,17,18,20,21]. Fos expression, used as a marker of neuronal activity, increases after sodium depletion and also after sodium consumption in cells of the SFO and OVLT. However, in serotonergic DRN cells, c-fos expression decreases when animals are sodium depleted and increases when animals are either in sodium balance or in the process of restoring sodium balance through ingestion [3,17,20,21]. These results are consistent with the idea that serotonergic cells originating in the DRN tonically inhibit sodium appetite, maybe via the LT. Such an inhibitory influence would likely be reduced in states of fluid deficiency and be increased as animals ingest water and NaCl to restore hydromineral balance. Data obtained by Reis and his colleagues [6] have also implicated the DRN as an inhibitory structure for the control of water and sodium intake in males. In these works, male rats with lesions of the DRN had enhanced sodium consumption under both ad libitum (i.e., need-free) conditions and after different experimental procedures that stimulate sodium appetite. These also suggest that the DRN tonically inhibits sodium intake. On the other hand, studies analyzing estrogen modulation of body fluid balance have been conducted both during the estrous cycle as well as in ovariectomized animals with hormone replacement and, although sometimes contradictory, all together suggest an inhibitory effect of estrogen on sodium consumption [1,7,25,43]. For example, Antunes-Rodrigues et al. (1963) [1] reported that spontaneous sodium intake decreases during estrus, a stage with high estrogen concentrations [35], and increases during diestrus. Furthermore, Scheidler et al. (1994) [43] showed that adult male rats and ovariectomized females ingested 50 to 150% more saline than intact females, when pronounced sodium appetite was induced by a prolonged sodium-free diet or mineralocorticoid administration. In addition, estrogen replacement in ovariectomized animals also decreased sodium intake levels. A series of studies also support an estrogen modulation upon the serotonergic system; it has been shown that estrogen: i) modulates 5-HT synthesis and release [39,40], ii) increases the basal firing rate of serotonergic neurons in female rats [38], iii) augments 5-HT transporter mRNA in the DRN of ovariectomized animals [30,52], and iv) chronically administered to ovariectomized rats, increases tryptophan hydroxylase-2 (TPH2) mRNA in the DRN [13,17]. In summary, as a number of studies have postulated the involvement of estrogen and the serotonergic systems in the inhibition of sodium appetite and the existence of physiological interactions between these, the following question arises: Is this inhibitory effect of estrogen on induced sodium intake in females mediated by the serotonergic system? We hypothesize that female rats in estrus and ovariectomized animals treated with estrogen replacement (OVX + E2) may drink less saline solution after fluid depletion than ovariectomized (OVX) and intact rats during diestrus, in part because the inhibitory serotonergic systems of the DRN are more active in animals with elevated plasma estrogen concentrations. To test this idea, we quantified the number of cells that are double-immunolabeled with Fos-5-HT immunoreactivity within the DRN in diestrus, estrus and ovariectomized animals (with and without estradiol replacement) subjected to sodium depletion by a Furosemide and low sodium diet treatment (DEP). Estrogen also modulates AVP synthesis and release [4,9,10,14,16,22, 23,38,46–50,55,61]. An increase has been reported in plasma AVP concentrations as well as in osmotically stimulated AVP mRNA, in female rats in estrus and ovariectomized animals treated with estrogen replacement [10,16,23,46]. However, the effects of estrogen on AVP synthesis and release are not conclusive; it seems to depend on the methodological approach utilized, since other authors have shown that estrogen has no effect on AVP release and osmotically stimulated AVP mRNA expression in rats [4,9,38]. Taking into account the above mentioned evidences we aim to analyzed the pattern of Fos immunoreactivity within vasopressinergic hypothalamic nuclei and along the
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neuronal groups of the Lamina terminalis nuclei. Additionally, a different set of females were subjected to sodium depletion by DEP, and water and sodium ingestion and excretion were analyzed. 2. Materials and methods 2.1. Animals Sixty-day-old Wistar-derived female rats, born and reared in the vivarium of the Instituto Ferreyra (INIMEC, Córdoba, Argentina) were used in the experiments. Animals weighing 190–230 g were individually housed in metabolic cages, in a temperature-controlled environment, with a 12:12 h light/dark cycle, beginning at 08:00 h. Food, distilled water and 2% sodium solution were available ad libitum during the training period. All experimental protocols were approved by the appropriate animal care and use committee of our institute, following the guidelines of the Public Health Service Guide for the Care and Use of Laboratory Animals. 2.2. Experimental design General considerations: Animals were randomly assigned to different groups defined by the combination of furosemide treatment (animals were administered either furosemide combined with low sodium diet [DEP] or saline with normal sodium diet [CON] and hormonal status (females either in diestrus [D], estrus [E], ovariectomized [OVX] or ovariectomized + estradiol replacement [OVX + E2]). The final groups were arranged as follows: (i) diestrus females with saline and normal sodium diet (D-CON); (ii) diestrus females with furosemide and low sodium diet (D-DEP); (iii) estrus females with saline and normal sodium diet (E-CON); (iv) estrus females with furosemide and low sodium diet (E-DEP); (v) ovariectomized with saline and normal sodium diet (OVX-CON); (vi) ovariectomized with furosemide and low sodium diet (OVX-DEP); (vii) ovariectomized + estradiol replacement with saline and normal sodium diet (OVX + E2-CON); (viii) ovariectomized + estradiol replacement with furosemide and low sodium diet (OVX + E2-DEP). Sodium and water intake tests were performed with a minimum of ten subjects. Additionally, for immunohistochemical analysis, a different set of animals with at least five subjects of each group, were sacrificed before the intake test. 2.3. Surgical procedure and estradiol (E2) replacement Bilateral ovariectomy was performed under aseptic conditions in sixty-day-old rats. Anesthesia was induced by ketamine (60 mg/ kg, i.p.) in combination with xylazine (7.5 mg/kg, i.p.). Estrogen replacement began two weeks after ovariectomy. Specifically, estradiol (E2) (20 μg 17-β-estradiol-3-benzoate/0.2 ml/animal, s.c., Schering Laboratories) or vehicle (neutral corn oil, 0.2 ml/animal, s.c., Gador Laboratories) was injected in OVX + E2 and OVX groups, respectively, twice a day during five consecutive days. Intact female rats were sham-operated and injected with neutral corn oil under the same administration schedule as OVX + E2 and OVX groups. The reproductive status of each animal was determined by microscopic analysis of vaginal smears; only females having two or more regular estrous cycles were used for these experiments. 2.4. Sodium depletion Sodium appetite was stimulated by acute treatment with furosemide (Lasix, Aventis Pharma, Brazil) in combination with a low sodium diet (DEP). Animals were injected subcutaneously with furosemide (20 mg/ kg in isotonic saline vehicle). Immediately after treatment, the animals were placed into clean individual metabolic cages, and they were allowed access to distilled water and low sodium diet (corn flour: 0%
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NaCl) for a 24 h period. Control rats were subjected to a similar procedure, receiving isotonic saline injections and a standard diet (0.2% NaCl), instead. After 24 h of sodium depletion, a two-bottle preference test was performed. Food was removed and two bottles containing distilled water or 0.34 M NaCl solution were offered. Ingested fluid volumes were recorded repeatedly during the intake test, (at 15, 30, 60, 90 and 120 min time points). For statistical analysis, intake scores were expressed as ml/100 g of body weight. Intake measures are corrected for body weight taking into account that there are differences based on treatment (depleted vs. non-depleted) and also due to ovariectomy and hormone replacement.
avidin–biotin peroxidase complex (Vector, 1:200 dilution in 1% NHS-PB 0.1 M) for 2 h at room temperature. The peroxidase label was detected using diaminobenzidine hydrochloride (DAB; Sigma). This method produces a brown cytoplasmatic reaction product, clearly distinguishable from the typical black nuclear stain. Finally, the free-floating sections were mounted on gelatinized slides, air-dried overnight, cleared in xylene, and placed under a coverslip with DePeX. Controls for Fos-ir, Fos-AVP-ir and Fos-5HT-ir were conducted by processing sections without the primary antiserum. No Fos-ir, Fos-5HT-ir or FosAVP-ir neurons were observed after this control procedure. 2.7. Cytoarchitectural and quantitative analysis
2.5. Analysis of urine and plasma osmolarity and electrolytes Urine volume of each rat was recorded 3 h after saline or furosemide injection. Urine osmolarity was measured using an Advanced ® MicroOsmometer Model 3300. This osmometer uses the technique of freezing-point depression to measure osmolarity, expressed as mOsm/ kg H2O. Urine electrolytes were measured using an Ion Selective Electrode (Hitachi Modular P + ISE. Roche Diagnostic). The amount of sodium excreted in urine was calculated by multiplying the sodium of urine by its volume. Urine volumes are corrected for body weight taking into account that there are differences based on treatment (depleted vs. non-depleted) and also due to ovariectomy and hormone replacement. 2.6. Staining procedure for Fos, Fos-5-HT and Fos-AVP immunoreactivity (ir) As mentioned previously, for immunohistochemical analysis, a different set of depleted and non-depleted animals were used. It is important to note that this set of animals was not allowed access to the intake test; we analyzed the brain neural activity during the appetitive phase. The rats were subjected to furosemide or control injection and 22 h later were anesthetized with chloral hydrate (0.6 ml/ 100 g bw) and perfused transcardially with normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2). The brains were removed, fixed in the perfusion solution overnight, and stored at 4 °C in PB 0.1 M containing 30% sucrose. Two equivalent sets of coronal sections (40 μm) were cut with a freezing microtome. One of the sets was processed for Fos-5HT immunodetection. The remaining set was processed for Fos-AVP immunostaining. The staining procedures followed the previously described doublelabeling technique [17,18]. Briefly, sections were placed in a mixture of 10% H2O2 and 10% methanol until oxygen bubbles ceased appearing. They were then incubated in 10% normal horse serum (NHS, Gibco, Auckland, NZ)) in PBS for 2 h to block non-specific binding. After blocking, sections were incubated overnight at room temperature in a rabbit anti-Fos antibody (against a synthetic 14-amino acid sequence corresponding to residues 4–17 of human Fos, Oncogene Science, Manhasset, NY, USA) diluted 1:10,000 in PBS 0.1 M containing 2% NHS and 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO,USA). Twenty-four hours later, sections were incubated in biotin-labeled anti-rabbit immunoglobulin (1:200) for 2 h at room temperature. Afterwards, sections were incubated with avidin–biotin peroxidase complex (Vector, 1:200 dilution in 1% NHS-PB 0.1 M) for 2 h at room temperature. The peroxidase label was detected using diaminobenzidine hydrochloride (DAB; Sigma Chemical Co., St. Louis, MO, USA) intensified with 1% cobalt chloride and 1% nickel ammonium sulfate. This method produces a blue-black nuclear reaction product. After washing in PBS 0.01 M, sections were blocked in 10% NHS in PBS, for 2 h, and then one of the sets was incubated in rabbit anti-5-HT polyclonal antibody (ImmunoStar) diluted 1:5000 and the other in rabbit anti-vasopressin polyclonal antibody (Chemicon) diluted 1:10,000, in a solution containing PBS 0.1 M, 2% NHS and 0.3% Triton X-100. After 48 h of incubation, all sections were washed in PBS 0.01 M, and then incubated in biotin-labeled anti-rabbit immunoglobulin (1:200) for 2 h at room temperature. Then they were incubated with
The brain nuclei exhibiting Fos-ir were identified and delimited according to the rat brain atlas [37]. The Fos-ir neurons of all nuclei were counted at one level. The distance from the bregma of the corresponding plates is as follows: for OVLT= −0.20 mm; SFO =−0.92 mm; MnPO = −0.30 mm; SON=−.3 mm; PaLM=−1.80 mm; DRN =−8.00 mm. Immunostaining cells were quantified with a computerized system that included a Zeiss microscope equipped with a DC 200 Leica digital camera attached to a contrast enhancement device. Images were digitalized and analyzed by Scion Image PC, based on the National Institutes of Health Image 1997 version. Representative sections in each group were acquired at exactly the same level, with the aid of the Adobe Photoshop image analysis program (version 5.5). The counting was done in at least five animals of each condition and was repeated at least twice on each section analyzed, to ensure that the number of profiles obtained was similar. The investigator who conducted the cell counting was blind to the experimental condition. 2.8. Statistical analysis All data are expressed as means ± SEM. Data were analyzed, using Statistica (StatSoft, Tulsa, OK), by means of a 2-way ANOVA, with hormonal status (D, E, OVX or OVX + E2) and furosemide treatment (saline or furosemide) as independent factors. Whenever the analysis showed either main effects or statistically significant interactions, posthoc comparisons were performed (Least Significant Difference -LSDtest, with significance levels set at p b 0.05). In experiment 1, the dependent variables under analysis were urine osmolarity as well as electrolyte concentration. Urine osmolarity and electrolytes obtained 3 h after sodium depletion were analyzed by means of a 1-way ANOVA with hormonal status as the independent factor. Given that no urine samples were obtained from non-depleted animals 3 h after treatment because they did not urinate, only furosemide-treated animals were included in the analysis. In experiment 2, consumption levels of 0.34 M NaCl solution and distilled water were included as repeated measures. In experiment 3, immunohistochemical data derived from Fos and double labeling Fos-5-HT and Fos-AVP immunohistochemistry were also analyzed. 3. Results 3.1. Experiment 1: Effects of hormonal status during estrous cycle and estradiol replacement in OVX rats on water and sodium excretion induced by DEP treatment Urine volume, osmolarity and electrolyte concentration 3 h after DEP treatment: Five different one-way ANOVAs were performed in order to analyze urine volume, osmolarity, and urinary sodium, chloride and potassium concentrations of animals subjected to DEP treatment. The significant main effect of hormonal status was recorded in all cases. [Urine volume: F(3,79) = 6.60; osmolarity: F(3,75) = 26.31; urinary sodium concentration: F(3,75) = 21.90; urinary chloride concentration: F(3,75) = 19.56; urinary potassium concentration: F(3,77) = 12.53. All p values b .005].
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Post-hoc analysis of urinary volume (Fig. 1) showed that E, OVX and OVX + E2 excreted significantly lower volumes than D animals. Moreover OVX + E2 females excreted less urine when compared with the OVX group. Post-hoc tests of urine osmolarity (table 1) indicated that E, OVX and OVX + E2 groups exhibited higher osmolarity than D rats. Interestingly, E and OVX + E2 groups registered greater osmolarity than that observed in OVX animals. Similarly, urine sodium concentration in E, OVX and OVX + E2 females was higher than in D rats. Furthermore, urine sodium concentration in OVX was significantly lower than in E and OVX + E2 groups. Besides, OVX + E2 females excreted the highest concentration of this electrolyte (Table 1). To sum up, these results indicate that, after sodium depletion, sodium concentration varies considerably across hormonal status, even when all animals have excreted the same total amount of this electrolyte. Specifically, E and OVX + E2 rats excreted the lowest urine volumes compared with the remaining groups and showed the highest sodium concentration scores.
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Table 1 Urine osmolarity (mOsm/kg) and urine Na+ concentration (mEq/l) of D, E, OVX and OVX + E2 animals accumulated 3 h after furosemide/low sodium diet (DEP) treatment. DEP
D E OVX OVX + E2
Osm (mOsmol/kg)
Na+ (mEq/l)
245.22 ± 17.17 356.79 ± 8.11 277.33 ± 9.11 341.76 ± 5.48
69.38 ± 6.77 98.66 ± 2.59 86.51 ± 2.69 109.06 ± 1.44
Results are expressed as mean ± SE. DEP: animals subjected to furosemide along with low sodium diet. D: female rats in diestrus; E: female rats in estrus; OVX: ovariectomized female rats; OVX + E2 ovariectomized female rats + estradiol replacement.
Water consumption was also affected by sodium depletion (Fig. 2). Furosemide combined with a low sodium diet induced a significant increase in water intake (F(1,82) = 133.75; p b .0001) in E-DEP, OVXDEP and OVX + E2-DEP. However, when considering hormonal status, there were no differences between the groups. 3.3. Experiment 3
3.2. Experiment 2: Effects of hormonal status during estrous cycle and estradiol replacement to OVX rats on sodium and water intake induced by DEP treatment The statistical analysis showed a main effect of furosemide factor (F(1.82) = 133.75; p b .001), hormonal status (F(3,82) = 11.93; p b .0001) and solution (F(1,82) = 49.17; p b .0001). Additionally, a significant interaction between the three factors was observed (F(3,82) = 5.35; p b .005). Post-hoc comparisons of sodium intake (Fig. 2) indicated that this was increased by ovariectomy in non-depleted animals: OVX rats showed a significant increase in sodium intake compared with E and OVX + E2 groups. It is important to note that these groups (E-CON and OVX + E2-CON) seemed to consume smaller amounts of sodium than D-CON subjects, although this difference did not reach significant levels. As expected, it was observed that furosemide depletion induced an increase in sodium intake in comparison to non-depletion treatment. After 120 min intake test, E-DEP animals ingested significantly lower sodium volumes compared with D-DEP and OVX-DEP groups. In addition, estradiol replacement significantly decreased sodium intake in comparison to D-DEP and OVX-DEP. Ovariectomized-depleted animals ingested the highest sodium volumes registered. These results are consistent with an inhibitory effect of estrogen on sodium consumption.
Fig. 1. Urine volume obtained 3 h after furosemide administration in the different groups: diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.05 compared with D-DEP. : p b 0.05 compared with OVX-DEP.
3.3.1. Effects of hormonal status during estrous cycle and estradiol replacement to OVX rats on Fos-ir along the lamina terminalis induced by DEP treatment Organum vasculosum of the lamina terminalis (OVLT): The inferential analysis showed a main effect of furosemide treatment (F(1,40) = 85.97; p b .0001) as well as a main effect of hormonal status (F(3,40) = 12.52; p b .0001). A significant interaction was also observed between the included factors (F(3,40) = 22.29; p b .0001) (Fig. 3).
Fig. 2. Sodium intake (panel A) and water intake (panel B) (ml/100 g bw; 2 h) of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON; white bars) or furosemide/low sodium diet (DEP; black bars). Vertical lines represent SEM. +: p b 0.001 compared with CON. : p b 0.05 compared with E-CON and OVX + E2-CON. : p b 0.05 compared with D-DEP, OVX-DEP. *: p b 0.005 compared with D-DEP.
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Fig. 3. Average number of Fos immunoreactive (Fos-ir) neurons in the OVLT, SFO and MnPO of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.05 compared with CON. : p b 0.05 compared with D-DEP. : p b 0.01 compared with E-DEP and OVX-DEP. *: p b 0.05 compared with D-CON and E-CON.
A posteriori comparisons showed that in D-DEP, E-DEP, and OVXDEP groups, sodium depletion induced a significant increase in Fos immunoreactivity compared with their respective controls without depletion. In contrast, furosemide treatment did not alter the OVLT basal activity of the OVX + E2 group (Figs. 3, 4). Regardless of the hormonal status exhibited, non-depleted animals did not differ in terms of Fos expression. After sodium depletion, it was observed that the D-DEP group exhibited the highest number of Fos immunostained neurons and the OVX + E2-DEP group the lowest. Subfornical Organ (SFO): The analysis of variance showed a main effect of furosemide factor (F(1,35) = 638.02; p b .0001), and a main effect of hormonal status (F(3,35) = 5.09; p b .005). Furthermore, a significant interaction between the factors was observed (F(3,35) = 4.20; p b .05). Post-hoc comparisons indicated that non-depleted OVX + E2-CON animals exhibited a higher number of Fos positive cells when compared with D-CON and E-CON groups (Fig. 3). The analysis also showed that sodium depletion induced a significant increase in Fos immunoreactivity in all depleted groups in relation to their respective controls without depletion. Additionally, furosemide treatment significantly increased the number of Fos immunostaining cells in E-DEP and OVX-DEP rats, compared with the D-DEP group.
Median Preoptic nucleus (MnPO): In this nucleus, the analysis of the data showed a main effect of furosemide (F(1,42) = 41.32; p b .0001) and a main effect of hormonal status (F(3,42) = 3.51; p b .05). The interaction between both factors did not reach significant levels. Post-hoc comparisons indicated that sodium depletion induced a decrease in the activity of this nucleus, as evidenced by diminished Fos immunostaining (Fig. 3). Considering hormonal status, the analysis showed that OVX group exhibited the highest activation, while E animals presented the lowest Fos-ir. 3.3.2. Effects of hormonal status on DEP-induced activity in serotonergic neurons of the dorsal raphe nucleus (DRN) Fig. 5 shows a schematic representation of the DRN detailing the subnuclei that were evaluated in the analysis of Fos-5-HT immunoreactive pattern. The ANOVA showed a main effect of furosemide factor (F(3,28) = 32.83; p b .0001) as well as a main effect of hormonal status (F(1,28) = 7.50; p b .01). An interaction between the factors (F(3,28) = 13.01; p b .0001) was also observed. A posteriori comparisons (Fig. 6) indicated that OVX-CON presented significantly lower number of Fos positive cells than the remaining nondepleted groups. In D-DEP and OVX-DEP groups, furosemide depletion induced a significant decrease in Fos-5-HT immunoexpression
Fig. 4. Pattern of Fos immunoreactivity in the OVLT of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Plates A, B, C and D (10x) show D, E, OVX and OVX + E2 groups treated with CON. Plates E, F, G and H (10x) show D, E, OVX and OVX + E2 groups treated with DEP. Scale bar = 100 μm.
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3.3.3. Effects of hormonal status on DEP-induced activity in vasopressinergic neurons of the supraoptic (SON) and magnocellular subdivision of the paraventricular nucleus (PaML) It is important to note that we analyzed the neural activity of vasopressinergic neurons of non-depleted and depleted animals before any fluid reestablishment. As expected during this stage, we found a relatively low neural activation in terms of Fos-AVP-ir. Within the SON, the ANOVA showed a main effect of furosemide treatment (F(1,38) = 70.15; p b .0001) as well as a main effect of hormonal status (F(3,38) = 150.08; p b .0001), and also an interaction between the factors considered (F(3,38) = 13.89; p b .0001). Post-hoc comparisons (Fig. 8) indicated that, under non-depleted condition, E and OVX + E2 animals exhibited higher double-labeled cells than D and OVX. It should be noted that OVX + E2 showed the highest number of Fos-AVP-ir cells. Sodium depletion caused an increase in Fos-AVP immunostaining only in E, OVX and OVX + E2 females (Fig. 9). With regard to the neural activity observed in vasopressinergic neurons of the PaLM subdivision, inferential analysis showed a main effect of furosemide treatment (F(1,37) = 7.27; p b .01) as well as a main effect of hormonal status (F(3,37) = 8.15; p b .0005). Regarding furosemide treatment, as expected, depleted animals exhibited a significant increase in Fos-AVP neurons compared with their respective controls (Fig. 8). Post-hoc comparisons showed significantly higher activation (operationalized as an increase in Fos-AVP double-labeled immunoreactivity) in E and OVX + E2 females than in D and OVX rats. In summary, the vasopressinergic activity changes observed between the different groups may reflect hormonal status influences, and can be correlated with the renal excretory response observed in those groups of animals 3 h after furosemide treatment, since E and OVX + E2 rats excreted the lowest urine volumes compared with the remaining groups and showed the highest sodium concentration scores. Fig. 5. Schematic representation of the dorsal raphe nucleus showing the specific subnuclei that were evaluated in the analysis of Fos-5-HT immunoreactive pattern (upper). Microphotography (5x) of the DRN that illustrates the corresponding schematic representation (lower).
compared with their respective non-depleted controls, while provoking a significant increase in the number of double-labeled cells in OVX + E2-DEP in comparison with OVX + E2-CON. In addition E-DEP and OVX + E2-DEP females exhibited significantly higher activation than D-DEP and OVX-DEP groups (Fig. 6, 7).
Fig. 6. Average number of double-immunolabeled Fos-serotonin (Fos-5-HT) neurons in the DRN of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.01 compared with CON. *: p b 0.01 compared with D-CON, E-CON and OVX + E2-CON. : p b 0.01 compared with D-DEP and OVX-DEP.
4. Discussion The main result of this work is consistent with involvement of the serotonergic system as a possible mechanism in the inhibitory action of estrogen on induced sodium intake in female rats. This behavioral inhibition is related to changes in Fos activation observed in serotonergic cells of the DRN and with reduced Fos activity found within OVLT neurons. In other words, hormonal status during the estrous cycle and estradiol replacement after ovariectomy change the neural activity induced by sodium depletion in cells of the OVLT and serotonergic cells of the DRN. In particular, there is an interesting correlation between the reduced Na intake (approximately a 50% reduction) in response to sodium depletion observed in estrus and ovariectomized rats treated with estradiol and the neural activity found in the stimulatory neurons of the OVLT and the inhibitory serotonergic neurons of the DRN. Thus, taking into account our previous observations in males, our results in females showed that the expected sodium depletion-induced activity of the OVLT is absent in ovariectomized rats treated with estradiol, while the usual inhibitory tonic activity of serotonergic neurons of the DRN increases or remains unchanged, rather than decreases, after sodium depletion. Our results also demonstrate a hormonal status modulation of stimulated sodium appetite, suggesting, like prior studies, an inhibitory effect of estrogen on sodium consumption [1,43] and providing new evidence about a possible mechanism involved in such inhibition. It is important to note that one of the lamina terminalis nuclei, the OVLT, often associated with sodium appetite regulation [15,42] showed a correlated neural activity with the level of stimulated sodium ingestion in the present study. The intact females in estrus and the OVX + E2 rats, with relatively lower basal and induced sodium intake, also showed a relatively lower or absent
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Fig. 7. Pattern of double immunoreactivity Fos-5-HT in the DRN of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Plates A, B, C and D (20x) show D, E, OVX and OVX + E2 groups treated with CON. Plates E, F, G and H (20x) show D, E, OVX and OVX + E2 groups treated with DEP. Plates in the upper right corner show higher magnifications (40x) of the neurons pointed by arrows. Scale bar = 50 μm.
activation of the OVLT. On the other hand, diestrus and ovariectomized animals showed higher basal and induced sodium consumption and also had greater OVLT activation induced by sodium depletion. Along the other nuclei of the lamina terminalis, the SFO and the MnPO, we found a similar pattern of activity to that observed in male rats after sodium depletion [18]: specifically, an increased number of Fos-ir cells along the SFO and a diminished number within the MnPO, which we believe signify, respectively, a stimulatory and inhibitory role of these nuclei on sodium intake behavior. The furosemide-low sodium diet treatment is a well-known animal model in which the renin–angiotensin system is stimulated. Numerous previous reports propose an interaction between the estrogen and angiotensinergic receptors along the LT nuclei as an explanation of the estrogen-mediated effect on drinking behavior [19,26,27,31,57,58] (Fig. 10). Lind (1986) has anatomically demonstrated a neural angiotensinergic connection originating in the LT nuclei projecting to the DRN. In addition, ANG II injected via the carotid artery or into the SFO enhances the electrical activity of SFO neurons that project to the DRN [58–60]. A recent microdialysis study [58] indicates that ANG II activation of SFO neurons projecting to the
DRN results in inhibition of neurons in this nucleus to effect a reduction in local 5-HT release. These results suggest that SFO neurons projecting to the DRN may be involved in monitoring circulating levels of ANG II and in carrying such information to the DRN. A comparable projection originating in the OVLT and terminating in the DRN may play a similar role. Our recent connectional studies using retrograde tracers in sodium-depleted male rats ingesting salt suggest that structures of the LT inform the DRN of sodium status or sodium consumption, by a descending neural pathway [4]. Thus, the LT structures may act to inform the DRN of body sodium and hormonal status and to regulate sodium consumption and volume expansion through a serotonergic mechanism (Fig. 10). Taking into account our present results and that: i) serotonin and the DRN have been implicated in the inhibitory control of salt intake in males, ii) estrogen has an inhibitory effect on sodium consumption in females, iii) signals arriving from the LT evoked by fluid depletioninduced sodium ingestion interact with this inhibitory serotonergic system, and iv) estrogen modulates 5-HT synthesis and release, it is possible to postulate serotonergic system involvement in the inhibitory action of estrogen on induced sodium appetite in female rats. Estrogenic modulation of serotonergic system activity may be
Fig. 8. Average number of double-immunolabeled Fos-vasopressin (Fos-AVP) neurons in the SON and PaML of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Vertical lines represent SEM. +: p b 0.01 compared with CON and D-DEP. : p b 0.01 compared with D-CON and OVX-CON. : p b 0.01 compared with D, E and OVX.
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Fig. 9. Pattern of double immunoreactivity Fos-AVP in the SON of diestrus (D), estrus (E), ovariectomized (OVX) and ovariectomized + estradiol replacement (OVX + E2) groups, treated with saline/normal sodium diet (CON) or furosemide/low sodium diet (DEP). Plates A, B, C and D (10x) show D, E, OVX and OVX + E2 groups treated with CON. Plates E, F, G and H (10x) show D, E, OVX and OVX + E2 groups treated with DEP. Plates in the upper right corner show higher magnifications (40x) of the neurons pointed by arrows. Scale bar = 100 μm.
involved in both tonic and phasic inhibition of sodium appetite [3,17,20,21,32–34], since several studies have shown that estrogen modulates tryptophan–hydroxylase enzyme activity and expression [13,24,41]. Another possibility, also considered in the schematic representation (Fig. 10), is that changes in estrogen levels may modulate ANGsensitive DRN projecting neurons in the OVLT, to suppress their response to circulating ANG II and consequently inhibit sodium intake. Our results also demonstrate that estradiol influences vasopressinergic neural activity and the associated diuresis after fluid depletion, before drinking. The increased basal vasopressinergic-neural activity observed in the SON of E and OVX + E2 animals may explain the different renal response to furosemide treatment in these two groups, since both excreted significantly lower urine volumes than diestrus rats, exhibiting higher sodium concentration in urine
compared with the remaining groups. After sodium depletion, E, OVX + E2 and ovariectomized rats without estradiol replacement (OVX) showed increased activity; however, it should be noted that the furosemide-induced overnight water drinking of OVX animals was significantly decreased compared with all groups (data not shown). Thus, this evidence suggests the existence in ovariectomized animals of differences in underlying osmoregulatory mechanisms, such as the threshold sensitivity to induce thirst and AVP release. The increased vasopressinergic activity observed in the hypothalamic cells of E and OVX + E2 animals is consistent with previous studies showing an increase in plasma AVP concentration as well as AVP-mRNA in female rats in estrus and in OVX rats after estradiol replacement [9,10,38]. Similarly, our data also support reports of an estrogen-mediated increase of AVP in normally menstruating women and post-menopausal women receiving estradiol [54]. Several studies claim that estrogens favour fluid retention by activating the renin–
Fig. 10. Schematic representation showing the possible mechanisms that may contribute to the inhibitory action of estrogen on induced sodium intake in female rats.
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angiotensin–aldosterone and vasopressinergic systems. During the luteal phase, estrogen elevation is associated with fluid redistribution from vascular to the extravascular space and changes in the osmotic threshold for AVP release [48,51,53]. Estrogen may be acting directly on vasopressinergic neurons, modulating estrogen receptor beta density expressed in these neurons [28,48,49,55,56]. Likewise, estrogen may be modulating estrogen alpha receptor density localized in the SFO and OVLT, and, by means of these projections, be acting on vasopressinergic neurons [22,25,32,50,57–59]. On the other hand, taking into account our data regarding the vasopressinergic neural activity and the inhibitory action of estrogen on induced sodium intake, it is possible to speculate that vasopressinmediated fluid retention during high estrogen states may be another physiological mechanism limiting sodium ingestion. Finally, recent evidence demonstrates that estrogen can impair brain cell volume regulation to hyponatremia through the inhibition of Na–K– ATPase activity [2]. In addition, previous reports indicate that estrogen may regulate the activity of the Na–K–ATPase pump in different systems [12,44]. Thus, it is possible to postulate a similar mechanism of estrogen modulation of the Na–K–ATPase activity in another system such as the present in sodium-sensitive cells of the OVLT and SFO, where a Nachannel coupled to the α-subunit of Na–K−ATPase may initiate the signal transduction to regulate salt-intake behavior [45]. In summary, our results demonstrate an estrogen-dependent inhibition of induced sodium appetite in normally cycling rats and ovariectomized animals with estradiol replacement that may involve an interaction between excitatory neurons of the OVLT and inhibitory serotoninergic cells of the DRN key brain cells underlying the responses to hyponatremia and hypovolemia. Acknowledgments This work was, in part, supported by grants from CONICET, ANPCyT and SECyT. Carolina Dalmasso is the recipient of a fellowship from CONICET. References [1] Antunes Rodrigues J, Covian MR. Hypothalamic control of sodium chloride and water intake. Acta Physiol Lat Am 1963;13:94–100. [2] Ayus JC, Achinger SG, Arieff A. Brain cell volume regulation in hyponatremia: role of sex, age, vasopressin, and hypoxia. Am J Physiol Renal Physiol 2008;295(3):F619–24. [3] Badauê-Passos Jr D, Godino A, Johnson AK, Vivas L, Antunes-Rodrigues J. Dorsal raphe nuclei integrate allostatic information evoked by depletion-induced sodium ingestion. Exp Neurol 2007;206(1):86–94. [4] Barron WM, Schreiber J, Lindheimer MD. Effect of ovarian sex steroids on osmoregulation and vasopressin secretion in the rat. Am J Physiol 1986;250 (4Pt1):E352–361. [5] Castro L, Athanazio R, Barbetta M, Ramos AC, Angelo AL, Campos I, Varjao B, Ferreira H, Fregoneze J, De Castro-e-Silva E. Central 5-HT2B/2 C and 5-HT3 receptor stimulation decreases salt intake in sodium-depleted rats. Brain Res 2003;981:151–9. [6] Cavalcante-Lima HR, Lima HR, Costa-e-Sousa RH, Olivares EL, Cedraz-Mercez PL, Reis RO, Badaue-Passos Jr D. de-Lucca Jr W, de Medeiros MA, Cortes WS, Reis LC. Dipsogenic stimulation in ibotenic DRN-lesioned rats induces concomitant sodium appetite. Neurosci Lett 2005;374:5–10. [7] Chow SY, Sakai RR, Witcher JA, Adler NT, Epstein AN. Sex and sodium intake in the rat. Behav Neurosci 1992;106(1):172–80. [8] Colombari DS, Menani JV, Johnson AK. Forebrain angiotensin type 1 receptors and parabrachial serotonin in the control of NaCl and water intake. Am J Physiol 1996;271:R1470–6. [9] Crofton J, Baer P, Share L, Brooks D. Vasopressin release in male and female rats: Effects of gonadectomy and treatment with gonadal steroid hormones. Endocrinology 1985;117: 1195–200. [10] Crowley WR, O'Donohue TL, George JM, Jacobowitz DM. Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones, evidence for mediation by norepinephrine. Life Sciences 1978;23:2579–86. [11] De Luca Jr L, Vivas L, Menani JV. Controle neuroendócrino da ingestao de agua e sal. Cap. 10. Neuroendocrinologia básica e aplicada. Eds. José Antunes-Rodrigues Ayrton Custodio Moreira Lucila Leico Kagohara Margaret De Castro, Rio de Janeiro, Brasil. 2005. [12] Del Castillo AR, Battaner E, Guerra M, Alonso T, Mas M. Regional changes of brain Na+, K+-transporting adenosine triphosphate related to ovarian function. Brain Res 1987;416:113–8.
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