**3. Results**

150 Neuroendocrinology and Behavior

weight.

**2.4. Tissue preparation** 

**2.5. Immunohistochemistry** 

antibody (1: 5000 dilution).

**2.3. Determination of water fluxes** 

Water fluxes were determined by direct analysis following the principles described by Holleman and Dieterich [30]. Rates of water flux represent the loss of water via excretion and evaporation and the simultaneous input of water, via metabolic water production and pre-formed water via food and drink (Nagy and Costa 1980). Free water content of the food determined by drying to constant weight at 60 °C was 3 %. The metabolic water content was determined from carbohydrate, fat and protein composition [33]. Thus 1 g of given food contains 0.509 mL of water. The intact unshaven carcasses were sublimated to dryness. The

After determining urine volume and feces weight, urine samples were frozen at -30 °C while the feces were dried for 72 h. Water efflux was calculated as the difference between the influx and total body water. Water fluxes are expressed in H2O mL per day. Finally these fluxes were normalized to the average body weights and expressed in kg-0.82. In small mammals an allometric relationship exists between the water efflux or influx and body weight (W) in kilograms, which is Fin=K.W 0.82 ([34-35], expressed as mL/day/100 g body

*Meriones* were anesthetized with sodium pentobarbital (70 mg/kg, i.p; Sanofi, Libourne,France) and perfused transcardially with heparin in physiological saline, followed by 500 mL of a freshly prepared solution of 4 % (wt / vol) paraformaldehyde in phosphate – buffered saline (PBS ; pH = 7.4). The brains were rapidly removed and postfixed overnight in 4 % paraformaldehyde at 4 °C. Forty micrometer thick coronal sections were cut with a Vibratome (VT 1000S; Leica, Nussloch, Germany). Brain sections were collected in cold PBS.

Free-floating sections were pretreated for 20 min with 3 % hydrogen peroxide in PBS to quench endogenous peroxidase. They were then washed with PBS (3 x 10 min), preincubated for 90 min at room temperature in PBS containing 0.05 %. Triton X-100 and 3 % normal horse serum. Sections were incubated for 36 h at 4 °C with Mouse anti-AVP

After incubation, sections were rinsed extensively with PBS (four times, 15 min) and incubated for 1.5 h in a 1/100 dilution of biotin conjugated horse anti-goat antibody and other secondary antibodies. Texas Red conjugated rabbit anti-mouse antibody (1/200; dilution; Jackson ImmunoResearch). For amplification, we used tyramide signal amplification fluorescence system technology (NEN, Boston, MA, USA). For details see Banisadr et al. [36]. After washing, sections were mounted onto gelatin-coated slides in Vectashield (Vector) and observed on fluorescent microscope (BX61; Olympus, Melville,

NY) and a connected image-acquisition software (Analysis) was used.

difference between live and dry weight was taken as total body water (TBW).
