AVP V1a-R expression in the rat hypothalamus around parturition: relevance to antipyresis at term

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Experimental Neurology 183 (2003) 338 –345

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AVP V1a-R expression in the rat hypothalamus around parturition: relevance to antipyresis at term Marie-Ste´phanie Clerget-Froidevaux and Quentin J. Pittman* Neuroscience Research Group, Department of Physiology and Biophysics, University of Calgary, Canada T2L 4N1 Received 6 August 2002; revised 23 December 2002; accepted 6 January 2003

Abstract An endogenous antipyresis has been observed around parturition in several species, including rats. It has been proposed that the neuropeptide vasopressin is responsible for this antipyresis via an action on the V1a receptor subtype, but this concept is controversial. We therefore addressed the question of the regulation of V1a receptor expression within the rat hypothalamus around parturition, to assess its possible involvement in the antipyresis phenomenon observed at term. We analyzed V1a receptor mRNA and protein levels in the hypothalamus/preoptic area of female rats at Days 15 and 22 (parturition) of gestation, and at Day 5 of lactation. We used quantitative RT-PCR to assess the mRNA levels and designed a semiquantitative Western blot assay to analyze changes in protein levels between the three stages studied. No significant changes either in V1a receptor mRNA or protein levels were observed between the three stages, suggesting that variations in the hypothalamic V1a receptor expression levels alone cannot account for the endogenous antipyresis observed at term. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Hypothalamus; AVP V1a-R; Fever; Antipyresis; Gestation; Parturition; Lactation; Quantitative RT-PCR; Western blot

Introduction Fever is one of the most important indicators of disease, and plays a crucial role in the neuroimmune response to infection. It is a coordinated endocrine, autonomic, and behavioral response organized by the brain in response to inflammatory stimuli (Elmquist et al., 1997). When fever is abolished experimentally, animals show an increased morbidity and mortality in response to infection, emphasizing the importance of fever as a defense for the protection of the organism (Moltz, 1993). However, there are physiological conditions in which the fever response is reduced or even abolished. This phenomenon has been termed “endogenous antipyresis” (reviewed in Pittman and Wilkinson, 1992), and can be seen in certain neonates, in some forms of hypertension, in acute hypotension, and in parturient animals. With respect to parturition, results from several labora* Corresponding author. Fax: ⫹403-283-2700. E-mail address: [email protected] (Q.J. Pittman).

tories (Kasting et al., 1978; Martin et al., 1995; Simrose and Fewell, 1995; Zeisberger et al., 1981) have shown that pyrogen-induced fever was suppressed around the time of delivery (i.e., from about 96 h before to 24 h after parturition). Prostaglandin E2 (PGE2), a naturally occurring mediator of fever within the brain (reviewed in Blatteis and Sehic, 1997), also induced a lower fever when administered around parturition (Chen et al., 1999; Martin et al., 1996). The impact of this reduced febrile responsiveness on the maternal–infant unit has not been elucidated and, up to now, the mechanism responsible for this antipyresis has not been characterized, even though several hypotheses have been proposed. However, several authors have suggested that the antipyretic arginine vasopressin (AVP) may be a candidate for the increased endogenous antipyretic activity observed at term (Cooper et al., 1988; Roth and Zeisberger, 1992). The antipyretic activity of AVP is well documented (reviewed in Pittman et al., 1998). When introduced into the ventral septal/preoptic area or amygdala, AVP reduces fever via an action on V1a AVP receptors (V1a-R) (Cooper et al., 1987; Naylor et al., 1986). Moreover, this laboratory has

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M.-S. Clerget-Froidevaux, Q.J. Pittman / Experimental Neurology 183 (2003) 338 –345

observed elevated secretion of AVP in the same region around parturition (Landgraf et al., 1991) and both AVP mRNA levels (Zingg and Lefebvre, 1988) and immunoreactivity (Caldwell et al., 1987) are increased during late pregnancy in the hypothalamus. These findings provided correlative evidence suggesting that AVP may be responsible for fever suppression and endogenous antipyresis observed around parturition. However, efforts to identify an obligatory role for AVP in the antipyresis associated with parturition have not resolved the issue. For example, whereas Chen et al. (1999) were unable to provide evidence implicating endogenous AVP in this phenomenon, others (Eliason and Fewell, 1998) reported evidence that endogenous AVP was indeed involved in reduced fevers to some, but not all (Eliason and Fewell, 1999) pyrogens. As an additional way of examining this issue, it is possible that an understanding of the dynamics of AVP receptor expression around the term of parturition may shed some light on the possible involvement of the vasopressin system. As the sequence for the rat AVP V1a-R is known (Morel et al., 1992) it is possible to identify the area of the brain in which it is expressed, as well as the dynamics of its expression. Of relevance is the fact that V1a receptors (Poulin et al., 1988) and V1a-R mRNA (Hurbin et al., 1998; Szot et al., 1994) have been localized to hypothalamic neurons. Thus, to address the question of the expression of V1a-R in the rat hypothalamus around the term of gestation, we used quantitative RT-PCR to measure the level of V1a-R transcripts in the hypothalamus and preoptic area of female rats on Days 15 and 22 (parturition) of gestation and on Day 5 of lactation. To evaluate the level of expression of V1a-R protein in the same animals, we designed a semiquantitative Western blot assay, in which we were able to detect simultaneously specific bands for V1a-R and a specific band for actin protein, as an internal control.

Material and methods 1. Primers and chemicals Oligonucleotide primers were from GIBCO BRL/Life Technologies (Gaithersburg, MD, USA). Restriction endonucleases were purchased from New England Biolabs (Beverly, MA, USA), Moloney murine leukemia virus (MMLV) was from GIBCO BRL/Life Technologies, and Thermus aquaticus (Taq) DNA polymerase and radiolabeled compounds, were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ, USA). RT-PCR reactions were carried out using a Perkin–Elmer thermal cycler (Foster City, CA, USA). For Western blot experiments, acrylamide-bis acrylamide solution and protein standards were from Bio-Rad Laboratories (Hercules, CA, USA), ECL and ECF detection kits were from Amersham Pharmacia Biotech, Inc., V1a-R antibody (AVP1A11-A) and control peptide (AVP1A11-P) were from Alpha Diagnostic International (San Antonio,

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TX, USA), actin antibody was from Sigma Immuno Chemical (St. Louis, MO, USA), and horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated goat antirabbit IgG (secondary antibodies) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals were from Sigma. 2. Animals Pregnant (D1, sperm positive) Sprague–Dawley rats were purchased from Charles River Canada (Lasalle, Que, Canada). They were housed in the vivarium at 20 –22°C on a 12/12-h light– dark cycle with free access to food and water. They were killed at 09:00 AM on Day 15 of gestation (G15, gestation), Day 22 (G22, just before parturition), or Day 5 of lactation (L5, 5 days after parturition) (five animals per group). All experimental protocols were approved by the University of Calgary Animal Care Committee and were carried out in accordance with the Canadian Council of Animal Care Guidelines. 3. Tissue isolation Under pentobarbital anesthesia (5 mg/100 g body weight (bw)), animals were decapitated and the hypothalamus and preoptic area were removed quickly, immersed in TBS solution (25 mM Tris–HCl, 2.7 mM KCl, 137 mM NaCl, pH 7.4) at 4°C, and separated at the midline into two halves. One was immediately minced and frozen into liquid nitrogen for later RNA extraction. The other half was put into lysis buffer (RIPA buffer ⫹ proteolysis inhibitors, see below) for subsequent protein isolation. 4. RNA extraction and quantitative RT-PCR analysis Total RNAs were extracted from hypothalami using Trizol (GIBCO BRL/Life Technologies) according to the manufacturer’s protocol. Briefly, tissue was homogenized with a glass potter into Trizol solution. Chloroform was added, and tubes were centrifuged. After recovery of the aqueous phase, the RNAs were precipitated with isopropyl alcohol and pellets rinsed with ethanol, before being dissolved into TE buffer (pH 7.4) and frozen at ⫺80°C. Quantitative RT-PCR was carried out as described previously (Clerget et al., 1997; Firsov et al., 1994). Briefly, serial dilutions (1.5, 6, and 24 ng) of each RNA were reversetranscribed and amplified simultaneously to a fixed amount of mutated cRNA (3000 molecules) in the same tube as an internal standard. The mutated cRNA acts as an internal standard because it is all derived from the same master sample in which all components are present but the wildtype (wt)RNA; as it is amplified with the same efficiency as the wild-type cRNA, this makes tube-to-tube variations irrelevant for quantification. This mutated cRNA displays the same sequence as V1a-R, except for the substitution of two bases that create a new HindIII restriction site in the

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amplified mutated DNA fragment (see details in Firsov et al., 1994). As validated previously (Clerget et al., 1997), the mutated and wild-type cRNA will be amplified with the same efficiency and thus tube-to-tube variations are irrelevant for quantification. This cRNA was obtained by in vitro transcription of a mutated cDNA, with Riboprobe system-T3 from Promega (Madison, WI, USA), using trace amounts of [␣32P]UTP to measure the amount of cRNA synthesized. Reverse transcription was carried out on a total volume of 50 ␮l in presence of 6.25 pmol of the downstream primer (5⬘-CTGCGTGAACGTGGGGCTCAAGT3⬘, base 1293 relative to the ATG codon), and 400 ␮M dNTP, at 46°C for 45 min. The amplification reaction was carried out in the same tube by adding to each sample 50 ␮l of a mix containing 6.25 pmol of the upstream primer (5⬘-AGCAAGGGTGACAAGGGCTCTGG-3⬘, base 775 relative to the ATG codon), 5 ␮Ci of [␣32P]dCTP, and 1.25 U of Taq DNA polymerase. Tubes were subjected to the following steps: 94°C, 30 s; 62°C, 30 s; 72°C, 1 min, for 28 cycles. The final elongation step lasted 10 min. To discriminate between the DNA fragments formed from the WT and the mutated (Mut) RNAs, the RT-PCR products were digested with HindIII and electrophoresed through a 3% agarose gel. After fixation in 10% acetic acid, the gel was dried and exposed with a storage phosphor screen (Molecular Dynamics, Amersham Pharmacia Biotech). Band intensity was measured using a Storm 860 apparatus and the ImageQuant software (Molecular Dynamics), and the number of V1a-R mRNA molecules calculated using the following formula: molecule of wt mRNA ⫽ (wt/[M1 ⫹ M2]) ⫻ M, where wt is the number of counts measured for the band corresponding to the wt; M1, the counts for the first band of the Mut; M2, the counts for the second band of the Mut, and M, the number of Mut molecules introduced in the reaction. Results are expressed as number of mRNA molecules per nanogram of total RNA. 5. Protein extraction and semiquantitative Western blot analysis Hypothalami/preoptic areas were homogenized at 4°C into RIPA buffer (1⫻ PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) containing proteolysis inhibitors (phenylmethylsulfonyl fluoride (0.5 mM), Aprotinin (30 ␮l/ml), sodium orthovanadate (1 mM), sodium fluoride (1 mM)). After a centrifugation at 10,000 g for 10 min at 4°C, supernatant was removed and centrifuged again. The supernatant contains the total cell lysate. Total protein amount was measured using BCA protein assays from Pierce (Rockford, IL, USA), according to the manufacturer’s instructions. Whole-cell lysate was then mixed with 2⫻ sample buffer (200 mM Tris–HCl, pH 6.8, 4% SDS, 200 mM DTT, 0.2% bromphenol blue, and 20% glycerol), heated at 95°C for 5 min, and frozen at ⫺20°C until use. Thirty micrograms of total proteins was loaded on a 10% SDS–PAGE denaturing gel, electrophoresed in SDS–PAGE

buffer (25 mM Tris base, pH 8.3, 192.5 mM glycine, and 0.1% SDS) using prestained Precision Protein Standards as molecular size marker, and then electrophoretically transferred to a nitrocellulose membrane by semidry transfer with a 4.8 mM Tris base–3.9 mM glycine–20% methanol– 0.0375% SDS buffer. These membranes were incubated for 2 h at room temperature with 10% low-fat powder milk– TBS-T buffer (20 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) to block the nonspecific binding. The membrane was then incubated overnight at 4°C in the same buffer with 5 ␮g/ml of rabbit polyclonal antibody against V1a-R (Hurbin et al., 2002) simultaneously with a 1/5000 dilution of rabbit anti-actin antibody. After 4 ⫻ 5-min washes in TBS-T, the membrane was incubated for 1 h at room temperature with a goat anti-rabbit IgG-HRP or AP conjugate. After 4 ⫻ 5-min washes in TBS-T, immunoreactive proteins on the blot were revealed either with ECL (HRP conjugate) or ECF (AP conjugate) Western blotting detection system. For ECL, the membrane was exposed with X-ray film from 30 s to 7 min. For ECF detection, a Storm 860 apparatus (Molecular Dynamics) was used according to the manufacturer’s instructions. Briefly, the wet membrane was covered with the reagent and put on a plastic sheet and the fluorescence immediately scanned by the Storm 860. The quantification was done using Molecular Dynamics ImageQuant software. The results are expressed as a ratio of the intensity of each band compared to the intensity of the band corresponding to actin. To test the specificity of the V1a-R antibody, the membrane was hybridized as described above with a mix of 5 ␮g/ml of V1a-R antibody and 10 ␮g/ml of the peptide against which the antibody was raised. ECL was used for the detection. To ascertain that V1a-R antibody and actin antibody were not cross-reacting, on the same gel, we used half of a membrane for hybridization with V1a-R antibody alone, and the other half for hybridization with both antibodies, and compared the signals with ECL. The membrane that was hybridized with V1a-R alone was stripped 1 h at 60°C in buffer ␤-mercaptoethanol, Tris, SDS, and rehybridized with actin antibody. ECL was used for detection. 6. Statistics Data are given as means ⫾ SEM. Statistical comparisons between groups were made by ANOVA followed by a Bartlett’s test, and P ⬍ 0.05 was considered significant.

Results 1. Quantification of V1a-R mRNA in rat hypothalamus at G15, G22, and L5 In order to address whether increased expression of V1a-R could be responsible for the antipyresis observed in

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Fig. 1. Quantitative analysis of V1a-R mRNA levels in representative female rat hypothalamus at Days 15 and 22 of gestation, and at Day 5 of lactation. Serial dilutions (1.5, 6, and 24 ng) of totRNA extracted from hypothalami of female rats either at Day 15 (G15) or 22 (G22, parturition) of gestation, or at Day 5 (L5) of lactation were amplified with a constant amount (3000 molecules) of mutated V1a-R synthetic cRNA. The DNA fragments were then digested by HindIII, fractionated on a 3% agarose gel, and analyzed by a phosphorimager. Mut, serial dilutions (1000, 3000, or 9000 molecules) of the mutated V1a-R synthetic cRNA were amplified alone and analyzed as above. RT, the reaction was carried out in the absence of reverse transcriptase on 24 ng of each RNA. WT, wild-type V1a-R PCR product, 519 bp. Mut V1a-R, mutated V1a-R PCR product, 268 and 255 bp after enzymatic restriction.

female rats at the end of gestation, we first determined whether the level of transcripts coding for V1a-R in the hypothalamus was altered between Day 15 of gestation (G15), at parturition (G22), and 5 days after parturition (Day 5 of lactation; L5). Total RNAs were extracted as described under Materials and Methods from hypothalami dissected from rats at G15, G22, or L5 (five animals per group). A mutated V1a-R cRNA was used with the wild-type V1a-R mRNA for quantification. The amplified sequences for Mut and wt RNA were identical, except for the substitution of two bases to create a HindIII restriction site in the amplified mutated DNA fragment. Consequently, the RTPCR products obtained from the wt and the mutated cRNA could be discriminated by HindIII digestion and gel electrophoresis. Mutated DNA was cut into two fragments of 268 and 255 bp, whereas wt was insensitive to HindIII digestion and measured 519 bp. Fig. 1 illustrates the result of an experiment designed to determine the absolute V1a-R mRNA level in the hypothalamus on G15, G22, and L5. Serial dilutions (1.5, 6, and 24 ng) of total RNA extracted from hypothalamus were co-reverse-transcribed and coamplified with 3000 molecules of mutated cRNA. The wt signal was clearly dependent on the amount of hypothalamic RNA introduced in the reaction. This indicates that there was no competition between the two targets during the reaction. Mut and wt signals could be safely compared to calculate the absolute V1a-R mRNA levels. Fig. 2 summarizes the results of V1a-R mRNA quantifications in the hypothalamus/preoptic area at G15, G22, and L5. The expression levels were very high and were constant throughout the gestation and lactation. There was no significant difference between the level of transcripts measured at G15, G22, and L5 (in number of molecules/ng of total RNA ⫾ SEM, being, respectively, 572 ⫾ 140, 507 ⫾ 64, and 509 ⫾ 95).

2. Relative levels of V1a-R protein measured by Western blot Western analysis by use of V1a-R antibody and ECL reagent revealed two major bands (around 50 and 105 kDa) and two secondary bands (around 65 and 80 kDa) instead of the one expected (theoretical molecular weight calculated from V1a-R cDNA sequence: 47.5 kDa). Consequently, we tested the specificity of the V1a-R antibody as described under Materials and Methods. As shown in Fig. 3, addition of control peptide with the antibody completely abolished the detection of three of the four bands observed before. The main band was still lightly detected but the intensity was very faint compared to that observed without peptide. Moreover, long exposure times were needed to reveal this weak band (2 h). Therefore, the detected bands were specific to the epitope of the V1a-R that was used to raise the antibody.

Fig. 2. Comparison of V1a-R mRNA levels in female rat hypothalamus at Days 15 (G15) and 22 of gestation (G22), and at Day 5 of lactation (L5). Values were generated by a phosphorimager and analyzed by ImageQuant software. Values are mean ⫾ SEM, n ⫽ 5. There is no significant difference between values at the three stages of gestation/lactation.

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Discussion

Fig. 3. Specificity of the V1a-R antibody. The specificity of the V1a-R antibody was tested using the peptide against which it was raised. Proteins (30 ␮g) extracted from rat hypothalami were subjected to electrophoresis on a 10% SDS–polyacrylamide denaturing gel and transferred by semidry transfer on a nitrocellulose membrane. When the membrane is hybridized with a mix of V1a-R antibody (5 ␮g/ml) and the control peptide (10 ␮g/ml) (lane ␣V1a-R AB ⫹ pept), the four specific bands revealed by V1a-R antibody alone (␣V1a-R AB) are mostly abolished after 2 h of exposure, confirming the specificity of the antibody.

These bands can reflect different states of glycosylation of the receptor, as described by others (Ancellin et al., 1999; Carnazzi et al., 1997; Phalipou et al., 1997). To further verify that these bands represent V1a-R, we also extracted protein from A7r5 cell lines which express this receptor (but not the V2-R or V1b-R) in large quantities (reviewed in Fahrenholz et al., 1993). A similar pattern of bands was revealed from this tissue (data not shown). To validate our method of codetection of V1a-R and actin, we needed to be sure that the two antibodies were not cross-reacting or interfering with each other. Fig. 4 shows that the patterns displayed in different gels by the successive hybridization of V1a-R and actin antibodies were similar when both antibodies were present at the same time. Therefore, both proteins could be detected in the same experiment, which eliminated the interassay variability introduced by the stripping of the membrane. Fig. 5A is representative of an experiment designed to evaluate the relative expression of each specific band for V1a-R compared to the expression of actin as an internal control. The internal control was used to limit the sampleto-sample variation as arising, for instance, from loading differences. We used ECF detection to allow the direct data acquisition and analysis on the Storm 860 apparatus. Thirty micrograms of protein extract from hypothalamus on G15, G22, or L5 was submitted to Western blot analysis with 5 ␮g/ml V1a-R antibody and a 1/5000 dilution of actin antibody. Four specific bands for V1a-R (approximately 50, 65, 80, and 105 kDa) and one band for actin (42 kDa) were detected. Fig. 5B shows the relative expression of each V1a-R band compared to the expression of actin, at G15, G22, and L5 (five animals per group). The major V1a-R band was at 50 kDa, for each group. No significant difference was observed in the level of expression of V1a-R between groups, either for each band taken individually or for the total V1a-R expression, which was very constant throughout the stages of gestation and lactation studied.

The aim of this study was to explore whether the antipyresis observed at term in the rat was due to changes in the V1a-R expression within the hypothalamus. Our data show that there are no significant changes either in the V1a-R mRNA levels or in the protein levels between G15, G22, and L5 in the rat hypothalamus. Moreover, using Western blot analysis, we detected four bands with an antibody raised against V1a-R, suggesting different states of glycosylation of this receptor. The proportion of each form was constant at the three studied stages. It is now well documented that thermoregulatory control is altered in several species around parturition. In rats, this is manifested as a decrease in core temperature near term (around 0.8°C) followed by an increase (1°C) throughout lactation (Fewell, 1995), as well as an endogenous antipyresis observed at term (Martin et al., 1995, 1996; Simrose and Fewell, 1995). It was in the light of this latter result showing a lower induced fever between 96 h before and 24 h after parturition that we decided to place our studies on Days 15 (before the time of endogenous antipyresis) and 22 (parturition) of gestation (maximal antipyresis) and on Day 5 of lactation (postantipyresis period). We focused on V1a-R expression, because AVP is one of the antipyretic agents thought to be responsible for the different forms of endogenous antipyresis. It is classed as an antipyretic because it decreases induced fever but does not regulate normal body temperature (Ruwe et al., 1985). However, its role in endogenous antipyresis at term is somewhat controversial. Eliason and Fewell have reported that AVP attenuates the febrile response at term to an intracerebroventricular injection of PGE1 (Eliason and Fewell, 1998), thought to be the final common mediator of pyrogen-induced fever. In contrast, they could not implicate AVP in suppression of IL-1␤

Fig. 4. Actin antibody does not compete with V1a-R antibody. Proteins (30 ␮g) extracted from rat hypothalami were subjected to electrophoresis on a 10% SDS–polyacrylamide denaturing gel and transferred by semidry transfer onto nitrocellulose membranes. The membranes were then hybridized with 5 ␮g/ml of V1a-R antibody (␣V1a-R AB), 1/5000 dilution of actin antibody (␣ actin AB), or both of them at the same time (␣V1a-R ⫹ ␣ actin AB) in independent experiments. The presence of ␣ actin antibody does not interfere with the detection of ␣ V1a-R antibody.

M.-S. Clerget-Froidevaux, Q.J. Pittman / Experimental Neurology 183 (2003) 338 –345

Fig. 5. Semiquantitative analysis of V1a-R protein levels in female rat hypothalamus at Days 15 and 22 of gestation, and at Day 5 of lactation using actin as an internal standard. (A) Hypothalamic proteins (30 ␮g) extracted from representative female rats at Day 15 (G15), Day 22 (G22, parturition) of gestation, and at Day 5 (L5) of lactation were subjected to electrophoresis on a 10% SDS–polyacrylamide denaturing gel and transferred by semidry transfer on a nitrocellulose membrane. The membrane was then hybridized with a mix of 5 ␮g/ml of V1a-R antibody and 1/5000 dilution of actin antibody. ECF detection substrate was applied on the membrane and the data were directly acquired by the Storm 860 apparatus. (B) Results are expressed as means ⫾ SEM (n ⫽ 5) of the percentage of the signal of each V1a-R band (respectively 105, 80, 65, and 50 kDa; total ⫽ sum of the individual bands) compared to actin signal for each stage studied (Gestation G15, Day 15 of gestation; Gestation G22, Day 22 of gestation; Lactation L5, Day 5 of lactation). No significant differences were observed between the three groups.

fever at term (Eliason and Fewell, 1999) and Chen et al. 1999 could not detect alterations in prostaglandin feverinduced AVP release as a function of gestational status. These discrepant results raise the possibility that changes in V1a-R expression could account for an AVP-induced antipyresis that would not be made evident by measuring release. As neither the levels of message nor the levels of protein in the hypothalamus/preoptic area changed throughout the latter stages of pregnancy and early lactation, our results suggest that alterations in V1a-R mRNA expression or synthesis are unlikely to account for antipyresis at term. However, several caveats must be considered. A first possibility is that the methodologies we used were not sensitive enough to detect small changes. However, the validity of the quantitative RT-PCR assay has already been amply assessed (Clerget et al., 1997). Briefly, the use of an internal standard differing from the wild-type by only 2

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bases, first, allows for the correction of tube to tube variations, and second, this method circumvents the problem of variations in RT-PCR (RT and PCR reaction efficiencies). While the highly accurate RT-PCR measurements revealed unambiguous stability of the message, stability of message levels does not eliminate the possibility that there is an alteration in protein levels. Thus we repeated all studies with the semiquantative Western analysis. Results show impressive similarity with the two methods. While it could be argued that antibodies against G-protein coupled receptors may poorly discriminate among different receptors, the antibody that we used has been validated in immunohistochemical studies (Hurbin et al., 2002) and the pattern of staining in the gel is similar to that which has been reported elsewhere for partially or highly glycosylated V1a-R (Ancellin et al., 1999; Carnazzi et al., 1997). Furthermore, independent quantification of each band in the gel gave similar results, as did the total receptor protein measurement. In addition, for the semiquantitative Western blot assays, we used a very sensitive chemifluorescent methodology for detection. This chemifluorescent detection method associates all the advantages of fluorescence and chemiluminescence. Its major advantage is that its extreme sensitivity permits quantification (Yanagida et al., 2000). This is due to the fact that it presents a wider dynamic range of detection than an autoradiographic film. Moreover, the simultaneous detection of the actin signal provides normalization of V1a-R expression, as actin levels are constant at the three stages studied. Thus, this detection method based on comparison of two protein levels by chemifluorescence provides relative and accurate quantification of protein expression in a given tissue (as shown by the low standard error of means in Fig. 5B). It thus permits comparison between different physiological or pathological states. Nonetheless, it is impossible to determine using even these sensitive methods whether there is a change in distribution of receptors from the membrane to intracellular compartments or in the coupling of receptors to the intracellular signaling pathways such as has been reported for AVP under some conditions (Poulin and Pittman, 1993). We also cannot eliminate the possibility that there are highly localized changes within the preoptic/hypothalamic tissue chunk which were masked by stable levels (or even changes in the opposite direction) in the remainder of the tissue. A second possibility is that other brain areas are involved, in addition to the hypothalamic/preoptic areas we examined. For example, it is possible that the antipyretic sites responsive to endogenously released AVP may lie in the medial amygdala. However, while AVP injected into these areas is able to decrease induced fever, Federico et al. (1992) were unable to reveal any action of endogenous AVP on V1a-R within this area. Moreover, the tissue chunk in which we measured the V1a-R includes a substantial part of the preoptic area and ventral septal regions where exogenously applied (Ruwe et al., 1985) and endogenously re-

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leased AVP (Naylor et al., 1988) has been shown to affect temperature and fever. These findings of the stability of the V1a-R throughout these stages of reproduction are in agreement with previous data from the uterus at parturition (Clerget et al., 1997) but contrast with the rather dramatic alterations in oxytocin receptor regulation observed throughout pregnancy and as a function of steroid milieu (reviewed in Ivell and Walther, 1999; Zingg et al., 1998). For example, in the hypothalamus, estrogen (Breton and Zingg, 1997) and progesterone (Bale and Dorsa, 1995; Johnson et al., 1991) upregulate oxytocin receptor mRNA and expression, an effect probably responsible for the alterations in such receptors throughout pregnancy and lactation (Insel, 1990; Kremarik et al., 1991). Furthermore, there is now good evidence that progesterone interferes directly with oxytocin but not AVP binding to their respective receptors (Grazzini et al., 1998). Nonetheless, both levels of the V1a-R (Hurbin et al., 2002) and its downstream signaling pathways (Burnard et al., 1983) have been demonstrated to change on a more short-term basis, possibly in response to receptor occupancy. Despite the invariant levels of V1a-R message and protein in the hypothalamus/preoptic area near term, there is little doubt that the expression and release of AVP itself does change significantly at this time (Landgraf et al., 1992; Zingg and Lefebvre, 1988). However, the dynamics of this release do not parallel the alterations in febrile responsiveness seen around the time of parturition. These data, together with that from previous studies using receptor antagonists (Chen et al., 1999) and our results on hypothalamic V1a-R expression, provide little support for a role for AVP in the antipyresis of term. Thus, mechanisms other than the vasopressin system would appear to be responsible for this antipyresis.

Acknowledgments This work was supported by the Canadian Institutes of Health Research. M.S.C.-F. was a Del Duca Foundation (France) and Canadian Institutes of Health Research/Canadian Hypertension Society postdoctoral fellow. We thank Pr. Barbara Demeneix for useful comments during the preparation of this article, Dr. Hans Van de Sande for access to his laboratory, Dr. Mark Brown for technical tips, and Dr. Jean-Marc Elalouf for providing the V1a-R mutant.

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