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Endocrinology Vol. 143, No. 2 456-466
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

The Vasopressin Receptors Colocalize with Vasopressin in the Magnocellular Neurons of the Rat Supraoptic Nucleus and Are Modulated by Water Balance

Amandine Hurbin, Hélène Orcel, Gérard Alonso, Françoise Moos and Alain Rabié

Centre National de la Recherche Scientifique—Unité Mixte de Recherche 5101, Biologie des Neurones Endocrines, Centre de Pharmacologie-Endocrinologie, Montpellier F-34094, France

Address all correspondence and requests for reprints to: A. Rabié, CNRS-UMR 5101, CCIPE, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France. E-mail: rabie{at}ccipe.montp.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activity of the magnocellular neurons that synthesize vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus is modulated by local release of the neuropeptide within the nuclei. V1a and V1b vasopressin receptor genes are expressed in these cells. The present study reports the localization of V1a and V1b receptors using multiple labeling immunocytochemistry. Both receptors are mainly located in vasopressinergic magnocellular neurons and colocalized with vasopressin in cytoplasmic vesicles dispersed throughout the cell. Possible functional modifications of the mRNA and protein levels of the V1a receptor, the major isoform, were also investigated by semiquantitative in situ hybridization and immunocytochemistry in rats submitted to reduced or increased water intake. V1a mRNA and receptor levels varied with water balance. V1a mRNA level dropped in rats submitted to high water intake. Conversely, dehydration up-regulated the V1a receptor content. These observations suggest that the pathways that regulate the expression of the genes encoding vasopressin and the V1a receptor are linked, which fits the present findings that the two partners are colocalized in cytoplasmic vesicles. Colocalization might explain how V1 autoreceptors are controlled by cell activity and/or local concentration of vasopressin (released locally by the neurons themselves), allowing fine adjustment of magnocellular neuron activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOPRESSIN SYNTHESIZED BY the magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei is released at their axon terminals in the neurohypophysis and reaches the bloodstream to act on peripheral targets and regulate plasma volume, plasma osmolality, and liver glycogenolysis (see review in Ref. 1). Many factors control the release of the neuropeptide, the most significant being the alteration of plasma osmolality, detected by osmoreceptors mainly localized in the organ vasculosum of the lamina terminalis (2), which projects to the magnocellular neurons.

Numerous studies have provided evidence that vasopressin and vasopressin mRNA vary in the hypothalamus of osmotically stimulated animals. Immunostaining for vasopressin drops in the hypothalamus of dehydrated rat (3, 4), likely as a consequence of the abundant release of the neuropeptide under this stimulus. On the contrary, vasopressin mRNA detected by either Northern blot (5), dot blot ( 6), quantitative in situ hybridization (5, 7), and competitive RT-PCR (8) is roughly doubled after water deprivation for a few days. This suggests that transcription of the vasopressin gene is enhanced to face the increased release of the neuropeptide. Similarly, a chronic osmotic stress (e.g. a saline load induced either by replacing drinking water with 2% NaCl or by ip injections of a hyperosmotic solution for several days) increases the vasopressin mRNA level, as reported using Northern blot (9, 10), dot blot (6, 11), and quantitative in situ hybridization (10, 12, 13, 14, 15). Conversely, hyponatremia rapidly decreases the vasopressin mRNA level (16, 17, 18), whereas vasopressin content progressively increases in the hypothalamo-neurohypophysial system, consequently leading to a slower turnover (19). Taken together, these experiments involving rapid or sustained osmotic stimulations show that the levels of vasopressin mRNA and vasopressin in supraoptic and paraventricular nuclei are under the tight control of water balance.

Vasopressin receptors expressed by some peripheral tissues have also been shown to be under this control of water balance. Water deprivation for 3 d down-regulates the V2 vasopressin receptor in the kidney, a major peripheral target for systemic vasopressin (20). The authors suggest that the high plasmatic concentration of vasopressin induced by dehydration may have long-lasting influence on the expression of the receptor in the kidney cells. Similarly, rats treated with 1-desamino-8-D-arginine vasopressin (DDAVP), a V2 receptor agonist, display a decrease of the number of kidney V2 receptor binding sites. The decrease is even higher when, in addition, rats are water overloaded, confirming that the expression of the kidney V2 receptor gene depends on the plasmatic concentration of vasopressin, which is in turn regulated by plasma osmolality (21).

In the antehypophysis, the facilitating effect of vasopressin on the ACTH secretion by corticotrophs is mediated by V1b vasopressin receptors and is also modulated by plasma osmolality. Water deprivation and replacement of the drinking water by a saline solution, both conditions that lower the ACTH response to vasopressin, down-regulate the binding sites for vasopressin on corticotroph cells (22), however, without affecting significantly the V1b mRNA level (23). Conversely, treatments which enhance the ACTH response to vasopressin, like chronic ip injection of hypertonic saline solutions, increase the number of binding sites for vasopressin (22) and, in this case, also the level of V1b mRNAs (23).

The genes encoding the V1a and to a lesser extent the V1b vasopressin receptors are also transcribed in the magnocellular neurons of the hypothalamus, as shown by RT-PCR and in situ hybridization (24). There, these autoreceptors modulate, by means of local somatodendritic release of the neuropeptide by the neurons themselves, the electrical activity of the cells and, in this way, the systemic vasopressin release that in fine counteracts the plasma osmolality changes (25, 26, 27, 28, 29). Little is known, however, of the hypothalamic distribution of these vasopressin receptors and their regulation in relation to changes in plasma osmolality. In the present report, immunocytochemistry was used to localize the V1a and V1b vasopressin receptors in the rat hypothalamus. Semiquantitative analysis by in situ hybridization and immunocytochemistry was also used to investigate whether water balance influences the level of the V1a receptor, the major isoform, as well as the mRNA which encodes it.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Most of the standard chemicals were purchased from Sigma (St. Louis, MO), Roche Molecular Biochemicals (Mannheim, Germany), or Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany), unless otherwise indicated. The oligonucleotides were purchased from Genosys (Pampisford, UK). Cy3-labeled tyramide was obtained from NEN Life Science Products (Brussels, Belgium) as component of the Tyramide Signal Amplification Kit. The Cy3-labeled donkey antirabbit IgG, the Cy3-labeled donkey anti-guinea pig, and the Cy5-labeled donkey antimouse IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), the Alexa fluor 488-labeled goat antirabbit IgG from Molecular Probes, Inc. (Eugene, OR), and the guinea pig antivasopressin from Peninsula Laboratories, Inc. (San Carlos, CA). The mouse monoclonal oxytocin-neurophysin PS-38 antibody was produced and purified using the mouse hybridoma purchased from the American Type Culture Collection (Manassas, VA). The rabbit anti-V1a and anti-V1b vasopressin receptor antibodies and the corresponding blocking peptides were purchased from Alpha Diagnostic (San Antonio, TX). Both antibodies were raised using synthetic peptides whose sequence are located in the extracellular N-terminal domain of the rat receptors. They do not cross-react neither between them nor with the V2 isoform of the vasopressin receptor.

Experimental animals
Adult male Wistar rats (Charles River Laboratories, Inc., L’Arbresle, France) were used in accordance with the European laws governing the care and use of experimental animals. The animals (280–300 g) were divided into three groups of three animals (control; reduced water intake, RWI; high water intake, HWI) and placed in individual metabolic cages. The RWI rats had a limited daily amount of drinking water (they drank a mean of 14.1 ml/d) while control and HWI rats had a free access to drinking water (the control rats drank a mean of 30.3 ml/d, the HWI rats a mean of 4.4 ml/d). The HWI rats were treated according to Bouby et al. (30): the food in powder form (UAR A04, Epinay sur Orge, France) was melted with Agar powder (Sigma) and added to hot water (25/1/95, wt/wt/vol) to prepare a gel. Each HWI rat received daily and actually ate 120 g gel and thereby 95 ml/d water (plus a little drinking water). The control and RWI rats received daily 25 g food plus 1 g Agar powders. With this protocol, the only difference between controls and RWI rats was the amount of drinking water, and between controls and HWI rats, essentially the water incorporated in the gel. Urine was collected daily; its volume was measured and its osmolality determined using a Roebling osmometer (Berlin, Germany).

In situ hybridization
After 1 wk treatment, the rats were decapitated and their brains were rapidly removed, frozen at -40 C in isopentane and stored at -80 C. Frontal sections (12 µm-thick) comprising the right and left supraoptic nuclei were cut at -20 C with a CM3000 cryostat (Leica Corp., Nusslach, Germany) and thaw mounted on polylysine-treated slides. Three sections (one for each treatment; control, RWI, HWI) were mounted in a random order on each slide.

The sections were air-dried and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min at room temperature, washed with PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4), dehydrated in increasing concentrations of ethanol (70% and 95%), and air-dried. Two oligonucleotidic probes, specific for the V1a vasopressin receptor, 3'-end-labeled with biotin 16-deoxyuridine 5-triphosphate using terminal transferase (Roche Molecular Biochemicals), were hybridized simultaneously (24): the sections were flooded with the two probes (10 nM final concentration) in hybridization buffer made of 50% deionized formamide, 0.08 M Tris buffer, pH 7.5, 0.6 M NaCl, 4 mM EDTA, 0.05% Na2H2P2O7, 0.05% Na4P2O7, 0.2% N-lauroylsarcosine, and 1x Denhardt’s solution (0.02% polyvinylpyrrolidone, 0.02% BSA, 0.02% Ficoll). Hybridization was performed in a humid chamber overnight at 42 C. The slides were then washed at 45 C in decreasing concentrations of SSC (2x, 0.5x, and 0.1x for 30 min each; 1x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7) and immersed in Buffer A (0.1 M Tris buffer, pH 7.5, 1 M NaCl, 2 mM MgCl2). Then, the hybridized biotinylated probes were revealed by 30 min incubation with horseradish peroxidase-conjugated streptavidin [1/100 diluted in 0.1 M Tris buffer, 0.15 M NaCl, 0.5% blocking reagent (NEN Life Science Products)]; the peroxidase detection was performed by addition of Cy3-tyramide stock solution (1/50 in 1x amplification diluent; NEN Life Science Products) for 8 min at room temperature. The sections were then washed with 0.1 M Tris buffer, 0.15 M NaCl, and 0.05% Tween 20 and mounted in Mowiol.

Immunocytochemistry
After 1 wk treatment, the rats were fixed-perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains and the hypophysis were dissected, postfixed for 3 d in the same fixative and rinsed with PBS. Frontal sections of the brain (comprising the supraoptic nuclei) and hypophysis (50 µm-thick) were cut with a Lancer 1000 vibratome and processed for immunocytochemistry.

The sections used in the semiquantitative study were floated at 4 C for 65 h in a 1/1000 dilution in PBS-BSA buffer (PBS plus 2% BSA) of the V1a or V1b vasopressin receptor antibody, rinsed three times for 10 min in PBS, incubated for 2 h at room temperature in a 1/1000 dilution of Cy3-labeled donkey antirabbit IgG in PBS-BSA, rinsed three times for 10 min in PBS, and mounted in Mowiol. The sections used for studying colocalization were incubated simultaneously with the rabbit vasopressin receptor antibody (as above), the guinea pig vasopressin antibody (1/500 dilution in PBS-BSA), and the mouse monoclonal oxytocin-neurophysin PS-38 antibody (1/10 000 dilution in PBS-BSA). They were revealed using Alexa fluor 488-labeled donkey antirabbit (1/500 in PBS-BSA), Cy3-labeled donkey anti-guinea pig IgG (1/1000 dilution in PBS-BSA), and Cy5-labeled donkey antimouse IgG (1/500 dilution in PBS-BSA) secondary antibodies, respectively, and mounted in Mowiol.

Semiquantitative analysis
In situ hybridization.
Four slides were prepared for each triplet of animals (control, RWI, HWI). Three of them were hybridized with the V1a vasopressin receptor specific probes and provided a total of six test images of supraoptic nucleus per animal. The fourth slide was hybridized without the probes and provided for each animal two images of the nonspecific labeling.

Immunocytochemistry.
Vibratome sections from each triplet of animal (control, RWI, HWI; three sections of each) were floated in the same well containing the anti-V1a receptor in PBS-BSA. They provided a total of six test images of supraoptic nucleus per animal. A second well containing sections from each triplet of animal (control, RWI, HWI; one section of each) was incubated without the antibody and provided for each animal two images of the nonspecific labeling.

Analysis.
Image acquisition was made using a Bio-Rad Laboratories, Inc. (Hercules, CA) MRC 1024 confocal microscope (x60 objective). Each image corresponded to about 20% of the supraoptic nucleus area. The settings of the confocal microscope (laser power, diaphragm, exposure time, thickness of the section) were made on a control section and kept unchanged to digitize the sections of the corresponding treated animals.

The images were processed using the public domain program NIH Image 1.62 (National Institute of Health, Bethesda, MD) with a macro-command written for this purpose. The final result is the measure of the mean pixel intensity in the image, after thresholding (to eliminate the general background) and subtraction of the mean of the values obtained on the section hybridized without probes or antibody (to eliminate the nonspecific labeling of the tissue). This result can thus be considered as a measure, expressed in arbitrary units, of the labeling intensity per unit area of the supraoptic nucleus. Each experimental point is the mean of measures made on six images per animal. Three (in situ hybridization) or four (immunocytochemistry) animals have been studied for each experimental group (control, RWI, HWI). The results are expressed as means ± SEM.

The analysis was performed using two-way (treatment vs. time) or one-way (treatment) ANOVA; pairwise comparisons among means were thereafter performed using the Duncan’s multiple range test (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
V1a and V1b receptors colocalize with vasopressin in magnocellular neurons
Within the hypothalamus, immunostaining for V1a and V1b vasopressin receptors was found in the supraoptic (Fig. 1Go, A and D; Fig. 2Go, A and D), paraventricular (Fig. 1Go, G and J), and suprachiasmatic (not shown) nuclei. Immunostaining was also observed in extrahypothalamic regions where vasopressin is known to act, for example in the pyramidal and granular cells of the hippocampal formation (not shown). Nevertheless, the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus were the most heavily labeled cells throughout the forebrain. All the magnocellular neurons immunostained for vasopressin were also immunolabeled for the vasopressin receptors. Immunostaining was, however, somewhat different between both types of receptors: first, intensity of the labeling detected throughout these nuclei was obviously lower for V1b than V1a receptors (Fig. 1Go, compare D with A, J with G; Fig. 2Go, compare D with A); second, beside the labeling of cell bodies and large dendrites of magnocellular neurons, signal for V1b receptors was also observed in nerve fibers throughout the forebrain, which is not the case for the V1a receptors.



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  		Figure 1. Low-magnification views of supraoptic and paraventricular nuclei, showing that V1a and V1b vasopressin receptors are essentially located in magnocellular neurons expressing vasopressin. Triple labeling confocal microscope general views of the supraoptic (A–F) and paraventricular (G–L) nuclei, showing the colocalization of vasopressin, but not oxytocin, and V1a or V1b receptor. A and G, V1a vasopressin receptor (V1a-R); D and J, V1b vasopressin receptor (V1b-R); B, E, H, and K, vasopressin; C, F, I, and L, oxytocin-neurophysin. Note that the labeling is more intense for the V1a than the V1b receptor, and that both are less intense than the immunostaining for the neuropeptides. Images are the mean of five confocal planes (0.8 µm-thick). Bar, 50 µm.

 


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  		Figure 2. Detail of the supraoptic nucleus, showing that V1a and V1b vasopressin receptors are differentially expressed in vasopressin magnocellular neurons. Triple-labeling confocal microscope images: A, V1a vasopressin receptor (V1a-R); D, V1b vasopressin receptor (V1b-R); B and E, vasopressin; C and F, oxytocin-neurophysin. The arrows in A–C and D–F point to the rare cells that synthesize both vasopressin and oxytocin, which are also positive for the V1a or V1b receptor. The arrowheads in F point to oxytocin magnocellular neurons, clearly not immunostained for the V1b receptor and vasopressin. Note in D that the ventral portion of the supraoptic nucleus contains nerve fibers immunostained for the V1b receptor that are not labeled for vasopressin (E) or oxytocin (F), which is obviously not the case for the V1a antibody (compare A and B). Images are the mean of five confocal planes (0.8 µm-thick). Bar, 10 µm.

 
When triple immunocytochemical labeling of V1 receptors, vasopressin, and oxytocin-neurophysin was performed, the intensity of the signals obtained for the V1 receptors were always lower than those obtained for the neuropeptides (Fig. 1Go, compare A with B or C; D with E or F; G with H or I; J with K or L; see also details in Fig. 2Go). Nevertheless, the images clearly showed that immunostaining for both types of vasopressin receptors (V1a or V1b) in the supraoptic and paraventricular nuclei is mainly located in the vasopressin magnocellular neurons (Fig. 1Go; compare A and B, D and E; see details of the supraoptic nucleus in Fig. 2Go). With both vasopressin receptor antibodies, labeling of the oxytocin magnocellular neurons was very faint or nil (Fig. 2FGo, arrowheads, with no staining in D and E), excepted for the rare cells which contained both neuropeptides simultaneously (Fig. 2Go, arrows in A–C and D–F).

Confocal images of supraoptic magnocellular neurons taken at higher magnification showed that the V1a receptors were mainly associated with cytoplasmic vesicles (Fig. 3AGo) that also contained vasopressin (Fig. 3BGo; compare with 3AGo) and are dispersed throughout the cytoplasm. The same colabeling of vasopressin containing vesicles was observed using the anti-V1b receptor antibody (Fig. 3Go, C and D).



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  		Figure 3. High-magnification views of supraoptic magnocellular neurons, showing that V1a and V1b vasopressin receptors are colocalized with vasopressin in cytoplasmic vesicles within the cell body and dendrites. Double-labeling confocal microscope images of magnocellular neurons in the supraoptic nucleus: the punctate immunocytochemical signals for either the V1a (A) or V1b (C) receptors are clearly located in vesicles also stained for vasopressin (B, D). Single confocal plane (0.8 µm-thick). Bar, 5 µm.

 
Confocal images of the neurohypophysis showed that the V1a receptors in the axon terminals of this area were colocalized with vasopressin and not oxytocin (Fig. 4AGo; compare with 4BGo and 4CGo). The signal for the V1b receptors was faint in these terminals (Fig. 4DGo; compare with 4EGo), whereas it was intense in fine processes surrounding them (Fig. 4DGo). These processes are probably extensions of the pituicytes, on which the presence of V1b receptors has already been reported (32).



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  		Figure 4. Detailed views of the neurohypophysis, showing that V1a and V1b vasopressin receptors are mostly associated with vasopressin and not oxytocin axon terminals. Triple-labeling confocal microscope images: A, V1a vasopressin receptor (V1a-R); D, V1b vasopressin receptor (V1b-R); B and E, vasopressin; C and F, oxytocin-neurophysin. The arrows in A, B, D, and E point to vasopressin axon terminals. The arrowheads in C and F point to oxytocin axon terminals. Note that the signal is faint for the V1b vasopressin receptors in the axon terminals, whereas intense labeling is observed in fine processes. Images are the mean of three confocal planes (0.8 µm-thick). Bar, 5 µm.

 
Lastly, preabsorption of the antibodies with the corresponding blocking peptides eliminated the immunocytochemical signals (not shown).

Water balance modulates the V1a receptor mRNA and V1a receptor levels in the supraoptic nucleus
The study was performed only on the V1a receptor isoform, i.e. on the major isoform expressed in the vasopressin magnocellular neurons (24). The signals obtained for the V1b isoform, especially by in situ hybridization, were indeed too low to expect reliable results from a quantitative approach.

Obtaining animals with modified water balance.
Adult male rats were subjected for 1 wk to a relatively mild protocol leading to reduced (RWI) or high water intake (HWI). The volume (Fig. 5AGo) and osmolality (Fig. 5BGo) of the urine they produced varied as expected for animals with a modified water balance. From the first day of treatment onwards, urine of the HWI rats had a much lower osmolality (a mean of -77%) and a volume considerably increased (up to +410%), when compared with control rats. Conversely, urine of the RWI rats was less abundant (a mean of -52%) and progressively more concentrated (up to +177% osmolality), when compared with control rats. These animals with mildly modified water balance have thus been used in a semiquantitative study of the V1a receptor mRNA and V1a receptor.



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  		Figure 5. Water balance affects urine parameters. Daily changes in the urine volume (A) and osmolality (B) in control rats and rats submitted to RWI or HWI. Means ± SEM (n = 3). Two-way variance analysis (treatment vs. time): treatment effect, P < 0.01; time effect, not significant. Duncan’s test: * and +, significant difference (P < 0.01 and P < 0.05, respectively) between HWI and control or RWI; {ddagger}, significant difference (P < 0.01) between control and RWI.

 
Semiquantitative analysis of the V1a receptor mRNA and V1a receptor.
The densitometric analysis of the supraoptic nucleus images obtained after in situ hybridization of V1a specific probes (Fig. 6Go, A–C) showed that modifying water balance led to important changes in the V1a mRNA levels (Fig. 7AGo). The signal was significantly decreased (-59%, P < 0.01) in the HWI rats after 1 wk treatment compared with controls. Conversely, the signal was increased in the RWI rats (+26%), in proportions similar to those observed for the mean osmolality of urine, however without reaching the significance level.



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  		Figure 6. Examples of images used in the semiquantitative study of the V1a mRNA and V1a receptor levels in the supraoptic nucleus. V1a mRNA was detected by in situ hybridization (A–C) and V1a receptor by immunocytochemistry (D–F) in control rats and rats submitted to 1 wk on RWI or HWI. The original dark-field fluorescence images were inverted for the semiquantitative study. Note the drop of the V1a mRNA in HWI rats and the up-regulation of the V1a receptor in RWI rats. Images are the mean of three confocal planes (0.8 µm-thick) taken near the section surface. Bar, 20 µm.

 


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  		Figure 7. Water balance modulates the level of the V1a mRNA and V1a receptor in the supraoptic nucleus. V1a mRNA (A) and receptor (B) levels were measured in control rats and rats submitted to 1 wk on RWI and HWI. Means ± SEM (n = 3 in A, n = 4 in B). One-way variance analysis (treatment): P < 0.01. Duncan’s test: *, significant difference (P < 0.01) between HWI and control or RWI in A, between RWI and control or HWI in B.

 
Changes in water balance do not have marked effects on the pattern of immunostaining: namely, the colocalization of vasopressin and V1a receptor in cytoplasmic vesicles was not disturbed. Visual comparison of the RWI and control animals indicated that dehydration increased the immunocytochemical signal for V1a receptor (Fig. 6Go, compare D and E). Conversely, HWI animals appeared nearly normal (Fig. 6Go, compare D and F). The semiquantitative analysis confirmed that V1a receptors are significantly up-regulated (+35%, P < 0.01) in the RWI rats (Fig. 7BGo). On the contrary, they were only slightly (-10%) and not significantly reduced in the supraoptic nuclei of the HWI rats (Figs. 6FGo and 7BGo), in spite of the large (-59%) and significant decrease in the mRNA signal detected under such conditions (see above and Figs. 6CGo and 7AGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides evidence, for the first time to our knowledge, that in the magnocellular neurons of the hypothalamus V1a and V1b vasopressin receptors are essentially expressed by vasopressinergic neurons, and that within these neurons they are mainly colocalized with their ligand vasopressin in cytoplasmic vesicles. Furthermore, our data show that, in the supraoptic nucleus, the level of the mRNA encoding the V1a vasopressin receptor (the major vasopressin receptor isoform expressed in the vasopressinergic magnocellular neurons; Ref. 24) varies significantly with water balance, in parallel with the receptor content itself.

Ligand/receptor colocalization
The first aim of this study was to localize the vasopressin receptors in the rat hypothalamus. Our results show that although immunolabeling was also found in other hypothalamic (suprachiasmatic nuclei) or extrahypothalamic (hippocampal formation) regions where vasopressin has actions, the vasopressinergic magnocellular neurons of the supraoptic and paraventricular nuclei show the highest immunostaining for both the V1a and V1b receptor subtypes throughout the forebrain. They also show that the immunocytochemical signal is higher for the V1a than the V1b subtype. These observations corroborate our recent report showing by in situ hybridization that the corresponding mRNAs were located in the vasopressinergic neurons of the hypothalamus and that the V1a receptor was the major isoform (24). The fact that oxytocinergic neurons were sometimes very faintly labeled is also in agreement with the presence of V1a receptors (probably at very low level) demonstrated by single cell RT-PCR in these neurons (24). It was also observed, as expected, that high levels of V1 receptors were found in the rare magnocellular cells that were immunostained for both vasopressin and oxytocin.

A recent report (32) described labeling of nerve fibers throughout the central nervous system using an antibody directed against the rat V1b receptor. Previous treatment of the animals with colchicine was necessary to detect labeling of cell bodies in some areas; however, the V1b receptor was not detected in the magnocellular neurons of the supraoptic nuclei. Our results, obtained with a different antibody, confirm that neurites immunostained for the V1b receptor are visible in many brain regions. However, we show here that, with this different antibody, the neuritic labeling was not the most prominent because labeling could also be observed into neuronal cell bodies, and namely in magnocellular neurons of the supraoptic and paraventricular nuclei. The differences between the two studies probably arise from 1) the epitopes chosen to raise the antibodies, and 2) the fixative used. In the study from Hernando et al. (32), the peptide was selected in the C-terminal domain of the receptor while it is in the N-terminal domain in our case. Apparently, the C-terminal epitope was sensitive to fixation since it was not detectable using the common 4% paraformaldehyde used in the present report but only using Bouin’s fixative. Nevertheless, because immunostaining for the V1a receptor was, in our hands, low in neurites, it is possible that V1a and V1b receptors are sorted differently within the magnocellular neurons. Another example is the intense staining obtained for the V1a receptor in the axon terminals of the neurohypophysis, compared with the faint signal for the V1b receptor. Double immunostaining, using antibodies against V1a and V1b receptors raised in different species, is needed to confirm the differential localization of these receptors within magnocellular vasopressin neurons, which could be important to understand the respective functional roles of the two receptors and thus to explain why the same cell needs to synthesize both.

A consistent result of this study is the colocalization of the receptors with vasopressin in cytoplasmic vesicles dispersed throughout the perikaryon and the proximal dendrites. Most likely, these vesicles are the secretory granules (or clusters of secretory granules) stored in high number in the cell body and the dendrites of the magnocellular neurons (33). This fits the present observation that immunostaining for vasopressin receptors was also associated with vasopressin in the neurohypophysial axon terminals of these neurons, which are filled with neurosecretory granules ready to be released. Similarly, vesicular oxytocin receptors have been described in the cell bodies of oxytocin magnocellular neurons and their nerve terminals in the neurohypophysis (34). The cytoplasmic vesicles could however also be, for a part, endosomal and lysosomal vesicles, which efficiently recycle the vasopressin receptors that are rapidly internalized after ligand binding (35). Another example of colocalization of a receptor (the {kappa} opioid receptor) and its ligand (dynorphin) has been recently described in secretory vesicles of the nerve terminals of the vasopressin magnocellular neurons (36, 37). Our immunocytochemical images suggest that such a vesicular ligand/receptor colocalization also exists for vasopressin and its two V1a and V1b autoreceptors. As already explained in the papers cited above, the biochemical conditions (pH, ions, concentrations) within the secretory or recycling vesicles would not allow receptor-ligand interaction. Nevertheless, these conditions change dramatically as soon as the vesicle is fused to the plasma membrane and is opened to the extracellular space: immediately, high local amounts of the ligand are ready to bind the receptor. In the present case, vasopressin released by granule exocytosis could rapidly saturate the V1a and V1b receptors newly incorporated to the plasma membrane via fusion of the same granule membrane. Both the rapid endocytosis of the receptors upon ligand binding (within a few minutes in HEK 293 cells transfected with V1a vasopressin receptors; Ref. 35) and the slow recycling of the receptors to the plasma membrane (within 1 h in the same cells, without significant degradation of the receptor; Ref. 35), could thus explain why the numerous experiments attempting to visualize binding sites for vasopressin on magnocellular neurons have failed up to date (38, 39, 40, 41). Likely, very few free binding sites are available for experimentally added vasopressin receptor ligands, either when the cell is at rest (when local and axonal vasopressin release are almost nil) or highly active (when they are maximal). The mechanism of simultaneous delivery on the cell membrane of the receptor and its ligand is also of major importance for the understanding of the autocrine regulation of these cells described many years ago and exerted via the somatodendritic release of neurosecretory granules containing vasopressin (33 ; for a recent review, see Ref. 42). It could for example explain why the locally released neuropeptide rapidly stimulates the inactive magnocellular cells: vasopressin binding to the local receptors inserted simultaneously into the membrane increases the cytoplasmic calcium concentration (43), which in turn could enhance the release of additional vesicles to the plasma membrane in an auto-amplified mechanism that rapidly leads to high cell activity. Thereafter, the processes that return the cytoplasmic calcium concentration to basal levels and internalize the ligand/receptor complexes slow down the release of new vesicles and therefore the vasopressin signaling and eventually the cell activity.

Physiological regulation of receptor expression
The second aim of this report was to determine whether the most powerful means to modulate the electrical activity of the vasopressinergic magnocellular neurons, i.e. the hydration state, was able to affect the expression of the V1a vasopressin autoreceptor. In the present study, water balance was modified by acting only on the daily water intake, all the other parameters such as food composition and quantity, or animal handling, being similar for the three groups of animals studied. In mammals, water balance is very efficiently regulated by adjustment of water intake (controlled by thirst sensation) or excretion (controlled by the systemic secretion of vasopressin and its lasting effects on urine concentration) (44). The equilibrium between these two processes can easily be moved: when water availability is reduced, the plasmatic level of vasopressin is increased, whereas it is reduced when water intake is high (44, 45). The protocol used here was chosen to limit the stress that inevitably occurs with the other experimental approaches currently used to induce water balance changes, e.g. severe dehydration by complete suppression of drinking water, or ingestion of salt-loaded water, and especially daily ip injection of water or saline. Nevertheless, important differences in urine volume and osmolality were obtained here, indicating that clear modifications of water balance were effectively obtained, and therefore that plasma osmolality, plasma vasopressin, and vasopressin level in the magnocellular nuclei have been altered as abundantly described in the literature (4, 6, 12, 15, 20, 30, 46, 47, 48, 49, 50).

The level of the V1a vasopressin mRNA was determined by semiquantitative in situ hybridization in the three groups of animals studied. A classical quantitative approach, using for example radioactive probes, was excluded because such probes have never allowed the detection of V1a mRNAs in the magnocellular part of supraoptic or paraventricular nuclei (51). To visualize in situ the V1a mRNAs, we therefore used the only method available to date: simultaneous in situ hybridization of two oligonucleotidic probes, revealed by fluorescent labeling deposited by the tyramide amplification system (24). Maximal precautions were taken to overcome possible drawbacks associated with such a labeling technique: 1) each individual value was the mean of six measures made on six images of the supraoptic nucleus (right and left) taken on three different and nonconsecutive sections; 2) three animals were studied for each experimental point; 3) the sections of the three treatments studied (control, RWI, HWI) were mounted on the same slide to ensure that they were processed exactly under the same hybridization and labeling conditions; 4) the three sections were mounted in a random order on the replicate slides to avoid a possible positional effect during slide processing; 5) it was verified in preliminary experiments that the amplification reaction did not lead to a very rapid saturation of the signal and thus that, with the short amplification time used (8 min), a good linear relationship between the labeling and the actual level of the V1a mRNA could be expected; 6) confocal images of the three groups were acquired with exactly the same settings of the microscope; 7) lastly, the densitometric analysis of the images was performed without any manual intervention, using a program running a macro-command developed for this purpose. A similar semiquantitative study of the V1a vasopressin receptor was performed using fluorescence immunocytochemistry and the antibody directed against this receptor.

Our results showed that experimentally modifying the water balance for 1 wk in adult rats significantly affects (P < 0.01) the level of both the V1a mRNA and the V1a receptor in the supraoptic nucleus. The mRNA level was reduced by 59% in the animals overloaded with water (and therefore expected to have a very low circulating concentration of vasopressin, resulting from a drastic inhibition of the secretory activity of the vasopressin magnocellular neurons). After the same treatment, the level of V1a receptor was not significantly affected, which suggests that the turnover of the receptor is reduced. This is not surprising because the receptor is contained in the same vesicles as vasopressin, and as these vesicles were stored under this physiological condition. Conversely, both the V1a receptor mRNA (+26%, although not significantly) and the receptor immunostaining (+35%, P < 0.01) were increased in the dehydrated rats (expected to have a high circulating concentration of vasopressin, due to the high secretory activity of the vasopressin magnocellular neurons). This is also not surprising given the high turnover of the secretory vesicles as a consequence of dehydration. Perhaps the highest increase in the receptor vs. mRNA can be related to the recycling of the former after internalization following vasopressin binding. Similarly, {kappa} opioid receptors were increased in vasopressin magnocellular neurons after hyperosmotic ip injection (36). In fact, many other genes are up-regulated in these neurons to face the high cellular activity induced by dehydration (52, 53, 54).

Many physiological conditions modulate the electrical activity of the vasopressin magnocellular neurons. Water overload completely inhibits these neurons and therefore the resulting release of the neuropeptide (55, 56). Dehydration has the reverse effect (55, 56). Numerous afferents to supraoptic and paraventricular nuclei, projecting from osmosensible structures (2, 57), as well as intrinsic osmosensibility of the magnocellular neurons (2, 57), participate in water balance regulation. The somatodendritic release of vasopressin by the magnocellular neurons themselves, within their own extracellular space (and therefore toward the vasopressin autoreceptors on their cell membrane and toward the receptors worn by the neighboring neurons) is also involved. This autocontrol mechanism is of major importance for the fine tuning of the secretory activity in the neurohypophysis (29), and in this way for the peripheral efficacy of the neuropeptide (58, 59, 60). The modulation in the level of the V1a vasopressin receptor mRNA by water balance suggests that activity of excitatory and inhibitory afferents and/or extracellular concentration of vasopressin arising from the somatodendritic release are somehow linked to the regulation of the gene encoding the major vasopressin autoreceptor in the magnocellular vasopressin neurons. It now remains to investigate the molecular mechanisms underlying this link. One important component could be the colocalization of the neuropeptide with its two subtypes of receptors within the secretory or recycling vesicles, which physically links the fate of the three molecules.


   Acknowledgments
 
Thanks are due to C. Serradeil-Le Gal for helpful discussions, to L. Bankir and N. Bouby for their help in the design of the animal treatment, and to A. Desoeuvre and D. Haddou for animal care. Prof. J. Brochier has kindly provided the facilities and his experience in the preparation of the PS-38 monoclonal antibody. Confocal microscopy was performed at the Centre Régional d’Imagerie Cellulaire (Montpellier), with the expert assistance of N. Lautredou-Audouy. We are also grateful to V. Tobin for critical review of the manuscript.


   Footnotes
 
The work was partly supported by the Centre National d’Etudes Spaciales (FCM, 793/CNES/98/7346) and the Fondation pour la Recherche Médicale (FRM/RA no. 000001/3). A. Hurbin present address: EMI 9924, INSERM, Institut Albert Bonniot, 38706 La Tronche Cedex, France.

Abbreviations: HWI, High water intake; RWI, reduced water intake.

Received June 12, 2001.

Accepted for publication October 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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