Increase of vasopressin mRNA in the hypothalamus of inbred polydipsic mice

Brain Research Bulletin, Vol. 50, No. 1, pp. 47–51, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter

PII S0361-9230(99)00082-9

Increase of vasopressin mRNA in the hypothalamus of inbred polydipsic mice Rieko Nishi,1 Yoichi Ueta,1* Ryota Serino,1 Masayoshi Nomura,1 Yukiyo Yamamoto,1 Izumi Shibuya,1 Kiyomi Koizumi,2 and Hiroshi Yamashita1 1

Department of Physiology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan; and 2Department of Physiology, State University of New York, Health Science Center at Brooklyn, NY, USA [Received 29 March 1999; Revised 31 May 1999; Accepted 31 May 1999]

ABSTRACT: The genetically inbred polydipsic mice, STR/N strain, are characterized by extreme polydipsia and polyuria without arginine vasopressin (AVP) deficiency. The expression of AVP gene in the hypothalamus of polydipsic and non-polydipsic mice was examined by Northern blot analysis and in situ hybridization histochemistry. Northern blot analysis revealed that the total amount of AVP mRNA in the hypothalamus of the STR/N mice was approximately three-fold of that in the control, ICR mice. In situ hybridization histochemistry showed that the signals of AVP mRNA in the paraventricular (PVN) and supraoptic nuclei (SON) of the STR/N were stronger than those in the ICR. Although AVP gene transcripts were detected in the anteroventral parts of the PVN (avPVN) in the STR/N, there was a few AVP transcripts in the same area (avPVN) in the ICR. There were no differences in plasma osmolality and hematocrit between STR/N and ICR mice. These results suggest that upregulation of AVP mRNA in the hypothalamus of STR/N may be involved in the central mechanism responsible for the polydipsia in genetically polydipsic mice. © 1999 Elsevier Science Inc.

the PVN and supraoptic nuclei (SON) of polydipsic mice are increased in numbers and distributed differently in the polydipsic mice, comparing to those in control mice [9]. Water deprivation causes a remarkable induction of c-fos protein in the circumventricular organs of the polydipsic mice [20]. In the present study, the expression of AVP gene in the hypothalamus of STR/N and ICR mice was examined by Northern blot analysis and in situ hybridization histochemistry. In addition, AVP-immunopositive fibers in the hypothalamus of STR/N mice were observed in detail. MATERIALS AND METHODS Male inbred polydipsic mice (STR/N) and their control, male ICR strain at 6 –12 months of age were used. They were individually housed in plastic cages under constant room temperature, humidity and 12-h light:dark cycle (lights on at 0700 h). Tap water and dry food were given ad libitum. Water intake of mice was measured individually once a day at 1100 h at least for 3 days before each experiment. The polydipsic mice that drank more than 20 ml/day were used for the experiment.

KEY WORDS: Hypothalamus, Inbred mice, mRNA, Polydipsia, Vasopressin.

Dehydration

INTRODUCTION

Tap water was deprived for 24 h and 48 h in control mice. The STR/N mice housed under normal condition were used for quantitative analysis of the hypothalamic AVP gene expression. Dry food was always available throughout the period of water deprivation. For Northern blot, whole hypothalamic areas obtained from four animals were mixed and used in each group (euhydrated, 24 h and 48 h dehydrated ICR mice and euhydrated STR/N). The experiment was carried out three times independently. For in situ hybridization histochemistry and immunohistochemistry for AVP, three euhydrated animals were used. When euhydrated animals were decapitated, trunkal blood was collected for measurement of plasma osmolality and hematocrit.

The inbred polydipsic mice (STR/N) that was originally described by Silverstein and his colleagues are characterized by extreme polydipsia and polyuria without arginine vasopressin (AVP) deficiency [16 –18]. Our previous studies have revealed the characteristics of the STR/N strain of mice as follows: Although daily water intake in the STR/N mice is five- to eight-fold more than that in control mice, food intake is similar [12,16 –18]. Plasma osmolality and ion concentrations are similar to those in control mice [16 – 18]. The polydipsia can be attenuated by some manipulations, such as centrally administered angiotensin antagonists [10,12] or a ␬-receptor antagonist [11,12], and by feeding sodium-free diet [13,14]. In vitro studies have revealed that responses of neurons in the paraventricular nucleus (PVN), the subfornical organ (SFO), and the anteroventral third ventricle (AV3V) region to angiotensin and opiates in polydipsic mice differ from those in control mice [6,7,15]. AVP- and oxytocin (OXT)-immunopositive neurons in

Tissue Preparation For Northern blot analysis, animals were decapitated and the whole hypothalamic areas were quickly removed by sterile mic-

* Address for correspondence: Dr. Y. Ueta, Department of Physiology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Fax: ⫹81-93-692-1711; E-mail: [email protected]

47

48 rotome blades. The tissues were immediately transferred into the denatured solution. For in situ hybridization histochemistry, the mice brains were collected on powdered dry ice immediately after decapitation. For immunohistochemistry, animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/ kg), and perfused transcardially with 0.9% saline containing heparin (1000 U/500 ml), followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). The brain was removed and postfixed in the fixative overnight at 4°C.

NISHI ET AL. TABLE 1 PLASMA OSMOLALITY AND HEMATOCRIT IN STR/N AND CONTROL MICE

Plasma osmolality (mOsm/kg H2O) Hematocrit (%)

ICR Mice

STR/N Mice

322 ⫾ 7.4 47.3 ⫾ 1.5

323 ⫾ 6.7 43.0 ⫾ 1.3

Values are the means ⫾ SEM. The number of each group was 10 mice.

RNA Preparation and Northern Blot Analysis Total RNA was extracted according to the guanidium thiocyanate procedure of Chirgwin et al. [3]. Five micrograms of total RNA was electrophoresed on 1% agarose gel and transferred to a nylon filter (Hybond N) (Amersham, Buckinghamshire, UK). The filter was hybridized with AVP cDNA probe, which had been labeled with [32P] dCTP by a random primer method (Amersham) to specific activity of approximately 109 cpm/␮g. Hybridization was performed at 42°C for 18 –20 h in hybridization buffer (50% deionized formamide, 4⫻ SSC [1⫻ SSC ⫽ 150 mM NaCl, 15 mM sodium citrate], 5⫻ Denhardt’s, 50 mM sodium phosphate buffer (pH ⫽ 6.8), 0.1% SDS, 5 mM EDTA and 20 ␮g/ml Herring sperm DNA). The filter was washed for 40 min in 2⫻ SSC and 0.2⫻ SSC containing 0.2% SDS at 42°C. The filter was apposed to imaging plate for autoradiogram. The filter was rehybridized with ␤-actin cDNA probe under the same hybridization conditions after the filter was thoroughly washed. The expression activities were assayed by using Bio-imaging Analyzer BAS-2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The expression activities of AVP mRNA was normalized to the expression of ␤-actin. The AVP gene probe which contain 638 bp of Pst I/Hind III insert in pUC9 of the rat AVP gene, was kindly provided from Dr. D. Richter (University of Hamburg, Germany). Rat AVP cDNA probe used here is more than 90% identical to mouse AVP gene. The ␤-actin probe was approximately 2000 bp of a Pst I insert in pUC19 of the rat ␤-actin gene (kindly provided from Dr. T. Matsui, Department of Molecular Biology, University of Occupational and Environmental Health, Japan).

hybridized to the AVP probe were dipped in nuclear emulsion (K-5, Ilford, Cheshire, UK) and exposed for 3 days. Immunohistochemistry Postfixed brains were cryoprotected in 20% sucrose in 0.1 M phosphate buffer for 12–24 h. Serial frozen sections were cut at 40 ␮m by a microtome and processed as free-floating sections. The primary AVP antibody purchased from Incstar (Stillwater, MN, USA) was a rabbit polyclonal antiserum, diluted 1:3000 in 0.1 M PBS containing 0.3% Triton X-100. Sections were incubated with the primary antibody solution at 4°C for 3 days. After washing for 1 h in 0.3% Triton X/PBS, the sections were further incubated for 90 min with biotinylated secondary antibody solution (1:250), and finally with avidin-biotin peroxidase complex (Vectastain ABC kit, Vector Labs. Inc., Burlingame, CA, USA). The peroxidase in the tissue sections was visualized with 0.02% 3-3⬘; diaminobenzidine (DAB) in Tris buffer containing 0.05% hydrogen peroxidase for 10 min. The sections were then mounted on slides, air dried, coverslipped, and examined under a light microscope. The AVP-immunopositive fibers were traced using a conventional light microscope with a drawing instrument.

In Situ Hybridization Histochemistry In situ hybridization histochemistry was performed on frozen 12-␮m thick coronal brain sections cut by cryostat at ⫺18°C and thaw-mounted onto gelatin/chrome alum-coated slides that were kept at ⫺80°C while awaiting further processing. The serial sections were cut throughout the hypothalamus, including the PVN and SON. Four sections were mounted onto the same slides. Approximately 20 slides were used for one brain. The slides were warmed to room temperature and allowed to dry for 10 min, then fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 5 min, washed twice in PBS, and incubated in 0.9% NaCl containing 0.25% acetic anhydride (vol./vol.) and 0.1 M triethanolamine (TEA) at room temperature for 10 min. Sections were then dehydrated through 70% (1 min), 80% (1 min), 95% (2 min), and 100% (1 min) ethanol and delipidated in 100% chloroform for 5 min. The slides were then partially rehydrated in 100% followed by 95% ethanol and allowed to dry briefly in air. Hybridization was carried out at 37°C overnight in 45 ␮l hybridization buffer under a Nescofilm coverslip. The probe used were 35S 3⬘-endlabeled 48-base deoxyoligonucleotide complementary to bases 1493–1540 of the mouse AVP sequence [5]. A total of 5 ⫻ 105 cpm/slide were used. After hybridization, sections were washed for 1 h in four changes of 1⫻ SSC at 55°C, and further for 1 h in two changes of 1⫻ SSC at room temperature. Dried sections

FIG. 1. Representative Northern blots of total RNA (10 ␮g) extracted from the hypothalamus of polydipsic (STR/N) and control (ICR) mice. They were hybridized with arginine vasopressin (AVP) cDNA probe (arrowhead) in (A) and ␤-actin cDNA probe (arrowhead) in (B). Lanes: 1, a four-hypothalamus pool in euhydrated ICR mice; 2, a four-hypothalamus pool in 24 h dehydrated ICR mice; 3, a four-hypothalamus pool in 48 h dehydrated ICR mice; 4, a four-hypothalamus pool in euhydrated STR/N. The expression activities (densities) of AVP mRNA was normalized to the expression of ␤-actin.

HYPOTHALAMIC AVP MRNA IN POLYDIPSIC MICE

49

FIG. 2. Representative computer-generated color images of autoradiograms of the brain sections, including the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) hybridized to a 35S-labeled oligodeoxynucleotide probe complementary to arginine vasopressin (AVP) mRNA. Red is the highest signal and dark blue is the lowest signal. Abbreviations: avPVN, anteroventral PVN; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus. Bar ⫽ 1 mm.

Plasma Osmolality and Hematocrit Plasma osmolality was measured by One-Ten Osmometer (Fiske Associates, Norwood, MA, USA). Hematocrit was measured by using hematocrit capillary tube (Terumo Co. Ltd., Tokyo, Japan).

RESULTS The amount of water drunk per day by animals used in the present study was 35.8 ⫾ 2.9 ml/day (mean ⫾ SEM) in polydipsic mice (n ⫽ 18) and 6.8 ⫾ 0.3 ml/day in ICR mice (n ⫽ 18). The body weights were 36.7 ⫾ 0.8 g in euhydrated STR/N mice,

FIG. 3. Bright field photomicrographs of emulsion-dipped slides hybridized to a 35S-labeled oligodeoxynucleotide probe complementary to arginine vasopressin (AVP) in the supraoptic nucleus (SON) (A, D), the anteroventral part of the paraventricular nucleus (avPVN) (B, E) and the PVN (C, F). (A), (B), and (C) from ICR; (D), (E), and (F) from STR/N. Sections were stained using cresyl fast violet. Bar ⫽ 20 ␮m.

50

NISHI ET AL. means that no difference in the size of AVP mRNA existed between control and polydipsic mice. There was no hybridization to AVP probe in the cerebellum of both polydispic and normal mice. In situ hybridization histochemistry showed that the signals of AVP mRNA in the PVN and the SON were stronger in STR/N mice than those in ICR mice (Fig. 2, 3). Although AVP gene transcripts were observed in the anteroventral parts of the PVN (avPVN) in the STR/N, there were a few AVP transcripts in the same area of the avPVN in the ICR mice (Figs. 2B, 2E, 3B, and 3E). AVP-immunopositive fibers in the hypothalamus of STR/N were traced by using a drawing instrument (Fig. 4). The distribution of AVP-immunopositive fibers in the hypothalamus of the STR/N was similar to that in control ICR mice, except in the avPVN and the suprachiasmatic nucleus (SCN). In the avPVN of STR/N mice fibers from AVP-immunopositive cells were abundant (Figs. 4A and 4B). In the suprachiasmatic nucleus of STR/N mice AVP-immunopositive cells were relatively less than those in control mice (Figs. 4C and 4D). This observation is in agreement with a previous study [9]. DISCUSSION

FIG. 4. The distribution of arginine vasopressin (AVP)-immunopositive cells and fibers from the rostral to the caudal hypothalamic areas, including the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of STR/N mouse. Bar ⫽ 500 ␮m.

43.5 ⫾ 1.2 g in control ICR mice, 39.5 ⫾ 1.2 g after 24 h dehydrated ICR and 37.5 ⫾ 1.2 g after 48 h dehydrated ICR mice (mean ⫾ SEM, n ⫽ 12 in each case). There were no differences in plasma osmolality and hematocrit between STR/N and ICR mice (Table 1). In euhydrated condition, Northern blot analysis revealed that total amount of AVP mRNA in the hypothalamus of the STR/N mice was approximately three-fold that in the control, ICR mice (Fig. 1, lanes 1 and 4). In ICR mice, water deprivation caused an increase in hypothalamic AVP mRNA to 210% (24 h dehydration) and 250% (48 h dehydration) of the value obtained in euhydrated condition (Fig. 1, lanes 1–3). The amount of AVP mRNA in the hypothalamus of euhydrated STR/N mice was slightly more than that observed in 48 h dehydrated ICR mice (Fig. 1, lanes 3 and 4). On the other hand, the band positions of AVP mRNA after electrophoresis on 1% agarose gel were similar among control, dehydrated ICR and STR/N mice (Fig. 1A, arrowhead). This

The present study demonstrates that the total amount of AVP mRNA and the AVP gene expression of individual cells in the hypothalamus are increased and stronger in the STR/N than those in control mice. In the avPVN of STR/N mice, strong signals of AVP mRNA and abundant AVP-immunopositive cells and fibers were observed. We have previously demonstrated that there were numerous AVP- and OXT-immunopositive neurons in the avPVN of the STR/N mice and their neurons in the avPVN were not found in the control mice [9,14]. In situ hybridization histochemistry revealed that the expression of AVP gene in the same site was detected under a water-satiated condition only in the STR/N mice. It is unclear why the expression of the AVP gene in the hypothalamus was excessive in STR/N, compared in control mice. Although osmotic stimulation and hypovolemia are known to stimulate AVP release and upregulate the expression of AVP gene in the hypothalamus, hyperosmolality of plasma and hypovolemia may not have occurred in STR/N because plasma osmolality and hematocrit were similar in STR/N and control. One possible explanation is that AVP-producing neurons in the hypothalamus may be tonically activated by afferent inputs from visceral receptors. Activities in the renal afferent fibers are known to excite AVPproducing cells in the SON and to stimulate secretion of AVP and OXT [2,4]. Anai et al. [1] demonstrated that the expression of AVP gene in the PVN and the SON was upregulated in lithium (Li)treated rats that develop polydipsia and hypotonic polyuria without changes of plasma osmolality. As STR/N mice showed hypotonic polyuria, afferents from renal chemo- and/or mechanoreceptors may be activated by the hypotonic polyuria and hemodynamic changes in the kidney. Activation of afferents from low pressure receptors or cancellation of inhibitory inputs to AVP-producing cells in the hypothalamus from high pressure baroreceptor are thought to be factors to upregulate the expression of AVP gene in the hypothalamus. Our preliminary study showed that mean arterial blood pressure in STR/N was slightly lower than that in control mice. This factor may be involved in the mechanism of upregulation of AVP mRNA in the hypothalamus in STR/N. Recent studies demonstrated that sensory information arising from the penis [8], melatonin [21], and kyotorphin (L-tyrosyl-L-arginine) [19] were involved in regulating the secretion of AVP and OXT in rats. The possible involvement of these factors on polydipsia in STR/N mice should be clarified by further study.

HYPOTHALAMIC AVP MRNA IN POLYDIPSIC MICE Another possible explanation is that abnormality of transcriptional factors may induce upregulation of the expression of the AVP gene in the hypothalamus of the STR/N mice. Our preliminary experiments have shown that intracerebroventricular administration of V1 receptor analogue and AVP antiserum remarkably attenuated spontaneous drinking in the STR/N mice compared to that of the control mice [14]. Although this result indicates that central AVP may contribute to the cause of the polydipsia, there is no direct evidence that upregulation of AVP in the hypothalamus is involved in polydipsia. Transgenic mice and rats may be useful genetic models to understand the relationship between polydipsia and excessive expression of AVP gene. In conclusion, our observation in the present study may imply that abnormalities in function of AVP in the hypothalamus may contribute to the central mechanism responsible for polydipsia in STR/N mice. ACKNOWLEDGEMENTS

We thank Ms. Sayuri Uesugi and Ms. Yuko Hara for their technical assistance. This work was supported in part by Grants-in-Aids for Scientific Research nos. 10218210 and 10470019 (to H.Y.) from the Ministry of Education, Science, Sports and Culture, Japan, by Special Grant by the Ministry of Labor for “Occupational Health Studies,” and by grants from the Uehara Memorial Foundation; from the Ministry of Health and Welfare; from Ajinomoto (Kawasaki, Japan) and the Salt Science Research Foundation.

REFERENCES 1. Anai, H.; Ueta, Y.; Serino, R.; Nomura, M.; Kabashima, N.; Shibuya, I.; Takasugi, M.; Nakashima, Y.; Yamashita, H. Upregulation of the expression of vasopressin gene in the paraventricular and supraoptic nuclei of the lithium-induced diabetes insipidus rat. Brain Res. 772: 161–166; 1997. 2. Caverson, M. M.; Ciriello, J. Contribution of paraventricular nucleus to afferent renal nerve pressor response. Am. J. Physiol. 254:R531– 543; 1988. 3. Chirgwin, J. M.; Przybyla, A. E.; MacDonald, R. J.; Rutter, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294 –5299; 1979. 4. Day, T. A.; Ciriello, J. Effects of renal receptor activation on neurosecretory vasopressin cells. Am. J. Physiol. 253:R234 –241; 1987. 5. Hara, Y.; Battey, J.; Gainer, H. Structure of mouse vasopressin and oxytocin genes. Mol. Brain Res. 8:319 –324; 1990. 6. Hattori, Y.; Katafuchi, T.; Koizumi, K. Characterization of opioidsensitive neurons in the anteroventral third ventricle region of polydipsic inbred mice in vitro. Brain Res. 538:283–288; 1987.

51 7. Hattori, Y.; Koizumi, K. Sensitivity to angiotensin II of neurons in the circumventricular organs of polydipsic inbred mice. Brain Res. 524: 181–186; 1990. 8. Honda, K.; Yanagimoto, M.; Negoro, H.; Narita, K.; Murata, T.; Higuchi, T. Excitation of oxytocin cells in the hypothalamic supraoptic nucleus by electrical stimulation of the dorsal penile nerve and tactile stimulation of the penis in the rat. Brain Res. Bull. 48:309 –313; 1999. 9. Ison, A.; Yuri, K.; Ueta, Y.; Leng, G.; Koizumi, K.; Yamashita, H.; Kawata, M. Vasopressin- and oxytocin-immunoreactive hypothalamic neurones of inbred polydipsic mice. Brain Res. Bull. 31:405– 414; 1993. 10. Katafuchi, T.; Hattori, Y.; Nagatomo, I.; Koizumi, K.; Silverstein, E. Involvement of angiotensin II in water intake of genetically polydipsic mice. Am. J. Physiol. 260:R1152–1158; 1991. 11. Katafuchi, T.; Hattori, Y.; Nagatomo, I.; Koizumi, K. ␬-Opioid antagonist strongly attenuates drinking of genetically polydipsic mice. Brain Res. 546:1– 8; 1991. 12. Koizumi, K.; Hattori, Y.; Katafuchi, T.; Silverstein, E. Studies of spontaneously polydipsic inbred mice. In: Yoshida, S.; Share, L., eds. Recent progress in posterior pituitary hormones. Amsterdam: Elsevier; 1988:403– 410. 13. Koizumi, K.; Inenaga, K.; Akamatsu, N.; Yamashita, H. Sodium sensitivity and origins of the primary polydipsia of the inbred mice. Soc. Neurosci. Abstr. 16:1242; 1990. 14. Koizumi, K.; Zeballos, M.; Kawata, M.; Kannan, H.; Yamashita, H. The hypothalamic vasopressinergic neurons of the inbred polydipsic mouse. The neurohypophysis: A window on brain function. Ann. N.Y. Acad. Sci. 689:612– 615; 1993. 15. Nagatomo, I.; Katafuchi, T.; Koizumi, K. Effects of the opiates on the paraventricular nucleus in genetically polydipsic mice. Brain Res. 598:23–32; 1992. 16. Silverstein, E. Effect of hybridization on the primary polydipsic trait of an inbred strain of mice. Nature 191:523; 1961. 17. Silverstein, E.; Sokoloff, L.; Mickelsen, O.; Jay, G. E., Jr. Polyuria, polydipsia and hydronephrosis in inbred strain of mice. Fed. Proc. 17:457; 1958. 18. Silverstein, E.; Sokoloff, L.; Mickelsen, O.; Jay, G. E., Jr. Primary polydipsia and hydronephrosis in an inbred strain of mice. Am. J. Pathol. 38:143–159; 1961. 19. Summy-Long, J. Y.; Bui, V.; Gestl, S.; Koehler-Stec, E.; Liu, H.; Terrell, M. L.; Kadekaro, M. Effects of central injection of kyotorphin and L-arginine on oxytocin and vasopressin release and blood pressure in conscious rats. Brain Res. Bull. 45:395– 403; 1998. 20. Ueta, Y.; Yamashita, H.; Kawata, M.; Koizumi, K. Water deprivation induces regional expression of c-fos protein in the brain of inbred polydipsic mice. Brain Res. 677:221–228; 1995. 21. Yasin, S. A.; Forsling, M. L. Mechanisms of melatonin inhibition of neurohypophysial hormone release from the rat hypothalamus in vitro. Brain Res. Bull. 45:53–59; 1998.