Cell Tiss. Res. 185, 175-181 (1977) Cell and Tissue Research �9 by Springer-Verlag 1977

The Role of the Subfornical Organ in Drinking Induced by Angiotensin in the Japanese Quail, Coturnix cotumix japonica

Yoshio Takei

Misaki Marine Biological Station, University of Tokyo, Misaki, Miura-shi, Kanagawa-ken, Japan

Summary. Synthetic 5-valine angiotensin II (AII) induced copious drinking when applied directly to the subfornical organ (SFO) in the Japanese quail. Reliable response was obtained with as little as 1 ng of AII. The amount of water intake increased dose-dependently from 5 ng to 1 gg. A latent period of 73.0 - 11.0 seconds at 100ng was noted. The electrical destruction of the SFO significantly reduced the amount of water intake induced by both intra- venous and intracranial AII injections. The decrease was proportional to the extent of the SFO lesion. It is conceivable, therefore, that the SFO plays an important role in elicitation of drinking by AII in birds as suggested in mammals.

Key words: Subfornical organ - Angiotensin - Drinking behavior - Preoptic area - Japanese quail.

Introduction

It has been demonstrated in mammals that drinking behavior is induced by angiotensin II (AII) when injected intracranially. It has further been suggested that sensitive site(s) to AII in the brain are the preoptic area (POA) (Epstein, Fitz- simons and Rolls, 1970) and/or the subfornical organ (SFO) (Simpson and Routtenberg, 1973). Thus, the site principally responsible for AII-induced drink- ing has not yet been clearly demonstrated (see Severs and Summy-Long, 1975).

In avian species, dipsogenic action of AII has also been demonstrated (White- crowned Sparrow, Wada et al., 1975; Japanese quail, Takei, 1977). These studies have shown that the most sensitive site in the brain is the POA. They have suggest-

Send offprint requests to." Yoshio Takei, Misaki Marine Biological Station, University of Tokyo, Misaki, Miura-shi, Kanagawa-ken, 238-02 Japan

Acknowledgements. The author would like to express his gratitude to Professor H. Kobayashi for his valuable advice in the course of this study. Hypertensin Ciba was generously supplied by Ciba-Geigy Ltd., Basel. This work was supported by grants from the Ministry of Education, Japan, and the Ford Foundation to Professor H. Kobayashi

176 Y. Takei

ed further that the SFO may also be responsible for AII-induced drinking, since injection of AII into the ventricle or sites near the SFO induced drinking. Thus, the actual responsive site to AII in birds is not fully clarified. In the present studies, an attempt was made to examine the role of the SFO in the induction of drinking by AII in the Japanese quail. Special attention was paid to the functional rela- tionship between the SFO and the POA.

Materials and Methods

1. Histological Study on SFO

Male Japanese quail (Coturnix coturnixjaponica) were purchased from a commercial source at the age of 50 days. They were reared indoors at 25 + I~ under a short daily photoperiod (8L 16D). After one week of acclimation, six birds were decapitated and brains were fixed in Bouin's fluid. After paraffin embedding, serial sections 7-15 lam in thickness were cut and stained with hematoxylin-eosin, aldehyde fuchsin-toluidine blue (Asai et al., 1969) or chrome hematoxylin-phloxine (Gomori, 1941).

IL Physiological Studies

General Procedures. Male Japanese quail were obtained at the age of 21 of 35 days. The older birds were used for intravenous injection of AII only. They were kept individually under the same conditions as ment ioned above. Before the experiment, they were trained to drink from the tap of a glass tube at tached to a 20 ml graduated cylinder. Food and water were available ad libitum throughout the ex- perimental period. Water intake was measured by reading the scale of the cylinder 30 minutes after AII injection. 5-valine angiotensin II amide (Hypertensin Ciba) dissolved in saline was used for injec- tions.

1. Direct Application o f AH to SFO. Nine birds at the age of 35-40 days were implanted stereotaxically with a guide eannula (o.d. = 0.5 ram, length = 12 ram), the tip being aimed 1 mm above the body of the SFO. The details of the implantat ion procedures have been described elsewhere (Takei, 1977). On the fifth day following the implantation, AII was injected through an injector (o.d. = 0.2 ram, length = 13 ram) guided by the implanted cannula. The injection was made every other day with different doses of 1, 5, 10, 100 ng and 1 lag in 0.5 lal saline. The doses were injected in random order. Saline served as a control solution. Drinking behavior was recorded with a videorecorder. The latent period after injec- tion until the commencement of drinking was measured. After the experiment was completed, injection sites were examined histologically. The lowest dose which induced significantly more water intake than that of control birds was estimated as the min imum effective dose. For statistical analysis, the Student 's t-test was applied. The dose-response curve was analyzed using the analysis of variance for linear regression.

2. Effect o f SFO Lesions on Water Intake Induced by Intravenous A H Injection. Forty birds at 45-50 days of age were injected into the right external jugular vein with 50 or 100 lag of AII in 0.1 ml of saline. According to a previous paper (Takei, 1977), birds injected with saline intravenously drank 0.36 -+ 0.11 ml (n = 9) for 30 min; the upper limit of the 99~o confidence level being 0.64 ml. Therefore, 21 birds which drank more than 1 ml of water for 30min after either 50 or 100lag of AII were subjected to electric lesioning. A stainless steel pin (o.d. = 0.3 ram) insulated except for the tip was inserted stereo- taxically into the brain. The tip of the pin was guided to the SFO by referring to lateral and frontal radiographs. An anodal electric lesion was made with 1.0 m A for 7 to 10 s in 17 birds. The remaining tbur birds were subjected to the same procedure without electric current and served as sham controls. One week after lesioning, AII was injected with the same dose as that used before the lesion. After the injection, birds were decapitated for histological verification of the lesion sites. Percent changes in water intake before and after the lesion were compared between experimental and sham control groups by means of the Mann-Whi tney ' s U-test.

Subfornical Organ and Angiotensin-Induced Drinking 177

3. Effect of SFO Lesions on Water Intake Induced by AH Injection into the Medial POA. Forty birds were implanted stereotaxically with a guide cannula (o.d. = 0.5mm, length = 12mm), the tip being placed 1 mm above the medial POA. Five days after the implantation, 1 or 2 lag of AII in 0.5 lal of saline were injected through an injector (o.d. = 0.2 ram, length = 13 mm). According to a previous paper (Takei, 1977), birds injected with saline into the medial POA drank 0.63 -0.11 ml (n = 17) for 30 rain; the upper limit of the 9 9 ~ confidence level being 0.91 ml. Twenty-four out of 40 birds which drank more than 3 ml for 30 rain in response to AII were used for the experiment. Twenty of 24 birds were subjected to electric lesioning and the remaining four birds served as sham controls. One week after lesioning, they received the same dose of AII as before the lesion. After the injection, birds were decapi- tated for histological verification of the lesion sites. Changes in water intake before and after the lesion were compared between experimental and sham control groups by means of the Mann-Whitney's U-test.

Results

L Histological Stud),' on the Subfornical Organ ( S F O )

I n t h e J a p a n e s e q u a i l , t h e S F O is l o c a t e d o n t h e a n t e r i o r wa l l o f t h e t h i r d v e n t r i c l e

b e t w e e n t h e b a s e o f t h e c h o r o i d p l e x u s ( C P ) a n d t h e a n t e r i o r c o m m i s s u r e . T h e

pa l l i a l c o m m i s s u r e is l o c a t e d i m m e d i a t e l y b e l o w t h e S F O . T h e S F O is c h a r a c t e r -

i zed b y t h e p r e s e n c e o f a l a r g e c e n t r a l s i n u s (Fig . 1 a). T h i s s i n u s is a p a r t o f a l a rge

Fig. l . a Mid-sagittal section of the SFO of the Japanese quail. Many red blood cells (B) are observed in the sinus (S). Note AF-positive glial cells (GC) in the vicinity of the sinus. AF-toluidine blue. x 80. b Sagittal section of the SFO about 0.1 mm lateral to the mid-sagittal plane. Note toluidine blue- positive nerve cells (NC) surrounding the organ. AF-toluidine blue. x 96. AC anterior commissure; PC pallial commissure; V vacuole; III, third ventricle

E

c_

10

I oC<i

3 lb 10o c~o

Dose(ng/O,SMI)

Fig. 2. Photomicrograph of the brain showing location of the injection site (arrow). Hematoxylin- eosin, x 120

Fig. 3. Regression line of the dose-response relationship in birds receiving AII into the SFO. Each point indicates the mean intake of four birds. The vertical line indicates the s tandard error of the mean

E

c~

E

o_

200-

150`

100`

50-

O --.-"---- �9 BG LG UeG

5 G r o u p

SG

|

I G Sham

Fig. 4. Photomicrograph of the brain showing electric lesion in the SFO (Group BG). AC anterior commissure, CP choroid plexus. Hematoxylin-eosin. x 32

Fig. 5. Change in water intake after SFO lesion. Relative value of water intake after the lesion is ex- pressed in terms of percentage to that before the lesion. For abbreviations, see text

Subfornical Organ and Angiotensin-Induced Drinking

Table 1. Effect of SFO lesion on water intake induced by intravenous AII injection

179

Group Number Change in water intake (Percentage of of birds (~) damage in SFO)

Median

0 0 0 0 Group I (80-100~) 8 0 7.1 19 45 0

Group II (30- 80~) 3 0 5.1 31 5.1 Group III (0- 30~) ~ 3 73 220 330 220 Sham controls" 4 5.1 140 160 221 150

a Significantly different (p < 0.05) from Group I by Mann-Whitney's U-test

blood vessel running between the CP and the POA. Many blood vessels communicating with the sinus form a rich vascular net inside the SFO. Tall cylindrical ependymal cells comprise the ventricular surface of the SFO. The anterior boundary is distinguished from more interior lying tissue by large nerve cells of the SFO (Fig. 1 b). The Nissl substance of these nerve cells is darkly stained with toluidine blue. Gomori-posit ive glial cells are often encountered near the sinus (Fig. 1 a). Cells are frequently observed in which large vacuoles compress the nucleus and cytoplasmic against the cell membrane (Fig. 1 b).

II. Physiological Studies

1. Direct Application of AH to SFO. Histological verification showed that the tip of the injector hit the SFO in four of nine operated birds (Fig. 2). The dose-response curve observed on these four birds was linear with more than 9 9 ~ confidence from 5 ng to 1 ~tg (Fig. 3). The latent period was 73.0 -+ 11.0 s (n = 4) at 100 ng. The minimum effective dose was approximately 1 ng. Water intake induced by 1 ng (2.28-+0.87ml, n = 4 ) was significantly (p<0.05) more than that induced by saline (0.77 -+ 0.24 ml, n = 4).

2. Effect of SFO Lesions on Water Intake Induced by Intravenous AH Injection. Three out o f t 7 birds did not survive the SFO lesion, while all four sham operated birds survived. The 14 birds bearing lesions were classified into three groups according to the extent of damage to the SFO: Group I (n = 8) with more than 8 0 ~ damage, Group II (n = 3) with 30 to 8 0 ~ damage and Group II I (n = 3) with less than 3 0 ~ damage. Changes in water intake induced by AII after lesioning are shown in Table 1. The values for Group I are significantly (p < 0.05) smaller than those of the sham control group and Group III.

3. Effect of SFO Lesions on Water Intake Induced by AH Injection into the Medial POA. Three out of 20 birds died soon after lesioning, while all sham operated birds survived. Among the birds bearing lesions, three had lesion outside the SFO. These birds were designated as Group IG. In seven of the remaining 14 birds, the SFO was almost completely destroyed (Group BG) (Fig. 4); in two birds the

180 Y. Takei

upper half was damaged (Group UG); in three animals the posterior half including the surface was damaged (Group SG); and in the two remaining birds the lower half was damaged (Group LG). Changes in water intake after lesioning are shown in Fig. 5. Water intake decreased significantly (p < 0.05) in Group BG compared with those of the sham control group and Group IG. Although the number of birds is small, it appears that water intake decreased considerably in Groups UG, SG and LG.

Discussion

The subfornical organ (SFO) of the Japanese quail has a similar location and structure to that of the gull (Wetzig and Palkovits, 1968). Large intracellular vacuoles observed in this organ may indicate its secretory function. It was sugges- ted in the rat and rabbit that such vacuoles are formed by the coalescence of clear vesicles in the endoplasmic reticulum of nerve cells and that the vacuolar content may be discharged into the ventricle (Rohr, 1966; Rudert et al., 1968; Weindl, 1973). The functional role of the Gomori-positive glial cells found near the sinus is not known.

The SFO of the quail is tairly vascularized; especially characteristic is the large sinus in its center. According to Spoerri (1963), two arteries from the choroid plexus (CP) and the arteria subfornicalis converge into one relatively large artery in the SFO of the rat. This large artery appears to be traced to the organum vascu- losum laminae terminalis. Therefore, a vessel observed between the CP and the POA in the quail may correspond to the large artery reported in the rat. However, differences exist in that the vessel forms a large sinus in the SFO of the Japanese quail, while it forms a dense network of capillaries in the rat. No explanation can be given for this difference in the SFO vasculature in the two species.

By applying AII directly to the SFO, the present study has demonstrated that the SFO of the Japanese quail represents a sensitive site to AII. Further it has been shown that the destruction of the SFO significantly reduces water intake induced by intravenous AII. These findings indicate that the SFO is concerned with the elicitation of drinking by intravenously injected AII. In other words, AII injected intravenously may reach the SFO through blood vessels instead of the cere- brospinal fluid. It is known that the SFO has a low threshold blood-brain barrier (Weindl, 1965).

It has also been shown in the present study that dipsogenic action of AII in- jected into the medial POA is impaired after lesion to the SFO. Similar results have been reported previously in the rat (Simpson and Routtenberg, 1973). This indicates that the AII-induced information given to the POA is transferred to the SFO via some route(s). Johnson and Epstein (1975) have suggested that the POA in the rat may not be a responsive site and other periventricular regions such as the SFO may have receptors responsible for AII-induced drinking. They have shown that AII injected into the anterior diencephalon including the POA diffuses up along the cannula shaft into the ventricle. In this way, the diffused peptide may reach the SFO. If this be the case, it is likely that the SFO possesses dipsogenic receptors in its surface structures to receive intraventricular AII. However,

Subfornical Organ and Angiotensin-Induced Drinking 181

complete destruction of the surface structures of the SFO failed to eliminate drinking induced by AII injected into the medial POA. Further, in the present study the injection site was 1 mm below the cannula tip in order to minimize diffusion along the cannula shaft. Thus, it appears that a route other than ventric- ular diffusion must exist for AII-induced information transfer from the POA to the SFO. One possible route may be nerve fibers connecting both sites, since nerve connections between the POA and the SFO have been reported in some vertebrate species (rat, Hernesniemi et al., 1972; chicken, Legait and Legait, 1958; frog, Rudert, 1965).

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Accepted July 12, 1977