Pharmacology Biochemistry & Behavior, Vol. 5, pp. 591-595. Copyright © 1976 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
BRIEF COMMUNICATION Carbachol, Angiotensin-II, Ventricular Spread and Water Balance in Rats 1 J E F F R E Y W. P E C K
Department o f Psychology, University o f Utah, Salt Lake City, Utah 84112 (Received 10 May 1976) PECK, J. W. Carbachol, angiotensin-II, ventricular spread and water balance in rats. PHARMAC. BIOCHEM. BEHAV. 5(5) 591-595, 1976. - Injections into the preoptic areas and anterior hypothalamus of as little as 1.0 ng]ul carbachol (5.5 × 10-6M, 11 pmols total dose) and 0.1 ng/~l angiotensin-II (lffTM, 0.2-0.4 pmols) were dipsogenic or antidiuretic-natriuretic. Lateral ventricular (VL) thresholds for drinking also were 11 pmols for carbachol and 0.2 pmols for angiotensin. The low VL threshold for carbachol supports, without proving, arguments that a limbic cholinergicallycoded thirst circuit could reflect leakage form placements near VL to the third ventricle. In contrast, VL thresholds for antidiuresis-natriuresis were 110 pmols for carbachol and 2 pmols for angiotensin. Carbachol was more effective rostral to the paraventricular nucleus and angiotensin dorsal to the supraoptic nucleus. However, lower thresholds in these areas were insufficient to localize receptors, since all cannulas positive for antidiuresis-natriuresis traversed ventricles, and cannulas not traversing ventricles were negative. Intracranial injection Intracerebroventricular injection Cholinergic Preoptic areas Anterior hypothalamus
Antidiuresis
R O U T T E N B E R G [ 1 2 , 1 4 ] argued t h a t leakage of carba m y l c h o l i n e chloride ( c a r b a c h o l ) to the ventricles f r o m i n j e c t i o n or i m p l a n t a t i o n loci p r o b a b l y c o m p r o m i s e d studies [ 1, 6, 7, 15] p r o p o s i n g a limbic cholinergicallycoded thirst circuit. D r i n k i n g f o l l o w e d as little as 0 . 0 1 - 0 . 2 ug c a r b a c h o l at p o i n t s on this p r o p o s e d circuit [ 6 , 7 ] . Yet, 0 . 2 5 - 4 . 0 ug of c a r b a c h o l in the lateral ventricle of rats were n o t dipsogenic in some studies [ 1 , 4 ] , and 0.055 ug was dipsogenic b u t o n l y with atypically long l a t e n c y in a n o t h e r [ 7 ] . These facts have flawed R o u t t e n b e r g ' s argument. The present report reexamined whether c a r b a c h o l in a lateral ventricle elicited drinking. In a d d i t i o n , d r i n k i n g was c o m p a r e d w i t h antidiuresisnatriuresis elicited b y v e n t r i c u l a r or p r e o p t i c - a n t e r i o r h y p o t h a l a m i c angiotensin-II or carbachol. Peck and E p s t e i n r e p o r t e d [9,11 ] t h a t t h e p r e o p t i c areas were the sites of t h e dipsogenic a n d a n t i d i u r e t i c - n a t r i u r e t i c actions of intracranially-injected angiotensin-II, b u t J o h n s o n and E p s t e i n [2] s h o w e d t h a t spread to t h e ventricles was necessary for angiotensin-II i n j e c t e d i n t o the p r e o p t i c areas to be dipsogenic. Such spread also m a y have m e d i a t e d t h e a n t i d i u r e t i c - n a t r i u r e t i c actions of a n g i o t e n s i n .
Natriuresis
Vasopressin
METHOD
Animals and Surgery Six male Long-Evans rats were i m p l a n t e d w i t h o n e d o u b l e - b a r r e l e d i n t r a c r a n i a l c a n n u l a t e r m i n a t i n g in a lateral ventricle a n d a s e c o n d c a n n u l a angled to r e a c h the c o n t r a l a t e r a l p r e o p t i c areas or a n t e r i o r h y p o t h a l a m u s w i t h o u t traversing a ventricle. T w e n t y o t h e r rats were i m p l a n t e d w i t h t w o a s y m m e t r i c a l l y - p l a c e d c a n n u l a s aimed for these same regions b u t i n t r o d u c e d p e r p e n d i c u l a r to t h e dorsal surface o f the cerebral cortex. All rats also h a d a chronic n a s o p h a r y n g e a l intragastric t u b e , a n d 5 of t h e rats, a c a t h e t e r in t h e right jugular vein. T h e s e o p e r a t i o n s have been described [ 1 0 ] .
Procedure Drinking tests. See [ 1 0 ] . Briefly, d r i n k i n g tests were conducted cages, w i t h to eat a n d before the
once weekly. T h e rats were tested in t h e i r h o m e b o t h f o o d a n d w a t e r p r e s e n t , and were allowed d r i n k freely a f t e r t h e i n j e c t o r s were inserted, test p r o p e r began. I n j e c t i o n s were n o t m a d e
1 Supported by University Research Committee and by USPHS Biomedical Sciences Support Grant RR 07092 to the University of Utah. Thanks to Dr. R. Straight, Director, Medical Research Laboratory, Veterans Administration Hospital, Salt Lake City, Utah for making available the Varian Techtron atomic absorption spectrophotometer, and to J. Denbutter for preparing the histology. 591
592 u n t i l t h e rats h a d a s s u m e d a p o s t u r e i n d i c a t i n g sleep, a n d were m a d e using r e m o t e syringes a n d w i t h o u t d i s t u r b i n g the rats. I n j e c t i o n s were unilateral, 2 ul in 1 8 - 2 2 sec. Chemicals were dissolved in i s o t o n i c saline ( S o d i u m C h l o r i d e Irrigation, A b b o t t ) . Tests using a n g i o t e n s i n - I I ( H y p e r t e n s i n , Ciba) were c o m p l e t e d first. If 1.0 n g / u l (10-6 M; 2 u l o f s o l u t i o n c o n t a i n e d 2 p m o l s ) was dipsogenic, 0.1 n g / u l was tried, t h e n 0.01 ng/ul. W h e n a c o n c e n t r a t i o n failed, t h e last effective c o n c e n t r a t i o n was tried; if effective, this c o n c e n t r a t i o n n o w was called t h e t h r e s h o l d . Only one c o n c e n t r a t i o n o f a n g i o t e n s i n - I I was tried o n a n y test day, so a m i n i m u m o f 3 tests (3 weeks) were r e q u i r e d to establish t h r e s h o l d s . T h r e s h o l d - f i n d i n g p r o v i d e d c o n t r o l s for t h e i n j e c t i o n itself causing drinking. First, a doser e s p o n s e curve r e s u l t e d ; second, an i n e f f e c t i v e i n j e c t i o n always was b r a c k e t e d b y effective i n j e c t i o n s differing o n l y in i n t r o d u c i n g a h i g h e r c o n c e n t r a t i o n of the dipsogen. Since rats a p p a r e n t l y were sleeping at t h e t i m e of i n j e c t i o n , arousal f o l l o w e d b y d r i n k i n g of a n y a m o u n t w i t h i n 10 m i n of the o n s e t of the i n j e c t i o n was called a positive r e s p o n s e , if r e p e a t e d o n the n e x t test day w i t h t h e dosage. In negative tests rats did n o t a w a k e n at all. R a t s a r o u s e d a f t e r i n j e c t i o n s were o b s e r v e d u n t i l t h e y again assumed a p o s t u r e i n d i c a t i n g sleep, a l t h o u g h in p r a c t i c e all drinking did o c c u r in o n e b o u t i n t e r r u p t e d b y relatively brief pauses. T h e d u r a t i o n of d r i n k i n g r e p o r t e d includes these pauses. A similar p r o c e d u r e for d e t e r m i n i n g t h r e s h o l d s was f o l l o w e d for c a r b a c h o l , the initial c o n c e n t r a t i o n being either 10 n g / ~ l (5.5 x 10-SM; 2 ~1 c o n t a i n e d 110 p m o l s ) or 1.0 n g / u l . T h e m a x i m u m c o n c e n t r a t i o n s used were 10 ng/~ 1 ( a n g i o t e n s i n ) a n d 100 ng/u 1 ( c a r b a c h o l ) . Analysis o f natriuresis and antidiuresis. See [ 1 0 ] . Briefly, rats were h y d r a t e d b y an initial i n t r a g a s t r i c i n j e c t i o n (12 ml) a n d s u s t a i n e d i n f u s i o n (3 m l / 1 0 0 g B W / h r typically c o n t i n u i n g for 3 - 4 h r ) of tap water. T h e t i m e o f each s p o n t a n e o u s void of u r i n e ( n o r m a l l y , 10 20 m i n a p a r t ) was r e c o r d e d a n d its v o l u m e m e a s u r e d . All voids were a n a l y z e d for s o d i u m c o n c e n t r a t i o n b y flame p h o t o m e t r y ( V a r i a n - T e c h t r o n M o d e l AA5). E x c e p t for a few i n s t a n c e s a f t e r t e r m i n a t i o n of d r i n k i n g tests, tests for antidiuresis-natriuresis were c o n d u c t e d o n c e weekly, 3 days a f t e r d r i n k i n g tests. W h e n w a t e r - l o a d i n g o c c u r r e d o n c e weekly, rats ate a n d d r a n k n o r m a l l y a n d gained weight. More f r e q u e n t w a t e r - l o a d i n g o f t e n led to weight loss. S o d i u m c o n c e n t r a t i o n of the rats' urine 3 days a f t e r w a t e r - l o a d i n g was n o r m a l . B o t h i n t r a c r a n i a l c a n n u l a s were t e s t e d a n d / o r several i n t r a v e n o u s i n f u s i o n s of vasopressin (Pitressin, Parke-Davis) were m a d e d u r i n g each test. T h r e s h o l d s were d e t e r m i n e d as for t h e d r i n k i n g tests. A n y increased s o d i u m c o n c e n t r a t i o n or decreased u r i n e flow, w i t h i n the limits of e r r o r of collection, could have b e e n a positive response, if r e p e a t e d on t h e n e x t test day w i t h t h a t dosage. In practice, o n l y large changes in urine c o n c e n t r a t i o n or flow were replicable. Most tests w i t h a n g i o t e n s i n involved r e p e a t i n g the i n j e c t i o n 5 m i n later, w h i c h was f o u n d necessary for renal changes to result f r o m t h r e s h o l d dosages. T h e s e c o n d i n j e c t i o n was f o u n d u n n e c e s s a r y for carbachol. Histology. C o m m u n i c a t i o n o f e a c h lateral v e n t r i c u l a r c a n n u l a w i t h the v e n t r i c l e was verified b y i n j e c t i o n of India ink (2 ul in 1 9 - 2 2 sec) p r i o r to p e r f u s i o n , a f t e r the rats h a d b e e n a n e s t h e t i z e d w i t h p e n t o b a r b i t a l . All o t h e r c a n n u l a p l a c e m e n t s were verified o n f r o z e n and stained s e c t i o n s as described p r e v i o u s l y [ 10].
PECK RESULTS C a n n u l a s t e r m i n a t e d in t h e lateral ventricle ( V L ) at the r o s t r o c a u d a l level of t h e a n t e r i o r c o m m i s s u r e (n = 6), medial p r e o p t i c area (POM, n = 3), lateral p r e o p t i c area (POL, n = 7), t h e bed n u c l e u s of the stria t e r m i n a l i s (n = 3), dorsal to or in t h e a n t e r i o r h y p o t h a l a m i c area (HA, n = 9), and a n t e r i o r l y in t h e lateral h y p o t h a l a m i c area (HL, n = 11). C a n n u l a s s h o w i n g i n f e c t i o n or in brains a p p a r e n t l y h y d r o c e p h a l i c on histological e x a m i n a t i o n were o m i t t e d . India ink was f o u n d t h r o u g h o u t the ipsilateral lateral ventricle for all V L cannulas, b u t only for 4 of the 6 c a n n u l a s was ink also f o u n d in the third ventricle. A m o d e r a t e b u t n o t yet gross e x p a n s i o n of t h e ipsilateral V L of the two e x c e p t i o n a l rats suggested the i n t e r v e n t r i c u l a r f o r a m e n was b l o c k e d , a l t h o u g h n e i t h e r rat a p p e a r e d ill. These two rats initially r e s p o n d e d to a n g i o t e n s i n at the t h r e s h o l d s r e p o r t e d b e l o w for t h e o t h e r 4 rats, b u t by the time of sacrifice n e i t h e r a n g i o t e n s i n n o r c a r b a c h o l elicited d r i n k i n g or a n t i d i u r e s i s - n a t r i u r e s i s in e i t h e r rat. Thus, r e p o r t s of V L p l a c e m e n t s are for f o u r rats, the results f r o m the o t h e r t w o VL rats being t a k e n to i n d i c a t e t h a t c o m m u n i c a t i o n w i t h t h e t h i r d ventricle was necessary for the effects r e p o r t e d . As little as 1.0 n g / u l c a r b a c h o l (2.0 ng t o t a l dose, 11 p m o l s ) in V L caused d r i n k i n g ( T a b l e 1). T h e same t h r e s h o l d , latencies, a n d v o l u m e s d r u n k were f o u n d for p r e o p t i c and a n t e r i o r h y p o t h a l a m i c p l a c e m e n t s s c a t t e r e d a m o n g higher t h r e s h o l d p l a c e m e n t s ( T a b l e 1). T h e r e was a dose-response r e l a t i o n s h i p for v o l u m e d r u n k for the latter p l a c e m e n t s , t ( 7 ) = 2.47, p < 0 . 0 5 . V L rats did n o t receive 10.0 n g / u l c a r b a c h o l . As little as 0.1 n g / u l a n g i o t e n s i n II (0.2 ng t o t a l dose, 0.2 p m o l s ) in V L caused d r i n k i n g (Table 1). The same t h r e s h o l d and v o l u m e d r u n k was f o u n d for p r e o p t i c -
TABLE I DRINKING CAUSED BY VENTRICULAR OR PREOPTIC-ANTERIOR HYPOTHALAMIC INJECTIONS OF CARBACHOL OR ANGIOTENSIN-II THRESHOLD (ng/pl)
1,0
n
DOSAGE LATENCY (ng/pl} (sec)
DURATION (xc)
VOLUME (ml)
LATERAL VENTRICULAR CARBACHOL 4 hO 85.+50 = 2 6 0 + 1 3 3 3,6 +- 0,7 PREOPTIC- HYPOTHALAMIC CARBACHOL
hO
8
hO I0,
150+122 60--.34
190 -+ 65 440-+108
3,0 -+ 1,3 7,2_+ 2.0
I0.
6
I0,
205+-81
2 6 0 +- 173
2.7 -* 1,5
0,1
LATERAL VENTRICULAR ANGIOTENSIN 4 0,1 210 -I- 128 105 +- 6 0 1,0 145 + 6 0 5 0 5 -+ 150 PREOPTIC- HYPOTHALAMIC ANGIOTENSlN
1,6 -+ 0,6 3,3 __4-1,4
0,1
5
0,1 hO
8.5+-32 70+-26
140--.64 240+-96
?-.,64"1,8 4,2 .-+ h8
1,0
8
1,0
120:1:44
200:1:65
2.8:1:0,8
*Mean + S.D.
V ENTR I C ULAR SPREAD OF CARBACHOL AND ANGIOTENSIN anterior hypothalamic placements scattered among higher threshold placements, but with slightly shorter latencies, t(7) = 2.0, p < 0 . 1 0 at 0.1 ng/ul; t(7) = 2.46, p
BRIEF COMMUNICATION Carbachol, Angiotensin-II, Ventricular Spread and Water Balance in Rats 1 J E F F R E Y W. P E C K
Department o f Psychology, University o f Utah, Salt Lake City, Utah 84112 (Received 10 May 1976) PECK, J. W. Carbachol, angiotensin-II, ventricular spread and water balance in rats. PHARMAC. BIOCHEM. BEHAV. 5(5) 591-595, 1976. - Injections into the preoptic areas and anterior hypothalamus of as little as 1.0 ng]ul carbachol (5.5 × 10-6M, 11 pmols total dose) and 0.1 ng/~l angiotensin-II (lffTM, 0.2-0.4 pmols) were dipsogenic or antidiuretic-natriuretic. Lateral ventricular (VL) thresholds for drinking also were 11 pmols for carbachol and 0.2 pmols for angiotensin. The low VL threshold for carbachol supports, without proving, arguments that a limbic cholinergicallycoded thirst circuit could reflect leakage form placements near VL to the third ventricle. In contrast, VL thresholds for antidiuresis-natriuresis were 110 pmols for carbachol and 2 pmols for angiotensin. Carbachol was more effective rostral to the paraventricular nucleus and angiotensin dorsal to the supraoptic nucleus. However, lower thresholds in these areas were insufficient to localize receptors, since all cannulas positive for antidiuresis-natriuresis traversed ventricles, and cannulas not traversing ventricles were negative. Intracranial injection Intracerebroventricular injection Cholinergic Preoptic areas Anterior hypothalamus
Antidiuresis
R O U T T E N B E R G [ 1 2 , 1 4 ] argued t h a t leakage of carba m y l c h o l i n e chloride ( c a r b a c h o l ) to the ventricles f r o m i n j e c t i o n or i m p l a n t a t i o n loci p r o b a b l y c o m p r o m i s e d studies [ 1, 6, 7, 15] p r o p o s i n g a limbic cholinergicallycoded thirst circuit. D r i n k i n g f o l l o w e d as little as 0 . 0 1 - 0 . 2 ug c a r b a c h o l at p o i n t s on this p r o p o s e d circuit [ 6 , 7 ] . Yet, 0 . 2 5 - 4 . 0 ug of c a r b a c h o l in the lateral ventricle of rats were n o t dipsogenic in some studies [ 1 , 4 ] , and 0.055 ug was dipsogenic b u t o n l y with atypically long l a t e n c y in a n o t h e r [ 7 ] . These facts have flawed R o u t t e n b e r g ' s argument. The present report reexamined whether c a r b a c h o l in a lateral ventricle elicited drinking. In a d d i t i o n , d r i n k i n g was c o m p a r e d w i t h antidiuresisnatriuresis elicited b y v e n t r i c u l a r or p r e o p t i c - a n t e r i o r h y p o t h a l a m i c angiotensin-II or carbachol. Peck and E p s t e i n r e p o r t e d [9,11 ] t h a t t h e p r e o p t i c areas were the sites of t h e dipsogenic a n d a n t i d i u r e t i c - n a t r i u r e t i c actions of intracranially-injected angiotensin-II, b u t J o h n s o n and E p s t e i n [2] s h o w e d t h a t spread to t h e ventricles was necessary for angiotensin-II i n j e c t e d i n t o the p r e o p t i c areas to be dipsogenic. Such spread also m a y have m e d i a t e d t h e a n t i d i u r e t i c - n a t r i u r e t i c actions of a n g i o t e n s i n .
Natriuresis
Vasopressin
METHOD
Animals and Surgery Six male Long-Evans rats were i m p l a n t e d w i t h o n e d o u b l e - b a r r e l e d i n t r a c r a n i a l c a n n u l a t e r m i n a t i n g in a lateral ventricle a n d a s e c o n d c a n n u l a angled to r e a c h the c o n t r a l a t e r a l p r e o p t i c areas or a n t e r i o r h y p o t h a l a m u s w i t h o u t traversing a ventricle. T w e n t y o t h e r rats were i m p l a n t e d w i t h t w o a s y m m e t r i c a l l y - p l a c e d c a n n u l a s aimed for these same regions b u t i n t r o d u c e d p e r p e n d i c u l a r to t h e dorsal surface o f the cerebral cortex. All rats also h a d a chronic n a s o p h a r y n g e a l intragastric t u b e , a n d 5 of t h e rats, a c a t h e t e r in t h e right jugular vein. T h e s e o p e r a t i o n s have been described [ 1 0 ] .
Procedure Drinking tests. See [ 1 0 ] . Briefly, d r i n k i n g tests were conducted cages, w i t h to eat a n d before the
once weekly. T h e rats were tested in t h e i r h o m e b o t h f o o d a n d w a t e r p r e s e n t , and were allowed d r i n k freely a f t e r t h e i n j e c t o r s were inserted, test p r o p e r began. I n j e c t i o n s were n o t m a d e
1 Supported by University Research Committee and by USPHS Biomedical Sciences Support Grant RR 07092 to the University of Utah. Thanks to Dr. R. Straight, Director, Medical Research Laboratory, Veterans Administration Hospital, Salt Lake City, Utah for making available the Varian Techtron atomic absorption spectrophotometer, and to J. Denbutter for preparing the histology. 591
592 u n t i l t h e rats h a d a s s u m e d a p o s t u r e i n d i c a t i n g sleep, a n d were m a d e using r e m o t e syringes a n d w i t h o u t d i s t u r b i n g the rats. I n j e c t i o n s were unilateral, 2 ul in 1 8 - 2 2 sec. Chemicals were dissolved in i s o t o n i c saline ( S o d i u m C h l o r i d e Irrigation, A b b o t t ) . Tests using a n g i o t e n s i n - I I ( H y p e r t e n s i n , Ciba) were c o m p l e t e d first. If 1.0 n g / u l (10-6 M; 2 u l o f s o l u t i o n c o n t a i n e d 2 p m o l s ) was dipsogenic, 0.1 n g / u l was tried, t h e n 0.01 ng/ul. W h e n a c o n c e n t r a t i o n failed, t h e last effective c o n c e n t r a t i o n was tried; if effective, this c o n c e n t r a t i o n n o w was called t h e t h r e s h o l d . Only one c o n c e n t r a t i o n o f a n g i o t e n s i n - I I was tried o n a n y test day, so a m i n i m u m o f 3 tests (3 weeks) were r e q u i r e d to establish t h r e s h o l d s . T h r e s h o l d - f i n d i n g p r o v i d e d c o n t r o l s for t h e i n j e c t i o n itself causing drinking. First, a doser e s p o n s e curve r e s u l t e d ; second, an i n e f f e c t i v e i n j e c t i o n always was b r a c k e t e d b y effective i n j e c t i o n s differing o n l y in i n t r o d u c i n g a h i g h e r c o n c e n t r a t i o n of the dipsogen. Since rats a p p a r e n t l y were sleeping at t h e t i m e of i n j e c t i o n , arousal f o l l o w e d b y d r i n k i n g of a n y a m o u n t w i t h i n 10 m i n of the o n s e t of the i n j e c t i o n was called a positive r e s p o n s e , if r e p e a t e d o n the n e x t test day w i t h t h e dosage. In negative tests rats did n o t a w a k e n at all. R a t s a r o u s e d a f t e r i n j e c t i o n s were o b s e r v e d u n t i l t h e y again assumed a p o s t u r e i n d i c a t i n g sleep, a l t h o u g h in p r a c t i c e all drinking did o c c u r in o n e b o u t i n t e r r u p t e d b y relatively brief pauses. T h e d u r a t i o n of d r i n k i n g r e p o r t e d includes these pauses. A similar p r o c e d u r e for d e t e r m i n i n g t h r e s h o l d s was f o l l o w e d for c a r b a c h o l , the initial c o n c e n t r a t i o n being either 10 n g / ~ l (5.5 x 10-SM; 2 ~1 c o n t a i n e d 110 p m o l s ) or 1.0 n g / u l . T h e m a x i m u m c o n c e n t r a t i o n s used were 10 ng/~ 1 ( a n g i o t e n s i n ) a n d 100 ng/u 1 ( c a r b a c h o l ) . Analysis o f natriuresis and antidiuresis. See [ 1 0 ] . Briefly, rats were h y d r a t e d b y an initial i n t r a g a s t r i c i n j e c t i o n (12 ml) a n d s u s t a i n e d i n f u s i o n (3 m l / 1 0 0 g B W / h r typically c o n t i n u i n g for 3 - 4 h r ) of tap water. T h e t i m e o f each s p o n t a n e o u s void of u r i n e ( n o r m a l l y , 10 20 m i n a p a r t ) was r e c o r d e d a n d its v o l u m e m e a s u r e d . All voids were a n a l y z e d for s o d i u m c o n c e n t r a t i o n b y flame p h o t o m e t r y ( V a r i a n - T e c h t r o n M o d e l AA5). E x c e p t for a few i n s t a n c e s a f t e r t e r m i n a t i o n of d r i n k i n g tests, tests for antidiuresis-natriuresis were c o n d u c t e d o n c e weekly, 3 days a f t e r d r i n k i n g tests. W h e n w a t e r - l o a d i n g o c c u r r e d o n c e weekly, rats ate a n d d r a n k n o r m a l l y a n d gained weight. More f r e q u e n t w a t e r - l o a d i n g o f t e n led to weight loss. S o d i u m c o n c e n t r a t i o n of the rats' urine 3 days a f t e r w a t e r - l o a d i n g was n o r m a l . B o t h i n t r a c r a n i a l c a n n u l a s were t e s t e d a n d / o r several i n t r a v e n o u s i n f u s i o n s of vasopressin (Pitressin, Parke-Davis) were m a d e d u r i n g each test. T h r e s h o l d s were d e t e r m i n e d as for t h e d r i n k i n g tests. A n y increased s o d i u m c o n c e n t r a t i o n or decreased u r i n e flow, w i t h i n the limits of e r r o r of collection, could have b e e n a positive response, if r e p e a t e d on t h e n e x t test day w i t h t h a t dosage. In practice, o n l y large changes in urine c o n c e n t r a t i o n or flow were replicable. Most tests w i t h a n g i o t e n s i n involved r e p e a t i n g the i n j e c t i o n 5 m i n later, w h i c h was f o u n d necessary for renal changes to result f r o m t h r e s h o l d dosages. T h e s e c o n d i n j e c t i o n was f o u n d u n n e c e s s a r y for carbachol. Histology. C o m m u n i c a t i o n o f e a c h lateral v e n t r i c u l a r c a n n u l a w i t h the v e n t r i c l e was verified b y i n j e c t i o n of India ink (2 ul in 1 9 - 2 2 sec) p r i o r to p e r f u s i o n , a f t e r the rats h a d b e e n a n e s t h e t i z e d w i t h p e n t o b a r b i t a l . All o t h e r c a n n u l a p l a c e m e n t s were verified o n f r o z e n and stained s e c t i o n s as described p r e v i o u s l y [ 10].
PECK RESULTS C a n n u l a s t e r m i n a t e d in t h e lateral ventricle ( V L ) at the r o s t r o c a u d a l level of t h e a n t e r i o r c o m m i s s u r e (n = 6), medial p r e o p t i c area (POM, n = 3), lateral p r e o p t i c area (POL, n = 7), t h e bed n u c l e u s of the stria t e r m i n a l i s (n = 3), dorsal to or in t h e a n t e r i o r h y p o t h a l a m i c area (HA, n = 9), and a n t e r i o r l y in t h e lateral h y p o t h a l a m i c area (HL, n = 11). C a n n u l a s s h o w i n g i n f e c t i o n or in brains a p p a r e n t l y h y d r o c e p h a l i c on histological e x a m i n a t i o n were o m i t t e d . India ink was f o u n d t h r o u g h o u t the ipsilateral lateral ventricle for all V L cannulas, b u t only for 4 of the 6 c a n n u l a s was ink also f o u n d in the third ventricle. A m o d e r a t e b u t n o t yet gross e x p a n s i o n of t h e ipsilateral V L of the two e x c e p t i o n a l rats suggested the i n t e r v e n t r i c u l a r f o r a m e n was b l o c k e d , a l t h o u g h n e i t h e r rat a p p e a r e d ill. These two rats initially r e s p o n d e d to a n g i o t e n s i n at the t h r e s h o l d s r e p o r t e d b e l o w for t h e o t h e r 4 rats, b u t by the time of sacrifice n e i t h e r a n g i o t e n s i n n o r c a r b a c h o l elicited d r i n k i n g or a n t i d i u r e s i s - n a t r i u r e s i s in e i t h e r rat. Thus, r e p o r t s of V L p l a c e m e n t s are for f o u r rats, the results f r o m the o t h e r t w o VL rats being t a k e n to i n d i c a t e t h a t c o m m u n i c a t i o n w i t h t h e t h i r d ventricle was necessary for the effects r e p o r t e d . As little as 1.0 n g / u l c a r b a c h o l (2.0 ng t o t a l dose, 11 p m o l s ) in V L caused d r i n k i n g ( T a b l e 1). T h e same t h r e s h o l d , latencies, a n d v o l u m e s d r u n k were f o u n d for p r e o p t i c and a n t e r i o r h y p o t h a l a m i c p l a c e m e n t s s c a t t e r e d a m o n g higher t h r e s h o l d p l a c e m e n t s ( T a b l e 1). T h e r e was a dose-response r e l a t i o n s h i p for v o l u m e d r u n k for the latter p l a c e m e n t s , t ( 7 ) = 2.47, p < 0 . 0 5 . V L rats did n o t receive 10.0 n g / u l c a r b a c h o l . As little as 0.1 n g / u l a n g i o t e n s i n II (0.2 ng t o t a l dose, 0.2 p m o l s ) in V L caused d r i n k i n g (Table 1). The same t h r e s h o l d and v o l u m e d r u n k was f o u n d for p r e o p t i c -
TABLE I DRINKING CAUSED BY VENTRICULAR OR PREOPTIC-ANTERIOR HYPOTHALAMIC INJECTIONS OF CARBACHOL OR ANGIOTENSIN-II THRESHOLD (ng/pl)
1,0
n
DOSAGE LATENCY (ng/pl} (sec)
DURATION (xc)
VOLUME (ml)
LATERAL VENTRICULAR CARBACHOL 4 hO 85.+50 = 2 6 0 + 1 3 3 3,6 +- 0,7 PREOPTIC- HYPOTHALAMIC CARBACHOL
hO
8
hO I0,
150+122 60--.34
190 -+ 65 440-+108
3,0 -+ 1,3 7,2_+ 2.0
I0.
6
I0,
205+-81
2 6 0 +- 173
2.7 -* 1,5
0,1
LATERAL VENTRICULAR ANGIOTENSIN 4 0,1 210 -I- 128 105 +- 6 0 1,0 145 + 6 0 5 0 5 -+ 150 PREOPTIC- HYPOTHALAMIC ANGIOTENSlN
1,6 -+ 0,6 3,3 __4-1,4
0,1
5
0,1 hO
8.5+-32 70+-26
140--.64 240+-96
?-.,64"1,8 4,2 .-+ h8
1,0
8
1,0
120:1:44
200:1:65
2.8:1:0,8
*Mean + S.D.
V ENTR I C ULAR SPREAD OF CARBACHOL AND ANGIOTENSIN anterior hypothalamic placements scattered among higher threshold placements, but with slightly shorter latencies, t(7) = 2.0, p < 0 . 1 0 at 0.1 ng/ul; t(7) = 2.46, p
