Differential actions on voluntary alcohol intake of tetrahydroisoquinolines or a β-carboline infused chronically in the ventricle of the rat

Pharmacology Biochemistry & Behavior, Vol. 7, pp. 381 --392. Copyright © 1977 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

Differential Actions on Voluntary Alcohol Intake of Tetrahydroisoquinolines or a fl-Carboline Infused Chronically in the Ventricle of the Rat R. D. M Y E R S a A N D C. L. M E L C H I O R 2 Departments o f Psychological and Biological Sciences, Purdue University, Lafayette, I N 47907 (Received 29 August 1977)

MYERS, R. D. AND C. L. MELCHIOR. Differential actions on voluntary alcohol intake of tetrahydroisoquinolines or a #-carboline infused chronically in the ventricle of the rat. PHARMAC. BIOCHEM. BEHAV. 7(4) 381-392, 1977. - The alcohol drinking pattern of rats of the Sprague-Dawley strain was determined initially in a free-choice situation where water was offered simultaneously with a solution of alcohol, which was increased in concentration from 3 to 30% over a 12-day interval. A special cannula for the chronic infusion of drugs was implanted subsequently in the lateral cerebral ventricle of each animal. Then, one of four tetrahydroisoquinolines (TIQ) or a 13-carboline (tryptoline) was infused directly into the ventricle in a dose ranging from 0.0004-4.0 ~tg per 4.0/A. Each amine-aldehyde metabolite, dissolved in a CSF vehicle, was delivered over a 58 sec interval every 30 rain around the clock throughout a second 12-day alcohol preference test. Control infusions of CSF failed to alter the typical aversion to alcohol exhibited by this strain. TIQ itself, infused similarly into the brain, was also without effect. The chronic intraventrieular infusion of 1-methyl-3-carboxy-6,7dihydroxy-TIQ (1M-3C-6,7DHTIQ) or 4,6,7-trihydroxy-TIQ (4,6,7 THTIQ) produced only a moderate increase in voluntary alcohol intake in terms of both the proportion measure and absolute grams of alcohol ingested. However, salsolinol, 6-methoxy-4,7-dihydroxy-TIQ (6M-4,7DHTIQ) and tryptoline all evoked a significant enhancement in the rats' alcohol preference within 2 to 6 days of the start of the infusions. When a solution of alcohol was offered to the rats in the upper range of concentrations, (i.e., 11 to 30%) the amount of alcohol consumed often reached 10 g per kg per day and in some animals exceeded 15 g per kg per day. Symptoms of intoxication and withdrawal were noted in many of the animals during the course of the infusion of a TIQ or #-carboline. Upon retest for alcohol preference 30 days later, the rats ~ exaggerated self-selection of alcohol persisted even though the chronic infusions of the amine metabolite had ceased. These findings provide further support for the theory that a family of specific alkaloid conjugates acting within the brain are involved in the etiology of the abnormal drinking of alcohol. Alcohol self-selection Amine-metabolite theory Cerebral ventricle infusions Drinking of alcohol Aldehyde condensation product /3-carboline in brain Brain alkaloid Tetrahydroisoquinoline infusions Addiction to alcohol Salsolinol Tryptoline

THE m e t a b o l i t e s p o s t u l a t e d t o be f o r m e d in a p o t e n t i a l l y efficacious a m o u n t b y a c o n d e n s a t i o n o f an a m i n e and a l d e h y d e a f t e r alcohol is ingested have b e e n i m p l i c a t e d in the: b i o c h e m i c a l m e c h a n i s m u n d e r l y i n g a l c o h o l a d d i c t i o n [4, 8, 23, 3 0 ] . One o f the t e t r a h y d r o i s o q u i n o l i n e s (TIQ) t h a t is p r o d u c e d by t h e P i c t e t - S p e n g t e r r e a c t i o n , tetrah y d r o p a p a v e r o l i n e (THP), i n d u c e s an exaggerated preference for a l c o h o l w h e n t h e c o m p o u n d is i n f u s e d c h r o n i c a l l y i n t o t h e cerebral ventricle o f t h e rat [ 3 0 ] . S h o u l d biological c o n d i t i o n s favor such a r e a c t i o n , several c o n d e n s a t i o n p r o d u c t s o t h e r t h a n THP m a y also be s y n t h e s i z e d in vivo. As early as 1961, McIsaac [21] d e m o n s t r a t e d t h a t a

c o n d e n s a t i o n p r o d u c t derived f r o m an i n d o l e a m i n e , a #-carboline, can be d e t e c t e d in the urine o f the rat. Similarly, C o h e n and colleagues [4] have r e p o r t e d t h a t TIQ derivatives o f n o r e p i n e p h r i n e and e p i n e p h r i n e are c o n d e n s ed w i t h a c e t a l d e h y d e or f o r m a l d e h y d e as the adrenal gland is p e r f u s e d w i t h t h e s e a l d e h y d e s , or t h e y are f o u n d in this gland following s y s t e m i c injections o f m e t h a n o l [ 3 ] . Salsolinol, the c o n d e n s a t i o n p r o d u c t o f d o p a m i n e and a c e t a l d e h y d e , has n o t only b e e n i d e n t i f i e d in the urine o f the h u m a n [ 3 8 ] , b u t also in the brain o f t h e rat a f t e r an i n t r a p e r i t o n e a l i n j e c t i o n o f pyrogallol plus alcohol [ 6 ] . In tissue slices or h o m o g e n a t e o f rat brain, the u p t a k e o f a

Send reprint requests to: R. D. Myers, SCAX, Purdue University, Lafayette, Indiana 47907 2 Present Address: C. L. Melchior, Department of Pharmacology, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80262 381

382

MYERS AND MELCHIOIq

catecholamine is inhibited by a TIQ derived from either dopamine or norepinephrine; serotonin uptake is similarly prevented by a t3-carboline [1, 5, 14, 17, 4 0 ] . The neuronal effects exerted by these metabolites in vivo and in vitro include the retardation of the degradation of a biogenic amine [2,7], their uptake into nerve terminals [20, 32, 39] and their action in releasing the endogenous amine [ 14,35]. Overall, however, there is no direct evidence at present to suggest that a condensation product other than THP acts in the CNS to augment the voluntary selection of alcohol. For example, when a #-carboline is injected systemically, the amount of 4% alcohol consumed by a rat is reduced [12]. The purpose of the present study was to determine whether the chronic exposure of the brain to specific TIQs and a ~-carboline would alter the pattern of a rat's alcohol intake. In these experiments, 1,2,3,4-TIQ or one of five monoamine-aldehyde condensation products was infused chronically into the ventricle of the rat while it had the choice of alcohol or water in its cage. The metabolites infused were 1-methyl-6,7-dihydroxy-TIQ (salsolinol); 4,6,7-trihydroxy-TIQ (4,6,7THTIQ); l-methyl-3-carboxy6,7-dihydroxy-TIQ (1M-3C-6,7DHTIQ); 6-methoxy-4,7dihydroxy-TIQ (6M-4,7DHTIQ); and 1,2,3,4-tetrahydro-~carboline (tryptoline). Individual patterns of alcohol selfselection were obtained not only during 12 days of chronic ventricular infusion, but also following a 30-day interval so that the longevity of a metabolite's initial action could be ascertained.

Each of the compounds used for infusion was dissolvec in an artificial cerebrospinal fluid (CSF) which consisted o: 7.46 g NaC1, 0.19 g KC1, 0.14 g CaCI~ (anhydrous), anc 0.19 g MgC12 .6 H 2 0 per liter of glass distilled water [26] In order to retard the degradation of the alkaloids, the pE of the solution was lowered to 3.8 with the addition of 0.1 mg/ml of ascorbic acid. After preparation in pyrogen-frec glassware, each solution was passed through a 0.22 urr Swinnex millipore filter. The infusion pump was programmed so that the substances were delivered automatically into the rat's lateral ventricle in a 4.0 ul volume every 3C min, during the 12-day alcohol preference sequence. The compounds thus infused were : 1-methyl-6,7-dihydroxy-1,2, 3,4-tetrahydroisoquinoline hydrobromide (salsolinol); 1 m ethyl-3-carboxy-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquin oline hydrochloride (1M-3C-6,TDHTIQ); 4,6,7-trihydroxy 1,2,3,4-tetrahydroisoquinoline hydrochlorid~ (4,6,TTHTIQ); 6-methoxy-4,7-dihydroxy- 1,2,3,4-tetra hydroisoquinoline hydrochloride (6M-4,7DHTIQ) 1,2,3,4-tetrahydroisoquinoline (TIQ); or 1,2,3,4-tetra hydro-¢-carboline hydrochloride (tryptoline, TLN). Sal solinol was purchased from Sigma, TIQ from Aldrich ant TLN from K and K Laboratories, whereas the othe: compounds were generously provided by Dr. M. Collins o: Loyola University. For approximately one hour or less eact morning, each rat was disconnected from the chronic infusion system in order that the tubing-needle assembb could be flushed with the CSF vehicle and reloaded with freshly prepared solution.

METHOD Male rats of the Sprague-Dawley strain weighing 3 0 0 - 5 0 0 g were housed in individual cages. They were maintained on powdered Wayne Lab Blox and kept on a 12 hr l i g h t - d a r k cycle at an environmental temperature of 2 1 - 2 3 ° C . Each rat's volitional selection of alcohol was tested by a standard three-bottle, two-choice technique [29]. For this procedure, three calibrated 100 ml Kimax drinking tubes were attached to the front of an animal's cage with one tube containing a solution of alcohol which was increased in concentration on successive days as follows: 3, 4, 5, 6, 7, 9, 11, 13, 15, 20, 25 and 30%. Each volume/volume solution was prepared in deionized water with 95% ethanol. The second tube was filled with wateL whereas the third tube served as a dummy and was empty. The position of the three tubes was interchanged on each day according to a predetermined schedule so that the rat did not develop a position habit.

Chronic Infusion Procedure Following its 12-day alcohol preference test, each rat was anesthetized with sodium pentobarbital and placed in a stereotaxic instrument in the DeGroot orientation. Following standard procedures [25], a 20 ga guide tube which extended through the base of a Khavari swivel [18] was implanted above the lateral cerebral ventricle at the coordinates of AP +5.8, Lat 1.5 and Hor +3.0. After 2 days had elapsed, a 27 ga injector needle was inserted through the guide tube so that the tip rested in the ventricle. The injector needle was held snugly in place by a cap in such a way that it rotated freely within the guide tube; thus, the rat had complete freedom of movement throughout the experiment [18]. As described earlier [27 ], the needle was connected via PE tubing to a syringe mounted on a chronic infusion pump.

Taste Tests and Drinking Patterns Upon completion of their 12-day alcohol test sequence the rats in which 4.0 ug/ul of salsolinol or tryptoline hac been infused intraventricularly were given a choice of wate: and a constant concentration of either 25% or 15% alcoho for six days; again the third tube was empty. Following thi: interval, the empty drinking tube was replaced with om containing a solution of a palatable solution of Sustagez (Mead Johnson) which was isocaloric with the solution o alcohol presented to the animal. This combination of thre~ fluid choices was presented for four days, after which th~ Sustagen solution was removed and the empty tub~ replaced for an additional three days of testing. Drinkometer records, collected to indicate the tempora distribution of drinking over a 24-hour period, were alsc obtained for selected animals during a preference test f o 25% solution of alcohol and water. Salsolinol in a dose o 4.0 ug]4.0 ul had been infused intraventricularly in one ra during the initial 12-day alcohol preference sequence whereas in two other animals 4.0 ug/4.0 ul of tryptolin~ had been similarly injected. Drinkometer records were als( collected during the entire course of a second alcoho preference test for these three animals as well as for two rats infused with 0.04 ug/4.0 ul of tryptoline and one ra given 6M-4,7DHTIQ intraventricularly during the firs preference sequence.

Behavioral Observations and Blood Alcohol Determinatiot Periodically throughout the day and night, routin( observations were made and signs of intoxication and/oJ withdrawal [ 1 1,1 5 ] were recorded. Symptoms of particulal significance included: "wet-dog shakes," tail stiffness hyperactivity, compulsive sniffing, weaving, ataxia, teetl~

AMINE METABOLITES AND ALCOHOL DRINKING

383 TABLE1

THE MEAN PROPORTION OF ALCOHOL TO TOTAL FLUID INTAKE AND MEAN G OF ALCOHOL PER KG OF BODY WEIGHT CONSUMED BY EACH GROUP OF ANIMALS DURING THE ALCOHOL PREFERENCE TESTS GIVEN BEFORE (CONTROL) AND DURING THE INTRAVENTRICULAR INFUSION OF 4.0/~1 EVERY 30 MIN OF VARIOUS SOLUTIONS Solution

Dose (/~g/4.0/~l)

N

Pre-Infusion Control Proportion gm/kg Total Fluid

During Infusion Proportion gm/kg Total Fluid

Salsolinol

4.0 0.04

4 3

0.18 0.09

0.7 0.4

34.0 29.3

0.72 0.37

4.9 2.9

38.0 33.0

IM-3C-6,7DHTIQ

4.0 0.04

3 1

0.37 0.42

1.3 1.4

30.7 32.0

0.64 0.78

3.5 4.7

35.3 33.0

4,6,7THTIQ

4.0 0.04

4 4

0.24 0.20

0.7 0.6

30.5 27.8

0.31 0.40

1.2 1.9

23.3 38.3

6M-4,7DHTIQ

4.0

1

0.50

2.2

33.0

0.60

5.0

35.0

TIQ

4.0

3

0.31

1.0

29.0

0.41

2.0

29.0

Tryptoline

4.0 0.04 0.0004

4 3 2

0.16 0.39 0.14

0.8 1.6 0.9

34.3 38.5 31.0

0.58 0.53 0.61

6.0 5.4 3.9

52.0 50.7 67.5

3

0.32

1.3

34.0

0.08

0.6

50.0

CSF

-

-

c h a t t e r i n g , b r o a d - b a s e d gait, gnawing, i n c o o r d i n a t i o n , t w i t c h i n g , t r e m o r or convulsions. R e p r e s e n t a t i v e b l o o d s a m p l e s were collected for selected rats in t h e m o r n i n g h o u r s b e t w e e n 12 p.m. a n d 3 a.m. during a peak drinking period after alcohol had been ingested d u r i n g t h e night. A f t e r 25.0 ul o f b l o o d were t a k e n in a h e p a r i n i z e d capillary p i p e t t e f r o m t h e tip of t h e r a t ' s tail, t h e m o d i f i e d e n z y m a t i c assay o f R o o s [ 3 7 ] was used t o e s t i m a t e t h e level o f b l o o d alcohol. RESULTS A c o m p o s i t e s u m m a r y is p r e s e n t e d in T a b l e 1 of t h e r a t ' s a l c o h o l d r i n k i n g in r e s p o n s e t o t h e c h r o n i c i n t r a v e n tricular i n f u s i o n of five T I Q c o m p o u n d s , t h e # - c a r b o l i n e ( T L N ) a n d t h e C S F c o n t r o l vehicle. T h e results are e x p r e s s e d in t e r m s o f m e a n s o f t h e p r o p o r t i o n of a l c o h o l to t o t a l fluid i n t a k e , t h e actual a m o u n t o f a l c o h o l c o n s u m e d in g p e r kg, a n d t h e t o t a l fluid ingested ( w a t e r plus alcohol). Values f o r t h e 12-day 3 t o 30% a l c o h o l p r e f e r e n c e s e q u e n c e p r i o r t o i n f u s i o n ( c o n t r o l ) as well as d u r i n g t h e 12 days o f c h r o n i c i n f u s i o n also are given. T h e g r e a t e s t increase in a l c o h o l d r i n k i n g was p r o d u c e d b y salsolinol a n d t r y p t o l i n e w i t h a virtual seven-fold increase in g per kg i n t a k e o c c u r r i n g w i t h t h e 4.0 ug dose o f each c o m p o u n d . Even t h e i n f u s i o n of t h e l o w e r doses o f t h e s e t w o m e t a b o l i t e s caused a s u b s t a n t i a l e l e v a t i o n in a l c o h o l ingestion. D u r i n g the 12 days o f c h r o n i c i n f u s i o n , t h e rats in b o t h t h e t r y p t o l i n e a n d c o n t r o l g r o u p s increased t h e i r t o t a l daily v o l u m e i n t a k e s b y as m u c h as 12 t o 35 ml, b u t t h e r e were essentially n o significant changes in t h e t o t a l fluid i n t a k e s in t h e o t h e r groups. A l t h o u g h t h e o t h e r TIQs also e v o k e d a rise in a l c o h o l p r e f e r e n c e , this shift was n o t as

great as t h a t p r o d u c e d b y salsolinol or #-carboline, a n d s e e m e d to be d e p e n d e n t u p o n t h e s t r u c t u r a l c o n f i g u r a t i o n of t h e infused c o m p o u n d .

Salsolinol T h e changes in t h e p r e f e r e n c e for alcohol d u r i n g t h e i n f u s i o n of e i t h e r dose of salsolinol given in t h e 4.0 ul v o l u m e were significantly d i f f e r e n t ( p < 0 . 0 1 , N e w m a n Keuls tests) f r o m t h e CSF controls. As illustrated in Fig. 1, t h e 4.0 ug dose of salsolinol i n d u c e d a greater shift in p r o p o r t i o n of alcohol to t o t a l fluid ingested t h a n t h e 0.04 ~tg dose (t = 3.34 ; df = 1 1 ; p < 0.01 ). In fact, it was n o t u n t i l t h e s i x t h day of c h r o n i c i n f u s i o n t h a t t h e l o w e r dose of salsolinol elicited t h e s h a r p shift in alcohol drinking. With respect to t h e actual i n t a k e of alcohol, Fig. 2 s h o w s t h a t t h e 4.0 ug g r o u p c o n s i s t e n t l y d r a n k close to 5 g p e r kg per day of t h e fluid, or a greater a m o u n t t h r o u g h o u t t h e course o f t h e 12-day p r e f e r e n c e s e q u e n c e . A l t h o u g h t h e -differences b e t w e e n t h e doses were significant (t = 3.01 ; df = 11 ; p < 0 . 0 1 ) , t h e l o w e r dose of salsolinol did n o t increase t h e i n t a k e to a c o r r e s p o n d i n g level u n t i l t h e l a t t e r h a l f of t h e s e q u e n c e w h e n t h e h i g h e r c o n c e n t r a t i o n s of a l c o h o l were offered. U p o n re-test 30 days a f t e r t h e c h r o n i c i n f u s i o n s of t h e 4.0 ug dose of salsolinol h a d b e e n t e r m i n a t e d , the rats n e v e r t h e l e s s e x h i b i t e d a nearly identical p a t t e r n of alcohol drinking. Figures 3 a n d 4 p r e s e n t t h e c o n c o r d a n t values for t h e p r o p o r t i o n of a l c o h o l to t o t a l fluid as well as t h e a c t u a l i n t a k e in t e r m s o f g p e r kg d u r i n g t h e r e p e a t e d 12-day s e q u e n c e . This result shows t h a t t h e effect of salsolinol o n t h e i n t e r n a l s t r u c t u r e s of t h e b r a i n persists long a f t e r t h e e x p o s u r e to t h e c o n d e n s a t i o n p r o d u c t is d i s c o n t i n u e d .

384

M Y E R S AND M E L C H I O R SALSOLINOL

I0

T

,",

SALSOLINOL

pg/4Opl 0--0

40

0--0

004

n--II

CSF

40 foo-

pg/ 40pl

U

PRE

[]

DURING

I"t

I MONTH

POST

o

LLI Z 0

,i

d

oo

\

r',

?\

%/

o

E c9

50

,,,o

n

\

x t

0

6 oo

0.0

ETOH

% -"

i

i

i

I

i

i

i

i

i

i

i

i

3

4

5

6

7

9

II

13

15

20

25

30

FIG. 1. The proportion of alcohol (ETOH) to the total fluid intake (alcohol plus water) plotted against the concentration of alcohol offered on each day. An infusion in a volume of 4.0 #1 of CSF (N = 3), of 4.0 #g (N = 4) or of 0.04 #g (N = 3) of salsolinol was given every 30 min throughout the 12 days. SALSOLINOL

Iq r] I

40 004 CSF

3

4

"~ 5o

ETOH

%

"*

5

6

9

7

II

1.5

15

20

25

.50

FIG. 2. G alcohol per kg body weight consumed at each concentration of alcohol offered during the chronic infusions of CSF or salsolinol for the three groups of rats as described in Fig. 1.

40

n--u

nO

/

121

12.

•

U

F--<

"o

o

U_ Z 0

/o~

o--20.-7-, o ~

o5

,,,

o,

/

pg/40pl

PRE

0--0

DUR ING

o--o

~ MONT. p o s t

o.

\.o./.

%

",o.%/--o,"

\,\,\,\,

\

o---

..

,

F-

rl O0

ETOH

%

--"

|

!

i

!

!

|

3

4

5

6

7

9

i

IP

--,-

I

I

I

I

I

I

!

3

4

5

6

7

9

II

I

15

I

15

I

20

I

I

25

30

FIG. 4. G alcohol per kg body weight consumed at each concentration of alcohol offered before (PRE), during, and after the salsolinol infusions for the 4.0 #g group of rats as described in Fig. 3.

As p r e s e n t e d in T a b l e 1, t h e a n i m a l s in w h i c h 0.04 or 4.0 ug per 30 m i n o f 1M-3C-6,7-DHTIQ was infused i n t r a v e n t r i c u l a r l y c o n s u m e d m o r e alcohol t h a n t h e c o n t r o l animals given the CSF vehicle. Figure 5 illustrates t h e p r o p o r t i o n curve for t h e t h r e e rats in w h i c h t h e h i g h e r close was infused. D u r i n g the first six days of the test sequence d u r i n g w h i c h t h e 3 - 9 % c o n c e n t r a t i o n s of alcohol were offered, nearly t h e entire fluid i n t a k e was in t h e f o r m of alcohol. As p o r t r a y e d in Fig. 6, t h e c o r r e s p o n d i n g i n t a k e of alcohol in g per kg g e n e r a t e d a s o m e w h a t u n u s u a l p a t t e r n . A progressive increase in alcohol c o n s u m p t i o n o c c u r r e d over t h e first six days, t h r o u g h t h e 9% c o n c e n t r a t i o n , a n d t h e n this stabilized at a b o u t 4.2 g per kg for several days. During this interval, each a n i m a l d r a n k 5.0 g p e r kg or m o r e o n at least o n e day o f the p r e f e r e n c e test sequence.

4, 6, 7-Trihydroxy-TIQ (4, 6, 7-THTIQ)

SALSOLINOL

I0

%

1-methyl-3-carboxy-6, 7-dihydroxy-TIQ (1M-3C-6, 7-DHTIQ)

pg/40pl I00

ETOH

i

15

i

i

i

i

15

20

25

30

FIG. 3. The proportion of alcohol to total fluid intake plotted against the concentration of alcohol offered before (PRE), during, and after (1 month POST) chronic intraventricular infusions of the 4.0 pg dose of salsolinol (N = 3).

T h e c h r o n i c i n t r a v e n t r i c u l a r i n f u s i o n of e i t h e r 4.0 or 0.04 ug of 4 , 6 , 7 - T H T I Q caused very little change in alcohol selection. As s h o w n in Fig. 7, t h e p r o p o r t i o n a l i n t a k e o f b o t h groups o f rats barely e x c e e d e d t h e 0.5 p r o p o r t i o n value d u r i n g t h e initial p a r t of t h e p r e f e r e n c e test. T h e r e a f t e r , t h e p r o p o r t i o n a p p r o x i m a t e d t h a t of t h e c o n t r o l group. Overall, t h e r e were n o statistical d i f f e r e n c e s b e t w e e n t h e curves of p r o p o r t i o n for these t h r e e groups. As d e p i c t e d in Fig. 8, t h e g per kg i n t a k e of t h e rats given t h e higher dose of 4 , 6 , 7 - T H T I Q n e v e r rose a b o v e t h e 2.4 g p e r kg level. F r o m this peak value r e c o r d e d o n t h e fifth day o f t h e test sequence, t h e c o n s u m p t i o n of alcohol fell to a level of a p p r o x i m a t e l y 1.0 g per kg for t h e r e m a i n d e r of the p r e f e r e n c e test. Again, t h e differences were n o t significant. It s h o u l d be n o t e d t h a t the slightly higher alcohol i n t a k e of the four rats w h i c h received t h e l o w e r dose of 4 , 6 , 7 - T H T I Q was a t t r i b u t e d to t h e d r i n k i n g r e s p o n s e of o n e animal. In this case, t h e rat displayed the progressive increase in a l c o h o l c o n s u m p t i o n typical of the o t h e r alkaloids in t h a t a m e a n of 6.0 g p e r kg of alcohol was ingested d u r i n g t h e l a t t e r half of t h e p r e f e r e n c e session.

AMINE METABOLITES AND ALCOHOL DRINKING

385 4,6,7 THTIQ

1M-5C-6,7 DHTIO pg/40pl f) 0

pg/4Opl

O~o__o/O--O--o

~

I-- :D LI.I _1 U_

Z (3

I--I

J

i

~

\

i

0 F--

I

CSF

E

°~o

oo ETOH %

o~ °

|

l

i

|

|

i

i

i

|

3

4

5

6

7

9

fl

13

15

m

20

i

i

25

30

FIG. 5. The proportion of alcohol to total fluid intake plotted against the concentration of alcohol offered on each day. An infusion in a volume of 4.0 #1 of CSF (N = 3) or 4.0 #g of 1M-3C-6,7DHTIQ (N = 3) was given every 30 min during the 12 days. 1M- 3C-6,7 DHTIQ pg/40pl I00

I-I

40

I

cSF

50

004;

% ~

3

,

4

5

6

9

II

I3

,

15

20

25

30

F][G. 6. O alcohol per kg body weight consumed at each concentration of alcohol offered during the chronic infusions of CSF or of 1M-3C-6,7DHTIQ for the two groups of rats as described in Fig. 5. 4,6,7 THTIQ

IO-

40

0--0

0--0 oo4 I--I

CSF

0

o

8,

"-.<>..-"X f %"-o.

013.. 2

~x

I--I

\

""~'.. "'0 ---O--. O"

% --

00-

ETOH %

i

i

i

,

i

i

i

i

i

|

i

i

3

4

5

6

7

9

II

I3

15

20

25

30

~

i__

i.....~ i i I ~

FIG. 8. G alcohol per kg of body weight consumed at each concentration of alcohol offered during the chronic infusions of CSF or of 4,6,7THTIQ for the three groups of rats as described in Fig. 7.

6-Methoxy-4, 7-Dihydroxy-TIQ (6M-4, 7DHTIQ) O f t h e t h r e e rats in w h i c h a 4.0 ug dose of 6M4 , 7 D H T I Q was infused i n t r a v e n t r i c u l a r l y , in o n l y o n e a n i m a l (Table 1) did this TIQ p e n e t r a t e t h e v e n t r i c u l a r cavity as revealed b y t h e dye v e r i f i c a t i o n analysis [ 2 7 ] . T h e relatively high p r e i n f u s i o n c o n t r o l values for its p r o p o r t i o n a n d g i n t a k e was e n h a n c e d s u b s t a n t i a l l y b y t h e i n f u s i o n of t h e TIQ derivative. Fig. 9 ( T O P ) s h o w s t h a t t h e p r o p o r t i o n of alcohol c o n s u m e d was s u s t a i n e d at the 0.5 level or greater t h r o u g h o u t t h e last 10 days of t h e p r e f e r e n c e s e q u e n c e (5% t h r o u g h 30% c o n c e n t r a t i o n s ) . In c o n t r a s t t o t h e p r e - i n f u s i o n c o n t r o l test, t h e s t e a d y rise in alcohol c o n s u m p t i o n is i l l u s t r a t e d in Fig. 9 ( B O T T O M ) . O f i n t e r e s t here is t h e fact t h a t a l t h o u g h t h e m e a n i n t a k e of alcohol for t h e first half of this test s e q u e n c e was 2.8 g per kg, the average over t h e last six days was 7.2 g per kg with a level of 9.1 g per kg of t h e 30% s o l u t i o n c o n s u m e d o n t h e final test day. W h e n s o l u t i o n s of 35, 4 0 , 45 a n d 50% were o f f e r e d ( n o t s h o w n ) , the a n i m a l c o n t i n u e d to d r i n k alcohol in t h e same a m o u n t , i.e., 8.3, 7.2, 5.6 a n d 10.3 g p e r kg, respectively.

Tetrahydroisoquinoline (TIQ )

,ug/40pl

la.I d It_ Z 0 J

50

~

ETOH % "*

ETOH

004

{.9

X O0

E

40

M

v 0

El:: EL 0 Eg CL

40 CSF

OiO

I0

FI

l ~ l ) I . . ~ l

i

i

i

i

|

w

!

|

|

|

i

l

3

4

5

6

7

9

II

13

f5

20

25

30

FIG. 7. The proportion of alcohol to total fluid intake plotted against the concentration of alcohol offered on each day. An infusion in a volume of 4.0 #1 of CSF (N = 3), or of 4.0 #g (N = 4) or 0.04 #g (N = 4) of 4,6,7THTIQ was given every 30 rain during the 12 days.

In t h r e e rats in w h i c h a 4.0 ug dose of TIQ was infused c h r o n i c a l l y i n t o t h e cerebral ventricles, t h e r e were n o n o t e w o r t h y changes in alcohol drinking. As s h o w n in T a b l e 1, o n l y a slight increase a b o v e t h e p r e - i n f u s i o n level o c c u r r e d in t e r m s o f b o t h p r o p o r t i o n a n d g per kg. Figure 10 reveals t h a t d u r i n g t h e i n t e r m e d i a t e stage of TIQ i n f u s i o n , t h e p r o p o r t i o n a l i n t a k e surpassed the 0.5 value at t h e 7%, 9% a n d 11% c o n c e n t r a t i o n s of alcohol. This t r a n s i t o r y p r e f e r e n c e is likewise r e f l e c t e d in t h e g per kg measures, for the same days, as p o r t r a y e d in Fig. 11. Over days, h o w e v e r , t h e changes in alcohol c o n s u m p t i o n were n o t statistically significant.

Tryptoline (TLN) O f t h e g r o u p of a m i n e - a l d e h y d e c o n d e n s a t i o n p r o d u c t s t e s t e d in these e x p e r i m e n t s , t h e t~-carboline, t r y p t o l i n e , e x e r t e d an e x c e e d i n g l y p o t e n t e f f e c t o n the alcohol c o n s u m p t i o n of t h e rat. I n f u s e d c h r o n i c a l l y i n t o t h e

386

MYERS AND MELCHIOR

TIQ

l OZ 0

pg/40pl

6b

JO0 kl_

[]

4O

I

CSF

~_ ~- o5 E r~ 0_

i b O/

50

m~'~i__i~i

6M-4,7 DHTIQ RAT ~117 oo

I0 0 I--I

PRE

(I)

0--0

40pg/4Opl

([3)

ETOH % ""

1

E 50

oo

ETOH % ""

,n

3

4

5

m 13

6

15

5

6

r

7

13

15

20

25

30

FIG. 11. G of alcohol per kg of body weight consumed at each concentration of alcohol offered during the chronic infusions of CSF or 4.0 ~g of TIQ as described for the groups in Fig. 10.

:2 t9

,

34

TRYPTOLINE

pg/4.o pi 0--0

4.0

0--<> 0,04 @....--o 0 . 0 0 0 4

!

20

25

bO-

30

FIG. 9. The proportion of alcohol to total fluid intake (TOP) and g of alcohol per kg body weight (BOTTOM) plotted at each concentration of alcohol offered to Rat No. 117 before infusion (PRE) and during the chronic infusions of 4.0 #g of 6M-4,7DHTIQ given in a volume of 4.0 #1 every 30 min to this rat during the 12-day preference test.

I ' ' ' ' ' l ' ' ' ' ' ~',

OSF

I--I

o.%

",,

\

I.~ d LI_ Z

0

F ~~

0-5-

0n 2

• x•

2.

\

13-

TIQ

,~',

~0

"~1..,..--.I .-......i

0-0-

DAYS ETOH % -*

pg/40pl PO I 0

r~

hl

d U-

Z 0

/

o

U

20

m ~

0--0~

0

0--0

40

ll--I

CSF

o

Xm

13_

~°~°--°~o

O©-

ETOH %

I

U

,

I

m

I

I

3

4

5

6

7

9

rl

m

n

n

n

n

v

n

u

m

n

I

2

3

4

5

6

7

8

9

I0

II

12

:5

4

5

6

7

9

II

13

15

20

25

30

FIG. 12. The proportion of alcohol to total fluid intake plotted against the concentration of alcohol offered on each day. An infusion in a volume of 4.0 #1 of CSF (N = 3), or of 4.0 #g (N = 4), 0.04 ~g (N = 3) or 0.0004 ug (N = 2) of tryptoline was given every 30 min during the 12 days.

\

i

n

I

13

n

m

u

m

15

20

25

30

FIG. 10. The proportion of alcohol to total fluid intake plotted against the concentration of alcohol offered during the chronic infusions of CSF (N = 3) or 4.0 #g TIQ (N = 3) given in a 4.0 #1 volume every 30 min. cerebral ventricle in doses ranging f r o m 4 0 0 p i c o g r a m s to 4.0 ug every 30 m i n , t r y p t o l i n e e v o k e d a p r e f e r e n c e for alcohol t h a t e x c e e d e d t h e p r o p o r t i o n value of 0.5 at every c o n c e n t r a t i o n of alcohol offered e x c e p t t h e t w o highest. This shift in p r e f e r e n c e is illustrated in Fig. 12. A n increase in the p r o p o r t i o n a l i n t a k e of a p p r o x i m a t e l y four-fold (Table 1) over t h e p r e - i n f u s i o n c o n t r o l levels (t = 3.3 a n d 30.7, respectively; df = 11 ; p < 0 . 0 5 ) o c c u r r e d in t h e rats in w h i c h t h e highest a n d lowest doses were infused. T h e

progressive e n h a n c e m e n t in t h e a b s o l u t e i n t a k e of alcohol d u r i n g t h e t r y p t o l i n e i n f u s i o n s is p o r t r a y e d in Fig. 13. Doses of t r y p t o l i n e of 4.0 a n d 0.04 ug i n d u c e d t h e m o s t i n t e n s e d r i n k i n g p a r t i c u l a r l y in t h e c o n c e n t r a t i o n s ranging f r o m 9% t o 30% alcohol. Nevertheless, t h e g r o u p of rats in w h i c h a 4 0 0 p i c o g r a m dose was given every 30 m i n likewise d r a n k 4.0 to 6.0 g p e r kg per day. T h e a m o u n t of alcohol i m b i b e d b y one rat was p a r t i c u l a r y r e m a r k a b l e . In Fig. 14, t h e striking increase above t h e p r e - c o n t r o l level is s h o w n for b o t h t h e p r o p o r t i o n a n d g per kg values. A t the 15 a n d 20% c o n c e n t r a t i o n , the q u a n t i t y o f a l c o h o l c o n s u m e d b y this rat e x c e e d e d 10 g per kg per day (Fig. 14 B O T T O M ) . O n the e l e v e n t h day o f t h e sequence, w h e n t h e 25% s o l u t i o n of alcohol was offered, this t r y p t o l i n e - i n f u s e d rat d r a n k 16.6 g per kg during t h e 24 h o u r interval. W h e n this a n i m a l was re-tested for its alcohol p r e f e r e n c e 30 days a f t e r t r y p t o l i n e i n f u s i o n s h a d e n d e d , the level of alcohol ingested had stabilized at a p p r o x i m a t e l y 3 to 6 g per kg per day (Fig. 14 B O T T O M ) . All of t h e rats given the 4.0 a n d 0.04 ug doses of

AMINE METABOLITES AND ALCOHOL DRINKING

387 tryptoline were retested 30 days after the discontinuation of the chronic infusion of the #-carboline. Figure 15 reveals that the proportional change over the precontrol level continued (p<0.05, Newman-Keuls tests). Correspondingly, the g per kg intake also persisted (Fig. 16) at a higher level than that of a control (p<0.01 Newman-Keuls tests) with the intakes of alcohol approaching or exceeding the 5.0 g per kg level at several concentrations.

TRYPTOLINE pg/4"0

K~.O

-

pl

FI

4.0

I'1

0.04

|

0.0004

I

CSF

13n °

E

Sustagen Test and Drinking Patterns

ID

0"0

DAYS ETOH

,

,

,

"*"

I

2

3

4

5

6

7

8

9

I0

II

12

% ""

3

4

5

6

7

9

II

13

15

20

25

50

FIG. 13. G alcohol per kg body weight consumed at each concentration of alcohol offered during the chronic infusions of CSF or three doses of tryptoline as described for the groups in Fig. 12.

I-O

0---£/0~

Q

o ~-

~

TLN

15.0

"" >,,,, .'<>---<>", x

O

Rot 4'67

• --•

PRE

(1)

O--O

4.0pg/4.0pl

(FI)

o--o

POST

(rl)

I0"0

E 0

u

|

u

l

I

2

4

5

t 6

7

8

9

I0

I

12

3

4

6

7

9

H

15

15

20

25

50

5.0

0.0

DAYS E-TOH

%

'-" '-,*

!

FIG. 14. The proportion of alcohol to total fluid intake (TOP) and g of alcohol per kg body weight (BOTTOM) plotted at each concentration of alcohol offered to rat No. 67 before infusion 0"RE), during the chronic infusions of 4.0 #g/4.0 #1 of tryptoline, and one month after the infusion sequence (POST).

In the eight animals in which either salsolinol (N = 4) or tryptoline (N = 4) was infused intraventricularly in the 4.0 ug dose, alcohol in concentrations of either 15% or 25% was consumed in spite of the presence of the highly palatable mixture of Sustagen which was isocaloric to the alcohol solution offered. As shown in Table 2, the proportion of alcohol to water remained relatively constant during the four-day period when Sustagen was presented. However, because of the enormous increase in the total fluid consumed, which ranged from 96 to 226 ml of fluid per day, the overall proportion of alcohol to total fluid (alcohol solution plus water plus Sustagen solution) declined sharply. In one animal given tryptoline (No. 68) the g per kg intake of alcohol rose from 4.8 prior to the Sustagen test to 6.1 g per kg per day while Sustagen was given. During the additional three days after the Sustagen test had been completed (Post) the g per kg intake in most of the animals once again returned to the level at which alcohol was consumed prior to the presentation of the Sustagen. These results indicate that the alternative choice of a highly preferred liquid food did not alter the ratio of alcohol to water taken and in several cases, the amount of alcohol drunk was somewhat greater than that observed when the animals were tested prior to the infusion of the metabolites. Drinkometer records obtained while six rats were either given a preference sequence or offered water and a constant concentration of 25% alcohol indicated that the drinking occurred mainly during the night-time hours; that is, approximately 80% of all alcohol drinking occurred at this time. In Fig. 17, the diurnal intake of alcohol offered on successive days in the 3 - 3 0 % range of concentrations (oblique axis) is plotted for a representative rat in which the 0.4 #g dose of tryptoline had been infused intraventricularly. It can be seen that this animal drank alcohol usually in two to four bouts during the interval from 7 p.m. to 8 a.m. In another animal in which 6M-4,7DHTIQ had been infused, most of the alcohol consumption again occurred during the night cycle when the rat was offered a choice of water or 30, 15, 7.5, 15 or 30% alcohol for the respective periods as denoted in Table 3. The proportion of alcohol to water consumed by the animal varied directly with the concentration of alcohol offered, as shown in the Table. Similarly, the g per kg values were related directly to the alcohol concentration presented, although during the last 12 days when the 30% concentration was available, the overall intake decreased. Thus, the consumption of alcohol was generally greater at higher concentrations, but this pattern decreased with time.

S y m p t o m s o f Withdrawal and Blood Alcohol In at least one or two animals of each group given the

388

MYERS AND MELCHIOR GROUPED

TRYPTOLINE

(0 0 4 AND 4 0

p g / 4 0 pl)

O

0--0

PRE DURING

/ ~

0--0

I MONTH POST

n--m

I0-

A ~,0,

c~__. w

j LL

Z 0

d

/?\ •

0 /

o2

•/

~

13_

0--.<~

m~n-~ n

,'x

% -"

!

i

|

|

|

i

m

3

4

5

6

?

9

II

• ""8

" " "-~'m~

O0

ETOH

%,0 ,,&

",,, ,

\

i

i

13

15

m~ i

20

~ i

i

25

30

FIG. 15. The proportion of alcohol to total fluid intake plotted against the concentration of alcohol offered on each day before (PRE), during, and after (POST) the chronic intraventricular infusions of tryptoline (N = 5) in doses of 0.04 and 4.0 #g/#l. GROUPED

TRYPTOLINE

(0 0 4 AND 4 0 p g / 4 0 pl) m PRE Iq DURING B MONTH POST

IO0-

E

so.

II

(.9

OO

ETOH

% --,'.

3

!

|

!

!

4

5

6

7

il Jl m

II

!

13

15

20

i

25

30

FIG. 16. G alcohol per kg body weight consumed at each concentration of alcohol offered before (PRE), during and after (POST) the chronic infusion of tryptoline for the groups as denoted in Fig. 15.

amine-aldehyde condensation products intraventricularly: signs of withdrawal, ataxia and other behavioral abnormalities were evident. For example, salsolinol induced "wet dog shakes" and hyperactivity within three days after the start of the alcohol test sequence. Following the cessation of 1M-3C-6,7DHTIQ infusions, one rat exhibited convulsions; all other animals given this compound intraventricularly exhibited stiffness of the tail within a few days of the beginning of the alcohol test sequence. Repeated convulsive episodes were observed in animals in which 4,6,7THTIQ as well as tryptoline were infused; epileptiform activity usually began on the fourth or fifth day after the alcohol drinking had commenced. In relation to this, a given animal often flattened itself on the floor, extended its limbs, elevated the tail rigidly, was hyper-reactive to touch, showed ataxia, sometimes vocalized and displayed tremors of the head muscles. In all rats given 4,6,7THTIQ infusions, teeth chattering also occurred ordinarily by the sixth day of the test sequence. Two of these animals also manifested " w e t dog shakes" as well as intermittent convulsions which were characterized by repetitive jerking movements of the forebody as the animal stood in a rearing posture or lay prone on the floor of the cage. Figure 18 (TOP) illustrates the pre-convulsive state of an animal given the higher dose of 4,6,7THTIQ; the animal's body is extended, the tail is elevated and ataxia is prevalent. Subsequent to this, a full-blown convulsion occurred in which hind and forelimb myoclonic movements were noted. This response is portrayed in Fig. 18 (BOTTOM). During the course of intraventricular infusions of salsolinol (N = 3) or tryptoline (N = 5), during which intervals the animals consistently drank either 20 or 25% alcohol, samples of blood were collected at either 1 a.m. or 2 a.m. Table 4 presents the results of an analysis of blood alcohol levels (BAL) as well as the g per kg intake for selected rats during the 24 hr period. In each case, the plasma levels were below the 0.1% value, presumably because of the rapid metabolism of alcohol in this species [24]. DISCUSSION

These experiments demonstrate that the presence of a minute amount of an amine-aldehyde condensation product in the brain of the rat induces the abnormal drinking of alcohol in preference to water, in a free-choice situation.

TABLE 2 PROPORTION OF A L C O H O L TO T O T A L F L U I D I N T A K E (PROPORTION), G OF A L C O H O L PER K G OF BODY W E I G H T A N D MEAN T O T A L F L U I D I N T A K E CONSUMED BY RATS I N F U S E D WITH 4.0 txg/4.0 gl OF E I T H E R S A L S O L I N O L (SAL) OR T R Y P T O L I N E (TLN), BEFORE, D U R I N G AND A F T E R T H E S U S T A G E N S O L U T I O N WAS OFFERED IN T H E T H I R D ( " D U M M Y " ) T U B E

Proportion

PRE g/kg

Mean Fluid

Sustagen Test Proportion of E T O H g/kg Mean to ETOH plus Water Fluid

Proportion to Total Fluid

POST Proportion g/kg

Animal

Compound Infused

% ETOH Offered

Mean Fluid

60 61 63 64

Sal Sal Sal S~

25 25 15 15

0.33 0.48 0.10 0.08

7.3 8.5 1.2 3.1

56 40 42 134

0.38 0.75 0.22 0.02

1.8 1.7 0.4 0.5

126 109 96 226

0.03 0.05 0.02 0.01

0.34 0.87 0.06 0.11

5.9 4.3 0.8 4.2

37 12 45 141

65 66 67 68

TLN TLN TLN TLN

15 15 25 25

0.37 0.30 0.50 0.23

5.0 4.2 11.6 4.8

52 50 49 53

0.27 0.53 0.19 0.56

! .8 2.6 1.1 6.1

145 99 134 109

0.04 0.08 0.02 0.11

0.30 0.58 0.22 0.18

4.8 6.3 3.4 4.6

65 41 33 57

AMINE METABOLITES AND ALCOHOL DRINKING

DIURNAL

389

ETOH

(3-30%)

800 \

INTAKE

\

7O0 600 5O0 (/3

400

(,.) __1

\

300 200 I00 30

n

IIT

9

t

II T 0 H

6 % I?1 -i I I I I~

8

12

I

I

4

8

I AM

I l I I I I I I

12

4

I PM

' AM

FIG. 17. Drinkometer record of alcohol intake in licks per hr (ordinate) for each hr of the day and night as denoted on the abscissa. The oblique axis denotes the concentration of alcohol presented to the rat on successive days. Tryptoline (0.4 #g every 30 min) was infused intraventricularly in this rat. T h r e e aspects o f this r e s p o n s e are n o t a b l e : first, even t h o u g h t h e c o n c e n t r a t i o n b e c o m e s m o r e g u s t a t o r i l y aversive, t h e rat n e v e r t h e l e s s selects increasing q u a n t i t i e s of alcohol w i t h t h e c o n t i n u a t i o n of i n t r a c e r e b r a l i n f u s i o n s o f an alkaloid; s e c o n d , s y m p t o m s t h a t have b e e n c h a r a c t e r i z e d as w i t h d r a w a l - l i k e [ 1 1 , 1 5 ] are e x h i b i t e d ; t h i r d , w h e n t h e TABLE3 THE MEAN PROPORTION OF ALCOHOL TO TOTAL FLUID INTAKE AND MEAN G OF ALCOHOL PER KG OF BODY WEIGHT CONSUMED lq~y A SINGLE RAT, WHICH HAD BEEN INFUSED WITH 6M4,7DHTIQ, DURING THE PRESENTATION OF CONSTANT CONCENTRATIONS OF ALCOHOL Concentration of ETOH offered

Duration (Days) on Concentration

Proportion

gm/kg

30 15 7.5 15 30

10 10 10 16 12

0.27 0.50 0.53 0.48 0.23

6.3 4.9 2.5 4.0 3.8

rat is o n c e again given t h e c h o i c e of a l c o h o l long a f t e r t h e i n t r a v e n t r i c u l a r a p p l i c a t i o n o f a given m e t a b o l i t e has ceased, t h e rat re-instates its i n g e s t i o n o f a n u n u s u a l a m o u n t of a l c o h o l even w h e n t h e r e was n o access to t h e fluid in t h e i n t e r i m . A l t h o u g h T H P acting c e n t r a l l y is t h e m o s t p o t e n t o f t h e a m i n e m e t a b o l i t e s in i n d u c i n g alcohol p r e f e r e n c e [ 2 3 , 3 0 ] , of t h e c o m p o u n d s t e s t e d in t h e p r e s e n t e x p e r i m e n t s , t h e ~-carboline, t r y p t o l i n e , e v o k e d t h e m o s t i n t e n s e drinking. This i n d o l e m e t a b o l i t e n o t o n l y served t o a u g m e n t the i m b i b i t i o n o f high c o n c e n t r a t i o n s of alcohol, b u t also e n h a n c e d t h e rat's t o t a l fluid i n t a k e . This o b s e r v a t i o n coincides w i t h t h e overall increase in fluid c o n s u m p t i o n w h i c h occurs w h e n t h e rat is given i n t r a p e r i t o n e a l i n j e c t i o n s of t r y p t o l i n e [ 1 2 ] . In t h e l a t t e r case, alcohol p r e f e r e n c e was n o t e n h a n c e d b y t h e p e r i p h e r a l t r e a t m e n t w i t h t r y p t o l i n e , w h i c h w o u l d i n d i c a t e t h a t t h e #-carboline m u s t act d i r e c t l y o n s t r u c t u r e s in the CNS in o r d e r to reverse t h e usual aversion t o alcohol. In c o n t r a s t to the i n t e n s i t y of d r i n k i n g p r o d u c e d b y t h e O-carboline, t h e derivatives of n o r e p i n e p h r i n e were far less p o t e n t w h e n t h e y were given c h r o n i c a l l y b y t h e v e n t r i c u l a r r o u t e . Only o n e o f eight rats increased its a l c o h o l i n t a k e

390

MYERS AND M E L C H I O R

FIG. 18. (TOP) Pre-convulsive prone position of rat in which 4,6,7THTIQ was infused into its lateral cerebral ventricle; the infusion line and swivel as well as food dispenser are shown. (BOTTOM) Same rat one min later undergoing convulsive attack (see text).

TABLE4 B L O O D A L C O H O L L E V E L S A C H I E V E D DURING A P E A K D R I N K I N G PERIOD AND 24 H O U R G/KG OF A L C O H O L C O N S U M E D F O R T H E ENTIRE DAY F O R A N I M A L S I N F U S E D I N T R A V E N T R I C U L A R L Y WITH T H E H I G H DOSE OF S A L S O L I N O L OR T R Y P T O L I N E

Animal

60 61 62 65 66 67 67 68

Compound Infused

salsolinol salsolinol salsolinol tryptoline tryptoline tryptoline tryptoline tryptoline

Time Collected

% ETOH Concentration Consumed

BAL

Daily g/kg

2 a.m. 1 a.m. 2 a.m. 2 a.m. 2 a.m. 2 a.m. I a.m. 2 a.m.

20 25 20 20 20 20 25 20

0.028 0.087 0.050 0.045 0.025 0.035 0.087 0.044

7.9 8.4 7.8 8.3 7.8 12.3 11.0 12.6

during the course of the chronic central infusion of 4,6,7THTIQ. In relation to this, b o t h of the TIQs derived from dopamine, salsolinol and 1M-3C-6,7DHTIQ, did enhance the c o n s u m p t i o n of alcohol when they were infused chronically into the rat's ventricles. Whether or not this dopa-acetaldehyde condensation product exerts its action on the brain after its decarboxylation to salsolinol cannot be ascertained f r o m these results. Nevertheless, the possibility is raised that should 1M-3C-6,7DHTIQ be formed peripherally, it might cross the blood-brain barrier more readily than salsolinol [ 2 2 ] , since dopa itself but not d o p a m i n e can penetrate this barrier. Since 6M-4,7DHTIQ was also capable of causing an increase in alcohol preference, a critical chemical variable in the induction of alcohol drinking would not appear to be the c o m p o u n d ' s availability for 0-methylation [71. TIQ itself, which contains no substituents on its ring structure, is ineffective, when infused in the brain, in altering alcohol selection. Structurally, this m a y be indicative of the relative biochemical i m p o r t a n c e of h y d r o x y l or other groups in the 6 and 7 positions of the isoquinoline molecule. Alternatively, the p o t e n c y of the ~-carboline analog, the unsubstituted tryptoline molecule, in enhancing alcohol c o n s u m p t i o n suggests that such a substitution is not essential for the indole structure to be active in the CNS. Because such a wide spectrum of condensation products derived from the biogenic amines may differentially influence alcohol intake as well as withdrawal, each one may contribute in some unique fashion to the etiology of the addictive state. Interestingly, the condensation product yielded by the reaction of dopamine with acetaldehyde, salsolinol, has a different central effect than its analog condensed from f o r m a l d e h y d e , 6 , 7 D H T I Q [7,14]. How the amine-aldehyde condensation products function to produce the withdrawal-like signs in the rat is n o t clear. Surprisingly, substances as diverse in chemical structure as narcotic antagonists, t h y r o i d releasing h o r m o n e , and a t e t r a h y d r o p y r i m i d i n e are all capable of inducing the shaking, t r e m o r and motorial responses characteristic of withdrawal [ 4 1 ] . A l t h o u g h catecholaminergic neurons are already implicated in the withdrawal state [ 13, 15, 19, 34, 3 6 ] , the stimulation of serotonin receptors in the brain of the rat or a rise in serotonin c o n t e n t , results in a s y n d r o m e resembling the stereotypical responses which we have observed following the administration of the condensation products [ 16] ; these include tremor, rigidity, head shaking and hyperactivity. In view of the influence of these alkaloids on serotonergic pathways in the CNS [ 4 0 ] , the possibility now exists that the indoleamine system is another neurochemical avenue through which they operate. Again, such withdrawal-like behavior elicited by the aminealdehyde condensation products acting directly within the brain offers some substantiation for the functional commonality between the addictive sequelae of m o r p h i n e and alcohol [4,8]. In agreement with our previous finding that an acute intraventricular injection of several alkaloids provokes alcohol preference, it is envisaged that a family of amine metabolites may in part be responsible for alcohol's addictive effects [3 I ] . That is, if a c o n d i t i o n exists in vivo which favors the formation of a condensation product via the Pictet-Spengler reaction, it is likely that c o m p o u n d s of this class are actually formed in amine-rich regions of the limbic-forebrain system. The kinetics of the reaction of the parent c o m p o u n d s as well as the availability of substrate

AMINE METABOLITES AND ALCOHOL DRINKING w o u l d seem t o be p r i n c i p a l d e t e r m i n a n t s o f t h e q u a n t i t y o f c o n d e n s a t i o n p r o d u c t ; s u c h as salsolinol o r THP, t h a t m a y b e s y n t h e s i z e d a f t e r a l c o h o l is ingested [ 9 , 4 2 ] . T h e o r e t i c a l l y , it is n o t n e c e s s a r y t o a s s u m e t h a t o n e or rn.ore of t h e alkaloids m u s t b e s y n t h e s i z e d in t h e b r a i n in o r d e r to b e biologically efficacious [ 1 0 , 2 2 ] . I n d e e d , it is r e a s o n a b l e t o assume t h a t a c o m p o u n d c o u l d b e m e t a b o l i z e d p e r i p h e r a l l y t o its active f o r m a n d t h e n r e a c h t h e b r a i n p a r e n c h y m e f o l l o w i n g its passage t h r o u g h t h e b l o o d b r a i n barrier. As p o s t u l a t e d p r e v i o u s l y , a d i f f e r e n c e in t h e p e n e t r a b i l i t y of t h e b l o o d - b r a i n barrier, w h i c h w o u l d selectively act t o p r e v e n t or p e r m i t t h e e n t r y o f s u c h a rn.etabolite i n t o t h e b r a i n , c o u l d b e a crucial f a c t o r in t h e d e v e l o p m e n t of t h e a d d i c t i v e s t a t e [ 2 8 ] . W i t h t h e p r o l o n g ed d r i n k i n g o f a l c o h o l in n e a r t o x i c q u a n t i t i e s , p a t h o l o g i c a l "]Roles" in t h e b l o o d - b r a i n barrier c o u l d d e v e l o p w h i c h w o u l d p e r m i t t h e i n f l u x o f t h e highly p o t e n t a m i n e a l d e h y d e c o n d e n s a t i o n p r o d u c t s i n t o c e r t a i n regions o f t h e CNS [ 2 8 ] . Paralleling this possibility is t h e v i e w p o i n t t h a t t h e d r i n k i n g of a l c o h o l at t h e same t i m e t h a t t h e circulating level o f an a m i n e is high, w o u l d favor t h e f o r m a t i o n of a c o n d e n s a t i o n p r o d u c t w h i c h c o u l d be t r a n s p o r t e d to specific r e c e p t o r sites in t h e CNS. In t h e case of p r o l o n g e d

391 stress a n d t h e c o n s e q u e n t a d r e n a l a c t i v a t i o n w h i c h ensues [ 3 3 ] , this s t a t e in c o m b i n a t i o n w i t h i m m o d e r a t e d r i n k i n g of a l c o h o l c o u l d t h e o r e t i c a l l y facilitate t h e s y n t h e s i s o f a n amine metabolite. Because of t h e v a r i a t i o n s seen in t h e r e s p o n s e o f an individual rat t o t h e c h r o n i c i n f u s i o n o f t h e m e t a b o l i t e s , it is likely t h a t a c e r t a i n region o f t h e b r a i n is m o r e reactive t o t h e c o n d e n s a t i o n p r o d u c t t h a n a n o t h e r , p r e s u m a b l y because of t h e p r e s e n c e of specific r e c e p t o r material. F u t u r e research, t h e r e f o r e , s h o u l d be d e v o t e d t o i d e n t i f y i n g t h e a n a t o m i c a l locus of a c t i o n of t h e family o f a m i n e m e t a bolites w i t h respect to t h e e v o c a t i o n o f a l c o h o l d r i n k i n g . Given s u c h a l o c a l i z a t i o n , it w o u l d t h e n be possible t o e x a m i n e in vivo t h e f u n c t i o n a l n a t u r e of t h a t s t r u c t u r e in t e r m s o f its u n i q u e c h e m i c a l p r o p e r t i e s .

ACKNOWLEDGEMENTS This research was supported in part by NSF Grant BMS 75-18441, U.S. Office o f Naval R e s e a r c h Contract N-00014-75-C-0203. C. L. Melchior was a David Ross Fellow of the Purdue Research Foundation. We thank R. Nattermann who provided excellent technical assistance.

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