Increased alcohol selection in rats after alcohol drinking paired with recovery from thiamine deficiency

Physiology & Behavior, Vol. 19, pp. 293-302. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.

Increased Alcohol Selection in Rats After Alcohol Drinking Paired With Recovery from Thiamine Deficiency MICHAEL B. BASS AND DAVID LESTER

Center o f Alcohol Studies, Rutgers University, N e w Brunswick, N J 08903 (Received 25 August 1976) BASS, M. B. AND D. LESTER. Increased alcohol selection in rats after alcohol drinking paired with recovery from thiamine deficiency. PHYSIOL. BEHAV. 19(2) 293-302, 1977. - Repeated pairings of novel alcohol solutions (5% or 7.5% w/v) with IP injections of 267 ug/kg thiamine in thiamine-deficient male Sprague-Dawley rats resulted in the ingestion of pharmacologically active doses of alcohol in association with recovery. Mean alcohol intake in a postrecovery fluid choice situation exceeded metabolic capacity for 7-10 days; such intake was not observed in nondeficient pair-fed rats or in formerly deficient rats whose recovery was not paired with alcohol drinking. Alcohol was self-selected in the presence of water under conditions where the rats had no experience with water while deficient, but the presence of 0.1% saccharin as a postrecovery alternative to alcohol was sufficient to abolish the elevated intake. Absolute alcohol intake and blood alcohol levels on recovery days and during self-selection were comparable with 5% and 7.5% alcohol solutions. Alcohol selection

Oral self-administration

Learned preferences

F A I L U R E to produce voluntary consumption of pharmacologically significant amounts of alcohol in the presence of alternative fluids without food deprivation has been a serious obstacle to the development of an animal model of alcoholism [ 19]. Oral intake of alcohol has been measured in many experimental situations, among them polydipsia [9, 17, 36], presentation of alcohol as the sole fluid [31] or in a liquid diet as the sole source of food and fluid producing physical dependence [3,14], food deprivation [12,25], periodic availability [29,42], sweetened solutions [ 12,36], lick-contingent hypothalamic stimulation [24], and incorporation of alcohol drinking into escape [30] and avoidance [26] contingencies. While several such procedures have produced alcohol ingestion in substantial quantities, none have altered patterns of alcohol selection in a home cage choice situation. Results of physiological manipulations to increase voluntary alcohol selection have been equivocal. Although intraventricular infusion of alcohol was reported to result in increased alcohol selection in rats [27], this has not been confirmed [10] and negative results have been reported following similar infusions to dogs [ 15 ] and monkeys [ 16 ]. Increased alcohol selection in rats has been reported following electrical stimulation of the lateral hypothalamus [1,2]; however, absence of tests with other fluids for specificity to alcohol consumption, as well as negative findings from other laboratories [24] have cast doubt on the significance of these findings. Deutsch and Koopmans

[5] reported increased alcohol selection by rats following intragastric intubation of amounts of alcohol which maintained continuous intoxication for six days, but these findings have not been replicated (Lester et al., unpublished results 1974, 1975). To date there are no procedures which reliably induce self-selection of intoxicating doses of alcohol by rats. Evidence from specific hungers indicates that rats develop preferences for diets whose ingestion is followed by recovery from thiamine deficiency [13, 34, 37]. Rats deficient in thiamine display an immediate preference for novel over familiar foods whether or not the novel diet contains the needed vitamin, but maintain this preference only if recovery occurs [32]. Although conditioned aversions can account for much of this data [33], under appropriate conditions appetitive conditioning can be demonstrated [46]. Thiamine-deficient rats cannot select the one diet of ten that contains the needed vitamin, but show a clear preference if offered that choice exclusively until recovery [13]. This preference is maintained even when the vitamin is removed and placed in another choice [ 13,37], but rats can be reeducated to the proper choice if offered it exclusively for a time. Learned associations apparently supercede recognition of the vitamin, if any. In the case of thiamine-specific hunger, oral ingestion of the vitamin is neither necessary nor sufficient to induce a preference for flavors paired with recovery from deficiency. Rats fail to display a preference of aqueous thiamine

Supported in part by a grant from the National Institute on Alcohol Abuse and Alcoholism (AA-00216 to D.L.), a grant from the Charles and Johanna Busch Memorial Fund of Rutgers University and a grant from the Biomedical Sciences Support Grant of Rutgers University. 293

BASS AND LESTER

294

solutions over water despite having recovered from deficiency from drinking such a solution and in spite of apparent ability to distinguish between the solutions [ 2 1,351. In contrast, thiamine-deficient rats display learned preferences for sapid fluids when such fluids are paired with injection of thiamine [ 11, 22, 40, 45, 461. The following experiments indicate that recovery from thiamine deficiency paired with alcohol drinking produces voluntary alcohol intake that results in intoxicating blood alcohol levels.

EXPERIMENT

1

Method Animals. The animals were 30 male Sprague-Dawley rats (Charles River, Wilmington, MA), 23 days of age on arrival (Day 1). The rats were housed five per cage until 50 days of age; thereafter, each was housed individually. A 12 hr light (0800&2000 hr), 12 hr dark cycle was employed. Apparatus. The animals were housed in galvanized wire mesh cages, 27.3 x 21.6 x 21.6 cm beginning at 50 days of age. The floor consisted of metal bars; fecal material fell through to bedding below the cage, discouraging coprophagia. Fluids were presented in 105 ml test tubes fitted with stoppers and stainless steel ball-bearing spouts. Each drinking tube was mounted by a clip in one of three positions on the front of the cage. Spouts extended 3.8 cm into the cage through openings 3.8 cm from the floor; the openings to the left or right were 5.6 cm from either end. Spout tips were approximately 1.3 cm above the floor. Leakage was less than 1 ml per day. Fluids. Distilled water was used to prepare all fluids. The ethanol solution was prepared from 95% (v/v) ethanol. All fluids were presented at room temperature. Diet. The composition of the thiamine-deficient diet was as follows:

% By Weight

Ingredient

Casein

(vitamin-free)

DL methionine B, -free vitamin Choline

chloride

Jones-Foster Corn oil

mix (70%)

salt mixture

20.0 0.2 0.2 0.3 5.0 5.0

Sucrose

16.3

Corn

50.0

starch

Cellulose

3.0

The diet was presented in food cups, 8 cm in diameter and 4 cm in height, A perforated disk rode atop the food; the small openings prevented the animals from cupping their paws in the food, retarding spillage. The cups were fastened to the rear wall of the cage at floor level by food cup holders (Wahmann LC-306).

Procedure Predeficiency stage. Beginning on Day 1, the rats were randomly assigned to six equal groups. Each group was housed in a 4 1.9 x 2 1.6 X 20.3 cm polycarbonate cage and given ad lib access to thiamine-deficient diet and the fluid to be presented during the deficiency stage (Table 1). Rats were weighed daily. Each rat received 267 fig/kg (based on the mean body weight of all rats) thiamine hydrochloride (Betalin S, Eli Lilly and Company) IP in 0.2 ml isotonic saline every third day beginning on Day 3. All subsequent thiamine doses were based on the mean weight of each group. An additional group of five rats was housed in a polycarbonate cage with tap water and Purina Lab Chow ad lib throughout the experiment; their weights were used to assess normal growth rate. Deficiency stage. At 50 days of age (Day 28), each rat was housed individually with the thiamine-deficient diet and a deficiency fluid (Table l), available ad lib to each group except ND-PF. Each rat in ND-PF was given the amount of food and water that had been consumed the previous day by a yoked rat in W-E. Fluid position was changed daily in accordance with a common predetermined random schedule. Intraperitoneal injections (0.2 ml) continued every third day; nondeficient control groups (ND-W-E and NDPF) received 800 pg/kg thiamine (HCl, while all other groups received 0.9% saline. Three recoveries from deficiency were carried out. Criteria for deficiency were mean weight loss in each deficient group and mean food and fluid intake (measured daily) of less than 50% of that of ND-W-E for three consecutive days. All groups were identically treated on recovery days, except for the recovery fluid (Table 1) paired with injection of thiamine. On recovery days this fluid (0.1 ml) was introduced by syringe into the mouths of the rats twice, preceding IP injection of thiamine. The interval between these forced tastes was 15 min. Twenty minutes later, each rat was injected with 267 pg/kg thiamine HCl in 0.2 ml isotonic saline. Immediately after the final rat of a group received thiamine, that group was given the recovery fluid ad lib as the sole fluid for 24 hr. All treatments for ND-PF occurred one day after other groups were treated. On days after recovery of deficient rats, each rat in ND-PF was offered the amount of alcohol consumed by its yoked rat in W-E the previous day; the food ration was presented five min later. The recovery fluid was removed after the 24 hr; the deficiency fluid was reinstated and remained the sole fluid between recoveries. The second and third recoveries were carried out in an identical manner when all deficient groups again met the criteria for deficiency. However, the dose of thiamine for the third recovery was 800 fig/kg. Postrecovery self-selection stage. Beginning 24 hr after the third recovery, the recovery fluid was available continuously as part of a three bottle, two fluid choice (Table 1); the third bottle was empty. Food and fluid intake were measured daily and positions of all bottles were varied daily in accordance with a common predetermined random schedule. All groups received 800 pg/kg thiamine in 0.2 ml isotonic saline IP every third day, obviating further deficiency. Thiamine-deficient diet was available ad lib to all groups except ND-PF, which continued to be pair-fed with members of W-E. However, this group now received fluids ad lib. Powdered Purina Lab Chow was offered in lieu of the deficient diet after 14 days of self-selection,

RECOVERY FROM DEFICIENCY AND ALCOHOL P R E F E R E N C E

throughout (p = 0.91), indicating adequacy of the deficient diet with exogenous thiamine. Alcohol intake. Behavioral intoxication (ataxia and flaccidity) was noted in many rats in W-E-SAC, W-E, and HAc-E on recovery days and in the latter two groups during the first two days of self-selection. Although large amounts of alcohol continued to be consumed, no gross signs of intoxication were evident thereafter. Mean daily absolute alcohol intake (g/kg) on recovery days and during self-selection is shown in Fig. 2. All members of W-E and HAc-E drank large amounts of alcohol. Analysis of variance indicated significant main effects of treatments, days, and the treatments x days interaction during self-selection (all p<0.0005). Pairwise comparisons (Duncan's New Multiple Range test) based on selection Days 1 - 2 0 employed the 0.05 level of significance. Alcohol intake by W-E did not differ from HAc-E, but was significantly greater than every other group. Intake by group HAc-E did not differ from W-E or W-W, but was significantly greater than all other groups. Results from other contrasts were not significant. The low absolute alcohol intake by ND-PF indicates that the intake by W-E and HAc-E cannot be accounted for by their low body weight after recovery. Consumption by the latter two groups is not the result of a nonassociative change in behavior resulting from alcohol's caloric utility to weight-restricted rats. Elevated alcohol intake by HAc-E indicates that alcohol intake by W-E is not attributable to avoidance of water; alcohol intake by both groups remained above metabolic capacity (approximately 8 g/kg/day, [43] ) during the first ten days of self-selection. Although the difference between HAc-E and W-W fell short of significance, intake by the latter was significantly lower than W-E and not different from nondeficient groups. A history of thiamine deficiency in itself does not elevate subsequent

TABLE 1 FLUIDS OFFERED TO EACH GROUP DURING EACH PHASE OF EXPERIMENT 1 Deficiency Fluid

Group* W-E ND-W-E ND-PF~ HAc-E W-W W-E-SAC

Recovery Fluid

Water Water Water Acetic acid§ Water Water

Postrecovery Choice Fluids

Ethanolt Ethanol Ethanol Ethanol Water Ethanol

Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol

vs vs vs vs vs vs

295

Water Water Water Water Water Saccharin//

*All groups other than ND-W-E and ND-PF were rendered deficient in thiamine. t7.5% w/v. ~Pair-fed with W-E. §0.2% v/v. //0.1% w/v. Parenteral administration of thiamine was discontinued at this point. This diet was replaced by pelleted Purina Lab Chow 8 days later. There were 27 days of self-selection for each group when the experiment was terminated.

Results and Discussion Deprivation of thiamine produced declines in feeding, drinking, and body weight. On recovery days food and fluid intake increased sharply to control levels, resulting in prominent weight gain (Fig. 1). Following the third recovery, all formerly deficient rats increased about 50% in weight over ten days. The growth curve of ND-W-E is comparable to that of a group fed standard lab food

500

"

00

""*'"',-., ***" "* . ****'"" *" * ***

m

,ooy I

i

6

i

!

12

!

!

18

I

I

24

J

!

30

!

|

36

J

I

I

42

DAY OF

l

48

I

!

54

J

i

60

I

I

66

I

I

72

|

a

78

*

*

84

a

a

90

a

i

95

EXPERIMENT

FIG. 1. Mean body weights, Experiment t. Solid stars represent the mean of all deficient groups; circles, group ND-W-E. Open stars represent rats fed standard lab food. Deficient groups received thiamine on Days 45, 54, and 66.

296

BASS A N D L E S T E R

1.0

15

W-E I0

0.8

'~ tr.

I

't

O

....

/

J

0-4

_J LL

~ . ~ A

-J

I0

~ND_

W

DO I--

pF\V~'~, c'E

-10 hi

I--Z "1" 0 w

,

0

S

t

',

0.6

~15 -..

,' ', /'"1

"

M

ND-W- E

,a¢

j

....... ND-W-E : ; HAC-E

^

>.J

15

0.2 I

Z

",~ / .~N

• ,~\

,~._j

_'

P, W-W t~ ~ ^

! J', /

,,,,,..-,.

',.',,g

t~

~,

v

,,~

,~,

\

, \

v

i

|

i

i

5

,~, . . . . . . .

I

0"6 I

i

4

I

I

I

W-E-SAC A ND-PF /~ w-w j

\A?v

_

0.4

\/

IO 15 20 SELECTION DAY

I

,

/"

W-E-SAC abc recover y day

I

0"8

a

Z

I

I

', /,.

,,,

,, _

02

25 I

5 FIG. 2. Mean alcohol intake (g/kg/day) on recovery days and during self-selection, Experiment 1. Standard errors were generally 1 - 2 g/kg/day for W-E and HAc-E and were lower for others. Recovery Days a, b and c refer to the first, second and third recoveries, respectively. alcohol intake. Presence of 0.1% saccharin as a p o s t r e c o v e r y alternative to alcohol (W-E-SAC) a b o l i s h e d the increased alcohol selection by recovered rats. Results of analyses of alcohol c o n s u m p t i o n (ml) and alcohol selection ratios ( t r a n s f o r m e d t o arc sine to b e t t e r m e e t statistical a s s u m p t i o n s ) over selection Days 1 - 2 0 i n d i c a t e d significant m a i n effects of t r e a t m e n t s , days, a n d t h e t r e a t m e n t s x days i n t e r a c t i o n (all p < 0 . 0 0 0 5 ) . Mean daily selection ratios ( a l c o h o l / t o t a l fluid) a p p e a r in Fig. 3. D u n c a n ' s New Multiple R a n g e test p r o d u c e d identical p a t t e r n s of results o n b o t h m e a s u r e s as follows: W-E s h o w e d greater i n t a k e t h a n ND-PF, W-E-SAC, and W-W; HAc-E s h o w e d greater i n t a k e t h a n ND-PF a n d W-E-SAC; ND-W-E significantly e x c e e d e d ND-PF and W-E-SAC. G r o u p W-W significantly e x c e e d e d ND-PF, b u t n o t W-E-SAC in ml c o n s u m e d and e x c e e d e d W-E-SAC, b u t n o t ND-PF in s e l e c t i o n ratios. A l c o h o l selection b y W-W increased gradually (Fig. 3) and t h u s does n o t a p p e a r to reflect a c o n d i t i o n e d preference. Results f r o m o t h e r comparisons were n o t significant ( p > 0 . 0 5 ) . Selection ratios and alcohol c o n s u m p t i o n (ml) by ND-W-E were n o t significantly d i f f e r e n t from W-E or HAc-E b u t were significantly greater t h a n ND-PF. Since t h i a m i n e is r e q u i r e d for c a r b o h y d r a t e m e t a b o l i s m [ 3 4 ] , rats in ND-W-E, fed the high c a r b o h y d r a t e diet ad lib, m i g h t

I

I

I

I0 15 20 SELECTION DAY

I

25

FIG. 3. Mean daily alcohol selection ratios, Experiment 1. Fluids offered to each group appear in Table 1. have c o m e closer to e x h a u s t i n g their t h i a m i n e stores t h a n ND-PF, w h i c h was fed o n l y the small a m o u n t s of f o o d c o n s u m e d by d e f i c i e n t g r o u p W-E. T h a t is, rats in ND-W-E m i g h t have b e e n sufficiently d e p l e t e d of t h i a m i n e to feel b e t t e r after the t h i a m i n e i n j e c t i o n paired w i t h alcohol. C o n s e q u e n t l y , these rats m i g h t have developed a p r e f e r e n c e for a l c o h o l while t h o s e in g r o u p ND-PF did not. While rats in ND-W-E d r a n k alcohol in v o l u m e s similar to W-E a n d HAc-E, t h e i r i n t a k e does n o t a p p e a r to be p h a r m a cologically significant. F o r E x p e r i m e n t 2 we a s s u m e d t h a t the selection p a t t e r n of ND-W-E resulted f r o m a s s o c i a t i o n o f alcohol w i t h t h i a m i n e ; pulsing levels of t h i a m i n e in c o n t r o l rats was e l i m i n a t e d by m a i n t a i n i n g t h e m o n an t h i a m i n e - s u f f i c i e n t diet, o t h e r w i s e identical to t h a t fed d e f i c i e n t rats. T h e r e l a t i o n s h i p b e t w e e n e t h a n o l c o n c e n t r a t i o n and m a g n i t u d e of self-selection was also e x a m i n e d . T h a t alcohol was actually c o n s u m e d was verified by m e a s u r e m e n t of b l o o d alcohol levels on recovery days and during self-selection. EXPERIMENT

2

Method Animals. F o r t y Sprague-Dawley rats (Charles River), 22 clays of age on receipt, served as subjects. T h i r t y - n i n e rats

RECOVERY FROM DEFICIENCY AND ALCOHOL PREFERENCE were male (the sex of one rat was found to be female after the experiment was well under way). All rats were housed four per cage until 40 days of age; thereafter they were maintained under the same conditions as in Experiment 1. Apparatus. Cages, food cups, drinking bottles, and spouts were those used in Experiment 1. Fluids. The available fluids were distilled water and 5.0% or 7.5% (w/v) ethanol prepared and offered as in Experiment 1. Diet. The diet was modified slightly to allow the use of vitamin mixtures (Vitamin Diet Fortification Mixture, ICN Pharmaceuticals, Inc., in dextrose) identical except for the presence or absence of thiamine. The deficient diet contained:

Ingredient

Casein (vitamin-free) DL methionine B~-free vitamin mix* Jones-Foster salt mixture Corn oil Sucrose Corn starch Cellulose

% By Weight

20.1 0.2 2.2 5.0 5.0 14.3 50.2 3.0

*Complete vitamin mix used for control diet

Procedure Predeficiency stage. On arrival at the laboratory, all rats were provided with the control diet and distilled water ad lib. Deficient Stage 1. At 40 days of age the rats were randomly assigned to five equal groups and housed individually. The diets of three of the groups (5E, 7.5E, and BA) were progressively changed to the deficient diet by offering a mixture of 20% deficient diet, 80% control diet and increasing the portion of deficient diet by 20% each day. Both diets were offered ad lib. Distilled water was the sole fluid during this stage, its position varied daily at random among three positions. The two remaining groups (5C and 7.5C) were fed thiamine-sufficient diet throughout. Therefore, it was not necessary to administer injections every third day, but since an injection procedure can function as a conditioned stimulus [41], saline injections were administered at irregular intervals to avoid selective pairing of injections with recovery from deficiency. These injections (0.2 ml, IP) were given three days and again one day before the first recovery, once between the first recovery and the second, and three times between the second and third recoveries. Recoveries from deficiency were performed when all deficient groups (5E, 7.5E, and BA) met the criteria for deficiency. However, fluid intake did not decline as precipitously as in Experiment 1; thus the criterion of fluid intake of 50% or less than that of the nondeficient groups (5C and 7.5C) for deficiency was dropped, the body weight and food intake (measured daily) criteria of Experiment 1

297

being employed. Group BA was used for blood alcohol determinations and was subdivided into two groups (n = 4 each); one subgroup (Group 5BA) was recovered in association with and tested for selection of 5% alcohol; the other subgroup (Group 7.SBA) was identically treated using 7.5% ethanol. On recovery days, alcohol (5% w/v for Groups 5E, 5C and 5BA; 7.5% w/v for Groups 7.5E, 7.5C and 7.5BA) was administered twice orally (0.1 ml), spaced 15 min apart, before the thiamine injection 15 to 20 min later. This assigned alcohol solution was given to each group immediately after its last member received thiamine; alcohol remained the sole fluid for 24 hr. All rats were given 267 ug thiamine/kg IP in 0.2 ml isotonic saline; the dose for the third recovery was 800 ~g/kg. Distilled water was reinstated as the sole fluid following each of the first two recovery periods. Fluid self-selection Stage 1. Following the 24 hr alcohol exposure of the third recovery, alcohol was offered in a three bottle, two fluid choice situation. Thiamine HC1 (800 ~g/kg) was injected IP in 0.2 ml saline three and six days following the third recovery. The assigned ethanol solution was available in a choice with distilled water. The assigned diet was available ad lib. Deficiency Stage 2. Fluid Self-Selection Stage 1 was terminated after eight days. Water was reinstated as the sole fluid. Isotonic saline was injected IP twice between the last dose of thiamine from the previous phase and the first recovery of this phase (Recovery4). The criteria for deficiency and procedures for recovery were those of Deficiency Stage 1. A fifth recovery marked the end of this phase. The dose of thiamine on Recovery Days 4 and 5 was 267 ~g/kg. The assigned ethanol solution was the sole fluid for 24 hr following injection of thiamine; distilled water was the sole fluid prior to and beginning 24 hr after the fourth recovery. Fluid self-selection Stage 2. Twenty-four hr following thiamine injection on the fifth recovery day, the three bottle, two fluid choice between water and the assigned ethanol solution was begun. All rats received injections of 267 ug/kg thiamine every third day. This dose was lowered in an attempt to maintain ethanol drinking by keeping the animals closer to the threshold of deficiency. The five thiamine injections in this phase, occurring at three day inervals, were paired with forced tastes of the assigned ethanol solution, as on recovery days. Following thiamine injections, alcohol and water were offered concurrently. All formerly deficient rats were gradually returned to the control diet beginning on the eleventh day of this stage. A mixture of 20% control diet, 80% deficient diet was introduced. After two days, the portion of control diet was increased to 40% and thereafter incremented in steps of 20% daily. Thiamine injections were discontinued after inclusion of the vitamin in the diet in adequate supply (Day 16 of this stage). The control diet was gradually changed to powdered Purina Lab Chow by mixing the two diets, increasing daily portion of chow in steps of 20%. Purina Lab Chow pellets were introduced three days after the powdered form was offered and was available along with the powder for the final 11 days of this 35 day period. Blood alcohol measurements. On recovery days and on self-selection days chosen for blood alcohol determinations, about 20 ml of air was injected subcutaneously in the backs of the rats of Group BA. After a minimum of 20 min to allow for equilibration between the air pocket and the

298

BASS AND LESTER

blood, air samples of approximately 2 ml were drawn off in a warm (50 °) air-tight syringe. The sample was introduced into a galvanic cell containing two platinum electrodes and a nitric acid-chromic acid electrolyte [4,18]. A Leeds and Northrop Speedomax H recorder measured the increase in potential difference between the electrodes produced by the air sample. This potential difference, proportional to blood alcohol level, was compared with that of an air sample of known alcohol concentration. When samples were taken during the dark phase, red light was used as a source of illumination to minimize disturbance of the animals.

p = 0.31) or between nondeficient controls 5C and 7.5C (Stage 1: p = 0.82; Stage 2: p = 0.69). Alcohol intake following association with recovery exceeds the metabolic capacity [43] and is equivalent at both alcohol concentrations. Preliminary analysis of alcohol consumption (ml) and selection ratios suggested that differences present during earlier days were masked by lack of differences during later days of prolonged observation. Thus, analyses of these two measures during the second self-selection stage are based on the first 25 days. Alcohol consumption (ml) during both self-selection stages (Fig. 5) showed significant treatments, days, and treatments x days effects (all p<0.005). Planned comparisons produced the same pattern of results in both stages: Group 5E drank significantly more than 5C ( S t a g e 1: F(1,28) = 18.25, p<0.001; Stage 2 F(1,28) = 6.38, p<0.02). Group 7.5E drank more than 7.5C (Stage 1: F(1,28) = 13.53, p<0.001; Stage 2: F ( 1 , 2 8 ) = 5.01, p<0.035). Increased alcohol drinking by recovered rats with respect to control levels is evident at each alcohol concentration. Analysis of alcohol selection ratios (Fig. 6) showed significant main effects of days and treatments x days interaction during both stages (all p<0.0005), but treatments produced significant main effects only during the first self-selection stage (Stage 1: p<0.015; Stage 2: p = 0.095). In spite of this, results of planned comparisons in each stage were mutually consistent: Group 7.5E showed higher selection ratios than 7.5C (Stage 1: F ( 1 , 2 8 ) = 8.13,

R e s u l t s a n d Discussion

Analysis of variance was carried out for each selfselection period on three measures of daily alcohol intake by Groups 5E, 5C, 7.5E and 7.5C: absolute alcohol intake (g/kg), alcohol consumption (ml) and selection ratios (arc sine transformation). Absolute alcohol intake (Fig. 4) showed significant effects of treatments, days, and treatments × days interaction during both periods of self-selection (all p<0.0003). Planned comparisons yielded identical conclusions in both self-selection stages: the recovered groups (5E and 7.5E) selected significantly higher daily doses of ethanol than nondeficient controls 5C a n d 7.5C (Stage 1: F ( 1 , 2 8 ) = 6 8 . 1 5 , p<0.00001; Stage2: F ( 1 , 2 8 ) = 2 6 . 1 8 , p<0.00005). Intake did not differ significantly between recovered groups 5E and 7.5E (Stage 1: p = 0.67; Stage 2:

>, o

12

-~

I0

v

ua

v

8

_z 6 -1O ua

[3"

4

~-zfiz'fi5-°

z ua

O

D-. O.-

.~, ~

_."

2 I

i

I

a b c recovery

|

|

i

i

I 2 3 4

//~

i

I

d e

i

*

I

i

i

I

I

i

I

i

I

I

I

,

i

I 2 5 4 5 6 7 8 9 I01112131415

reeovery

BLOCKS OF 2 DAYS FIG. 4. Mean daily alcohol intake (g/kg) on recovery days and averaged over two day periods of self-selection, Experiment 2. Recovery Days a, b and c refer to the first, second and third recoveries, respectively. Eight days of self-selection Stage 1 followed. Recoveries d and e followed self-selection Stage 1 and preceded self-selection Stage 2. The first 30 days of self-selection Stage 2 are shown. Closed circles represent Group 5E; open circles, Group 7.5E; stars, Group 5C; and squares, Group 7.5C.

RECOVERY FROM DEFICIENCY AND ALCOHOL P R E F E R E N C E

60

~ 50

/.

W

40 Z

-I-

~ 3o W

S

O

o/

-1

...,,,.,,

~ 2o Z <

o

o ~

~ o ~ c"°"°

.o--[~ .o

recovery

recOvery

BLOCKS OF 2 DAYS

FIG. 5. Mean daily alcohol consumption (ml) on recovery days and averaged over two day periods of self-selection, Experiment 2. Recovery days a, b and c refer to the first, second and third recoveries. Eight days of fluid self-selection Stage 1 followed. Recoveries d and c followed self-selection Stage 1 and preceded self-selection Stage 2. The first 30 days of self-selection Stage 2 are shown. Closed circles represent Group 5E; stars, Group 5C; open circles, Group 7.5E; and squares, Group 7.5C. 1.0

~

0.9

.J "0.8

J

<0.7 I-o p-0.6 "-0.5 o "~ 0 . 4

~o p

~o S

>-

--Jo.3

[3_ ..0-

-D-

*'C

,

,

<

0.2 Z

,,<, O. I ,

,

I 2 3 4

a¢

i

|,

i

|

.

.

.

.

.

.

.

•

'

•

'

I 2 3 4 5 6 7 8 9 I011 12 13 1415 BLOCKS

OF 2 DAYS

FIG. 6. Mean daily selection ratios (averaged over two day periods), Experiment 2. At the left of the figure is the first self-selection period; the second self-selection period is to the right of the break. Closed circles represent Group 5E; stars, Group 5C; open circles, Group 7.5E; and squares, Group 7.5C. p< 0. 0 1 ; Stage2: F ( 1 , 2 8 ) = 4 . 9 0 , p<0.035), but no significant differences in selection ratios were evident between Groups 5E and 5C (Stage 1: p = 0.28; Stage 2: p = 0.99). Despite large differences in absolute alcohol intake and consumption in ml, there is no significant difference in selection ratios at the 5% concentration. ~Ihe selection ratio appears to be the least sensitive indicator of elevated alcohol consumption.

299

Selection ratios and consumption in ml by control rats increased substantially from the first to the second stage of self-selection (Figs. 5 and 6); we attribute this to periodic availability [29,42]. Selection ratios and alcohol drinking by recovered subjects also increased. Effects of additional pairings and periodic availability cannot be separated but preparedness [38] is characterized by the minimal number of necessary acquisition trials. Absolute alcohol intake during the second self-selection stage was comparable to that of the first. The additional pairings failed to elevate alcohol consumption further; it is thus conceivable that one or two pairings might be as effective as three. Blood alcohol determinations. Absolute intake was examined to determine if there was an effect of gathering samples for blood alcohol determinations on alcohol selection. An unweighted means analysis of Groups 5E and 5BA of the first 30 days of Stage 2 self-selection indicated that the groups did not differ (p = 0.32). The effect of days was significant (p<0.00001), but the treatments x days interaction was not (p = 0.24). Similarly, Group 7.5E did not differ from 7.5BA ( p = 0.17). Days was significant (p<0.00001), unlike the treatments x days interaction ( p = 0.96). Sampling had no effect on daily absolute alcohol intake at either concentration. Results of blood alcohol measurements in Group BA performed on each of the five recovery days (Table 2) show that the rats drank substantial quantities of alcohol, resulting in intoxicating blood ethanol levels at most hours of the day. There is apparently no significant effect of the concentration of alcohol on blood alcohol levels. During Stage 1 self-selection, blood alcohol levels were measured twice, one day apart. They were measured on five consecutive days during the first week of Stage 2 selfselection. Results are presented in Table 3. While differences in the time of sampling and occasional failure to obtain samples make generalizations about the circadian distribution of alcohol drinking difficult, blood ethanol levels were generally highest during dark hours. Early in self-selection there was generally a substantial level of blood alcohol at all times of the day. Blood alcohol measurements verify that Group BA ingested alcohol in pharmacologically effective patterns on recovery days and under self-selection. There was no obvious gross behavioral intoxication beyond recovery days, but blood alcohol levels attained produce measurable behavioral effects in rats of the same strain (Bass and Lester, unpublished results, 1976). That concentration of the alcohol solution did not affect blood alcohol concentrations attained under self-selection is consistent with the finding of no difference in absolute alcohol intake of rats drinking 5% or 7.5% ethanol. Daily absolute intake was often significantly correlated with daytime blood alcohol levels and was always significantly correlated with nocturnal levels. These significant positive correlations, taken together with the homogeneity of absolute intake suggest that nonsampled groups reached comparable blood alcohol levels.

GENERAL DISCUSSION These findings contrast with those of Senter and Sinclair [39], who found significantly lower alcohol selection in rats recovered from thiamine deficiency. Their rats received a novel ethanol solution when t h e deficient diet was

300

BASS AND L E S T E R TABLE 2 MEAN (± SE) BLOOD ALCOHOL LEVELS (rag/100 ml) RECORDED AT VARIOUS TIMES OF THE DAY DURING RECOVERY DAYS IN EXPERIMENT 2 Recovery

Time*

Group 5BA

a

1930 (3) 2330 (7) 0330 (11) 1630 (24)

160 219 158 261

b

1545 (1) 2200 (7.25) 1045 (20)

c

Group 7.5BA

t

df

p

23 33 11 54

2.41 1.87 2.51 1.68

6 5 5 4

.053 .12 .054 .17

222 ± 42 218 ± 37 164 ± 31

136 ± 97 103 ± 44 146 ± 86

0.82 1.27 0.20

6 5 6

.44 .26 .85

2315 (9) 0615 (16) 1015 (20)

87 ± 43 170 ± 41 74 ± 15

129 ± 41 124 ± 42 122 ± 29

0.71 0.76 1.46

6 5 6

.51 .52 .19

d

1530 (2)

174 ± 64

95 ± 33

1.24

4

.28

e

1500 (1) 0900 (19)

124 ± 30 57 ± 30

234 ± 79 109 ± 42

1.14 1.01

5 4

.31 .37

± ± ± ±

33 43 23 33

62 110 100 162

± ~ ± ±

*Numbers in parentheses refer to number of hours access to alcohol at time of sample.

TABLE 3 COMPARISON OF MEAN (± SE) BLOOD ALCOHOL LEVELS (mg/100 ml) IN RATS DRINKING 5% OR 7.5% ALCOHOL AND CORRELATIONS BETWEEN BLOOD ETHANOL AND DALLY ABSOLUTE ALCOHOL INTAKE DURING SELF-SELECTION IN EXPERIMENT 2 Time

Group 5BA

Group 7.5BA

2100-0230 1200-1430 1700

143 ± 29 57 ± 23 108 ± 33

73 ± 18 12 _+ 1 36 ~ 16

0815 1300-1400 2300

66 ± 14 42 ± 11 139 ± 12

95 ± 20 56 ± 17 95 ± 17

df

p

r*

pt

Stage 1 2.05 1.72 1.97

22 22 14

.052 .099 .069

+ .637 - .097 + .468

< .001 .741 .068

Stage 2 1.22 0.68 2.11

37 22 34

.230 .504 .042

+ .421 + .557 + .729

< .008 < .005 < .00001

t

*Pearson correlation coefficient tProbability based on t distribution under null hypothesis of zero correlation.

replaced by an a d e q u a t e one, but failed to select alcohol over w a t e r in s u b s e q u e n t tests. The animals t h u s experienced t w o novel flavors in association with recovery, but changes in the diet were c o r r e l a t e d w i t h o n s e t of deficiency while the fluid r e m a i n e d c o n s t a n t . Such rats w o u l d be e x p e c t e d to associate f o o d r a t h e r t h a n fluids with deficiency and recovery [ 2 2 ] . A second group o f d e f i c i e n t rats given a t h i a m i n e - c o n t a i n i n g alcohol s o l u t i o n in a choice situation recovered as a c o n s e q u e n c e o f drinking but also failed to select alcohol ( w i t h o u t t h i a m i n e ) over water. A generalization d e c r e m e n t and failure to selectively pair alcohol with recovery m i g h t c o n t r i b u t e to the lack o f postrecovery preference. In c o n t r a s t with m o s t a t t e m p t s to p r o d u c e selfi n t o x i c a t i o n by r o d e n t s in a choice situation, intake in these e x p e r i m e n t s appears to be p h a r m a c o l o g i c a l l y effective. Eriksson [7] has bred rats on the basis o f voluntary alcohol c o n s u m p t i o n , with intake at times a p p r o x i m a t i n g m e t a b o l i c capacity, and r e p o r t e d m e a n

b l o o d alcohol levels o f 27 m g / 1 0 0 ml [ 8 ] . Levels r e c o r d e d in E x p e r i m e n t 2 are c o n s i d e r a b l y higher. In b o t h e x p e r i m e n t s , significant d i f f e r e n c e s in absolute alcohol intake were o b t a i n e d despite absence o f d i f f e r e n c e s in alcohol selection ratios. Most studies o f v o l u n t a r y alcohol intake e m p l o y selections ratios to indicate alcohol p r e f e r e n c e ; t h e y are p r e s e n t e d here, t h e r e f o r e , for p u r p o s e s o f c o m p a r i s o n . However, dosage may be related to i n t o x ication, tolerance and d e p e n d e n c e . In c o n t r a s t , selection ratios, which may be i n f l u e n c e d by o t h e r factors (e.g., diuresis), c a n n o t be directly related to alcohol's pharmacological effects. Thus, the levels o f absolute alcohol intake a t t a i n e d in these e x p e r i m e n t s indicate increased alcohol p r e f e r e n c e in spite of the fact t h a t relative m e a s u r e s did n o t reflect this. Many e x p e r i m e n t s have d e m o n s t r a t e d c o n d i t i o n e d p r e f e r e n c e s for h e d o n i c a l l y neutral or palatable flavors paired w i t h recovery f r o m t h i a m i n e deficiency [ 11, 22, 40, 4 5 , 4 6 ] , but p r e s e n t results e x t e n d such findings t o flavors

RECOVERY FROM DEFICIENCY AND ALCOHOL PREFERENCE

o r d i n a r i l y aversive. P h a r m a c o l o g i c a l [ 2 0 ] , a n d o r o s e n s o r y [43] effects of a l c o h o l are aversive t o rats. Rats r e c o v e r e d from thiamine deficiency, having consumed intoxicating q u a n t i t i e s o f alcohol, m i g h t be e x p e c t e d to develop a c o n d i t i o n e d aversion [6] b u t i n s t e a d s h o w a s u s t a i n e d h i g h i n t a k e . T h u s , c o n d i t i o n e d p r e f e r e n c e s , like c o n d i t i o n e d aversions [ 4 4 ] , are a t t a i n a b l e in spite o f o p p o s i n g u n c o n d i t i o n e d p r o p e r t i e s o f t h e fluid. G u s t a t o r y c o n d i t i o n i n g offers a r a t i o n a l a p p r o a c h to m o d i f y the aversive p r o p e r t i e s of alcohol. Physical d e p e n d e n c e can be p r o d u c e d in rats in as little as f o u r days [23] if t h e y are c o n t i n u o u s l y i n t o x i c a t e d . Previous a t t e m p t s t o p r o d u c e p h y s i c a l d e p e n d e n c e by oral i n g e s t i o n of a q u e o u s a l c o h o l s o l u t i o n s [ 9 , 3 1 ] have failed unless a l c o h o l is available for at least seven weeks. W h e n

301

c o n s u m p t i o n is self-paced, p r e l i m i n a r y w e i g h t r e d u c t i o n is n e c e s s a r y to reliably p r o d u c e physical d e p e n d e n c e [ 2 8 ] . T h e l o w b o d y w e i g h t o f o u r rats a f t e r recovery, a n a l o g o u s to w e i g h t r e d u c t i o n , a n d the s u s t a i n e d i n t o x i c a t i n g p a t t e r n o f alcohol i n t a k e m i g h t , c o n c e i v a b l y , result in physical d e p e n d e n c e . H o w e v e r , n o a t t e m p t was m a d e to p r o d u c e a w i t h d r a w a l s y n d r o m e b y a b r u p t cessation o f alcohol at the height of consumption. Pairing a l c o h o l d r i n k i n g w i t h r e c o v e r y from t h i a m i n e deficiency elevates v o l u n t a r y alcohol c o n s u m p t i o n , prod u c i n g i n t o x i c a t i n g b l o o d levels. S u c h an a p p r o a c h u n d e r scores the i m p o r t a n c e of using a r e i n f o r c e r c o m p a t i b l e w i t h t h e desired r e s p o n s e of ingestion. This w o u l d a p p e a r t o be a m a j o r c o n s i d e r a t i o n in e x p e r i m e n t s d i r e c t e d t o w a r d s increasing volitional a l c o h o l c o n s u m p t i o n .

REFERENCES 1. Amit, Z. and M. H. Stern. A further investigation of alcohol preference in the laboratory rat induced by hypothalamic stimulation. Psychopharmacologia 21: 3 1 7 - 3 2 7 , 1971. 2. Amit, Z., M. H. Stern and R. A. Wise. Alcohol preference in the laboratory rat induced by hypothalamic stimulation. Psychopharmacologia 17: 3 6 7 - 3 7 7 , 1970. 3. Begleiter, H. Ethanol consumption subequent to physical dependence. In: Alcohol Intoxication and Withdrawal, edited by M. M. Gross. New York: Plenum, 1975, pp. 3 7 3 - 3 7 8 . 4. Berton, A. Piles galvaniques sensibles a des traces de substances gazeuses liquides ou solides. Chim. Analyt. 41: 3 5 1 - 3 5 8 , 1959. 5. Deutsch, J. A. and H. S. Koopmans. Preference enhancement for alcohol by passive exposure. Science 179: 1242-1243, 1973. 6. Eckardt, M. J. The role of orosensory stimuli from ethanol and blood alcohol levels in producing conditioned taste aversion in the rat. Psychopharmacologia 44: 2 6 7 - 2 7 1 , 1975. 7. Eriksson, K. Rat strains specially selected for their voluntary alcohol consumption. Annls Med. exp. Biol. Fenn. 49: 6 7 - 7 2 , 1971. 8. Eriksson, K. Alcohol consumption and blood alcohol in rat strains selected for their behavior towards alcohol. In: Biological Aspects of Alcohol Consumption, edited by O. Forsander and K. Eriksson. The Finnish Foundation for Alcohol Studies 20: 121-125, 1972. 9. Falk, J. L., H. H. Samson and G. Winger. Behavioral maintenance of high concentrations of blood alcohol and physical dependence in the rat. Science 1 7 7 : 8 1 1 - 8 1 3 , 1972. 10. Friedman, H. J. and D. Lester. Intraventricular ethanol and ethanol intake: A behavioral and radiographic study.Pharmac. Biochem. Behav. 3: 3 9 3 - 4 0 1 , 1975. 11. Garcia, J., F. R. Ervin, C. H. Yorke and R. A. KoeUing. Conditioning with delayed vitamin injections. Science 155: 7 1 6 - 7 1 8 , 1967. 12. Gilbert, R. M. Effects of food deprivation and fluid sweetening on alcohol consumption by rats. Q. Jl Stud. Alcohol 35: 4 2 - 4 7 , 1974. 13. Harris, L. J., J. Clay, F. J. Hargreaves and A. Ward. Appetite and choice of diet. The ability of the vitamin B deficient rat to discriminate between diets containing and lacking the vitamin. Proc. R. Soc. (Series B) 113: 161-190, 1933. 14. Hunter, B, E., D. W. Walker and J. N. Riley. Dissociation between physical dependence and volitional ethanol consumption: Role of multiple withdrawal episodes. Pharmac. Biochem. Behav. 2: 5 2 3 - 5 2 9 , 1974. 15. Jones, B. E., C. F. Essig and W. Creager. Intraventricular infusion of ethanol in dogs. Q. J1Stud. Alcohol 31: 2 8 8 - 2 9 2 , 1970.

16. Koz, G. and J. Mendelson. Effects of intraventricular ethanol in free choice alcohol consumption by monkeys. In: Biochemical Factors in Alcoholism, edited by R. P. Maickel. Oxford: Pergamon Press, 1967, pp. 17-24. 17. Lester, D. Self-maintenance of intoxication in the rat. Q. JI Stud. Alcohol 22: 223-231, 1961. 18. Lester, D. Rapid methods for processing large numbers of tissue samples for ethyl alcohol. In: Alcohol and Road Traffic. Proceedings of the Third International Conference, London, 1963, pp. 231-235. 19. Lester, D. and E. X. Freed. Criteria for an animal model of alcoholism. Pharmac. Biochem. Behav. 1: 103-107, 1973. 20. Lester, D., M. Nachman and J. LeMagnen. Aversive conditioning by ethanol in the rat. Q. JI Stud. Alcohol 31: 5 7 8 - 5 8 6 , 1970. 21. Luria, Z. Behavioral adjustment to thiamine deficiency in albino rats. J. comp. physiol. Psychol. 46: 358-362, 1953. 22. Maier, S. F., D. M. Zahorik and R. W. Albin. Relative novelty of solid and liquid diet during thiamine deficiency determines development of thiamine-specific hunger. J. comp. physiol. Psychol. 74: 2 5 4 - 2 6 2 , 1971. 23. Majchrowicz, E. Induction of physical dependence upon ethanol and the associated behavioral changes in rats. Psychopharmacologia 43: 2 4 5 - 2 5 4 , 1975. 24. Martin, G. E. and R. D. Myers. Ethanol ingestion in the rat induced by rewarding brain stimulation. Physiol. Behav. 8: 1151-1160, 1972. 25. Meisch, R. A. and T. Thompson. Ethanol intake as a function of concentration during deprivation and satiation. Pharmac. Biochem. Behav. 2: 5 8 9 - 5 9 6 , 1974. 26. Mello, N. K. and J. Mendelson. The effects of drinking to avoid shock in primates. In: Biological Aspects o f Alcohol, edited by M. K. Roach, W. M. Mclsaac and P. J. Creaven. Austin: University of Texas Press, 1971, pp. 313-340. 27. Myers, R. D. Alcohol consumption in rats: Effects of intracranial injections of ethanol. Science 142: 2 4 0 - 2 4 1 , 1963. 28. Ogata, H., F. Ogato, J. Mendelson and N. K. Mello. A comparison of techniques to induce alcohol dependence and tolerance in experimental animals. ,L Pharmac. exp. Ther. 180: 216-230, 1972. 29. Pinel, J. P. J., R. F. Mucha and L. I. Rovner. Temporary effects of periodic alcohol availability. Behav. Biol. 16: 2 2 7 - 2 3 2 , 1976. 30. Ramsay, R. W. and H. van Dis. The role of punishment in the aetiology and continuance of alcohol drinking in rats. Behav. Res. Ther. 5: 2 2 9 - 2 3 5 , 1967. 31. Ratcliffe, F. Ethanol dependence in the rat: Its production and characteristics. A rchs int. Pharmacodyn. Ther. 196: 146-156, 1972. 32. Rodgers, W. and P. Rozin. Novel food preference in thiaminedeficient rats. J. comp. physiol. Psychol. 61: 1 - 4 , 1966.

302 33. Rozin, P. Specific aversions as components of specific hungers. J. comp. physiol. Psychol. 64: 237-242, 1967. 34. Rozin, P. Thiamine-specific hunger. In: Handbook o f Physiology, edited by W. Heidel. Section 6. Alimentary Canal. Washington, D.C.: American Physiological Society, 1967, pp. 411-431. 35. Rozin, P., C. Wells and J. Mayer. Specific hunger for thiamine: Vitamin in water versus vitamin in food. J. cornp, physiol. Psychol. 57: 7 8 - 8 4 , 1964. 36. Samson, I-I. H. and J. L. Falk. Schedule-induced ethanol polydipsia: Enhancement by saccharin. Pharmac. Biochem. Behav. 2: 835-838, 1974. 37. Scott, E. M. and E. L. Verney. Self-selection of diet: The appetite for thiamine. J. Nutr. 3 7 : 8 1 - 9 2 , 1949. 38. Seligman, M. E. P. On the generality of the laws of learning. Psychol. Rev. 77: 4 0 6 - 4 1 8 , 1970. 39. Senter, R. J. and J. D. Sinclair. Thiamin-induced alcohol consumption in rats. Q. Jl Stud. Alcohol 29: 337-341, 1968. 40. Seward, J. P. and S. R. Greathouse. Appetitive and aversive conditioning in thiamine-deficient rats. J. comp. physiol. Psychol. 83: 157-167, 1973.

BASS A N D L E S T E R 41. Siegel, S. Conditioning of insulin-induced glycemia. J. comp. physiol. Psychol. 78: 233-241, 1972. 42. Sinclair, J. D. and R. J. Senter. Development of an alcoholdeprivation effect in rats. Q. Jl Stud. Alcohol 29: 8 6 3 - 8 6 7 , 1968. 43. Wallgren, H. and H. Barry III. Actions of Alcohol. Amsterdam: Elsevier, 1970, pp. 47,428. 44. Zahorik, D. M. and R. E. Johnston. Taste aversions to food flavors and vaginal secretions in golden hamsters. J. comp. physiol. Psychol. 90: 5 7 - 6 6 , 1976. 45. Zahorik, D. M. and S. F. Maier. Appetitive conditioning with recovery from thiamine deficiency as the unconditioned stimulus. Psychon. ScL 17: 309-310, 1969. 46. Zahorik, D. M., S. F. Maier and R. W. Pies. Preferences for tastes paired with recovery from thiamine deficiency: Appetitive conditioning or learned safety? J. comp. physiol. Psychol. 87: 1083-1091, 1974.