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Different Behavioral Patterns Related to Alcohol Use in Rodents
Alcohol, Vol. 17, No. 2, pp. 157–162, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/99 $–see front matter
PII S0741-8329(98)00049-4
Different Behavioral Patterns Related to Alcohol Use in Rodents: A Factor Analysis RAMIZ M. SALIMOV Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia Received 1 June 1998; Accepted 10 July 1998 SALIMOV, R. M. Different behavioral patterns related to alcohol use in rodents: A factor analysis. ALCOHOL 17(2) 157– 162, 1999.—To estimate genetic correlations among behavioral measures from explorative crossmaze, inescapable slip funnel, as well as from drinking tests, the data from five pairs of high/low alcohol drinking rat (AA/ANA, P/NP, HAD1/LAD1, HAD2/LAD2, HIGH_IPH/LOW_IPH) and from three pairs of mouse (B10.AKM/B10.A(4R), HD/LD, Small_Brain/ Large_Brain) lines were evaluated by the use of principal component analysis. The analysis yielded a two-factor solution explaining 71.8% of total variability. Both the factors had high positive loadings on alcohol drinking. The first factor had sufficient positive loadings on latency of crossmaze exploration and total time of slip funnel immobility, whereas, there was a negative loading on slip funnel avoidance. The second factor had positive loadings on efficacy of crossmaze exploration and slip funnel escape attempts, whereas there was a negative loading on slip funnel immobility. The number of defecations in the crossmaze, time in open arms of the elevated plus-maze, time immobile during the forced-swim test, as well as intake of a saccharin solution, additionally available for a lesser number of the lines, were studied for correlations with the factor scores. The first factor of “alcohol drive with timidity and meekness” exhibited positive relation to saccharin intake. Time in the open arms of the elevated plus-maze showed significant negative correlation with a latency of crossmaze exploration. The second factor of “alcohol drive with novelty seeking and persistence” showed a negative link to crossmaze defecations and forced-swim immobility. © 1999 Elsevier Science Inc. All rights reserved. Rat
Mouse
Alcohol drinking
Exploration
Immobility
Avoidance
Escape
rin solution (3,23,41,54,57), in measures of explorative behavior, and, for few lines, in their behavior in inescapable situations (20,23,39,40,47,60). Recently, an attempt to take advantage of the methods of multivariate analysis of the behavioral predictors of alcohol use have been made. In that work, some of previously published variables of drinking behavior as well as those of open-field, elevated plus-maze, and forced swim tests from nine rat lines (six of them were genetically selected for differential alcohol drinking AA/ANA, P/NP, and HAD2/ LAD2 lines) have been reevaluated by the use of principal component analysis (40). The analysis has revealed a factor having sufficient (.0.5) positive loadings on the amount of alcohol and saccharin intake, with negative loading on a number of open-field defecation and ultrasonic vocalization, thus supporting the existence of common denominator(s) for behavioral predictors of alcohol use in rodents.
RODENT lines selected for high- and low-volitional intake of alcohol provide a useful tool for study of factors that influence alcohol consumption. Comparative works have shown a commonality in alcohol-seeking behavior among high alcoholdrinking rodent lines, when free choice between water and alcohol solution is given (3,12,18,55), with some dissimilarity among the lines when palatable liquid is offered as a third choice (9,29,30). In the past 3 decades a number of studies have reported differences between high- and low-alcohol lines in explorative and avoidance behavior evaluated before their first contact with alcohol (2,10–12,14,15,19–21,23,26,27,33,35, 39,40,43,46,47,50,52,56,58,60). Some works have suggested the existence of common behavioral traits that may be specific for high-alcohol lines (9,11,23,24,35,58). Further comparison of rodents of several high-alcohol lines have revealed a similarity in the preference of a saccha-
Requests for reprints should be addressed to Ramiz M. Salimov, Ph.D., Institute of Pharmacology, Russian Academy of Medical Sciences, P.O. Box 23, Moscow, 127572, Russia. Tel/Fax: +7 095⁄400-7612; E-mail: [email protected]
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SALIMOV
In the present study, the factor analysis was applied to behavioral measures from distinct explorative crossmaze and inescapable slip funnel tests performed with 16 rodent lines (10 rats and 6 mice) diverging in volitional drinking of alcohol. The variables and results of their factor analysis were finally tested for correlation with important variables from elevated plus-maze, forced swim, and drinking tests mentioned in previous works. METHOD
Animals At least 10 male rats or mice in each group of animals known for high or low alcohol drinking were used. Four pairs of lines were well known outbred AA/ANA, P/NP, HAD1/ LAD1, and HAD2/LAD2 rats genetically selected for differential alcohol consumption at the Alko Biomedical Research Center, Helsinki, and at the Indiana University Medical Center, Indianapolis, IN. The HIGH_IPH and LOW_IPH rats, which have been used as subjects at the Institute of Pharmacology, Moscow, for last 2 decades (6,24,45,51,59), were two subpopulations from heterogenous stock of randomly bred animals (Stolbovaya Animal Center, Moscow region; the stock is now maintained at the Institute of Pharmacology) selected for high and low alcohol intake. The B10.AKM and B10.A(4R) mice were inbred lines derived on the basis of the C57BL/10SnY strain at the Laboratory of Experimental Biological Models (Moscow region) (43). The HD and LD mice were two subpopulations from hybrid mice produced by crossing the C57BL and CBA lines at the Laboratory of Experimental Biological Models (49). The Small_Brain and Large_ Brain mouse lines were selected from heterogenous stock formed from an six-way cross of inbred strains based on the CBA, C57BL/6J, C57Br, DBA, BALB/c, and A/He strains at the Laboratory of Physiology and Genetics of Behavior of Moscow State University (33). The main difference between pairs of mouse lines [B10.AKM vs. B10.A(4R), HD vs. LD, and Small_Brain vs. Large_Brain] was in the magnitude of a so-called alcohol deprivation effect, a temporary increase in alcohol intake after a short abstinence. Mean values of volitional alcohol intake (g/kg/h) after at least a month of free access to alcohol and water (alcohol intake during continual access—AL_C, alcohol intake during the first 1–2 h of renewal access to alcohol after a short abstinence—AL_D) and those for consumption of 0.1% (w/v) saccharin were taken from previously published works (13,14,16, 17,21,24,27,29–34,36,37, 40,42,43,45,46,49,51,53–55,58–61). The animals were group housed, two to eight animals per cage, with free access to standard rodent chow and tap water. The animal facilities had a room temperature of 20–248C, about 60% relative humidity, and 12:12-h light:dark cycle (lights on a 0600–0800, off at 1800–2000 h). The crossmaze and slip funnel tests were conducted with alcohol-naive animals between 1000 and 1400 h in an isolated general-purpose laboratory room (diffuse artificial light, 20–228C temperature, background noise approximately 60 dB[A]). Masking 70 dB[A] white noise was employed. Results from the tests as well as from elevated plus-maze and forced swim tests were taken from previously published works (20,22,27,28,33, 35,38, 40,43,45,47,48,50,52,54,56,58–60). Behavioral Test Employed Details of explorative crossmaze and inescapable slip funnel tests have been described previously (44,47,50). In this section, the behavioral tasks will be described in brief.
The crossmaze apparatus was made of Plexiglas and consisted of four closed empty arms (numbered 1, 2, 3, and 4) connected to the same central compartment. The animal was placed into the central compartment of the maze and allowed to explore. The floor was cleaned after each animal. The sequence and timing of arms visited were recorded directly into a personal computer by an observer until 13 visits had been made. The criterion for a visit is entry into a compartment with all four paws inside. Subsequent computer analysis was made to reveal several independent behavioral measures: 1. Latency to start exploration, i.e., the time before the first arm entry (FLAT). This variable is thought to reflect a balance between novelty-seeking of novelty-induced anxiety. 2. Length of first episode of maze patrolling, i.e., number of entries before every arm has been first visited (FPAT). For instance, if the arm-entering sequence for the 13 entries was 1241413344321, then the length of first patrolling is 7, because initial exploration was completed with entry into arm 3 on visit 7. The more visits takes a patrolling episode the less efficient is exploration. 3. The total number of patrolling episodes observed (PAT_N). In the example above, this would be two, because the second patrolling episode was completed on the 13th trial. The more patrolling episodes made the more efficient is exploration. 4. Immediate reentry to the arm just visited previously (IRE_N). This is considered to be a measure of working memory errors. 5. Stereotyped visits—scored if the rat visits two arms in an alternating manner (SV_N). In the example above, there is only one episode of four stereotyped visits (the 4141 pattern starting with the third entry). 6. Number of defections (fecal boli) considered to be a measure of general emotionality. This measure was only studied during the final analysis of correlations because it was not available for all 16 rodent lines.
TABLE 1 FACTOR LOADINGS ON BEHAVIORAL MEASURES
AL_C AL_D FLAT FPAT FIMM FAVO FESC Eigenvalues Variance explained (% of total)
Factor 1
Factor 2
0.63* 0.67* 0.79* 20.41 0.70* 20.83* 20.24
0.62* 0.37 0.16 20.52* 20.70* 0.35 0.86*
2.87 41.0
2.16 30.8
Alcohol drinking test (intake during continual access—AL_C, intake during first 1–2 hours after abstinence—AL_D), explorative crossmaze test (latency of first arm entry—FLAT, number of visit during first patrolling expisode—FPAT), and inescapable slip funnel test (time immobile at the bottom of the funnel—FIMM, time in sprawling posture on the slopping walls—FAVO, time climbing and jumping— FESC) from eight pairs of high and low alcohol-drinking rodent groups. Only those factors that had eigenvalues more than 1 were included. *Marks sufficient (.0.50) factor loadings.
BEHAVIORAL PATTERNS RELATED TO ALCOHOL USE The slip funnel apparatus consisted of an upper cylindrical portion with vertical walls, a center funnel section with walls sloping inward at an angle of approximately 408 from horizontal, and a lower cylindrical portion with vertical walls. Water (approximately 228C) filled the bottom cylinder and was replaced after each test. The depth of water was such that when standing immobile at the bottom, the hind legs of the animal would be immersed in the water. The test started with the animal being placed into the lower cylinder. The duration of the test was 3 min. The observer entered information directly into a computer, which was analyzed for time spent: 1) immobility in the water at the bottom of the funnel (FIMM). This measure correlated to immobility in the Porsolt’s forced-swim test (45), which measures what has been called “behavioral despair.” 2) In a sprawling posture on the sloping walls with all four paws out of the water, seen as a measure of passive avoidance (FAVO). 3) Climbing and jumping to get out of the funnel, interpreted as a measure of active escape (FESC). Statistical Analysis The statistical analysis was made with the help of a STATISTICA package. Mean values from distinct rodent groups served as a data for multivariate analysis. First, the results from explorative crossmaze and inescapable slip funnel tests were studied for correlation with those from alcohol drinking tests. The variables having statistically significant (p , 0.05) correlations with those of alcohol drinking and/or with each other were retained for the further principal component analysis with factor extraction based on eigenvalues greater than or equal to 1. Then factor loadings for the variables and factor scores for each group of rodents were calculated. Finally, vari-
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ables from the crossmaze and slip funnel tests as well as factor scores obtained were studied for correlation with number of defecation in the crossmaze, volitional saccharin intake, percent of time spent in open arms of elevated plus-maze, and perc‹ent of time immobile during forced-swim tests.
RESULTS
Principal component analysis yielded a two-factor solution explaining 71.8% of total variability among the variables analyzed (Table 1). The first factor had sufficient positive loadings on both measures of alcohol drinking, latency to start exploration in crossmaze, and total time of slip funnel immobility. The first factor had sufficient negative loading on total time of slip funnel avoidance. The second factor had sufficient positive loadings on alcohol drinking during continual access and on total time attempting to escape out of slip funnel. Sufficient negative loading of the second factor were on number of visits during first patrolling in the cross maze and on total time of slip funnel immobility. Analysis of factors scores for each pair of the animal groups (Table 2) has shown that, for HAD1/LAD1, HAD2/ LAD2, and Small_Brain/Large_Brain pairs the differences among high/low alcohol counterparts were greater at the first factor, while for AA/ANA, P/NP, B10.AKM/B10.A(4R) , and HD_hybrid/LD_hybrid pairs the differences were greater at the second factor. Variables from the crossmaze and slip funnel tests as well as factor scores exhibited relation to behavioral measures reportedly known as predictors of high alcohol intake among rodents (Table 3). Number of defecations correlated positively with number of visits during first patrolling and immedi-
TABLE 2 FACTOR SCORES PROVIDED BY PRINCIPAL COMPONENTS ANALYSIS FOR EACH MEMBER OF EIGHT PAIRS OF HIGH (H) AND LOW (L) ALCOHOL-DRINKING RODENT GROUPS AND DIFFERENCE BETWEEN THE HIGH/LOW ALCOHOL COUNTERPART GROUPS Factor 1
Group of Animal
ANA AA NP P LAD1 HAD1 LAD2 HAD2 LOW_IPH HIGH_IPH B10.A(4R) B10.AKM LD_hybrid HD_hybrid Large_Brain Small_Brain
Low or High Alcohol Group Membership
Score
L H L H L H L H L H L H L H L H
21.44 21.39 20.10 0.16 20.26 0.52 20.38 1.43 21.32 21.52 0.46 0.72 0.55 0.89 0.08 1.59
Difference Between Counterpart Groups
0.05 0.26 0.78* 1.81* 20.20 0.26 0.34 1.51*
Factor 2
Score
0.71 1.22 21.04 20.10 20.78 20.64 21.08 20.57 20.43 20.14 0.18 1.27 0.92 2.25 21.11 20.65
Difference Between Counterpart Groups
0.51* 0.94* 0.14 0.51 0.29 1.09* 1.33* 0.46
*Marks highest contrast between the factor scores for the counterpart groups.
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SALIMOV The difference in factor scores among counterpart high/ low alcohol rodents lines is higher at the first factor for the HAD1, HAD2, and Small_Brain lines, while at the second factor for the AA, P, HD_hybrid, and B10.AKM ones. The diversity corresponds to the facts that HAD1, HAD2, and Small_Brain lines are more timid during initiation of crossmaze exploration, and passive in the slip funnel than their low-alcohol counterpart lines, while AA, P, HD_hybrid, and B10.AKM demonstrate greater escape/avoidance attempts to get out of slip funnel, and a relatively lower difference in crossmaze timidity compared with their low-alcohol counterparts (43,47,50). The factor scores did not diverge much between the HIGH_IPH/LOW_IPH pair of rats. Compared with their counterpart LOW_IPH rats , the HIGH_IPHs are characterized by higher immobility in the forced-swim test (5,22,25,59), showing a trait attributed to the first factor, and relatively little difference in latency of crossmaze exploration (45,52), which seems to be a trait of the second factor. In accord to previous works (3,23,40,41,54,57), saccharin intake, time in open arms of elevated plus-maze, immobility in the forced-swim test, and defecations in novel situation were related; hence differentially, to factors revealed in the present study. Saccharin intake had a direct relation to alcohol intake, as well as to forced-swim immobility and to the first alcohol factor. There was significant negative correlation of the time in open arms of elevated plus-maze with the latency of crossmaze exploration; hence, its relation with the first alcohol factor that did not reach a significance level. Superficially, the coincidence of drive for saccharin with the first type of alcohol drive may help to explain why, when palatable liquid is offered as a third choice, a reduction in alcohol intake takes place in HAD1 rats (29), but not in P rats (30). The number of defecations in a novel situation of the crossmaze and forced swim immobility negatively correlated to the second factor, and exhibited connection to low efficient crossmaze exploration. These additionally evaluated correlations seem to confirm interpretation, given to the second factor, and suggest that attribution of “timidity and meekness” to the first factor, however, does not
ate reentries, and negatively with number of patrolling episodes in the crossmaze. Pearson correlations of the number of defecations with factor scores do not reach a significance level; hence, Spearman’s rank order correlation of the variable with the second factor was significant (r 5 20.65, n 5 12, p , 0.05). Saccharin consumption significantly correlated with alcohol intake after abstinence, with total time of slip funnel immobility and with the first factor. Time spent in open arms of the elevated plus-maze showed significant negative correlation with latency of first visit to an arm in the crossmaze. Immobility in the forced-swim test exhibited significant positive correlation with number of crossmaze stereotyped visits, and negative with the number of crossmaze patrolling episodes and total time of escape attempts out of slip funnel. The Pearson correlation of forced-swim immobility with the factors did not reach a significance level; however, Spearman’s rank order correlation of the variable with the second factor was significant (r 5 20.67, n 5 9, p , 0.05). DISCUSSION
A major finding in the present study is the existence of two factors explaining 71.8% of the variability among behavioral measures of alcohol drinking, crossmaze, and slip funnel test. Interestingly, the two factors are almost alike in their relation to alcohol drinking, but are differentially linked with crossmaze variables and have an opposite relation to the slip funnel measures. As far as the first factor is related to high alcohol intake, the long latency to start exploration in a novel situation of a crossmaze, high immobility, and low avoidance during inescapable slip funnel test, it may be interpreted as an alcohol drive that coincides with initial timidity and meekness. The second factor may be interpreted as an alcohol drive coinciding with the initial novelty-seeking and persistence because the factor is related to high alcohol intake, low number of visits during first patrolling in the crossmaze (which means high explorative efficacy), high escape, and low immobility during the inescapable slip funnel test.
TABLE 3 PEARSON CORRELATION OF BEHAVIOR MEASURES FROM ALCOHOL DRINKING, EXPLORATIVE CROSSMAZE, INESCAPABLE SLIP FUNNEL TESTS, AND SCORES OF FACTOR 1 AND FACTOR 2 FROM ONE SIDE WITH DEFECATION IN CROSSMAZE, SACCHARIN (0.1%, W/V) INTAKE, TIME IN OPEN ARMS OF ELEVATED PLUS-MAZE (%), AND TIME IMMOBILE (%) IN FORCED-SWIM TEST FROM THE OTHER SIDE
Defecations in Crossmaze
Number of pairs AL_C AL_D FLAT FPAT PAT_N IRE_N SV_N FIMM FAVO FESC Factor 1 Factor 2
12 20.36 20.48 0.10 0.62* 20.66* 0.60* 0.05 0.27 20.23 20.28 20.10 20.53
Saccharin Intake
12 0.09 0.60* 0.52 20.07 20.01 20.11 0.19 0.65* 20.49 20.51 0.65* 20.29
*Marks correlations significant at p , 0.05.
Time in Open Arms of Elevated Plus-Maze
9 20.53 20.41 20.68* 0.33 20.05 20.23 0.42 20.16 0.45 20.29 20.55 20.30
Immobility in Forced Swim Test
9 20.42 20.27 20.16 0.33 20.72* 0.17 0.74* 0.39 20.04 20.67* 20.05 20.62
BEHAVIORAL PATTERNS RELATED TO ALCOHOL USE necessarily involve a state like so-called “behavioral despair” believed to happen during the forced-swim test. The distinction between two alcohol factors, found in the present study, may be compared to some differential traits reported for type 1 and 2 (as well as for types A and B) alcoholics, namely, low or high novelty seeking, high or low harm avoidance, and high or low reward dependence, respectively (1,7,8). Higher efficacy of exploration corresponds to higher novelty seeking, whereas a longer latency to initiate crossmaze exploration parallel the more prominent tendency to avoid the situation (4,7,8). Because behavioral extinction is
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thought to reflect reward dependence (7,8), higher immobility in the water at the bottom of the slip funnel, which develops after extinction of initial attempts to avoid the water, may be seen as a higher reward dependence. ACKNOWLEDGEMENTS
The author would like to express his gratitude to Ting-Kai Li, William J. McBride, James M. Murphy, John D. Sinclair, and David H. Overstreet for helpful information regarding results previously published by them.
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PII S0741-8329(98)00049-4
Different Behavioral Patterns Related to Alcohol Use in Rodents: A Factor Analysis RAMIZ M. SALIMOV Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia Received 1 June 1998; Accepted 10 July 1998 SALIMOV, R. M. Different behavioral patterns related to alcohol use in rodents: A factor analysis. ALCOHOL 17(2) 157– 162, 1999.—To estimate genetic correlations among behavioral measures from explorative crossmaze, inescapable slip funnel, as well as from drinking tests, the data from five pairs of high/low alcohol drinking rat (AA/ANA, P/NP, HAD1/LAD1, HAD2/LAD2, HIGH_IPH/LOW_IPH) and from three pairs of mouse (B10.AKM/B10.A(4R), HD/LD, Small_Brain/ Large_Brain) lines were evaluated by the use of principal component analysis. The analysis yielded a two-factor solution explaining 71.8% of total variability. Both the factors had high positive loadings on alcohol drinking. The first factor had sufficient positive loadings on latency of crossmaze exploration and total time of slip funnel immobility, whereas, there was a negative loading on slip funnel avoidance. The second factor had positive loadings on efficacy of crossmaze exploration and slip funnel escape attempts, whereas there was a negative loading on slip funnel immobility. The number of defecations in the crossmaze, time in open arms of the elevated plus-maze, time immobile during the forced-swim test, as well as intake of a saccharin solution, additionally available for a lesser number of the lines, were studied for correlations with the factor scores. The first factor of “alcohol drive with timidity and meekness” exhibited positive relation to saccharin intake. Time in the open arms of the elevated plus-maze showed significant negative correlation with a latency of crossmaze exploration. The second factor of “alcohol drive with novelty seeking and persistence” showed a negative link to crossmaze defecations and forced-swim immobility. © 1999 Elsevier Science Inc. All rights reserved. Rat
Mouse
Alcohol drinking
Exploration
Immobility
Avoidance
Escape
rin solution (3,23,41,54,57), in measures of explorative behavior, and, for few lines, in their behavior in inescapable situations (20,23,39,40,47,60). Recently, an attempt to take advantage of the methods of multivariate analysis of the behavioral predictors of alcohol use have been made. In that work, some of previously published variables of drinking behavior as well as those of open-field, elevated plus-maze, and forced swim tests from nine rat lines (six of them were genetically selected for differential alcohol drinking AA/ANA, P/NP, and HAD2/ LAD2 lines) have been reevaluated by the use of principal component analysis (40). The analysis has revealed a factor having sufficient (.0.5) positive loadings on the amount of alcohol and saccharin intake, with negative loading on a number of open-field defecation and ultrasonic vocalization, thus supporting the existence of common denominator(s) for behavioral predictors of alcohol use in rodents.
RODENT lines selected for high- and low-volitional intake of alcohol provide a useful tool for study of factors that influence alcohol consumption. Comparative works have shown a commonality in alcohol-seeking behavior among high alcoholdrinking rodent lines, when free choice between water and alcohol solution is given (3,12,18,55), with some dissimilarity among the lines when palatable liquid is offered as a third choice (9,29,30). In the past 3 decades a number of studies have reported differences between high- and low-alcohol lines in explorative and avoidance behavior evaluated before their first contact with alcohol (2,10–12,14,15,19–21,23,26,27,33,35, 39,40,43,46,47,50,52,56,58,60). Some works have suggested the existence of common behavioral traits that may be specific for high-alcohol lines (9,11,23,24,35,58). Further comparison of rodents of several high-alcohol lines have revealed a similarity in the preference of a saccha-
Requests for reprints should be addressed to Ramiz M. Salimov, Ph.D., Institute of Pharmacology, Russian Academy of Medical Sciences, P.O. Box 23, Moscow, 127572, Russia. Tel/Fax: +7 095⁄400-7612; E-mail: [email protected]
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In the present study, the factor analysis was applied to behavioral measures from distinct explorative crossmaze and inescapable slip funnel tests performed with 16 rodent lines (10 rats and 6 mice) diverging in volitional drinking of alcohol. The variables and results of their factor analysis were finally tested for correlation with important variables from elevated plus-maze, forced swim, and drinking tests mentioned in previous works. METHOD
Animals At least 10 male rats or mice in each group of animals known for high or low alcohol drinking were used. Four pairs of lines were well known outbred AA/ANA, P/NP, HAD1/ LAD1, and HAD2/LAD2 rats genetically selected for differential alcohol consumption at the Alko Biomedical Research Center, Helsinki, and at the Indiana University Medical Center, Indianapolis, IN. The HIGH_IPH and LOW_IPH rats, which have been used as subjects at the Institute of Pharmacology, Moscow, for last 2 decades (6,24,45,51,59), were two subpopulations from heterogenous stock of randomly bred animals (Stolbovaya Animal Center, Moscow region; the stock is now maintained at the Institute of Pharmacology) selected for high and low alcohol intake. The B10.AKM and B10.A(4R) mice were inbred lines derived on the basis of the C57BL/10SnY strain at the Laboratory of Experimental Biological Models (Moscow region) (43). The HD and LD mice were two subpopulations from hybrid mice produced by crossing the C57BL and CBA lines at the Laboratory of Experimental Biological Models (49). The Small_Brain and Large_ Brain mouse lines were selected from heterogenous stock formed from an six-way cross of inbred strains based on the CBA, C57BL/6J, C57Br, DBA, BALB/c, and A/He strains at the Laboratory of Physiology and Genetics of Behavior of Moscow State University (33). The main difference between pairs of mouse lines [B10.AKM vs. B10.A(4R), HD vs. LD, and Small_Brain vs. Large_Brain] was in the magnitude of a so-called alcohol deprivation effect, a temporary increase in alcohol intake after a short abstinence. Mean values of volitional alcohol intake (g/kg/h) after at least a month of free access to alcohol and water (alcohol intake during continual access—AL_C, alcohol intake during the first 1–2 h of renewal access to alcohol after a short abstinence—AL_D) and those for consumption of 0.1% (w/v) saccharin were taken from previously published works (13,14,16, 17,21,24,27,29–34,36,37, 40,42,43,45,46,49,51,53–55,58–61). The animals were group housed, two to eight animals per cage, with free access to standard rodent chow and tap water. The animal facilities had a room temperature of 20–248C, about 60% relative humidity, and 12:12-h light:dark cycle (lights on a 0600–0800, off at 1800–2000 h). The crossmaze and slip funnel tests were conducted with alcohol-naive animals between 1000 and 1400 h in an isolated general-purpose laboratory room (diffuse artificial light, 20–228C temperature, background noise approximately 60 dB[A]). Masking 70 dB[A] white noise was employed. Results from the tests as well as from elevated plus-maze and forced swim tests were taken from previously published works (20,22,27,28,33, 35,38, 40,43,45,47,48,50,52,54,56,58–60). Behavioral Test Employed Details of explorative crossmaze and inescapable slip funnel tests have been described previously (44,47,50). In this section, the behavioral tasks will be described in brief.
The crossmaze apparatus was made of Plexiglas and consisted of four closed empty arms (numbered 1, 2, 3, and 4) connected to the same central compartment. The animal was placed into the central compartment of the maze and allowed to explore. The floor was cleaned after each animal. The sequence and timing of arms visited were recorded directly into a personal computer by an observer until 13 visits had been made. The criterion for a visit is entry into a compartment with all four paws inside. Subsequent computer analysis was made to reveal several independent behavioral measures: 1. Latency to start exploration, i.e., the time before the first arm entry (FLAT). This variable is thought to reflect a balance between novelty-seeking of novelty-induced anxiety. 2. Length of first episode of maze patrolling, i.e., number of entries before every arm has been first visited (FPAT). For instance, if the arm-entering sequence for the 13 entries was 1241413344321, then the length of first patrolling is 7, because initial exploration was completed with entry into arm 3 on visit 7. The more visits takes a patrolling episode the less efficient is exploration. 3. The total number of patrolling episodes observed (PAT_N). In the example above, this would be two, because the second patrolling episode was completed on the 13th trial. The more patrolling episodes made the more efficient is exploration. 4. Immediate reentry to the arm just visited previously (IRE_N). This is considered to be a measure of working memory errors. 5. Stereotyped visits—scored if the rat visits two arms in an alternating manner (SV_N). In the example above, there is only one episode of four stereotyped visits (the 4141 pattern starting with the third entry). 6. Number of defections (fecal boli) considered to be a measure of general emotionality. This measure was only studied during the final analysis of correlations because it was not available for all 16 rodent lines.
TABLE 1 FACTOR LOADINGS ON BEHAVIORAL MEASURES
AL_C AL_D FLAT FPAT FIMM FAVO FESC Eigenvalues Variance explained (% of total)
Factor 1
Factor 2
0.63* 0.67* 0.79* 20.41 0.70* 20.83* 20.24
0.62* 0.37 0.16 20.52* 20.70* 0.35 0.86*
2.87 41.0
2.16 30.8
Alcohol drinking test (intake during continual access—AL_C, intake during first 1–2 hours after abstinence—AL_D), explorative crossmaze test (latency of first arm entry—FLAT, number of visit during first patrolling expisode—FPAT), and inescapable slip funnel test (time immobile at the bottom of the funnel—FIMM, time in sprawling posture on the slopping walls—FAVO, time climbing and jumping— FESC) from eight pairs of high and low alcohol-drinking rodent groups. Only those factors that had eigenvalues more than 1 were included. *Marks sufficient (.0.50) factor loadings.
BEHAVIORAL PATTERNS RELATED TO ALCOHOL USE The slip funnel apparatus consisted of an upper cylindrical portion with vertical walls, a center funnel section with walls sloping inward at an angle of approximately 408 from horizontal, and a lower cylindrical portion with vertical walls. Water (approximately 228C) filled the bottom cylinder and was replaced after each test. The depth of water was such that when standing immobile at the bottom, the hind legs of the animal would be immersed in the water. The test started with the animal being placed into the lower cylinder. The duration of the test was 3 min. The observer entered information directly into a computer, which was analyzed for time spent: 1) immobility in the water at the bottom of the funnel (FIMM). This measure correlated to immobility in the Porsolt’s forced-swim test (45), which measures what has been called “behavioral despair.” 2) In a sprawling posture on the sloping walls with all four paws out of the water, seen as a measure of passive avoidance (FAVO). 3) Climbing and jumping to get out of the funnel, interpreted as a measure of active escape (FESC). Statistical Analysis The statistical analysis was made with the help of a STATISTICA package. Mean values from distinct rodent groups served as a data for multivariate analysis. First, the results from explorative crossmaze and inescapable slip funnel tests were studied for correlation with those from alcohol drinking tests. The variables having statistically significant (p , 0.05) correlations with those of alcohol drinking and/or with each other were retained for the further principal component analysis with factor extraction based on eigenvalues greater than or equal to 1. Then factor loadings for the variables and factor scores for each group of rodents were calculated. Finally, vari-
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ables from the crossmaze and slip funnel tests as well as factor scores obtained were studied for correlation with number of defecation in the crossmaze, volitional saccharin intake, percent of time spent in open arms of elevated plus-maze, and perc‹ent of time immobile during forced-swim tests.
RESULTS
Principal component analysis yielded a two-factor solution explaining 71.8% of total variability among the variables analyzed (Table 1). The first factor had sufficient positive loadings on both measures of alcohol drinking, latency to start exploration in crossmaze, and total time of slip funnel immobility. The first factor had sufficient negative loading on total time of slip funnel avoidance. The second factor had sufficient positive loadings on alcohol drinking during continual access and on total time attempting to escape out of slip funnel. Sufficient negative loading of the second factor were on number of visits during first patrolling in the cross maze and on total time of slip funnel immobility. Analysis of factors scores for each pair of the animal groups (Table 2) has shown that, for HAD1/LAD1, HAD2/ LAD2, and Small_Brain/Large_Brain pairs the differences among high/low alcohol counterparts were greater at the first factor, while for AA/ANA, P/NP, B10.AKM/B10.A(4R) , and HD_hybrid/LD_hybrid pairs the differences were greater at the second factor. Variables from the crossmaze and slip funnel tests as well as factor scores exhibited relation to behavioral measures reportedly known as predictors of high alcohol intake among rodents (Table 3). Number of defecations correlated positively with number of visits during first patrolling and immedi-
TABLE 2 FACTOR SCORES PROVIDED BY PRINCIPAL COMPONENTS ANALYSIS FOR EACH MEMBER OF EIGHT PAIRS OF HIGH (H) AND LOW (L) ALCOHOL-DRINKING RODENT GROUPS AND DIFFERENCE BETWEEN THE HIGH/LOW ALCOHOL COUNTERPART GROUPS Factor 1
Group of Animal
ANA AA NP P LAD1 HAD1 LAD2 HAD2 LOW_IPH HIGH_IPH B10.A(4R) B10.AKM LD_hybrid HD_hybrid Large_Brain Small_Brain
Low or High Alcohol Group Membership
Score
L H L H L H L H L H L H L H L H
21.44 21.39 20.10 0.16 20.26 0.52 20.38 1.43 21.32 21.52 0.46 0.72 0.55 0.89 0.08 1.59
Difference Between Counterpart Groups
0.05 0.26 0.78* 1.81* 20.20 0.26 0.34 1.51*
Factor 2
Score
0.71 1.22 21.04 20.10 20.78 20.64 21.08 20.57 20.43 20.14 0.18 1.27 0.92 2.25 21.11 20.65
Difference Between Counterpart Groups
0.51* 0.94* 0.14 0.51 0.29 1.09* 1.33* 0.46
*Marks highest contrast between the factor scores for the counterpart groups.
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SALIMOV The difference in factor scores among counterpart high/ low alcohol rodents lines is higher at the first factor for the HAD1, HAD2, and Small_Brain lines, while at the second factor for the AA, P, HD_hybrid, and B10.AKM ones. The diversity corresponds to the facts that HAD1, HAD2, and Small_Brain lines are more timid during initiation of crossmaze exploration, and passive in the slip funnel than their low-alcohol counterpart lines, while AA, P, HD_hybrid, and B10.AKM demonstrate greater escape/avoidance attempts to get out of slip funnel, and a relatively lower difference in crossmaze timidity compared with their low-alcohol counterparts (43,47,50). The factor scores did not diverge much between the HIGH_IPH/LOW_IPH pair of rats. Compared with their counterpart LOW_IPH rats , the HIGH_IPHs are characterized by higher immobility in the forced-swim test (5,22,25,59), showing a trait attributed to the first factor, and relatively little difference in latency of crossmaze exploration (45,52), which seems to be a trait of the second factor. In accord to previous works (3,23,40,41,54,57), saccharin intake, time in open arms of elevated plus-maze, immobility in the forced-swim test, and defecations in novel situation were related; hence differentially, to factors revealed in the present study. Saccharin intake had a direct relation to alcohol intake, as well as to forced-swim immobility and to the first alcohol factor. There was significant negative correlation of the time in open arms of elevated plus-maze with the latency of crossmaze exploration; hence, its relation with the first alcohol factor that did not reach a significance level. Superficially, the coincidence of drive for saccharin with the first type of alcohol drive may help to explain why, when palatable liquid is offered as a third choice, a reduction in alcohol intake takes place in HAD1 rats (29), but not in P rats (30). The number of defecations in a novel situation of the crossmaze and forced swim immobility negatively correlated to the second factor, and exhibited connection to low efficient crossmaze exploration. These additionally evaluated correlations seem to confirm interpretation, given to the second factor, and suggest that attribution of “timidity and meekness” to the first factor, however, does not
ate reentries, and negatively with number of patrolling episodes in the crossmaze. Pearson correlations of the number of defecations with factor scores do not reach a significance level; hence, Spearman’s rank order correlation of the variable with the second factor was significant (r 5 20.65, n 5 12, p , 0.05). Saccharin consumption significantly correlated with alcohol intake after abstinence, with total time of slip funnel immobility and with the first factor. Time spent in open arms of the elevated plus-maze showed significant negative correlation with latency of first visit to an arm in the crossmaze. Immobility in the forced-swim test exhibited significant positive correlation with number of crossmaze stereotyped visits, and negative with the number of crossmaze patrolling episodes and total time of escape attempts out of slip funnel. The Pearson correlation of forced-swim immobility with the factors did not reach a significance level; however, Spearman’s rank order correlation of the variable with the second factor was significant (r 5 20.67, n 5 9, p , 0.05). DISCUSSION
A major finding in the present study is the existence of two factors explaining 71.8% of the variability among behavioral measures of alcohol drinking, crossmaze, and slip funnel test. Interestingly, the two factors are almost alike in their relation to alcohol drinking, but are differentially linked with crossmaze variables and have an opposite relation to the slip funnel measures. As far as the first factor is related to high alcohol intake, the long latency to start exploration in a novel situation of a crossmaze, high immobility, and low avoidance during inescapable slip funnel test, it may be interpreted as an alcohol drive that coincides with initial timidity and meekness. The second factor may be interpreted as an alcohol drive coinciding with the initial novelty-seeking and persistence because the factor is related to high alcohol intake, low number of visits during first patrolling in the crossmaze (which means high explorative efficacy), high escape, and low immobility during the inescapable slip funnel test.
TABLE 3 PEARSON CORRELATION OF BEHAVIOR MEASURES FROM ALCOHOL DRINKING, EXPLORATIVE CROSSMAZE, INESCAPABLE SLIP FUNNEL TESTS, AND SCORES OF FACTOR 1 AND FACTOR 2 FROM ONE SIDE WITH DEFECATION IN CROSSMAZE, SACCHARIN (0.1%, W/V) INTAKE, TIME IN OPEN ARMS OF ELEVATED PLUS-MAZE (%), AND TIME IMMOBILE (%) IN FORCED-SWIM TEST FROM THE OTHER SIDE
Defecations in Crossmaze
Number of pairs AL_C AL_D FLAT FPAT PAT_N IRE_N SV_N FIMM FAVO FESC Factor 1 Factor 2
12 20.36 20.48 0.10 0.62* 20.66* 0.60* 0.05 0.27 20.23 20.28 20.10 20.53
Saccharin Intake
12 0.09 0.60* 0.52 20.07 20.01 20.11 0.19 0.65* 20.49 20.51 0.65* 20.29
*Marks correlations significant at p , 0.05.
Time in Open Arms of Elevated Plus-Maze
9 20.53 20.41 20.68* 0.33 20.05 20.23 0.42 20.16 0.45 20.29 20.55 20.30
Immobility in Forced Swim Test
9 20.42 20.27 20.16 0.33 20.72* 0.17 0.74* 0.39 20.04 20.67* 20.05 20.62
BEHAVIORAL PATTERNS RELATED TO ALCOHOL USE necessarily involve a state like so-called “behavioral despair” believed to happen during the forced-swim test. The distinction between two alcohol factors, found in the present study, may be compared to some differential traits reported for type 1 and 2 (as well as for types A and B) alcoholics, namely, low or high novelty seeking, high or low harm avoidance, and high or low reward dependence, respectively (1,7,8). Higher efficacy of exploration corresponds to higher novelty seeking, whereas a longer latency to initiate crossmaze exploration parallel the more prominent tendency to avoid the situation (4,7,8). Because behavioral extinction is
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thought to reflect reward dependence (7,8), higher immobility in the water at the bottom of the slip funnel, which develops after extinction of initial attempts to avoid the water, may be seen as a higher reward dependence. ACKNOWLEDGEMENTS
The author would like to express his gratitude to Ting-Kai Li, William J. McBride, James M. Murphy, John D. Sinclair, and David H. Overstreet for helpful information regarding results previously published by them.
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