Psychological Reports, 1975, 37, 683-692. @ Psychological Reports 1975 EFFECTS O F SITUATIONAL VARIABLES O N PERFORMANCE O F INBRED MICE I N ACTIVE- A N D PASSIVE-AVOIDANCE SITUATIONS1 RICHARD L. SPROTT AND KAREN STAVNES3 The Jack~onLaborato~~' Summary.-A group of experiments were conducted to assess the interaction between environmental factors and specific genetic loci upon the performance of mice i n avoidance learning situations. The performance of all mice was better i n passive-avoidance than in active-avoidance situations. The performance of C57BL/GJ mice was generally superior to that of DBA/2J mice. C57BL/6J mice usually avoided foot-shocks, while DBA/2J mice attempted to escape them. DBA/2J mice performed poorly in the presence of mild foot-shocks, while C57BL/6J mice performed poorly in the presence of intense foot-shocks. Analysis of the data suggested that the loci involved probably exerted their influence by affecting the subject's response to its environment. T h e emphasis of behavior-genetic research is currently changing. Genotype related differences in performance i n learning situations are now well documented phenomena (Fuller & Thompson, 1960; Hirsch, 1970; Spuhler, 1967; Collins, 1 9 7 0 ) . Avoidance learning situations, both active and passive, are commonly used to assess learning i n rodents (Sprott & Stavnes, 1975). While individual and strain differences in active and passive avoidance are now firmly established (Wimer, et al., 1968; Sprott, 1972 ), and i n some cases tied to single genetic loci (Oliverio, et al., 1973; Spratt, 1974; Stavnes & Sprott, 1975b), we know little about the mode of genetic influence upon performance. Genes could, and very likely do, act upon memory, motivation, sensory thresholds, and reactions to task difficulty. It is therefore appropriate that attention is shifting from demonstrations of the existence of genetic effects to studies of the nature of gene actions. W e have been using two inbred mouse strains and two tasks ( a n active and a passive task) in a n attempt to determine the manner in which specific gene alleles are affecting mouse avoidance performance. So far we have observed that in a passive-avoidance situation the performance of mature DBA/2J mice ( D 2 ) is usually superior to that of comparable C57BL/6J mice ( B 6 ) . T h e reverse relationship was observed in younger mice at certain shock levels. Both 'This investigation was supported by NIH Research Grant HD 05523 from the National Institute of Child Health and Human Development, Research Grant GM 21266 from the National Insticute of General Medical Sciences, and Training Grant MH 12126 from the National Instimte of Mental Health. 'Requests for reprints should be sent to Richard L. Sprott, The Jackson Laboratory, Bar Harbor, Maine 04609. 3Present address: Department of Psychiatry and Behavioral Sciences, Universiry of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104. T h e Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.
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age and shock level appeared to affect the performance of BG mice, while only shock level appeared to affect the performance of D2 mice in these experiments (Sprott, 1972). A subsequent genetic analysis of the performance of mature mice in this situation led to the conclusion that the strain differences in performance could be accounted for on the basis of the existence of alternative alleles at a single locus ( P p ) in the two strains. Two alleles at this locus have been demonstrated: Pp" the dominant allele associated with poor performance, and Ppd, the recessive allele associated with superior performance (Sprotc, 1974). Similar experiments in an active avoidance situation have shown that the performance of D2 mice is characterized predominantly by escape responses rather than avoidances. Increases in the intensity of the foot-shock result in an increased number of escape responses. C57BL/6J mice consistently exhibit avoidance responses in this situation, and subsequent increases in shock intensity increase the number of avoidance responses (Stavnes & Sprott, 1975a). The strain differences in performance in this situation can also be accounted for on the basis of the existence of alternative alleles at a single locus ( A * ) in the two strains. Two alleles at this locus have been demonstrated: Aapb, the recessive allele associated with superior performance, and Aapd, the dominant allele associated with inferior performance (Stavnes & Sprott, 1975b). This locus does not appear to be the same as P p described above. Transfer of training experiments which included both active- and passiveavoidance demonstrated a positive transfer effect from active- to passive-avoidance. This effect was equally large in both strains, and suggested that the observed genetic effects might exert their influence upon performance by affecting the subjects' responses to learning situations rather than by affecting learned patterns of motor responses (Sprott & Stavnes, 1974). However, several other mechanisms of influence are possible, such as sensitivity to foot-shock (a sensory threshold effect), genocypic differences in activity level, genocypic differences in motivational variables, or genotypic differences in learning ability. W e are invest~gatingenvironmental factors which interact with genotype to increase our understanding of genotype-environment interaction. Specifically, in the experiments reported here we are ( a ) manipulating shock intensity as a reflection of motivation (Wahlsten, 1972), ( b ) attempting to make motivation (shock conditions) in the passive task more like those in the accive task (intermittent condition in passive), and ( c ) attempting to equate task difficulty in passive and accive by using shelf extensions and ladders.
METHOD Subjects Six hundred and forty inbred male mice, 320 C57BL/6J (B6) and 320 DBA/2J ( D 2 ) mice, were trained to criterion or for 30 trials in either an active-
ACTIVE- AND PASSIVE-AVOIDANCE IN INBRED MICE
685
or a passive-avoidance learning task. The mice were obtained from the Production Department of The Jackson Laboratory at 6 to 8 wk. of age, 3 days prior to the first day of training. During training all animals were maintained on a 12-hr. light-dark cycle under standard laboratory conditions, housed five to a cage, with ad libitum access to Old Guilford 911-R mouse food and tap water. A radio played 12 hr. per day in the housing colony room and during training in the testing cubicle. The training apparatus used for passive-avoidance testing was identical to that described by Wimer, ct al. (1968), while the apparatus used for activeavoidance testing was modified slightly from that described by Wimer, et nl. (1968). The conditioning chamber consisted of a clear Plexiglas box (15.3 X 15.9 X 16.6 cm) with a stainless steel grid floor. The chamber could be modified for use in active- or passive-avoidance by changing the shelves mounted within it. In passive-avoidance training a removable black Plexiglas shelf (2.5 X 13.5 cm) was mounted against one wall, 2.2 cm above the grid floor. This platform could be folded up against one wall to prevent the mouse's return after it had stepped down. In active-avoidance training a clear Plexiglas shelf (3.3 cm wide), which was continuous around all 4 sides of the box, was mounted 6 cm above the grid floor. Two small "ladders" (1.5 cm wide) fashioned from %-in. hardware cloth were artached to [he shelf on opposite sides of the box. Each ladder extended down approximately 2 cm from the top of the shelf. In the shelf-extension condition, a clear Plexiglas extension was attached to the inner rim along the length of each of the 4 sides of the shelf. This extension projected down to the grid floor, prevented the mice from entering the area under the shelf before jumping up and also reduced the grid floor area. The grid floor was electrified by a Grason-Stadler (E1064GS) shock generator and scrambler. The grid floor was designed so that shorting would not occur even if large quantities of urine were present. Meylan elecuic timers were used to measure latencies and to monitor the duration of foot-shock.
Procedure One hour before training each mouse was placed in an individual, cottonlined "milk shake" carton that had airholes punched in the top. Each mouse was assigned to a particular carton which was used throughout training. A separate avoidance chamber was used to train mice of each strain. The chamber and grid were not cleaned between subjects within a group, but boli were removed after each trial and the avoidance chamber was washed with a strong detergent and laboratory glassware cleaner at the end of training for each group each day. Passive-avoidance training was conducted between 9 AM and 11: 30 AM and active-avoidance training was conducted between 12:30 PM and 3: 30 PM.
686
R. L. SPROTT a K. STAVNES
Passive-avoidance procedure.-At the beginning of each trial a mouse was removed from its carton and placed gently on the chamber shelf. As soon as the mouse stepped down, placing all 4 feet on the grid floor, foot-shock was administered. Simultaneously, the shelf was rotated up against one wall of the chamber to prevent the mouse from escaping the shock by returning to the shelf. An avoidance response occurred when the mouse remained on the shelf for 60 sec. without stepping down to the grid floor. At the end of each trial the mouse was grasped by the tail with a large forceps and returned to its carton for 2 hr. and then returned to its home cage. Each mouse was given 1 trial per day, 5 days per week, until it reached the criterion by remaining on the shelf for 60 sec. on 3 consecutive trials or for a maximum of 30 training trials. Mice were trained in the passive-avoidance task under 6 different foot-shock conditions as follows: 20 D2 and 20 B6 mice at 0.0 mA (no foot-shock), 40 D2 and 40 B6 at 0.1 mA, 40 D2 and 40 B6 mice at 1.0 mA, 40 D2 and 40 B6 at 2.0 mA, 40 D2 and 40 B6 mice at 4.0 mA, and finally 20 D2 and 20 B6 mice at 0.1 mA administered intermittently ( 1 sec. on, 1 sec. off) for 1 min. Portions of the data from 8 of these groups (0.1, 1.0, 2.0, and 4.0 mA for each strain) have appeared previously (Sprott, 1972 ) . Active-avoidance procedure.-At the beginning of each trial a mouse was removed from its carton and placed on the grid floor. The 5-sec. period immediately following placement on the grid was defined as the avoidance period and foot-shock was not administered. This avoidance period was followed by a period of foot-shock which lasted until the subject jumped up to the shelf or until 60 sec. had elapsed. The trial ended when the mouse avoided or escaped the shock by jumping to the shelf or failed to escape the shock within the 60-sec. shock period. The mouse was then grasped by the tail with a large forceps and returned to ics carton for 1 co 2 hr. and then returned to its home cage. Each mouse was given 1 trial per day, 5 days per week, until ic reached a criterion of 8 avoidance responses in 10 trials or for a maximum of 30 trials. Four different foot-shock conditions were used in active-avoidance training; 20 D2 and 20 B6 at 0.0 mA (no foot-shock), 40 D2 and 40 B6 at 0.1 rnA, 20 D2 and 20 B6 at 0.6 mA, 20 D2 and 20 B6 at 1.0 mA. In a fifth condition, 20 D2 and 20 B6 mice were trained at 1.0 mA with the shelf-extension.
RESULTS The performances of all groups (mean trials to criterion) in the active and passive situations are summarized in Table 1. All comparisons of experimental groups were based on distributions of trials to criteria and evaluated with the Kolmogorov-Smirnov two-sample test (Siegel, 1956, pp. 127-136). In the passive situation the performance of B6 mice was good under every condition except 4.0mA foot-shock. The best performance was observed when 0.1-mA foot-shock
ACTIVE. A N D PASSIVE-AVOIDANCE IN INBRED MICE
687
was administered intermittently for 1 min. (mean trials to criterion = 4.55 f 0.21) followed by 1.0 d, 2.0 mA, 0.1 mA, 0.0 mA, and 4.0 mA in that order. Performance in the no-shock condition (0.0 mA) was inferior to that at 0.1 mA (D = ,375, crit. D = ,372, p < .O5). The performance of D2 mice in the passive situation was best at 1.0 mA (mean trials to criterion 4.85 +- 0.36) followed by 0.1 mA-intermittent, 2.0 mA: 4.0 mA, 0.0 mA, and 0.1 mA in that order. D2 performance at 0.1 mA-intermittent was significantly better than that at 0.1 mA (D = 0.675, crit. D = 0.372, p < .01), significantly poorer than . O 5 ) , and not signifperformance at 1.0 mA ( D= 0.375, crit. D = 0.372, p icantly different from performance at 2.0 mA.
684
R. L. SPROTT & K. STAVNES
age and shock level appeared to affect the performance of BG mice, while only shock level appeared to affect the performance of D2 mice in these experiments (Sprott, 1972). A subsequent genetic analysis of the performance of mature mice in this situation led to the conclusion that the strain differences in performance could be accounted for on the basis of the existence of alternative alleles at a single locus ( P p ) in the two strains. Two alleles at this locus have been demonstrated: Pp" the dominant allele associated with poor performance, and Ppd, the recessive allele associated with superior performance (Sprotc, 1974). Similar experiments in an active avoidance situation have shown that the performance of D2 mice is characterized predominantly by escape responses rather than avoidances. Increases in the intensity of the foot-shock result in an increased number of escape responses. C57BL/6J mice consistently exhibit avoidance responses in this situation, and subsequent increases in shock intensity increase the number of avoidance responses (Stavnes & Sprott, 1975a). The strain differences in performance in this situation can also be accounted for on the basis of the existence of alternative alleles at a single locus ( A * ) in the two strains. Two alleles at this locus have been demonstrated: Aapb, the recessive allele associated with superior performance, and Aapd, the dominant allele associated with inferior performance (Stavnes & Sprott, 1975b). This locus does not appear to be the same as P p described above. Transfer of training experiments which included both active- and passiveavoidance demonstrated a positive transfer effect from active- to passive-avoidance. This effect was equally large in both strains, and suggested that the observed genetic effects might exert their influence upon performance by affecting the subjects' responses to learning situations rather than by affecting learned patterns of motor responses (Sprott & Stavnes, 1974). However, several other mechanisms of influence are possible, such as sensitivity to foot-shock (a sensory threshold effect), genocypic differences in activity level, genocypic differences in motivational variables, or genotypic differences in learning ability. W e are invest~gatingenvironmental factors which interact with genotype to increase our understanding of genotype-environment interaction. Specifically, in the experiments reported here we are ( a ) manipulating shock intensity as a reflection of motivation (Wahlsten, 1972), ( b ) attempting to make motivation (shock conditions) in the passive task more like those in the accive task (intermittent condition in passive), and ( c ) attempting to equate task difficulty in passive and accive by using shelf extensions and ladders.
METHOD Subjects Six hundred and forty inbred male mice, 320 C57BL/6J (B6) and 320 DBA/2J ( D 2 ) mice, were trained to criterion or for 30 trials in either an active-
ACTIVE- AND PASSIVE-AVOIDANCE IN INBRED MICE
685
or a passive-avoidance learning task. The mice were obtained from the Production Department of The Jackson Laboratory at 6 to 8 wk. of age, 3 days prior to the first day of training. During training all animals were maintained on a 12-hr. light-dark cycle under standard laboratory conditions, housed five to a cage, with ad libitum access to Old Guilford 911-R mouse food and tap water. A radio played 12 hr. per day in the housing colony room and during training in the testing cubicle. The training apparatus used for passive-avoidance testing was identical to that described by Wimer, ct al. (1968), while the apparatus used for activeavoidance testing was modified slightly from that described by Wimer, et nl. (1968). The conditioning chamber consisted of a clear Plexiglas box (15.3 X 15.9 X 16.6 cm) with a stainless steel grid floor. The chamber could be modified for use in active- or passive-avoidance by changing the shelves mounted within it. In passive-avoidance training a removable black Plexiglas shelf (2.5 X 13.5 cm) was mounted against one wall, 2.2 cm above the grid floor. This platform could be folded up against one wall to prevent the mouse's return after it had stepped down. In active-avoidance training a clear Plexiglas shelf (3.3 cm wide), which was continuous around all 4 sides of the box, was mounted 6 cm above the grid floor. Two small "ladders" (1.5 cm wide) fashioned from %-in. hardware cloth were artached to [he shelf on opposite sides of the box. Each ladder extended down approximately 2 cm from the top of the shelf. In the shelf-extension condition, a clear Plexiglas extension was attached to the inner rim along the length of each of the 4 sides of the shelf. This extension projected down to the grid floor, prevented the mice from entering the area under the shelf before jumping up and also reduced the grid floor area. The grid floor was electrified by a Grason-Stadler (E1064GS) shock generator and scrambler. The grid floor was designed so that shorting would not occur even if large quantities of urine were present. Meylan elecuic timers were used to measure latencies and to monitor the duration of foot-shock.
Procedure One hour before training each mouse was placed in an individual, cottonlined "milk shake" carton that had airholes punched in the top. Each mouse was assigned to a particular carton which was used throughout training. A separate avoidance chamber was used to train mice of each strain. The chamber and grid were not cleaned between subjects within a group, but boli were removed after each trial and the avoidance chamber was washed with a strong detergent and laboratory glassware cleaner at the end of training for each group each day. Passive-avoidance training was conducted between 9 AM and 11: 30 AM and active-avoidance training was conducted between 12:30 PM and 3: 30 PM.
686
R. L. SPROTT a K. STAVNES
Passive-avoidance procedure.-At the beginning of each trial a mouse was removed from its carton and placed gently on the chamber shelf. As soon as the mouse stepped down, placing all 4 feet on the grid floor, foot-shock was administered. Simultaneously, the shelf was rotated up against one wall of the chamber to prevent the mouse from escaping the shock by returning to the shelf. An avoidance response occurred when the mouse remained on the shelf for 60 sec. without stepping down to the grid floor. At the end of each trial the mouse was grasped by the tail with a large forceps and returned to its carton for 2 hr. and then returned to its home cage. Each mouse was given 1 trial per day, 5 days per week, until it reached the criterion by remaining on the shelf for 60 sec. on 3 consecutive trials or for a maximum of 30 training trials. Mice were trained in the passive-avoidance task under 6 different foot-shock conditions as follows: 20 D2 and 20 B6 mice at 0.0 mA (no foot-shock), 40 D2 and 40 B6 at 0.1 mA, 40 D2 and 40 B6 mice at 1.0 mA, 40 D2 and 40 B6 at 2.0 mA, 40 D2 and 40 B6 mice at 4.0 mA, and finally 20 D2 and 20 B6 mice at 0.1 mA administered intermittently ( 1 sec. on, 1 sec. off) for 1 min. Portions of the data from 8 of these groups (0.1, 1.0, 2.0, and 4.0 mA for each strain) have appeared previously (Sprott, 1972 ) . Active-avoidance procedure.-At the beginning of each trial a mouse was removed from its carton and placed on the grid floor. The 5-sec. period immediately following placement on the grid was defined as the avoidance period and foot-shock was not administered. This avoidance period was followed by a period of foot-shock which lasted until the subject jumped up to the shelf or until 60 sec. had elapsed. The trial ended when the mouse avoided or escaped the shock by jumping to the shelf or failed to escape the shock within the 60-sec. shock period. The mouse was then grasped by the tail with a large forceps and returned to ics carton for 1 co 2 hr. and then returned to its home cage. Each mouse was given 1 trial per day, 5 days per week, until ic reached a criterion of 8 avoidance responses in 10 trials or for a maximum of 30 trials. Four different foot-shock conditions were used in active-avoidance training; 20 D2 and 20 B6 at 0.0 mA (no foot-shock), 40 D2 and 40 B6 at 0.1 rnA, 20 D2 and 20 B6 at 0.6 mA, 20 D2 and 20 B6 at 1.0 mA. In a fifth condition, 20 D2 and 20 B6 mice were trained at 1.0 mA with the shelf-extension.
RESULTS The performances of all groups (mean trials to criterion) in the active and passive situations are summarized in Table 1. All comparisons of experimental groups were based on distributions of trials to criteria and evaluated with the Kolmogorov-Smirnov two-sample test (Siegel, 1956, pp. 127-136). In the passive situation the performance of B6 mice was good under every condition except 4.0mA foot-shock. The best performance was observed when 0.1-mA foot-shock
ACTIVE. A N D PASSIVE-AVOIDANCE IN INBRED MICE
687
was administered intermittently for 1 min. (mean trials to criterion = 4.55 f 0.21) followed by 1.0 d, 2.0 mA, 0.1 mA, 0.0 mA, and 4.0 mA in that order. Performance in the no-shock condition (0.0 mA) was inferior to that at 0.1 mA (D = ,375, crit. D = ,372, p < .O5). The performance of D2 mice in the passive situation was best at 1.0 mA (mean trials to criterion 4.85 +- 0.36) followed by 0.1 mA-intermittent, 2.0 mA: 4.0 mA, 0.0 mA, and 0.1 mA in that order. D2 performance at 0.1 mA-intermittent was significantly better than that at 0.1 mA (D = 0.675, crit. D = 0.372, p < .01), significantly poorer than . O 5 ) , and not signifperformance at 1.0 mA ( D= 0.375, crit. D = 0.372, p icantly different from performance at 2.0 mA.
