Neurotoxicologyand Teratology, Vol. 13, pp. 129-133. ©PergamonPress plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
Activity in a Modified Open-Field Apparatus: Effect of Diazepam and Prenatal Stress L. A. P O H O R E C K Y
A N D P. R O B E R T S
Center o f Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969 R e c e i v e d 4 July 1988
POHORECKY, L. A. AND P. ROBERTS. Activity in a modified open-field apparatus: Effect of diazepam and prenatal stress. NEUROTOXICOL TERATOL 13(2) 129-133, 1991.--We examined the effect of prenatal stress exposure on sensitivity to diazepam. The stress exposure consisted of handling pregnant rats 5 minutes dally from the 14th to 21st day of gestation. Male offspring were tested at 60 days of age in a modified open-field apparatus 30 minutes after injection with diazepam (0, 1, 5 mg/kg). The 5 mg/kg dose of diazepam depressed the frequency and duration of crossover, rearing, headpoke and comer activities. Rearing was not affected by the 1 mg/kg dose of diazepam. Defecation was increased by the 1 mg/kg dose, but was decreased by 5 mg/kg of diazepam. Prenatal stress exposure altered responsiveness to diazepam on only crossover activity and on defecation. Prenatally stressed animals exhibited an increase in crossover at the low dose of diazepam, while control offspring were insensitive to this dose level. Also, there were more boli in prenatally stressed rats treated with 1 mg/kg diazepam, while the 5 mg/kg dose of the drug decreased the number of boli. The other measures showed a trend in the same direction but the differences were not statistically significant. Thus our results indicate that prenatal stress has very specific effects on the sensitivity of adult offspring to diazepam. Prenatal stress
Diazepam
Open-field activity
Defecation
over 5-10 mg/kg (6). Activity in the open field is a sensitive behavioral measure which has been shown to vary as a function of the subject's age and to be influenced by such factors as the age of weaning, degree of handling of the subjects, etc. (10,16). This measure has also been found to be influenced by prenatal drug treatment. For instance prenatal treatment with diazepam from days 4-20 of gestation did not alter the effect of an acute dose of diazepam on measures of learning in the offspring (11). In the open field, however, these offspring showed decreased incidence of rearing activity and no effect on crossover activity. We felt, therefore, that open-field activity would be sensitive to the long-term effect of prenatal stress exposure.
IT is well known that the response of an organism to a drug can be modified by a variety of factors. Some of these are subject-related factors (e.g., genetics, age) while others are not (e.g., time of day, exposure to other drugs, diet/food ingestion). The range of individual variability encountered in the response to drugs can mostly be accounted for by such factors. Stress is one of the exogenous factors that has profound effects on the homeostasis of an organism; it has therefore received considerable attention during the past few years. Acute stress has been shown to modify the response to some drugs (9, 13, 14, 25). This is not surprising in view of the extensive neurochemical and neuroendocrine changes produced by stress (12, 24, 26). Two issues concerning the effect of stress on drug sensitivity are not well understood, however. One of these is the long-term effect of stress. That is, will stress exposure alter the responsiveness of an organism long after termination of the stressor? The other issue is, what are the consequences of stress exposure during the developmental period? This report focuses on the latter issue. Namely, we have examined the effect of prenatal stress exposure on the response of rodents to diazepam, a widely used drug. Diazepam has been shown to depress a variety of behaviors in rodents. For example, doses up to 3 mg/kg diazepam suppressed defensive burying behavior in rats (27), saccharin-induced drinking (5), fear-enhanced startle response (3), and rearing activity in a modified open field tailored to evaluate the anxiolytic properties of drugs (4). Diazepam has also been found to have a biphasic effect on locomotor and exploratory behavior. In mice tested in a two-compartment chamber (one brightly illuminated and the other dark), diazepam increased locomotor and exploratory behaviors at low to moderate doses and decreased them with doses
METHOD
Subjects Male and female Sprague Dawley rats were purchased from Hilltop Laboratories (Scottdale, PA). Animals were housed individually in stainless steel cages in a vivarium which was temperature (21°C) and light (on 9:30 p.m. to 9:30 a.m.) controlled. Food (Purina Rat Chow) and water were available ad lib. Animals were mated one week after their arrival. Paper cage liners were checked dally and presence of a vaginal plug was designated as day 1 of gestation. From days 14 to 21 of gestation, the prenatally stressed animals were handled for a period of 5 minutes daily by making them walk repeatedly through an openended wire mesh cylinder 20 cm long and 7 cm wide. After birth all pups were cross-fostered to nonstressed females giving birth within plus or minus one day. All litters were culled to 10 pups. Pups were not handled until weaning at the age of 21 days, at
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which time they were individually caged. Male offspring were tested in the open field when they were 60 days of age, at 30 minutes after injection, after random assignment to the various treatment groups. A total of 5 randomly selected litters was assigned to each treatment group. Each litter consisted of 3--4 subjects. Randomly selected representative offspring from each litter were used for testing. Diazepam (dissolved in peanut oil) was administered by intraperitoneal injection (IP). Control animals were injected with an equivalent volume of the vehicle.
Open-Field Testing The modified open-field apparatus was 40.0 cm long by 31.5 cm wide and 46.0 cm high. Its floor was divided into four equal squares each containing four equidistant holes 3.0 cm in diameter. The removable floor of the open field consisted of a plastic tray which was elevated 7.0 cm above the ground. Under the floor was a small lit bulb. There was no overhead illumination. Testing was carried out between 10:00 a.m. and 1:00 p.m. Behavioral activity (headpokes, rearing, crossing, and comer activity) were recorded using an automated system. A headpoke was counted when the animal dipped his head into one of the holes past the level of the eyes. Rearing was counted when the animal assumed an upward posture standing on only his back legs. Crossover was counted when the animal crossed from one square to another with all 4 legs. Comer activity consisted of the animal poking or nosing one of the comers. Gnawing activity was counted when the animal gnawed at the edges of the holes in the floor. The automated recording system consisted of an IBM-XT personal computer equipped with a digital interface. The interface contained 4 buttons each assigned to a particular behavior (crossing, rearing, headpoke and comer). A program was written to monitor the latency to the first press for each key as well as the duration and frequency each key was pressed for during the test period. In addition, the program performed a time block analysis so that the development and maintenance of each behavior could be assessed. Animals were placed in the apparatus for a period of 15 minutes during which time their activity was recorded for a one-minute period every 5 minutes. At the end of the open-field test, the removable floor tray of the apparatus was carefully removed and the presence of urine and fecal boli was recorded. All the scoring was carried out by one observer present in the room during testing. The observer was blind as to the treatment of the subjects.
Data Analysis Data analysis was carried out not on individual subjects but rather on means for litters. There were three to four subjects per litter, and 5 litters per experimental group. Results are presented as the mean-+ standard error of the mean. Data were analyzed using an appropriate analysis of variance (stress treatment × drug dose × time) using a computerized package. Linear trend analysis and post hoc Newman Keuls tests were also carried out. Differences between means are considered statistically significant when p--
0892-0362/91 $3.00 + .00
Activity in a Modified Open-Field Apparatus: Effect of Diazepam and Prenatal Stress L. A. P O H O R E C K Y
A N D P. R O B E R T S
Center o f Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969 R e c e i v e d 4 July 1988
POHORECKY, L. A. AND P. ROBERTS. Activity in a modified open-field apparatus: Effect of diazepam and prenatal stress. NEUROTOXICOL TERATOL 13(2) 129-133, 1991.--We examined the effect of prenatal stress exposure on sensitivity to diazepam. The stress exposure consisted of handling pregnant rats 5 minutes dally from the 14th to 21st day of gestation. Male offspring were tested at 60 days of age in a modified open-field apparatus 30 minutes after injection with diazepam (0, 1, 5 mg/kg). The 5 mg/kg dose of diazepam depressed the frequency and duration of crossover, rearing, headpoke and comer activities. Rearing was not affected by the 1 mg/kg dose of diazepam. Defecation was increased by the 1 mg/kg dose, but was decreased by 5 mg/kg of diazepam. Prenatal stress exposure altered responsiveness to diazepam on only crossover activity and on defecation. Prenatally stressed animals exhibited an increase in crossover at the low dose of diazepam, while control offspring were insensitive to this dose level. Also, there were more boli in prenatally stressed rats treated with 1 mg/kg diazepam, while the 5 mg/kg dose of the drug decreased the number of boli. The other measures showed a trend in the same direction but the differences were not statistically significant. Thus our results indicate that prenatal stress has very specific effects on the sensitivity of adult offspring to diazepam. Prenatal stress
Diazepam
Open-field activity
Defecation
over 5-10 mg/kg (6). Activity in the open field is a sensitive behavioral measure which has been shown to vary as a function of the subject's age and to be influenced by such factors as the age of weaning, degree of handling of the subjects, etc. (10,16). This measure has also been found to be influenced by prenatal drug treatment. For instance prenatal treatment with diazepam from days 4-20 of gestation did not alter the effect of an acute dose of diazepam on measures of learning in the offspring (11). In the open field, however, these offspring showed decreased incidence of rearing activity and no effect on crossover activity. We felt, therefore, that open-field activity would be sensitive to the long-term effect of prenatal stress exposure.
IT is well known that the response of an organism to a drug can be modified by a variety of factors. Some of these are subject-related factors (e.g., genetics, age) while others are not (e.g., time of day, exposure to other drugs, diet/food ingestion). The range of individual variability encountered in the response to drugs can mostly be accounted for by such factors. Stress is one of the exogenous factors that has profound effects on the homeostasis of an organism; it has therefore received considerable attention during the past few years. Acute stress has been shown to modify the response to some drugs (9, 13, 14, 25). This is not surprising in view of the extensive neurochemical and neuroendocrine changes produced by stress (12, 24, 26). Two issues concerning the effect of stress on drug sensitivity are not well understood, however. One of these is the long-term effect of stress. That is, will stress exposure alter the responsiveness of an organism long after termination of the stressor? The other issue is, what are the consequences of stress exposure during the developmental period? This report focuses on the latter issue. Namely, we have examined the effect of prenatal stress exposure on the response of rodents to diazepam, a widely used drug. Diazepam has been shown to depress a variety of behaviors in rodents. For example, doses up to 3 mg/kg diazepam suppressed defensive burying behavior in rats (27), saccharin-induced drinking (5), fear-enhanced startle response (3), and rearing activity in a modified open field tailored to evaluate the anxiolytic properties of drugs (4). Diazepam has also been found to have a biphasic effect on locomotor and exploratory behavior. In mice tested in a two-compartment chamber (one brightly illuminated and the other dark), diazepam increased locomotor and exploratory behaviors at low to moderate doses and decreased them with doses
METHOD
Subjects Male and female Sprague Dawley rats were purchased from Hilltop Laboratories (Scottdale, PA). Animals were housed individually in stainless steel cages in a vivarium which was temperature (21°C) and light (on 9:30 p.m. to 9:30 a.m.) controlled. Food (Purina Rat Chow) and water were available ad lib. Animals were mated one week after their arrival. Paper cage liners were checked dally and presence of a vaginal plug was designated as day 1 of gestation. From days 14 to 21 of gestation, the prenatally stressed animals were handled for a period of 5 minutes daily by making them walk repeatedly through an openended wire mesh cylinder 20 cm long and 7 cm wide. After birth all pups were cross-fostered to nonstressed females giving birth within plus or minus one day. All litters were culled to 10 pups. Pups were not handled until weaning at the age of 21 days, at
129
130
POHORECKY AND ROBERTS
which time they were individually caged. Male offspring were tested in the open field when they were 60 days of age, at 30 minutes after injection, after random assignment to the various treatment groups. A total of 5 randomly selected litters was assigned to each treatment group. Each litter consisted of 3--4 subjects. Randomly selected representative offspring from each litter were used for testing. Diazepam (dissolved in peanut oil) was administered by intraperitoneal injection (IP). Control animals were injected with an equivalent volume of the vehicle.
Open-Field Testing The modified open-field apparatus was 40.0 cm long by 31.5 cm wide and 46.0 cm high. Its floor was divided into four equal squares each containing four equidistant holes 3.0 cm in diameter. The removable floor of the open field consisted of a plastic tray which was elevated 7.0 cm above the ground. Under the floor was a small lit bulb. There was no overhead illumination. Testing was carried out between 10:00 a.m. and 1:00 p.m. Behavioral activity (headpokes, rearing, crossing, and comer activity) were recorded using an automated system. A headpoke was counted when the animal dipped his head into one of the holes past the level of the eyes. Rearing was counted when the animal assumed an upward posture standing on only his back legs. Crossover was counted when the animal crossed from one square to another with all 4 legs. Comer activity consisted of the animal poking or nosing one of the comers. Gnawing activity was counted when the animal gnawed at the edges of the holes in the floor. The automated recording system consisted of an IBM-XT personal computer equipped with a digital interface. The interface contained 4 buttons each assigned to a particular behavior (crossing, rearing, headpoke and comer). A program was written to monitor the latency to the first press for each key as well as the duration and frequency each key was pressed for during the test period. In addition, the program performed a time block analysis so that the development and maintenance of each behavior could be assessed. Animals were placed in the apparatus for a period of 15 minutes during which time their activity was recorded for a one-minute period every 5 minutes. At the end of the open-field test, the removable floor tray of the apparatus was carefully removed and the presence of urine and fecal boli was recorded. All the scoring was carried out by one observer present in the room during testing. The observer was blind as to the treatment of the subjects.
Data Analysis Data analysis was carried out not on individual subjects but rather on means for litters. There were three to four subjects per litter, and 5 litters per experimental group. Results are presented as the mean-+ standard error of the mean. Data were analyzed using an appropriate analysis of variance (stress treatment × drug dose × time) using a computerized package. Linear trend analysis and post hoc Newman Keuls tests were also carried out. Differences between means are considered statistically significant when p--
