Physiology & Behavior, Vol. 47, pp. 735-741. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0031-9384/90 $3.00 + .00
Neonatal Chronic Stress Induces Subsensitivity to Chronic Stress in Adult Rats. I. Effects on Forced Swim Behavior and Endocrine Responses 1 A. S. GONZ,~LEZ, 2 E. L. R O D R I G U E Z E C H A N D I A , R. C A B R E R A , M . R. F O S C O L O A N D L. N. F R A C C H I A
Laboratorio de Investigaciones Cerebrales ( L I N C E ) , F a c u l t a d de Ciencias M ( d i c a s Universidad N a c i o n a l de Cuyo, 5500 Mendoza, Argentina
R e c e i v e d 7 F e b r u a r y 1989
GONZ.~EZ, A. S., E. L. RODR.[GUEZ ECHAND~A, R. CABRERA, M. R. FOSCOLO AND L. N. FRACCHIA. Neonatal chronic stress induces subsensitivity to chronic stress in adult rats. 1. Effects on forced swim behavior and endocrine responses. PHYSIOL BEHAV 47(4) 735-741, 1990.--An influence of early stimulation on sensitivity to acute stress in adulthood has been reported. The purpose of the present work was to determine the effect of exposure of male and female rats to three models of chronic stress (unpredictable stress, cold stress and handling) from day 2 to day 15 of life on behavioral and endocrine sensitivity to chronic stresses in adulthood. The chronic stresses applied in adulthood were a model of intermittent cold stress (daily 30-min sessions at -20°C for 15 days) and the Katz's model of unpredictable chronic stress (15 days). Forced swim behavior and serum concentration of the stress-sensitive hormones, corticosterone and prolactin, were chosen to investigate stress sensitivity. It was found that all neonatal treatments stimulated body weight gain, did not cause infant mortality and did not affect forced swim behavior as adult. The repetitive exposure to cold stress in adulthood did not cause major impairment of forced swim behavior and did not affect basal levels of serum corticosterone and prolactin in either control or experimental rats. These findings support the view that repeated stressors can induce behavioral and endocrine adaptation in rats. The neonatal treatments did not affect this characteristic. The exposure of control rats to the unpredictable stress model severely impaired forced swim behavior and increased basal levels of serum corticosterone and prolactin. This observation conforms to the view that standard laboratory rats cannot adapt to unpredictable chronic stress. This has been reported to cause a behavioral depression syndrome comprising forced swim deficit and endocrine alterations. The exposure of rats to the neonatal stress models provided protection to these behavioral and endocrine effects of the unpredictable chronic stress in adulthood. Though minor between-sex and between-treatment differences were apparent, all neonatal treatments were effective. The results suggest that mild neonatal stresses might increase resistance of rats to some behavioral and endocrine effects of chronic aleatory stress in adulthood. Neonatal stress
Chronic stress
Behavioral depression
Prolactin
Corticosterone
Rats
Denenberg (5-11) and Levine (29) showed that both neonatal handling and daily exposure to footshock sessions induce hyperactivity and hypoemotionality in rats and mice. Early handling was shown to enhance the growth and development of the corpus callosum (12), to induce lateralized brain and can reduce the rate of mouse-killing behavior as adults (14). The first two postnatal weeks are a critical period for maturation of the neurotransmitter systems in rats (4, 17-19, 30). An influence of manipulation during the first weeks of life on sensitivity to acute stress in adulthood has been reported. Pfeifer et al. (33) showed that neonatal handling results in a lower corticosterone response as adults to footshocks and heat stress than
THE neural mechanisms involved in development of behavior are regulated by genetic and environmental interactions. The early sensory experience, therefore, can affect neural development and adult behavior [see (16,31)]. In general, neonatal manipulations have been found to give rise to a more specialized brain, as well as to enhance some physiological and behavioral characteristics in adulthood. Denenberg and Morton were the first to report that rats maintained in a free environment during infancy are more active in open field trials than rats reared in standard laboratory cages (5,7). Among the external environmental influences that can modify neural development are the various types of stressful stimuli experienced by the developing organism. The pioneering work of
XSupportod by a grant from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET, PID 3-053500/85). 2Requests for reprints should be addressed to Dr. A. S. Gonz~lez, LINCE, Casilla correo 425, 5500 Mendoza, Argentina.
735
GONZALEZ ET AL.
736
controls. It is likely, therefore, that neonatal manipulation might also cause long-lasting effects on sensitivity and adaptation to chronic stress. The purpose of the present experiments was to determine the influence of early exposure to mild chronic stresses on behavioral and endocrine adaptation to unpredictable and repetitive chronic stress in adulthood. In this work, the Katz's model of chronic aleatory stress (20,21) and two models of repetitive stresses (intermittent cold stress and handling) were daily applied to male and female pups from day 2 to day 15 of age. The influence of these neonatal treatments on sensitivity to the Katz's model of chronic aleatory stress and a model of chronic cold stress as adults was analyzed through forced swim behavior and the response of the stress-sensitive hormones, corticosterone and prolactin. METHOD
Subjects
TABLE 1 CHRONIC ALEATORY STRESS TREATMENTS
Day
Neonatal Stresses At day 2 of age the litters were divided in 4 groups and weighed before starting the exposure to 3 models of mild chronic stress for 14 consecutive days. Neonatal handling stress (neohandling). This group (males n = 2 9 ; females n = 2 6 ) was submitted to a daily session of separations of the dams, handling and body contact to a plastic surface during 5-10 min from day 2 to day 15 of age. Neonatal cold stress (neocold). This group (males n = 3 3 ; females n = 42) was daily exposed to a session of cold stress. The stress started with a 10-min exposure to a cold surface (15°C) from day 2 to 6, followed by a 5-min session at - 20°C (days 7-12) and a 10-min session at the same temperature from day 13 to 15. Neonatal aleatory stress (neoaleatory). This group (males n = 34; females n = 4 0 ) was daily exposed (day 2 up to day 15) to a variety of unpredictable mild physical stressors (see Table 1). Neonatal control group (neocontrol). This group (males n = 32; females n = 3 3 ) was kept undisturbed between birth and weaning, except for house cleaning and food-water provision. All groups were weaned at day 21 of age, weighed, divided by sex, reared in groups of 5 and maintained undisturbed under standard conditions up to day 65 of age.
Stress Treatments in Adulthood At day 65 of age the animals were weighed and each group was divided into 3 subgroups. These were assigned to a) the chronic aleatory stress model (CAS) according to Katz (20,21) over a 15-day period throughout the lighting cycle (Table 1 illustrates the schedule of the stress administered); b) chronic cold stress (CCS): daily 30-min session at - 20°C during 15 days; or c) maintained without any stressful exposure for 15 days. The subgroups obtained and the number of animals in each subgroup are summarized in Table 2.
Adult Aleatory Stress Treatment (Day 65 to Day 80 of Age)
1
Exposure to a cold surface (15°C), 10 min
Restraint stress, 5 min
2
Handling, 10 min
Cold stress, - 20°C, 15 min
3
Heat stress, 45°C, 10 min
Shaker stress (30-min horizontal high speed shakes)
4 5
Hanging by the tail, 5 min
Individual housing, 24 hr
Restraint stress, 5 min
Individual housing, 24 hr
Cage blows, 3 exposures to 3 blows in 3 min
Food deprivation, 24 hr
Footshocks avg. 1 mA 0.5 sec duration, 3 shocks, 4 min
Food deprivation, 24 hr
Heat stress, 45°C, 5 min
Group housing in a cage devoid of wooden shavings, 24 hr
Shake stress (10-min horizontal high speed shakes)
Footshocks avg. 1 mA, 1 sec duration, 3 shocks/min, 3 min
10
White noise, 95 dB, 3 exposures of 30 sec in 5 min
Tail pinch, 5 min
11
Novel cage, 24 hr
Water deprivation, 24 hr
12
Tail pinch, 5 min
Water deprivation, 24 hr
13
Mother deprivation, 6 hr
Increased house density (10 rats/cage), 24 hr
14
Individual housing, 6 hr
Heat stress, 40°C, 15 min
6 Female rats (Holtzman derived strain, 5 months of age) were mated and housed in groups of 4 in stainless steel cages (40 × 27 × 20 cm), provided with white poplar shavings and maintained under controlled temperature (22 +- 2°C) and lighting (light on from 0700 to 1900 hr), with rodent food pellets (Cargill) and water ad lib. Pregnancy was ascertained by the presence of spermatozoa in the vaginal smears. One or two days before parturition rats were individually housed in cages similar to the already described ones. One day after delivery the litters were normalized to 4 males and 4 female pups.
Neoaleatory Stress Treatment (Day 2 to Day 15 of Age)
7
8
9
15
Cage blows, 3 exposures to 3 blows in 3 min
Forced Swim Test Forced swim tests (38) were performed 24 hr after the last stress session (80 days of age). The test room was illuminated by two 40-W fluorescent lights placed 2.5 m above the testing apparatus. All animals were submitted to this test for the first time. Rats were individually introduced into a transparent acrylic cylinder (44 cm height, 16 cm diameter) filled with tap water (22°C) up to 19 cm for a 10-min trial. The following behavioral components were scored in 1-min time intervals: 1) frequency of jumps exceeding 5 cm over the water surface; 2) immobility duration (sec spent floating); and 3) frequency of immersions.
Prolactin and Corticosterone Assay It was found previously that the effects of CAS on serum corticosterone and prolactin (37) are maintained 48 hr after interruption of the stress (unpublished results). To avoid probable effects of the forced swim test itself, the hormone assays were performed at day 2 after testing, i.e., day 3 after the CCS or the CAS treatments were completed. Subjects from each subgroup of
NEONATAL STRESS
737
Murphy (32) (sensitivity: 5 ixg/ml; interassay variability: 7%).
TABLE 2 EXPERIMENTAL DESIGN
Statistics Groups
Sex
Subgroups
Males (n=34)
Neoaleatory + CAS (n= 11) Neoaleatory + CCS (n= 13) Neoaleatory (n = 10)
Females (n=40)
Neoaleatory + CAS (n= 13) Neoaleatory + CCS (n= 15) Neoaleatory (n = 12)
Males (n= 33)
Neocold Neocold Neocold
+ CAS (n = 11) + CCS (n = 8) (n = 14)
Females (n = 42)
Neocold Neocold Neocold
+ CAS (n= 13) + CCS (n = 13) (n = 16)
Males (n = 29)
Neohandling + CAS (n = 9) Neohandling + CCS (n = 12) Neohandling (n= 8)
Females (n = 26)
Neohandling + CAS (n = 11) Neohandling + CCS (n = 9) Neohandling (n= 6)
Males (n = 32)
Neocontrol + CAS (n = 13) Neocontrol + CCS (n = 8) Neocontrol (n = 11)
Females (n=33)
Neocontrol + CAS (n = 10) Neocontrol + CCS (n= 13) Neocontrol (n = 10)
Neoale~o~
Neocold
Neohandling
Neocontrol
1) Forced swim test comparisons: for comparisons of subgroups responses vs. time, the A N O V A II followed by the ScheffCs tau-test for multiple comparisons was used. 2) For body weight, serum corticosterone and prolactin comparisons between-subgroups differences were submitted to ANOVA I and Duncan's new multiple range test. Results are expressed as means - SEM. Probabilities less than 0.05 were considered significant. RESULTS
General Effects of Neonatal Treatments The neonatal treatments did not cause infant mortality. The neoaleatory group showed no neonatal mortality and mortality between days 1-21 was about 5 percent in the other groups. The surviving pups were healthy and did not show any sign of suffering. Table 3 shows that the mean body weight at day 2 of life was similar in all groups. At day 21, however, between-groups differences were significant [males: F ( 3 , 6 7 ) = 2 7 . 4 9 , p < 0 . 0 0 5 ; females: F ( 3 , 7 2 ) = 20.14, p < 0 . 0 0 5 ] . This was due to the higher body weight gain found in the neohandfing, neocold and neoaleatory groups (Duncan's test: p < 0 . 0 1 , for both sexes). Betweengroups differences remained significant at day 65 of age [males: F(3,45)---15.37, p < 0 . 0 0 5 ; females: F ( 3 , 4 9 ) = 6 . 2 2 , p < 0 . 0 0 5 ] . Table 3 shows that the neohandling and the neoaleatory groups of males weighed more than neocontrols (Duncan's test: p < 0 . 0 5 ) . In females there was a tendency for higher body weight at day 65 in the neocold and neoaleatory groups, but only the scores of the neohandling group were significantly higher than neocontrols (Duncan's test: p < 0 . 0 5 ) .
Effect of Neonatal Treatments on Forced Swim Behavior in Adulthood
CAS: chronic aleatory stress; CCS: chronic cold stress.
males (n in Fig. 5) were decapitated and blood from the trunk was collected into test tubes and centrifuged. The serum was rapidly frozen to - 2 5 ° C until required for assay. Serum prolactin was determined by standard double antibody radioimmunoassay according to the instructions of the kits provided by Dr. E. F. Parlow (NIAMD, NIH, Bethesda, MD). Results are expressed in ng/ml of serum in terms of the RP-II reference preparations (sensitivity: 0.25 ng/ml; interassay variability: 14%). Corticosterone was measured by competitive protein-binding radioassay according to
Figure 1 shows that the neonatal treatments did not affect significantly the behavior in the forced swim test performed at 80 days of age. Between-groups differences in the scores of jumping and immobility duration were not found. The neocold treatment, however, impaired the immersion response in the male group (Duncan's test: p < 0 . 0 5 vs. neocontrol scores).
Influence of Neonatal Treatments on the Effect of Chronic Cold Stress (CCS) on Forced Swim Behavior in Adulthood CCS exposure caused minor alterations in forced swim behav-
TABLE 3 BODY
Sex
WEIGHT GAIN (MEAN g __. SEM)
Age
Neocontrol
Neohandling
Neocold
Neoaleatory
Males
Day 2 Day 21 Day 60
7.8 ± 0.72 24.3 --- 0.80 192.3 ± 4.59
7.6 --- 0.27 37.0 ± 0.75t 236.1 ± 11.63"
7.1 ___ 0.23 32.2 ___ 0.74t 184.6 ± 6.13
7.9 +__0.68 32.1 ___ 0.80t 227.4 + 6.77*
Females
Day 2 Day 21 Day 60
7.6 ± 0.85 24.2 ± 0.54 146.9 ± 1.64
7.2 --- 0.29 35.1 ± 0.77t 171.6 ___ 2.77*
6.9 --- 0.45 32.5 ± 0.56I 153.0 ± 3.61
7.7 ± 0.55 32.0 ± 0.82# 162.7 ± 4.39
*p
0031-9384/90 $3.00 + .00
Neonatal Chronic Stress Induces Subsensitivity to Chronic Stress in Adult Rats. I. Effects on Forced Swim Behavior and Endocrine Responses 1 A. S. GONZ,~LEZ, 2 E. L. R O D R I G U E Z E C H A N D I A , R. C A B R E R A , M . R. F O S C O L O A N D L. N. F R A C C H I A
Laboratorio de Investigaciones Cerebrales ( L I N C E ) , F a c u l t a d de Ciencias M ( d i c a s Universidad N a c i o n a l de Cuyo, 5500 Mendoza, Argentina
R e c e i v e d 7 F e b r u a r y 1989
GONZ.~EZ, A. S., E. L. RODR.[GUEZ ECHAND~A, R. CABRERA, M. R. FOSCOLO AND L. N. FRACCHIA. Neonatal chronic stress induces subsensitivity to chronic stress in adult rats. 1. Effects on forced swim behavior and endocrine responses. PHYSIOL BEHAV 47(4) 735-741, 1990.--An influence of early stimulation on sensitivity to acute stress in adulthood has been reported. The purpose of the present work was to determine the effect of exposure of male and female rats to three models of chronic stress (unpredictable stress, cold stress and handling) from day 2 to day 15 of life on behavioral and endocrine sensitivity to chronic stresses in adulthood. The chronic stresses applied in adulthood were a model of intermittent cold stress (daily 30-min sessions at -20°C for 15 days) and the Katz's model of unpredictable chronic stress (15 days). Forced swim behavior and serum concentration of the stress-sensitive hormones, corticosterone and prolactin, were chosen to investigate stress sensitivity. It was found that all neonatal treatments stimulated body weight gain, did not cause infant mortality and did not affect forced swim behavior as adult. The repetitive exposure to cold stress in adulthood did not cause major impairment of forced swim behavior and did not affect basal levels of serum corticosterone and prolactin in either control or experimental rats. These findings support the view that repeated stressors can induce behavioral and endocrine adaptation in rats. The neonatal treatments did not affect this characteristic. The exposure of control rats to the unpredictable stress model severely impaired forced swim behavior and increased basal levels of serum corticosterone and prolactin. This observation conforms to the view that standard laboratory rats cannot adapt to unpredictable chronic stress. This has been reported to cause a behavioral depression syndrome comprising forced swim deficit and endocrine alterations. The exposure of rats to the neonatal stress models provided protection to these behavioral and endocrine effects of the unpredictable chronic stress in adulthood. Though minor between-sex and between-treatment differences were apparent, all neonatal treatments were effective. The results suggest that mild neonatal stresses might increase resistance of rats to some behavioral and endocrine effects of chronic aleatory stress in adulthood. Neonatal stress
Chronic stress
Behavioral depression
Prolactin
Corticosterone
Rats
Denenberg (5-11) and Levine (29) showed that both neonatal handling and daily exposure to footshock sessions induce hyperactivity and hypoemotionality in rats and mice. Early handling was shown to enhance the growth and development of the corpus callosum (12), to induce lateralized brain and can reduce the rate of mouse-killing behavior as adults (14). The first two postnatal weeks are a critical period for maturation of the neurotransmitter systems in rats (4, 17-19, 30). An influence of manipulation during the first weeks of life on sensitivity to acute stress in adulthood has been reported. Pfeifer et al. (33) showed that neonatal handling results in a lower corticosterone response as adults to footshocks and heat stress than
THE neural mechanisms involved in development of behavior are regulated by genetic and environmental interactions. The early sensory experience, therefore, can affect neural development and adult behavior [see (16,31)]. In general, neonatal manipulations have been found to give rise to a more specialized brain, as well as to enhance some physiological and behavioral characteristics in adulthood. Denenberg and Morton were the first to report that rats maintained in a free environment during infancy are more active in open field trials than rats reared in standard laboratory cages (5,7). Among the external environmental influences that can modify neural development are the various types of stressful stimuli experienced by the developing organism. The pioneering work of
XSupportod by a grant from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET, PID 3-053500/85). 2Requests for reprints should be addressed to Dr. A. S. Gonz~lez, LINCE, Casilla correo 425, 5500 Mendoza, Argentina.
735
GONZALEZ ET AL.
736
controls. It is likely, therefore, that neonatal manipulation might also cause long-lasting effects on sensitivity and adaptation to chronic stress. The purpose of the present experiments was to determine the influence of early exposure to mild chronic stresses on behavioral and endocrine adaptation to unpredictable and repetitive chronic stress in adulthood. In this work, the Katz's model of chronic aleatory stress (20,21) and two models of repetitive stresses (intermittent cold stress and handling) were daily applied to male and female pups from day 2 to day 15 of age. The influence of these neonatal treatments on sensitivity to the Katz's model of chronic aleatory stress and a model of chronic cold stress as adults was analyzed through forced swim behavior and the response of the stress-sensitive hormones, corticosterone and prolactin. METHOD
Subjects
TABLE 1 CHRONIC ALEATORY STRESS TREATMENTS
Day
Neonatal Stresses At day 2 of age the litters were divided in 4 groups and weighed before starting the exposure to 3 models of mild chronic stress for 14 consecutive days. Neonatal handling stress (neohandling). This group (males n = 2 9 ; females n = 2 6 ) was submitted to a daily session of separations of the dams, handling and body contact to a plastic surface during 5-10 min from day 2 to day 15 of age. Neonatal cold stress (neocold). This group (males n = 3 3 ; females n = 42) was daily exposed to a session of cold stress. The stress started with a 10-min exposure to a cold surface (15°C) from day 2 to 6, followed by a 5-min session at - 20°C (days 7-12) and a 10-min session at the same temperature from day 13 to 15. Neonatal aleatory stress (neoaleatory). This group (males n = 34; females n = 4 0 ) was daily exposed (day 2 up to day 15) to a variety of unpredictable mild physical stressors (see Table 1). Neonatal control group (neocontrol). This group (males n = 32; females n = 3 3 ) was kept undisturbed between birth and weaning, except for house cleaning and food-water provision. All groups were weaned at day 21 of age, weighed, divided by sex, reared in groups of 5 and maintained undisturbed under standard conditions up to day 65 of age.
Stress Treatments in Adulthood At day 65 of age the animals were weighed and each group was divided into 3 subgroups. These were assigned to a) the chronic aleatory stress model (CAS) according to Katz (20,21) over a 15-day period throughout the lighting cycle (Table 1 illustrates the schedule of the stress administered); b) chronic cold stress (CCS): daily 30-min session at - 20°C during 15 days; or c) maintained without any stressful exposure for 15 days. The subgroups obtained and the number of animals in each subgroup are summarized in Table 2.
Adult Aleatory Stress Treatment (Day 65 to Day 80 of Age)
1
Exposure to a cold surface (15°C), 10 min
Restraint stress, 5 min
2
Handling, 10 min
Cold stress, - 20°C, 15 min
3
Heat stress, 45°C, 10 min
Shaker stress (30-min horizontal high speed shakes)
4 5
Hanging by the tail, 5 min
Individual housing, 24 hr
Restraint stress, 5 min
Individual housing, 24 hr
Cage blows, 3 exposures to 3 blows in 3 min
Food deprivation, 24 hr
Footshocks avg. 1 mA 0.5 sec duration, 3 shocks, 4 min
Food deprivation, 24 hr
Heat stress, 45°C, 5 min
Group housing in a cage devoid of wooden shavings, 24 hr
Shake stress (10-min horizontal high speed shakes)
Footshocks avg. 1 mA, 1 sec duration, 3 shocks/min, 3 min
10
White noise, 95 dB, 3 exposures of 30 sec in 5 min
Tail pinch, 5 min
11
Novel cage, 24 hr
Water deprivation, 24 hr
12
Tail pinch, 5 min
Water deprivation, 24 hr
13
Mother deprivation, 6 hr
Increased house density (10 rats/cage), 24 hr
14
Individual housing, 6 hr
Heat stress, 40°C, 15 min
6 Female rats (Holtzman derived strain, 5 months of age) were mated and housed in groups of 4 in stainless steel cages (40 × 27 × 20 cm), provided with white poplar shavings and maintained under controlled temperature (22 +- 2°C) and lighting (light on from 0700 to 1900 hr), with rodent food pellets (Cargill) and water ad lib. Pregnancy was ascertained by the presence of spermatozoa in the vaginal smears. One or two days before parturition rats were individually housed in cages similar to the already described ones. One day after delivery the litters were normalized to 4 males and 4 female pups.
Neoaleatory Stress Treatment (Day 2 to Day 15 of Age)
7
8
9
15
Cage blows, 3 exposures to 3 blows in 3 min
Forced Swim Test Forced swim tests (38) were performed 24 hr after the last stress session (80 days of age). The test room was illuminated by two 40-W fluorescent lights placed 2.5 m above the testing apparatus. All animals were submitted to this test for the first time. Rats were individually introduced into a transparent acrylic cylinder (44 cm height, 16 cm diameter) filled with tap water (22°C) up to 19 cm for a 10-min trial. The following behavioral components were scored in 1-min time intervals: 1) frequency of jumps exceeding 5 cm over the water surface; 2) immobility duration (sec spent floating); and 3) frequency of immersions.
Prolactin and Corticosterone Assay It was found previously that the effects of CAS on serum corticosterone and prolactin (37) are maintained 48 hr after interruption of the stress (unpublished results). To avoid probable effects of the forced swim test itself, the hormone assays were performed at day 2 after testing, i.e., day 3 after the CCS or the CAS treatments were completed. Subjects from each subgroup of
NEONATAL STRESS
737
Murphy (32) (sensitivity: 5 ixg/ml; interassay variability: 7%).
TABLE 2 EXPERIMENTAL DESIGN
Statistics Groups
Sex
Subgroups
Males (n=34)
Neoaleatory + CAS (n= 11) Neoaleatory + CCS (n= 13) Neoaleatory (n = 10)
Females (n=40)
Neoaleatory + CAS (n= 13) Neoaleatory + CCS (n= 15) Neoaleatory (n = 12)
Males (n= 33)
Neocold Neocold Neocold
+ CAS (n = 11) + CCS (n = 8) (n = 14)
Females (n = 42)
Neocold Neocold Neocold
+ CAS (n= 13) + CCS (n = 13) (n = 16)
Males (n = 29)
Neohandling + CAS (n = 9) Neohandling + CCS (n = 12) Neohandling (n= 8)
Females (n = 26)
Neohandling + CAS (n = 11) Neohandling + CCS (n = 9) Neohandling (n= 6)
Males (n = 32)
Neocontrol + CAS (n = 13) Neocontrol + CCS (n = 8) Neocontrol (n = 11)
Females (n=33)
Neocontrol + CAS (n = 10) Neocontrol + CCS (n= 13) Neocontrol (n = 10)
Neoale~o~
Neocold
Neohandling
Neocontrol
1) Forced swim test comparisons: for comparisons of subgroups responses vs. time, the A N O V A II followed by the ScheffCs tau-test for multiple comparisons was used. 2) For body weight, serum corticosterone and prolactin comparisons between-subgroups differences were submitted to ANOVA I and Duncan's new multiple range test. Results are expressed as means - SEM. Probabilities less than 0.05 were considered significant. RESULTS
General Effects of Neonatal Treatments The neonatal treatments did not cause infant mortality. The neoaleatory group showed no neonatal mortality and mortality between days 1-21 was about 5 percent in the other groups. The surviving pups were healthy and did not show any sign of suffering. Table 3 shows that the mean body weight at day 2 of life was similar in all groups. At day 21, however, between-groups differences were significant [males: F ( 3 , 6 7 ) = 2 7 . 4 9 , p < 0 . 0 0 5 ; females: F ( 3 , 7 2 ) = 20.14, p < 0 . 0 0 5 ] . This was due to the higher body weight gain found in the neohandfing, neocold and neoaleatory groups (Duncan's test: p < 0 . 0 1 , for both sexes). Betweengroups differences remained significant at day 65 of age [males: F(3,45)---15.37, p < 0 . 0 0 5 ; females: F ( 3 , 4 9 ) = 6 . 2 2 , p < 0 . 0 0 5 ] . Table 3 shows that the neohandling and the neoaleatory groups of males weighed more than neocontrols (Duncan's test: p < 0 . 0 5 ) . In females there was a tendency for higher body weight at day 65 in the neocold and neoaleatory groups, but only the scores of the neohandling group were significantly higher than neocontrols (Duncan's test: p < 0 . 0 5 ) .
Effect of Neonatal Treatments on Forced Swim Behavior in Adulthood
CAS: chronic aleatory stress; CCS: chronic cold stress.
males (n in Fig. 5) were decapitated and blood from the trunk was collected into test tubes and centrifuged. The serum was rapidly frozen to - 2 5 ° C until required for assay. Serum prolactin was determined by standard double antibody radioimmunoassay according to the instructions of the kits provided by Dr. E. F. Parlow (NIAMD, NIH, Bethesda, MD). Results are expressed in ng/ml of serum in terms of the RP-II reference preparations (sensitivity: 0.25 ng/ml; interassay variability: 14%). Corticosterone was measured by competitive protein-binding radioassay according to
Figure 1 shows that the neonatal treatments did not affect significantly the behavior in the forced swim test performed at 80 days of age. Between-groups differences in the scores of jumping and immobility duration were not found. The neocold treatment, however, impaired the immersion response in the male group (Duncan's test: p < 0 . 0 5 vs. neocontrol scores).
Influence of Neonatal Treatments on the Effect of Chronic Cold Stress (CCS) on Forced Swim Behavior in Adulthood CCS exposure caused minor alterations in forced swim behav-
TABLE 3 BODY
Sex
WEIGHT GAIN (MEAN g __. SEM)
Age
Neocontrol
Neohandling
Neocold
Neoaleatory
Males
Day 2 Day 21 Day 60
7.8 ± 0.72 24.3 --- 0.80 192.3 ± 4.59
7.6 --- 0.27 37.0 ± 0.75t 236.1 ± 11.63"
7.1 ___ 0.23 32.2 ___ 0.74t 184.6 ± 6.13
7.9 +__0.68 32.1 ___ 0.80t 227.4 + 6.77*
Females
Day 2 Day 21 Day 60
7.6 ± 0.85 24.2 ± 0.54 146.9 ± 1.64
7.2 --- 0.29 35.1 ± 0.77t 171.6 ___ 2.77*
6.9 --- 0.45 32.5 ± 0.56I 153.0 ± 3.61
7.7 ± 0.55 32.0 ± 0.82# 162.7 ± 4.39
*p
