Neonatal handling increases fear and aggression in lactating rats

Neonatal handling reduces fear in male and cycling female rats, but increases maternal aggressive behavior against intruders to the nest area. Present study aimed to analyze the effects of neonatal handling on the maternal aggressive behavior and the activity in the open field with a predator of lactating rats on the 8th and the 18th postpartum days (periods of high and low aggressiveness). As pups, animals were divided into two groups: nonhandled (no neonatal manipulation) and handled (handling for 1 min during the first 10 days after delivery). As adults, females of both groups were impregnated and tested against a male intruder for aggressive behavior and in the open field with a cat inside a wire-meshed cage. Results showed that on the 8th day frequency of aggressive behaviors of handled females was higher than that of the nonhandled ones, but on the 18th day, no significant difference was detected. Surprisingly, in the open field test, handled females showed decreased locomotion and increased freezing on the 8th day compared to the nonhandled ones. The opposite relationship between increased aggressiveness with reduced fear is observed in the nonhandled control females in early and late lactation periods. However, neonatal handling abolishes this relationship. Apparently, the increased aggressiveness in neonatal handled lactating females does not depend on a decrease in fear. Our findings support the hypothesis that long lasting effects of early life stimulation is a dynamic function depending on the behavioral system and the period of life analyzed. Moreover, they caution the relationship between aggressive behavior and fear.     

Infantile handling and sex differences in shock-elicited aggressive responding of hooded rats

Infantile handled (Days 1-20) and nonhandled, male and female Long Evans hooded rats were tested at maturity (90-100 days) over 10 daily sessions for aggressive response to footshock. Individual jump and flinch thresholds for reactivity to shock, as well as paired aggressive responding to shock, were not significantly influenced by the handling procedure, although handled females ultimately adopted higher levels of fighting than nonhandled females. Handled rats were heavier than nonhandled rats of the same sex before and after testing for social aggression. Males fought significantly more than females; the discrepancy increased with additional sessions of paired exposure to shock. Male and female fight trends over sessions were linear and positive with a greater acceleration for males. The results were interpreted as indicative of a social learning variable occurring with repeated aggressive contact and primarily affecting males.     

Effects of neonatal handling on the behavior and prolactin stress response in male and female rats at various ages and estrous cycle phases of females

Neonatal handling induces behavioral and hormonal changes, characterized by reduced fear in novel environments, and lesser elevation and faster return to basal levels of plasma corticosterone, prolactin and adrenaline, in response to stressors in adulthood. The present study aimed to analyze the effects of neonatal handling from Days 1 to 10 postnatal on prolactin response to ether stress in male and female rats at three life periods: neonatal, peripubertal and adulthood. Moreover, adult females were tested in two different phases of the estrous cycle, i.e., diestrus and estrus. In another set of experiments, the behavior of peripubertal and adult males and females in estrus and diestrus was analyzed in the elevated plus maze test. Pups were either handled for 1 min (handled group) or left undisturbed (nonhandled group) during the first 10 days after delivery. In adults, in the handled females in diestrus, stress induced a lesser increase in plasma prolactin compared with nonhandled ones, as in males. However, in estrus, handled females showed no difference in the prolactin response to stress. In the elevated plus maze, handled females in diestrus, but not in estrus, showed higher locomotor activity compared with nonhandled ones. Peripubertal male and female rats handled during the neonatal period showed no difference in behavior in the elevated plus maze compared with nonhandled animals. Early-life stimulation can induce long-lasting behavioral and stress-related hormonal changes, but they are not stable throughout life and phases of the estrous cycle.     

The effects of early handling on latent inhibition in male and female rats

Latent inhibition (LI) is a behavioral paradigm in which repeated exposure to stimuli not followed by meaningful consequences renders these stimuli ineffective for subsequent learning. The development of LI is considered to reflect learning not to attend to, ignore, or tune out irrelevant stimuli. The present study investigated the differences in the development of LI between handled and nonhandled males and females. Infantile handled (Days 1-22) and nonhandled, male and female Wistar rats were tested in maturity in the LI paradigm. The LI procedure consisted of two stages: pre-exposure, where animals received 60 presentations of the to-be-conditioned stimulus (tone) and test, where the animals acquired a two-way active avoidance response with the tone serving as the warning signal. Handled animals reached higher percentage of avoidance responses as compared with nonhandled animals. Latent inhibition was obtained in both the handled and the nonhandled females, but only the handled males showed the LI effect. Nonhandled males failed to develop LI. The results indicate that the effects of handling are evident in learning tasks that do not involve motivational-emotional variables, i.e., learning to ignore irrelevant stimuli; handling differentially affects males and females, with a much greater impact on males and the nonhandling procedure has significant deleterious consequences on adult behavior.     

Programming life histories: Effects of maternal and environmental variables upon open-field behavior

Open-field behavior was studied as a function of three classes of variables: maternal characteristics, rearing environment, and sexual experience. Mothers of experimental subjects were either handled or nonhandled in infancy. Subjects were born and reared to weaning either in maternit cages or in Hebb-type free environments and were placed after weaning into either a laboratory cage or free enviroment. Sexual experience consisted either of bearing and raising a litter or no sexual experience. The results confirmed previous findings that offspring of mothers handled in infancy arc significantly less active in the open field than the offspring of nonhandled mothers. The maternal handling variable also interacted significantly with breeding experience: for rats which had not been bred, those raised by nonhandled mothers were more active than those raised by handled mothers; for females with breeding experience the reverse was true. Overall, thosc femalcs which had reared a litter were more active and defecated more than their nonbred littermates.     

Early handling and latent inhibition in the conditioned suppression paradigm

Latent inhibition (LI) is a behavioral paradigm in which repeated exposure to stimuli not followed by meaningful consequences renders these stimuli ineffective for subsequent learning. The development of LI is considered to reflect learning not to attend, to ignore, or tune out irrelevant stimuli. The present study investigated the differences in the development of LI between handled and nonhandled males and females. Infantile handled (days 1-22) and nonhandled, male and female Wistar rats were tested in maturity in the LI paradigm, using a conditioned emotional response (CER) procedure. The procedure consisted of three stages: preexposure, in which the to-be-conditioned stimulus, tone, was presented without being followed by reinforcement; acquisition, in which the preexposed tone was paired with shock; and test, in which LI was indexed by animals' suppression of licking during tone presentation. Handled animals exhibited less conditioned suppression as compared to nonhandled animals. LI was obtained in both the handled and the nonhandled females, but only the handled males showed the LI effect. Nonhandled males failed to develop LI. These results replicate our previous findings on LI in conditioned avoidance in demonstrating that: the effects of handling are evident in learning tasks that do not involve motivational-emotional variables, i.e., learning to ignore irrelevant stimuli; handling differentially affects males and females; the nonhandling procedure has deleterious consequences on adult behavior.     

Prenatal stimulation and postnatal testosterone affects infanticide in female rats

Prenatal handling, prenatal stress, and early postnatal exogeneous testosterone were examined in female rats for their effects on rat pup-killing and pup retrieval. During each of the last 5 days of pregnancy, Long-Evans rats received either 3 minutes of handling, 45 minutes of restraint and intense illumination or remained untouched. Half of the offspring of each group received testosterone from Day 1 after birth to Day 30. In adulthood, animals that received handling prenatally and testosterone postnatally killed pups more rapidly than any other group and a larger proportion did so than in the control groups. Animals not manipulated at any time retrieved pups more rapidly and a larger proportion did so than the combined other groups. The study suggests that prenatal handling interacts with testosterone presented immediately postnatally to increase infanticide in female rats. A variety of perinatal manipulations seem to suppress pup retrieval.     

Effects of pre- and postnatal stimulation on developmental,emotional, and cognitive aspects in rodents: a review

Interactions between the organism and its environment, during pregnancy as well as during the postnatal period, can lead to important neurobehavioral changes. We briefly review the literature, and successively present the main results from our laboratory concerning the behavioral effects of prenatal stress, differential rearing conditions, and postnatal handling. We show that submitting primiparous DA/HAN rats to an acute emotional stress (exposure to a cat) at gestational day10, 14, or 19 leads to greatly increased mortality of pups and to decreased body weight of surviving animals. The effects of such a stressor on emotional reactivity are less obvious. Cognitive processes are impaired depending on the learning task. Enriched environments restore abnormal behaviors (emotional reactivity, motor skills, motor and spatial learning) due to brain trauma or genetic deficiencies. In any case, environmental enrichment does prevent or slow down aging effects. The effects of postnatal handling noted when using classical tests of emotional reactivity also are clear when defensive reaction paradigms are used. Furthermore, pregnant females that are early handled are less anxious than nonhandled females. We hypothesize that, when subjected to a stressor, the offspring of early-handled females would be protected from the deleterious effects of this stress compared to pups of nonhandled females.     

Ontogeny of maternal behavior in the laboratory rat: factors underlying changes in responsiveness from 30 to 90 days.

Male and female rats that were nonhandled or that were handled from weaning, and that had intact or impaired olfaction (intranasal zinc sulfate), were sensitized through continuous pup exposure commencing at 30, 45, 60, or 90 days of age. Nonhandled males and females were alike in latencies to become maternal at Day 30 but thereafter latencies of females became shorter and latencies of males lengthened; by 90 days males had markedly longer latencies than females and only 1/3 became maternal. Handling facilitated sensitization at 30 days among males and females but only among males at 45 and 60 days. Intranasal zinc sulfate reduced latencies of both males and females at all ages tested, but appeared most effective after 45 days of age. Gender differences in latencies persisted in adult animals even after combined treatments. Results indicate that both timidity and olfaction inhibit the onset of maternal responses to pups in virgin males and females, but they differ in relative importance by age and gender.     

The effect of early handling is dependent upon the state of the rabbit (Oryctolagus cuniculus) pups around nursing.

We investigated the behavior toward humans in 4-week-old pups and adult rabbits handled daily at different times around the nursing visits during their 1st week of life. The timing of handling significantly influenced its efficiency in altering the subsequent behavior of rabbits. Animals handled around nursing readily approached a human hand when tested at weaning. Other pups, handled either 6, 12, or 18 hr after nursing, avoided the human hand. Our results show that there is a narrow sensitive period for successful stimulation, because only those rabbits that were handled within the interval starting 15 min before and ending 30 min after nursing became tame. The effect of early handling proved to be long-lasting because nonhandled rabbits tested as adults were afraid of humans and showed behavioral elements of avoidance, while the handled ones behaved fearlessly in the open field. The effect of handling proved to be specific toward humans because both handled and nonhandled animals showed avoidance toward a stuffed fox.     

Experimental programming of life histories: Toward an experimental science of individual differences

Four experimental variables were combined in a 2 × 2 × 2 × 2 factorial design. These were (1) infantile handling vs. no handling of the mothers of the subjects, (2) infantile handling vs. no handling of the subjects, (3)rearing of the subjects in either a maternity cage or a free environment between birth and weaning, and (4) rearing of the subjects in either a laboratory cage or a free environment between weaning and 42 days of age (N = 6 Purdue-Wistar rats per group). Starting at 220 days of age, the groups were given a battery of tests which measured emotional reactivity, exploratory behavior, and consumption-elimination. Analysis of the criterion data revealed the following: (1) handling pups in infancy reduced emotional reactivity and this reduction was found to be invariant with respect to different combinations of life history experiences; (2) exploratory behavior was markedly influenced by the animal's pattern of life experiences; (3) when mothers were handled during their infancy, their offspring explored significantly less than offspring of nonhandled mothers.     

Neonatal handling induces deficits in infant mother preference and adult partner preference

Neonatal handling is an experimental procedure used to understand how early‐life adversity can negatively affect neurobehavioral development and place animals on a pathway to pathology. Decreased preference for the maternal odor during infancy is one of many behavioral deficits induced by neonatal handling. Here, we hypothesize that deficits in maternal odor preference may interfere with partner preference in the adult. To test this hypothesis, we assessed infant maternal odor preference and adult partner preference in different reproductive stages in both male and female rats that received neonatal handling. Our results indicate that only neonatally handled females present deficits in maternal odor preference during infancy, but both male and females present deficits in adult partner preference. However, sexual experience was effective in rescuing partner preference deficits in males. These results indicate that, considering infant and adult social interactions, females are more susceptible to the effects of neonatal handling than males. © 2012 Wiley Periodicals, Inc. Dev Psychobiol 55: 496–507, 2013     

Visual exploratory behavior as a function of early handling, sex, and level of auditory prestimulation

Previous research has indicated that preexposing animals to intense stimulation leads to subsequent decrements in exploratory behavior. The present study was an attempt to determine if early handling could “buffer” animals against intense stimulation effects. At birth, rat pups were assigned to either a handling or a nonhandling condition. As adults, they received either intense, moderate, or no stimulation prior to being allowed 3 min of visual exploration. The predictions were: (1) handled animals will show significantly more exploration than nonhandled animals; (2) intensely stimulated animals will show significantly less exploration than animals receiving lower amounts of stimulation; (3) handled animals, under intense stimulation, will show significantly more exploration than nonhandled animals under intense stimulation. The results supported the 2nd and 3rd predictions. The first prediction was supported, but was complicated by a Sex x Handling interaction.     

Ovarian influences on the development of sexual behavior in neonatally androgenized rats

Female sexual behavior was studied in male and female rats. Males were castrated on the day of birth (Day 1); some received ovarian implants at that time; others were injected on Day 3 with oil, 5 μg testosterone propionate (TP), or 50 μg TP. Females were ovariectomized at birth, 20, or 60 days of age; on Day 3 all were injected with oil, 5 μg, TP, or 50 μg TP. Prepuberal ovarian tenancy in females tended to counteract the effects on sexual receptivity of TP administered during neonatal life. In males ovarian implants facilitated female sexual behavior at adulthood in oil-injected animals, but did not significantly influence the effect on neonatally injected TP.     

Differential effects of handling on exploration in male and female rats

In this study, sex differences in the exploratory behavior of handled and nonhandled animals were measured under conditions of both high and low stimulus variation. The apparatus used was a hole-board, which provided an exploratory response independent of ambulation. Significant effects of handling were found: nonhandled animals decreased head-dipping over days whereas handled animals maintained a high rate. Sex differences were also observed: patterns of head-dipping for males and females differed over days depending on whether objects were present (high stimulus variation) or absent (low stimulus variation) under the hole-board; females also head-dipped for longer durations than males when objects were present; further, males and females showed completely opposite patterns of exploration depending on handling treatment and on level of stimulus variation. We conclude that handling differentially affects males and females, particularly as level of stimulus variability changes.     

Sex differences in body growth in the rat.

Adult male rats are larger than females, due to a persistent difference in the growth rate from puberty onward. Gonadectomy at birth abolished, whereas gonadectomy on Day 21 caused a diminution of the sex differences. There were no differences in growth pattern between females spayed at birth and females spayed on Day 21. In male rats this was different: males castrated at birth became lighter and smaller than males castrated on Day 21. Thus males castrated at birth and females spayed at either age grew at comparable rates which were below the growth rate of males castrated at 21 days. This demonstrated the significant role of the neonatal testes on subsequent growth; prepuberal ovaries did not seem to play an important role. The administration of testosterone propionate (TP) to female rats prenatally suppressed growth of intact, but not of females spayed at birth. This TP effect is ovary-dependent. TP given neonatally promoted growth independently of the ovaries. It is concluded that neonatal androgens organize mechanisms which regulate subsequent body growth in the male rat, and that from puberty on ovarian secretions suppress the growth rate. These opposite actions of the gonads cause the sex differences in body growth of the rat.     

Neonatal handling reduces the number of cells in the locus coeruleus of rats

Neonatal handling induces long-lasting effects on behaviors and stress responses. The objective of the present study was to analyze the effects of neonatal handling (from the 1st to the 10th day after delivery) on the number of cells and volume of locus coeruleus (LC) nucleus in male and female rats at 4 different ages: 11, 26, 35, and 90 days. Results showed significant reductions in the number of cells and the volume of the LC nucleus in neonatally handled males and females compared with nonhandled rats. Environmental stimulation early in life induced a stable structural change in a central noradrenergic nucleus, which could be one of the causal factors for the behavioral and hormonal alterations observed in adulthood.     

Plasma testosterone concentration in adult male rats following acquisition of copulation-illness associations.

Prior research has identified stimuli and procedures that elicit increments in plasma testosterone in copulating male rats. In the present experiment, we demonstrate that associative inhibition of copulatory behaviors in male rats is not correlated with and cannot be attributed to a conditioned suppression of testosterone. Each male rat was paired with an inaccessible estrous female for 7 min and was then given an opportunity to copulate. Two groups received an injection of either lithium chloride (LiCl; 0.3 M, 20 ml/kg, IP) or saline (0.3 M, 20 ml/kg, IP) immediately after each of 11 pairings spaced at 3- to 4-day intervals. A third group received a noncontingent injection of LiCl 24 h after each pairing. After an initial screening for copulatory behaviors, a fourth group received only handling comparable to that received by the other three groups. Rats that received contingent LiCl gradually ceased to copulate; rats that received either noncontingent LiCl or saline remained vigorous copulators. Male rats were returned to their home cages on Trial 12 after 7-min exposure to an inaccessible female. Blood was collected by decapitation 38 min later. Testosterone levels, measured by radioimmunoassay, were significantly higher for saline than for handled control rats. Testosterone levels for handled control rats, however, were comparable to those of copulating and noncopulating rats that had received either noncontingent or contingent LiCl, respectively.     

Early-life environmental intervention may increase the number of neurons, astrocytes, and cellular proliferation in the hippocampus of rats

Neonatal handling reduces the stress response in adulthood due to a feedback mechanism. The present study analyzed the effects of repeated neonatal environmental intervention (daily handling during the first 10 days after birth) on neuron-, astroglial cell density, and cellular proliferation of the hippocampal (CA1, CA2, and CA3) pyramidal cell layers in female rats. Pups were divided into two groups, nonhandled and handled, which were submitted to repeated handling sessions between postnatal days 1 and 10. Histological and immunohistochemical procedures were used to determine changes in neuron density, astroglial cell density, and cellular proliferation. We found an increase in neuron density in each pyramidal cell layer of the hippocampus (CA1, CA2, and CA3) in female rats (11 and 90 day old) that were handled during the neonatal period. Furthermore, we found an increase in astroglial cell density in both hemispheres of the brain in the handled group. Finally, we observed an increase in cellular proliferation in both hippocampi (CA1, CA2, and CA3) of the brain in female pups (11 days old) handled during the neonatal period. This study demonstrates that an early-life environmental intervention may induce morphological changes in a structure involved with several functions, including the stress response. The results of the current study suggest that neonatal handling may influence the animals’ responses to environmental adversities later in life.     

Influence of neonatal androgen on the display of territorial marking behavior in the gerbil.

The effect of the presence or absence of androgen during the neonatal period on territorial marking behavior in the Mongolian gerbil was studied. Scent marking frequency was 20-40 fold greater in males than in females. Gonadectomy depressed marking in males but not in females. Testosterone propionate (TP) therapy completely restored marking in male but increased marking in intact and ovariectomized females to only one fourth that in males. Genetic males castrated within 2 days postpartum did not mark more frequently than TP-treated females after TP treatment in adulthood. Genetic females given a single TP injection within 6 days postpartum marked at male levels after TP treatment in adulthood. Males castrated after Day 2 and females given TP after Day 6 displayed marking frequencies intermediate between normal male and female levels after TP treatment in adulthood. This study suggests that sexual dimorphism in territorial marking behavior is due to a sex difference in the competency to respond to androgen, and it appears that development of this competency occurs during the neonatal period and is regulated, at least in part, by androgen. The onset of this differentiation process occurs earlier in the male than in the female.