Prenatal stress interacts with prepuberal social isolation to reduce male copulatory behavior

Male offspring of female rats exposed to the stress of restraint and high intensity light during Day 14–21 of gestation were weaned when 22 days old into individual cages (social isolation) or into group cages with 2 other males (social rearing). Copulatory behavior was reduced by prenatal stress or social isolation alone and was absent in males exposed to both stress and prepuberal isolation. Adult therapy consisting of living with young females restored copulatory behavior in some stressed males only if they were reared prepuberally in a social environment. Stressed isolates showed chronic deficits in sexual behavior relative to controls. The study indicates that prenatal stress and prepuberal social isolation can interact to determine adult copulatory potentials in male rats.     

Exploration, distraction, and habituation in rats reared in isolation

Rats reared in isolation have been reported to show increased motor activity; the purpose of the present experiment was to establish whether they also show enhanced exploratory and orienting responses. Rats were reared in isolation or in social groups from Day 20 to Day 45. Subgroups of each rearing condition were either left undisturbed, received daily handling, or received daily handling and were given objects in their cages. From Day 45 all the rats were rehoused in groups of 6, and testing began at Day 73. In a 1st test exploration was measured by the number of contacts with objects placed in the home cage; and a 2nd test exploration was measured by responses to a holeboard; and in a 3rd test orienting was measured by the distraction produced by the presentation of a tone. When tested in novel situations, in contrast to their enhanced motor activity, isolates showed reduced exploration and orienting. This may have been due to a discrepancy between their adaptation level (established during rearing) and their current level of sensory input. In all the tests the isolates showed normal habituation, both within sessions and between sessions. An inability to habituate is not, as has been claimed, a general characteristic of isolates.     

Behavioral, neurochemical and endocrinological characterization of the early social isolation syndrome

Rearing rats in isolation has been shown to be a relevant paradigm for studying early life stress and understanding the genesis of depression and related affective disorders. Recent studies from our laboratory point to the relevance of studying the social isolation syndrome as a function of home caging conditions. Accordingly, the present series of experiments assessed the contribution of each condition to the expression of the prepulse inhibition of the acoustic startle, food hoarding and spontaneous locomotor activity. In addition, ex vivo neurochemical changes in the brains of isolated and grouped rats reared either in sawdust-lined or in grid-floor cages were determined by measuring dopamine and serotonin as well as their major metabolites in a “psychosis circuit” that includes mainly the hippocampus and selected hippocampal efferent pathways projecting towards the anterior cingulate and infralimbic cortices, nucleus accumbens, dorsolateral caudate nucleus, amygdala and entorhinal cortex. The results of the present study demonstrate that rearing rats in isolation (i) produces a syndrome of generalized locomotor hyperactivity; (ii) increases the startle response; (iii) impairs prepulse inhibition; (iv) tends to increase food hoarding behavior; (v) increases basal dopamine turnover in the amygdaloid complex; (vi) decreases basal dopamine turnover in the infralimbic part of the medial prefrontal cortex; and (vii) decreases basal turnover of serotonin in the nucleus accumbens. In the entorhinal cortex, dopamine neurotransmission seemed to be more sensitive to the caging conditions since a decreased basal turnover of dopamine was observed in grid-reared animals. Plasma corticosterone levels were also increased in grid-reared animals compared with rats reared in sawdust cages. Finally, isolates reared on grids showed a significant positive correlation between plasma corticosterone levels and dopamine in the left nucleus accumbens.

Altogether, these results support the contention that there is a link between social isolation, attention deficit, spontaneous locomotor hyperactivity and reduced dopamine turnover in the medial prefrontal cortex. Furthermore, our data demonstrate that rearing rats in grid-floor cages represents a form of chronic mild stress associated with increased corticosterone levels, decreased basal turnover of entorhinal dopamine and increased dopamine activity in the left nucleus accumbens. Finally, a significant and selective decrease in the basal turnover of serotonin in the nucleus accumbens of isolated rats may be linked to the isolation-induced locomotor hyperactivity.     

Isolation-rearing retards the acquisition of schedule-induced polydipsia in rats

Rats were reared from weaning either in isolation or in social groups for 10 weeks and were then tested for the acquisition of schedule-induced polydipsia (SIP). Isolation-reared rats were impaired in the development of this behaviour when compared to socially-reared controls. This reduction in drinking was specific to this test situation. Home cage water consumption and drinking in the SIP test chamber without a schedule of food delivery were unaltered and home cage drinking following water deprivation was significantly higher in the isolates. Housing adult rats in isolation did not impair SIP. The locomotor hyperactivity induced by isolation was also specific to rearing conditions. The inverse relationship between water consumption on the last day of testing and plasma corticosterone levels observed in both the socially-reared and socially-housed rats was absent in both the isolation-reared and isolation-housed rats.     

The development of intersession habituation and emergence in socially reared and isolated rats.

Male and female hooded rats were weaned at 17 days and then reared in isolation or in social groups. Intersession habituation of locomotor activity in the open field was tested at 15, 25, and 45 days. Contrary to some previous reports, the 15-day-old animals showed significant habituation. At 45 days, however, the isolates showed very little habituation compared to the social animals. The isolates showed a similar pattern of development in their emergence into the open field. Apparently, isolation does not result in an arresting of development, but rather in the formation of behavior patterns otherwise absent in normally reared rats.     

Effect of social isolation on CB1 and D2 receptor and fatty acid amide hydrolase expression in rats

Rearing rats in isolation has been shown to produce behavioral and neurochemical alterations similar to those observed in psychoses such as schizophrenia. Also, a dysregulation in both the endocannabinoid and dopaminergic systems has been implicated in schizophrenia. The aim of this study was to determine if there are differences in CB1 receptor and fatty acid amide hydrolase (FAAH) protein expression, as well as D2 dopamine receptor expression in different brain regions in rats reared in different environmental conditions. Twenty-one-day-old male Sprague-Dawley rats were either reared in individual cages (isolated rats) or in group cages of six per cage (group-housed rats) for 8 weeks. Quantitative fluorescence immunohistochemistry was performed on brain slices using antibodies specific to the CB1 or D2 receptor, or the enzyme FAAH. Raising rats in isolation led to a significant decrease in CB1 receptor expression in the caudate putamen and the amygdala, a significant increase in FAAH expression in the caudate putamen and the nucleus accumbens core and shell, and no significant change in D2 receptor expression in any region studied. These results indicate that the endocannabinoid system is altered in an animal model of aspects of psychosis. This implies that rearing rats under different housing conditions may provide new insight into the role of the endocannabinoid system in the development of psychoses.     

Social isolation and chronic handling alter endocannabinoid signaling and behavioral reactivity to context in adult rats

Social deprivation in early life disrupts emotionality and attentional processes in humans. Rearing rats in isolation reproduces some of these abnormalities, which are attenuated by daily handling. However, the neurochemical mechanisms underlying these responses remain poorly understood. We hypothesized that post-weaning social isolation alters the endocannabinoid system, a neuromodulatory system that controls emotional responding. We characterized behavioral consequences of social isolation and evaluated whether handling would reverse social isolation-induced alterations in behavioral reactivity to context and the endocannabinoid system. At weaning, pups were single or group housed and concomitantly handled or not handled daily until adulthood. Rats were tested in emotionality- and attentional-sensitive behavioral assays (open field, elevated plus maze, startle and prepulse inhibition). Cannabinoid receptor densities and endocannabinoid levels were quantified in a separate group of rats. Social isolation negatively altered behavioral responding. Socially-isolated rats that were handled showed less deficits in the open field, elevated plus maze, and prepulse inhibition tests. Social isolation produced site-specific alterations (supraoptic nucleus, ventrolateral thalamus, rostral striatum) in cannabinoid receptor densities compared to group rearing. Handling altered the endocannabinoid system in neural circuitry controlling emotional expression. Handling altered endocannabinoid content (prefrontal and piriform cortices, nucleus accumbens) and cannabinoid receptor densities (lateral globus pallidus, cingulate and piriform cortices, hippocampus) in a region-specific manner. Some effects of social isolation on the endocannabinoid system were moderated by handling. Isolates were unresponsive to handling-induced increases in cannabinoid receptor densities (caudal striatum, anterior thalamus), but were sensitive to handling-induced changes in endocannabinoid content (piriform, prefrontal cortices), compared to group-reared rats. Our findings suggest alterations in the endocannabinoid system may contribute to the abnormal isolate phenotype. Handling modifies the endocannabinoid system and behavioral reactivity to context, but surmounts only some effects of social isolation. These data implicate a pivotal role for the endocannabinoid system in stress adaptation and emotionality-related disturbances.     

Adrenocortical, beta-endorphin and behavioral responses to graded stressors in differentially reared rats

Isolation rearing has long been suspected to alter hormonal and behavioral responses to stress. Two experiments were conducted to test the hypothesis that isolates are more timid or fearful than socially reared rats when exposed to novel test environments. In both, isolate response to 3 graded stressors was compared to that of socially-reared rats. In the first experiment, animals were handled, shocked or not treated prior to testing to produce three levels of conditioned fear. They were then tested on four paradigms previously shown sensitive to conditioned fear: open field activity, emergence latency, auditory startle, and latency to accept food from the experimenter. In the second experiment, rats were given a 0-, 5- or 20-min forced swim, then sacrificed for analysis of plasma corticosterone and pituitary and hypothalamic beta-endorphin. It was found that isolates showed little evidence of enhanced behavioral timidity, although rearing effects were seen on all 4 behavioral measures. Plasma corticosterone levels increased in a graded fashion over the course of the forced swim, but there was no effect of rearing conditions. While there were no effects of rearing or stress on hypothalamic beta-endorphin, pituitary beta-endorphin content was lower in females than in males, and isolate males had lower pituitary endorphin than social males. In summary, these experiments provide no evidence that isolation rearing produces a primary, global increase in fearfulness, but identify several behavioral and hormonal differences associated with differential housing in rats.     

The genetic liability to stress and postweaning isolation have a competitive influence on behavioral organization in rats

Rats housed in social isolation postweaning (isolates) show profound behavioral and neurobiological differences when compared to socially housed rats (socials). Fischer rats (F344) relative to Lewis rats are hyperresponsive and significantly more susceptible to stressful stimuli. This investigation tested the hypothesis that the behavioral effects of postweaning isolation are more pronounced in a strain of rats with high susceptibility to stress compared to a strain with low susceptibility to stress. Seventy male Sprague-Dawley, Lewis, and F344 rats were housed individually or in groups at weaning on Day 21 and tested on Day 85 in the Behavioral Pattern Monitor. There was no interaction between strain and postweaning isolation for measures of locomotor activity and exploratory behavior (holepoking). However, the postweaning isolation-induced increase in the frequency of repetitive straight movements, a measure of behavioral organization, was more pronounced in Lewis isolates compared to Sprague-Dawley and F344 isolates. These results do not support the hypothesis that rats with a higher susceptibility to stress show more pronounced changes in behavior following postweaning isolation; instead, increased susceptibility to stress may counteract the repetitive movement patterns induced by social isolation.     

Preoperative differential housing and dorsal hippocampal lesions in rats

Rats housed in impoverished environments often show greater behavioral deficits after receiving brain lesions than to rats housed in standard or enriched environments. However, the resemblance between the effects of social isolation and those of hippocampal lesions in rats prompted the suggestion that rats socially isolated at weaning rather than grouped counterparts may show less behavioral change after sustaining dorsal hippocampal lesions when adult. In socially reared rats, hippocampal lesions produced increased ambulation and object contact in an open field, reduced passive avoidance in a runway task, and produced faster acquisition of active avoidance in a shuttle box, but there were no such differences in isolation-reared rats. Ambulation and object contact in isolates were intermediate to those of rats with lesions and intact group-housed rats, and the behavior of isolates during passive and active avoidance training was generally similar to that of grouped rats with lesions. The introduction of a distractor during approach training in an alley reduced running speeds more in rats with lesions than in controls. The several significant interactions between housing state and lesion state suggest that neural pathways associated with the hippocampal formation may mediate some behavioral effects of differential housing.     

Neonatal novelty exposure, dynamics of brain asymmetry, and social recognition memory

Brief and transient early-life stimulation via neonatal handling and neonatal novelty exposure can lead to differential changes within the right and left brains. In rats, these lateralized changes have been demonstrated behaviorally, neuroanatomically, and neurophysiologically. Recently, we found that neonatal novelty exposure can prolong the duration of social recognition memory from less than 2 hr to at least 24 hr among male rats reared in social isolation and that this enhancement is associated with an initial right-turn preference in a novel testing cage. In contrast to stable forms of asymmetry, such as handedness, we show that this turning asymmetry is dynamic-decreasing as the animal adjusts to the novel testing environment over a 2-day period. This change in turning asymmetry was found only among animals that experienced neonatal novelty exposure during the first 3 weeks of their lives. Furthermore, individual differences in short-term social recognition memory for a conspecific can be predicted by this change in functional asymmetry.     

Devices for handling small mammals in laboratory conditions

Wild rodents, and especially rats, are significantly more difficult to handle, maintain and breed than laboratory rats and mice. They can be characterized as more skittish, neophobic and aggressive. While establishing a breeding colony of wild Norwegian rats (Rattus norvegicus) I developed some helpful devices and I will describe them here. In this paper I also describe the techniques for transporting animals between their home cages, separating them inside cages, and catching individuals that escape. These devices do not require any home cage modification. They may be modified for size. With size modification these devices may be used for handling animals in any type of laboratory cages. They may be used with all species of small mammals, for example, mice, gerbils, hamster, and opossums. They prove especially helpful when physical contact with humans is a problem (e.g. SPF conditions of breeding, and behavioral procedures that do not allow handling).     

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.     

Testing the cumulative stress and mismatch hypotheses of psychopathology in a rat model of early-life adversity

BackgroundIn the present study, we tested both the cumulative stress and the mismatch hypothesis of psychopathology. For this purpose the combined effects of early-life adversity and later-life stress exposure on behavioral markers of psychosis susceptibility were studied in male Wistar rats.MethodExperiment I: rat pups divided on the basis of the levels of their maternal care experience in low, medium or high maternal care groups, were reared post-weaning in groups (Exp. IA) or in social isolation (Exp. IB) and tested at adulthood under basal conditions or after an acute corticosterone (CORT) administration. Maternal care levels were assessed by measuring the dam's licking and grooming (LG) the first postnatal week of life.Experiment II: rat pups exposed as neonates to daily sessions of 8 h of maternal separation (MS) on postnatal days 3, 4 and 5 either altogether in their home cage (HOME SEP) or alone in a novel environment (NOVEL SEP), were reared post-weaning in groups and tested at adulthood under basal conditions.Adult testing included behaviors marking psychosis susceptibility: apomorphine-induced gnawing (APO-gnawing), acoustic startle response and its modulation by a prepulse stimulus (PPI). The behavior of the Medium LG offspring was used as baseline reference for all the three experiments.ResultsExperiment I: Low maternal LG history alone had limited effects on the behavior of Wistar offspring, although increased acoustic startle and increased PPI, at high prepulse intensity levels, were observed. When low maternal LG history was combined with post-weaning social isolation, basal APO-gnawing was decreased and PPI increased, compared to High LG and Med LG offspring. This reflects attenuated psychosis susceptibility. High LG offspring reared in isolation displayed, however, the highest APO-gnawing and the lowest PPI levels among rats reared in social isolation, which is indicative for increased psychosis susceptibility. These findings support the mismatch hypothesis. For demonstration of the cumulative stress hypothesis an injection of CORT in the adult Low LG offspring was required that increased APO-gnawing and reduced PPI. This CORT-induced PPI disruption was greatly enhanced after additional isolation rearing. The High LG group, either socially housed or reared in isolation, was resistant to the acute effects of CORT at adulthood.Experiment II: MS increased psychosis susceptibility only in NOVEL SEP rats that had experienced MS in the context of early social isolation. These individuals displayed increased adult APO-gnawing and reduced PPI, if reared post-weaning in a condition that does not match with their early life social environment (i.e. group housing). This finding supports the mismatch hypothesis.ConclusionThe outcome of environmental manipulations on developmental programming of psychosis susceptibility depends on the interplay of early-life adversity and later-life stressors in a manner that supports the mismatch hypothesis. However, evidence for the cumulative stress hypothesis arises if vulnerable individuals are exposed in later life additionally to excess of the stress hormone CORT.     

Activity and exploration in thyroid-deficient and socially-isolated rats.

Rats were treated with an anti-thyroid drug during infancy, and their behavior and brain weights were later compared with control rats, and with a group of animals reared in social isolation. The brain weight of the thyroid-deficient rats was considerably smaller than that of the other two groups, but their behavior in an open-field situation was not different from that of the socially-reared controls. Isolates, on the other hand showed a marked slowing down of habituation in the open field, confirming previous reports of a failure of inhibition in these animals. The behavioral effects of social isolation do not appear to be due to a slowing down of brain development.     

Effects of experience and context on 50-kHz vocalizations in rats

Rats can emit ultrasonic 50-kHz vocalizations which are generally assumed to reflect the animals' positive emotional state. However, some aspects question the reliability of 50-kHz calls as indicators of positive affective states. Firstly, rats also emit them in novel environments containing scents of other rats, or even while being victims of intra-species aggression. Secondly, huge inter-individual variability in call production can be observed. The present two studies were conducted to further determine factors other than reward, which may influence or even induce calling. Experiment A showed that 50-kHz calls were emitted in relatively high numbers during short isolation in test cages, and, to a lesser extent, also during testing in an open field and an elevated plus maze. Despite inter-individual variability, calling behavior was individually stable over days and occurred irrespective of whether rats were tested in a cage with or without familiar rat scents. These data indicate that 50-kHz calling is not necessarily a response to the presence of pleasurable or social stimuli. Additionally, it was observed that call emission during isolation is strongly affected by prior experience. Rats that had been trained repeatedly in an appetitive discrimination task emitted only few calls during short isolation in test cages, whereas naïve rats emitted high numbers of 50-kHz calls which decreased over time. The most likely explanation is that rats call in response to separation from the cage mate, as the first group was trained before the recordings, while the naïve rats were recorded immediately after separation. This explanation was supported by Experiment B, which showed that the rats that remained alone in the home cage also called at 50 kHz after separation from the cage mates. In both experiments, most of the 50-kHz calls were not frequency modulated, which lend support for the suggestion that this subtype has a social-coordinating function. The present findings urge sophisticated spectrographic analysis of ultrasonic vocalizations and caution when interpreting 50-kHz vocalizations, since specific subtypes of these calls can occur in contexts that are not necessarily pleasurable to rats, and are affected by prior experience and huge individual differences.     

Effects of postweaning social isolation and re-socialization on the expression of vasoactive intestinal peptide (VIP) and dendritic development in the medial prefrontal cortex of the rat

In the present study we evaluated the effects of early social isolation and re-socialization on dendritic development and the expression of the vasoactive intestinal peptide (VIP) in the medial prefrontal cortex (mPFC) of the rat. Sprague-Dawley male rats were reared either in isolation (IC) or social (SC) conditions from postnatal day 18 to 32. Rats were then behaviorally evaluated in the open field test, and approximately half of the animals were sacrificed. Their brains were processed either for immunocytochemical labeling against VIP or for the Golgi-Cox-Sholl staining. The remaining IC rats were re-socialized during 30 additional days. The results demonstrated that early social isolation impair neuronal dendritic arborization and increase the number of VIP-immunoreactive neurons. Furthermore, animals displayed hyperlocomotion in the open-field test. According to our structural, immunocytochemical and open-field data, the resocialization experience was unable to reverse neuronal and behavioral abnormalities.     

Effects of postweaning rearing condition on recovery of copulatory behavior from lesions of the medial preoptic area in rats

Bilateral lesions of the medical preoptic area (MPOA)–anterior hypothalamus of adult male rats markedly disrupt male copulatory behavior. In contrast, more group-reared then isolated male rats receiving bilateral MPOA lesions as juveniles copulated to ejaculation as adults. The present experiment was designed to analyze the role of rearing condition in promoting copulatory recovery from MPOA lesions in juvenile male rats. Juvenile male rats were given bilateral MPOA lesions or a sham operation and reared in isolation without handling, in isolation with daily handling, across a perforated Plexiglas divider from a male peer, or together with a male peer (social). Socially reared and handled males, but not isolated and divided males, with MPOA lesions showed evidence of copulatory recovery (combined socially reared and handled males vs combined isolated and divided males). These results support previous findings that postweaning rearing condition can affect copulatory recovery following juvenile MPOA lesions, but indicate that play experiences involving physical contact with peers are not necessary for such recovery to occur.     

Hyperactivity and obesity: The interaction of social isolation and cafeteria feeding

Rats were reared in social isolation or in social groups of 4 or 5 rats per cage from weaning and were fed either a lab chow diet or a diet of 4 palatable foods (cafeteria diet), in addition to the lab chow. The hyperactivity of isolation-reared rats appears to be a reactivity to novel environmental stimuli, since it was seen only in the 0.5 hr tests and not in the near 24 hr test. It was found that hyperactivity and increased body weight can develop within as few as 7 to 10 days in rats reared in isolation from weaning. Cafeteria feeding enhanced activity in isolation-reared rats, but suppressed it in group-reared rats. Isolation-reared rats fed a cafeteria diet had strong, stable preferences for their most preferred food over the 25 days of measurement. Rats reared in isolation had significantly different food preferences, as compared with rats reared in groups. Cafeteria fed rats had a significantly greater calorie intake and body weight than rats fed lab chow. On analysis, cafeteria fed rats had significantly greater carcass energy and an increased amount of parametrial white adipose tissue as compared with rats fed only lab chow. The interscapular brown adipose tissue (IBAT) weights of cafeteria fed rats were also greater. However, as there was no difference between the cafeteria and chow fed rats in the total amount of protein in the IBAT, it was concluded that the increased weight of the IBAT did not reflect a genuine hypertrophy of the tissue.     

The influence of handling and isolation postweaning on open field,exploratory and maternal behavior of female rats

Fifty-three black-hooded female rats, raised from the 28th to the 128th day of life under four types of environmental conditions differing in social and handling factors, had their behavioral performance evaluated in eight tests involving exploration of a complex environment (EX) in two open field (OF) tests and in three pup-retrieving tests. These tests were held before, during and after the period of lactation of their first brood. Females differed from (previously studied) males in that environmental raising conditions had a much smaller influence, especially during the lactation period. Nevertheless, females raised in isolation tended to remain for longer times inside a protected den in the exploration tests and to have greater locomotion in the open field. Previous handling allowed a greater exploration during the first contact with the novel complex environment as well as greater activity in the open field. The presence of small pups drastically reduced the exploratory motivation, but the presence of pups more than 20 days old tended to increase it. Maternal behavior evaluated in the pup retrieval test and in the test of time for the female to leave the nest side of the double box system was markedly refractory to previous environmental influences.