Ethanol-induced effects on the dopamine and serotonin systems in adult Wistar rats are dependent on early-life experiences.

Some individuals control their ethanol consumption throughout life, but others escalate their intake to levels that increase the risk for addiction. The early environment influences the individual response to ethanol and affects the underlying physiological processes that lead to a transition from a voluntary to a compulsive use of ethanol. However, the neurobiological substrates for these processes are not understood. The present study aimed to test the hypothesis that early environmental experiences affect the neurobiological effects that are induced by voluntary ethanol consumption. Rat pups were subjected to three different rearing environments: conventional animal facility rearing or separation from dam and littermates for either 15 or 360min. In adulthood, the rats were exposed to a two-bottle free choice between ethanol and water for seven weeks. Tissue levels of dopamine, 5-hydroxytryptamine (5-HT) and their metabolites were measured in brain areas that have been implicated in reward and addiction processes. Differences in ethanol-induced effects were noted in 5-HT-related measurements in the nucleus accumbens and ventral tegmental area and in dopamine-related measurements in the dorsal raphe nucleus (DRN). These results provided evidence of an early environmental impact on interactive neuronal circuits between the DRN and reward pathways. The amygdala, a key area in addiction processes, was particularly sensitive to early-life conditions. The animals that experienced the longest separation differed from the others; they had low basal 5-HT levels and responded with an increase in 5-HT after ethanol. These altered responses to initial ethanol consumption as a result of early environmental factors may affect the transition from habitual to compulsive drinking and contribute to individual vulnerability or resilience to addiction.

Short- and long-term consequences of different early environmental conditions on central immunoreactive oxytocin and arginine vasopressin levels in male rats.

Numerous studies have provided evidence for an important role for the neuropeptides oxytocin (OT) and arginine vasopressin (AVP) in establishment of social behaviour early in life, such as mother-pup interactions. However, there are few reports examining the consequences of early-life experiences on OT and AVP in male offspring. We have used the maternal separation (MS) model to study the effect of different early environmental conditions in rats. The purpose was to study OT and AVP in rats subjected to prolonged daily MS (360 min, MS360), short daily MS (15 min, MS15) and conventional animal facility rearing (AFR) during postnatal days 1-21. In addition, the influence of the presence or absence of littermates during MS, i.e. litter-wise (l) or individual (i) MS, was assessed. The immunoreactive (ir) peptide levels were measured in the hypothalamus, amygdala and pituitary gland of 3 and 10 weeks old male rats. Assessment in 3-week-old rats revealed that MS15 was associated with low ir OT levels in the hypothalamus and amygdala and high levels in the pituitary gland compared with the MS360 and AFR condition. In the amygdala, differences between groups were also detected in adulthood. MS studies commonly use either MS15 or AFR as a control for prolonged MS. The present results show differences in MS360 rats as compared to MS15 but not AFR rats. Consequently, comparisons between prolonged MS with either short periods of MS or AFR will generate divergent results, hence, making the outcome of MS difficult to compare between studies. Moreover, the different early environments had no effect on ir AVP levels. In conclusion, OT in the amygdala was most sensitive to MS. Besides both short- and long-term consequences, distinct effects were seen after litter and individual separation, respectively. We propose that environmentally induced alterations in OT transmission due to disrupted mother-pup interactions early in life may cause altered susceptibility to challenges later in life.

Postpartum Behavioral Profiles in Wistar Rats Following Maternal Separation - Altered Exploration and Risk-Assessment Behavior in MS15 Dams.

The rodent maternal separation (MS) model is frequently used to investigate the impact of early environmental factors on adult neurobiology and behavior. The majority of MS studies assess effects in the offspring and few address the consequences of repeated pup removal in the dam. Such studies are of interest since alterations detected in offspring subjected to MS may, at least in part, be mediated by variations in maternal behavior and the amount of maternal care provided by the dam. The aim of this study was to investigate how daily short (15 min; MS15) and prolonged (360 min; MS360) periods of MS affects the dam by examining postpartum behavioral profiles using the multivariate concentric square field (MCSF) test. The dams were tested on postpartum days 24-25, i.e., just after the end of the separation period and weaning. The results reveal a lower exploratory drive and lower risk-assessment behavior in MS15 dams relative to MS360 or animal facility reared dams. The present results contrast some of the previously reported findings and provide new information about early post-weaning behavioral characteristics in a multivariate setting. Plausible explanations for the results are provided including a discussion how the present results fit into the maternal mediation hypothesis.

Prolonged maternal separation decreases granule cell number in the dentate gyrus of 3-week-old male rats.

Maternal separation (MS) early in life affects many aspects of development and we have previously observed significant decreases in NMDA and AMPA receptor and elevated glutamate transporter expression in the hippocampus of MS360 animals relative to MS15. We hypothesized that this disruption of the glutamatergic system in adult animals was a sign of a reduction in hippocampal neuronal number in 3-week-old animals. Male Wistar rat pups were separated litter-wise for 15 min (MS15) or 360 min (MS360) from postnatal day 1 to 21. Conventional laboratory reared animals were also included. At postnatal day 22, brains were dissected and sliced on a cryostat. Immunohistochemistry labeled neurons (NeuN) and astrocytes (GFAP) and the number of neurons was quantified using the disector method and Cavalieri principle for stereology for the CA1, CA2, CA3 and dentate gyrus regions of the hippocampus. The volume of 4 regions did not differ across the 3 experimental groups. Numerical density of neurons, however, was significantly different in CA3 (one-way ANOVA; p=0.044) and the dentate gyrus (one-way ANOVA; p<0.0001) with post hoc differences MS360 vs. MS15. Finally, the total number of neurons was calculated and MS360 animals had significantly fewer neurons than MS15 animals in the dentate gyrus. Therefore, the maternal separation procedure affected development of the hippocampus directly at 3 weeks of age. The differences observed consequently place young MS360 animals in a vulnerable state for challenges later in life.

Two repeated maternal separation procedures differentially affect brain 5-hydroxytryptamine transporter and receptors in young and adult male and female rats.

Early environment is a known determinant for individual differences in vulnerability for adult psychopathology, e.g., ethanol addiction. One underlying mechanism could be dysfunction in serotonergic neurotransmission. This study focused on the methodological considerations regarding an animal model for studying effects of early environment, maternal separation (MS), using two different paradigms. Age- and sex-specific effects on brain stem 5-hydroxytryptamine (5-HT) transporter and receptors were examined. Male and female rat pups were assigned to either litter-wise MS for 15 or 360 min (MS15l or MS360l) or individual MS for 15 or 360 min (MS15i or MS360i) daily during postnatal days 1-21. Normal animal facility reared rats were used as controls. Analyses were performed in young and adult rats. As compared to the other males, MS15l males had lower 5-HT(1A) and 5-HT(2C) receptor mRNA expression at both ages, lower 5-HT(2A) receptor mRNA when young and lower 5-HTT mRNA expression when adult. In contrast, adult MS15l females had higher 5-HT(2C) receptor mRNA expression than other female rats. The strong impact of MS15l on 5-HT-related genes was either transient or persistent depending on sex and fewer effects on gene expression were observed in females than in males. This study shows the importance of tactile contact for the consequences of short but not prolonged MS, as evidenced by major differences between MS15l and MS15i. The results suggest that MS15i is less suitable than MS15l to simulate a protective environment in studies of, for instance, ethanol addiction processes.

The impact of postnatal environment on opioid peptides in young and adult male Wistar rats.

Early environmental influences can change the neuronal development and thereby affect behavior in adult life. The aim in the present study was to thoroughly examine the impact of early environmental factors on endogenous opioids by using a rodent maternal separation (MS) model. The endogenous opioid peptide system is not fully developed at birth, and short- and/or long-term alterations may occur in these neural networks in animals exposed to manipulation of the postnatal environment. Rat pups were subjected to one of five rearing conditions; 15 min (MS15) litter (l) or individual (i), 360 min (MS360) l or i daily MS, or housed under normal animal facility rearing (AFR) conditions during postnatal days 1-21. Measurements of immunoreactive (ir) Met-enkephalin-Arg6Phe7 (MEAP) and dynorphin B (DYNB) peptide levels in the pituitary gland and in a number of brain areas, were performed at three and 10 weeks of age, respectively. MS-induced changes were more pronounced in ir MEAP levels, especially in individually separated rats at three weeks of age and in litter-separated rats at 10 weeks of age. The enkephalin and dynorphin systems have different developmental patterns, dynorphin appearing earlier, which may point at a more sensitive enkephalin system during the early postnatal weeks. The results provide evidence that opioid peptides are sensitive for early environmental factors and show that the separation conditions are critical and also result in changes manifesting at different time points. MS-induced effects were observed in areas related to stress, drug reward and dependence mechanisms. By describing effects on opioid peptides, the study addresses the possible role of a deranged endogenous opioid system in the previously described behavioral consequences of MS.