Effects of Maternal Deprivation on the Prefrontal Cortex of Male Rats: Cellular, Neurochemical, and Behavioral Outcomes

Stressful events experienced during early life are associated with increased vulnerability of developing psychopathology in adulthood. In the present study, we exposed 9-day-old Wistar rats to 24 h maternal deprivation (MD) with the aim to investigate the impact of early life stress (ELS) on morphological, biochemical, and functional aspects of the prefrontal cortex (PFC), a brain region particularly sensitive to stress. We found that in the superficial medial orbital cortex (MO), young adult male rats had reduced density of GAD67 and CCK immunopositive cells, while the rostral part of the ventral lateral orbital cortex (roVLO) showed a decrease in the density of GAD67 immunopositive cells in both superficial and deep layers. In addition, the superficial rostral part of area 1 of the cingulate cortex (roCg1) and deep prelimbic cortex (PrL) was also affected by MD indicated by the reduction in PV immunopositive cellular density. Furthermore, MD induced upregulation of brain-derived neurotrophic factor (BDNF), while it did not affect the overall expression of Iba1 in neonatal or young adult PFC as measured by Western blot, however, microglial activation in young adult MD rats was detected immunohistochemically in deep layers of MO and infralimbic cortex (IL). Interestingly, when young adult male rats were subjected to a behavioral flexibility test in a T-maze, MD rats showed a subtle impairment in T-maze reversal learning indicating a mildly affected PFC function. Taken together, our findings demonstrated that MD reduced the density of interneurons and induced microglial activation, in particular, PFC areas at young adulthood, and could alter synaptic plasticity accompanied by PFC dysfunction.

Analysis of Molecular Networks in the Cerebellum in Chronic Schizophrenia: Modulation by Early Postnatal Life Stressors in Murine Models

Despite the growing importance of the cerebellum as a region highly vulnerable to accumulating molecular errors in schizophrenia, limited information is available regarding altered molecular networks with potential therapeutic targets. To identify altered networks, we conducted one-shot liquid chromatography–tandem mass spectrometry in postmortem cerebellar cortex in schizophrenia and healthy individuals followed by bioinformatic analysis (PXD024937 identifier in ProteomeXchange repository). A total of 108 up-regulated proteins were enriched in stress-related proteins, half of which were also enriched in axonal cytoskeletal organization and vesicle-mediated transport. A total of 142 down-regulated proteins showed an enrichment in proteins involved in mitochondrial disease, most of which were also enriched in energy-related biological functions. Network analysis identified a mixed module of mainly axonal-related pathways for up-regulated proteins with a high number of interactions for stress-related proteins. Energy metabolism and neutrophil degranulation modules were found for down-regulated proteins. Further, two double-hit postnatal stress murine models based on maternal deprivation combined with social isolation or chronic restraint stress were used to investigate the most robust candidates of generated networks. CLASP1 from the axonal module in the model of maternal deprivation was combined with social isolation, while YWHAZ was not altered in either model. METTL7A from the degranulation pathway was reduced in both models and was identified as altered also in previous gene expression studies, while NDUFB9 from the energy network was reduced only in the model of maternal deprivation combined with social isolation. This work provides altered stress- and mitochondrial disease-related proteins involved in energy, immune and axonal networks in the cerebellum in schizophrenia as possible novel targets for therapeutic interventions and suggests that METTL7A is a possible relevant altered stress-related protein in this context.

Assessment of the effects of maternal deprivation in offspring treated with ultra-high diluted Zincum metallicum in rats

Introduction: Clinical studies have shown that adverse events in childhood can lead to the development of psychiatric disorders, such as anxiety, in adolescence and adulthood in humans. Manipulations with laboratory animals, such as maternal deprivation (MD), are a source of stress for the offspring and can be a useful tool for the understanding how these events in the early period of development can lead to behavioral changes in adulthood. Studies about the use of homeopathic ultra-high dilutions as tools to minimize stress are found in the literature, i.e. Zincum metallicum is used for the treatment of neurological and behavioral symptoms, including: weakening of intellectual functions with brain and nervous exhaustion, loss of vitality, slow comprehension, memory disorder, general tremor and constant movements. These alterations can be characterized as stress-related phenomena in different species. Aim: The aim of this study was to evaluate the long-term effects of homeopathic treatment in animals subjected to stress in their early days (maternal deprivation). This study was approved by the Ethics Committee of the University of Santo Amaro (UNISA), according to Process number 11/2014. Materials and Methods: In this study, newborn female rats were subjected to maternal deprivation and treated from the 10th day of lactation (PND10) until weaning (PND21). The animals were divided in 4 groups: 8 treated with Zincum metallicum 30 cH (Zn30cH); 8 treated with Zincum metallicum 6 cH (Zn6cH); 8 treated with 10% hydroalcoholic solution (medicines in blind trials, identified by codes); and 8 animals who had neither taken anything (“blank control”) nor experienced deprivation. The animals were weighed weekly, from weaning until the end of the experiment, and evaluated in the Open Field (OF) and in the Plus Maze (PM) devices to measure mobility, emotionality and anxiety, in 3 moments: in PND21 (childhood), during puberty (PND 40) and adulthood (PND75). Data were analyzed statistically by ANOVA, followed by the Bartlett's Test and Bonferroni's Multiple Comparison Test, being p≤0.05. Results and Discussion: In the OF, it was observed reduction of immobility in Zn6cH group (p≤0.05) at PND21. At PND40, the Zn30cH group showed higher activity than the other groups, with increased rearing and decreased immobility (p≤0.05). Finely, at PND75 (adulthood) no change occurred, and Zincum metallicum treated rats presented similar behavior to the undeprived animals. In the PM, at PND21, the Zn30cH treated deprived group showed decrease in the open arm entry and retention period in the Maze, compared to the control undeprived group (p≤0.05). The time in the closed arm was higher than the undeprived control group and the number of head dips was lower (p≤0.05). The PM observation at 45 and 75 days showed no statistical difference among groups. The deprived animals which took Zn30cH obtained the same gain that the undeprived animals did (p≤0.05). Therefore, the deprived animals that presented anxiety during childhood were in accordance to other studies, showing that maternal deprivation is a stress factor that causes anxiety. However, the time of emotional unbalance was shorter in rats treated with Zincum metallicum. Conclusion: Zincum metallicum 30 cH seems to be a potential medicine to manage troubles in the childhood derived from stress caused by maternal deprivation. However, other studies are already underway with male offspring and neurochemical measurements, for a better parameter of results.

Complex Housing, but Not Maternal Deprivation Affects Motivation to Liberate a Trapped Cage-Mate in an Operant Rat Task

Early life environment influences the development of various aspects of social behavior, particularly during sensitive developmental periods. We studied how challenges in the early postnatal period or (early) adolescence affect pro-social behavior. To this end, we designed a lever-operated liberation task, to be able to measure motivation to liberate a trapped conspecific (by progressively increasing required lever pressing for door-opening). Liberation of the trapped rat resulted either in social contact or in liberation into a separate compartment. Additionally, a condition was tested in which both rats could freely move in two separate compartments and lever pressing resulted in social contact. When partners were not trapped, rats were more motivated to press the lever for opening the door than in either of the trapped configurations. Contrary to our expectations, the trapped configuration resulted in a reduced motivation to act. Early postnatal stress (24 h maternal deprivation on postnatal day 3) did not affect behavior in the liberation task. However, rearing rats from early adolescence onwards in complex housing conditions (Marlau cages) reduced the motivation to door opening, both in the trapped and freely moving conditions, while the motivation for a sucrose reward was not affected.

Adolescent cocaine induced persistent negative affect in female rats exposed to early-life stress

Rationale The combination of several risk factors (sex, a prior underlying psychiatric condition, or early drug initiation) could induce the emergence of negative affect during cocaine abstinence and increase the risk of developing addiction. However, most prior preclinical studies have been centered in male rodents, traditionally excluding females from these analyses. Objectives To ascertain the behavioral and neurochemical consequences of adolescent cocaine exposure when the combination of several risk factors is present (female, early-life stress). Methods Whole litters of Sprague–Dawley rats were exposed to maternal deprivation for 24 h on postnatal day (PND) 9. Cocaine was administered in adolescence (15 mg/kg/day, i.p., PND 33–39). Negative affect was assessed by several behavioral tests (forced swim, open field, novelty-suppressed feeding, sucrose preference). Hippocampal cell fate markers were evaluated by western blot (FADD, Bax, cytochrome c) or immunohistochemistry (Ki-67; cell proliferation). Results Maternal deprivation is a suitable model of psychiatric vulnerability in which to study the impact of adolescent cocaine in female rats. While adolescent cocaine did not alter affective-like behavior during adolescence, a pro-depressive–like state emerged during adulthood, exclusively in rats re-exposed to cocaine during abstinence. FADD regulation by cocaine in early-life stressed female rats might contribute to certain hippocampal neuroadaptations with some significance to the observed induced negative affect. Conclusions Adolescent cocaine induced persistent negative affect in female rats exposed to early-life stress, highlighting the risk of early drug initiation during adolescence for the emergence of negative reinforcement during abstinence likely driving cocaine addiction vulnerability, also in female rats.

Sex Differences in the Brain-Blood Barrier in Rats Exposed to Early life Stress and the Treatment with Antidepressants and Psychobiotic

Major depressive disorder is a debilitating mental disorder. Although the etiology is not fully understood, the impairment to the blood-brain barrier (BBB) integrity may be involved. Maternal deprivation was performed in the first 10 postnatal days for 3h/day. Male and female rats were divided into control and maternal deprivation. Maternal deprivation animals were subdivided and received treatment with saline, escitalopram, ketamine, or probiotic. The integrity of BBB was evaluated in the prefrontal cortex and hippocampus at postnatal days 11, 21, 41, and 61. Maternal deprivation caused BBB breakdown in the prefrontal cortex and hippocampus in female and male rats in all ages evaluated, except in the prefrontal cortex of females at postnatal day 41. In females, escitalopram, ketamine, and probiotic reversed BBB breakdown in all ages evaluated, except probiotic at postnatal day 21 (prefrontal cortex), and ketamine at postnatal days 21 and 41 (hippocampus). In males, escitalopram, ketamine, and probiotic reversed BBB breakdown in the prefrontal cortex in all ages evaluated, except escitalopram at postnatal days 41 and 61. In the hippocampus of males, BBB damage was reversed by escitalopram at postnatal day 21 and ketamine at postnatal day 41. Treatment with escitalopram, ketamine, or probiotics can prevent changes in the BBB integrity, depending on the age and sex of the animal. Clinically it is important to evaluate different treatments depending on age and sex.