The role of genes involved in stress, neural plasticity, and brain circuitry in depressive phenotypes: Convergent findings in a mouse model of neglect.

Early life neglect increases risk for the development of psychopathologies during childhood and adulthood, including depression and anxiety disorders. We recently reported epigenetic changes in DNA derived from saliva in three genes predicted depression in a cohort of maltreated children: DNA-binding protein inhibitor ID-3 (ID3), Glutamate NMDA Receptor (GRIN1), and Tubulin Polymerization Promoting Protein (TPPP). To validate the role of these genes in depression risk, secondary analyses were conducted of gene expression data obtained from medial prefrontal cortex (mPFC) tissue of mice subjected to a model of maternal neglect which included maternal separation and early weaning (MSEW). Anxiety and depression-like phenotype data derived using the elevated plus maze (EPM) and forced swimming test (FST), respectively, were also available for secondary analyses. Behavioral tests were conducted in MSEW and control adult male mice when they were between 65 and 80days old. ID3, GRIN1 and TPPP gene expression in the mPFC were found to significantly predict behavioral differences in the EPM and FST. These results further support the role of these genes in the etiology of depressive and anxiety phenotypes following early life stress.

Brief prenatal ethanol exposure alters behavioral sensitivity to the kappa opioid receptor agonist (U62,066E) and antagonist (Nor-BNI) and reduces kappa opioid receptor expression.

BACKGROUND: Approximately 10 to 15% of women consume alcohol (ethanol [EtOH]) during pregnancy in the United States. Even low amounts of EtOH consumption during pregnancy can elicit long-term consequences. Prenatal experience with as few as 3 drinks has been associated with increase problem drinking in adulthood. Such effects are corroborated in rodents; however, the underlying neural adaptations contributing to this effect are not clear. In the current set of experiments, we investigated whether changes in EtOH responding following prenatal EtOH exposure involved kappa opioid receptor activation and expression. METHODS: Sprague-Dawley rats were prenatally exposed to low levels of alcohol (1.0 g/kg) during late gestation (gestational days 17 to 20 [GD17-20]) via intragastric intubation of pregnant dams. Following birth, EtOH intake, kappa- and mu-opioid-induced place conditioning, and kappa opioid receptor expression in mesolimbic brain regions were assessed in infant rats (postnatal days 14 to 15 [PD14-15]) that were offspring of dams given EtOH, vehicle, or untreated, during pregnancy. RESULTS: Animals exposed to prenatal alcohol drank more alcohol later in life and exhibited significant changes in the kappa opioid system. While control subjects found kappa opioid activation aversive, animals exposed to EtOH prenatally exhibited either no aversion or appetitive responding. Further analysis revealed that synaptosomal kappa opioid receptor expression was significantly decreased in brain areas implicated in responding to EtOH. CONCLUSIONS: Overall, these data suggest that prenatal EtOH affects kappa opioid function and expression and that these changes may be involved in increased drinking later in life.

cAMP-PKA phosphorylation of tau confers risk for degeneration in aging association cortex.

The pattern of neurodegeneration in Alzheimer's disease (AD) is very distinctive: neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau selectively affect pyramidal neurons of the aging association cortex that interconnect extensively through glutamate synapses on dendritic spines. In contrast, primary sensory cortices have few NFTs, even in late-stage disease. Understanding this selective vulnerability, and why advancing age is such a high risk factor for the degenerative process, may help to reveal disease etiology and provide targets for intervention. Our study has revealed age-related increase in cAMP-dependent protein kinase (PKA) phosphorylation of tau at serine 214 (pS214-tau) in monkey dorsolateral prefrontal association cortex (dlPFC), which specifically targets spine synapses and the Ca(2+)-storing spine apparatus. This increase is mirrored by loss of phosphodiesterase 4A from the spine apparatus, consistent with increase in cAMP-Ca(2+) signaling in aging spines. Phosphorylated tau was not detected in primary visual cortex, similar to the pattern observed in AD. We also report electron microscopic evidence of previously unidentified vesicular trafficking of phosphorylated tau in normal association cortex--in axons in young dlPFC vs. in spines in aged dlPFC--consistent with the transneuronal lesion spread reported in genetic rodent models. pS214-Tau was not observed in normal aged mice, suggesting that it arises with the evolutionary expansion of corticocortical connections in primates, crossing the threshold into NFTs and degeneration in humans. Thus, the cAMP-Ca(2+) signaling mechanisms, needed for flexibly modulating network strength in young association cortex, confer vulnerability to degeneration when dysregulated with advancing age.

Maternal separation with early weaning: a rodent model providing novel insights into neglect associated developmental deficits.

Child neglect is the most prevalent form of child maltreatment in the United States, and poses a serious public health concern. Children who survive such episodes go on to experience long-lasting psychological and behavioral problems, including higher rates of post-traumatic stress disorder symptoms, depression, alcohol and drug abuse, attention-deficit/hyperactivity disorder, and cognitive deficits. To date, most research into the causes of these life-long problems has focused on well-established targets such as stress responsive systems, including the hypothalamus-pituitary-adrenal axis. Using the maternal separation and early weaning model, we have attempted to provide comprehensive molecular profiling of a model of early-life neglect in an organism amenable to genomic manipulation: the mouse. In this article, we report new findings generated with this model using chromatin immunoprecipitation sequencing, diffuse tensor magnetic resonance imaging, and behavioral analyses. We also review the validity of the maternal separation and early weaning model, which reflects behavioral deficits observed in neglected humans including hyperactivity, anxiety, and attentional deficits. Finally, we summarize the molecular characterization of these animals, including RNA profiling and label-free proteomics, which highlight protein translation and myelination as novel pathways of interest.

Neuroanatomical changes in a mouse model of early life neglect.

Using a novel mouse model of early life neglect and abuse (ENA) based on maternal separation with early weaning, George et al. (BMC Neurosci 11:123, 2010) demonstrated behavioral abnormalities in adult mice, and Bordner et al. (Front Psychiatry 2(18):1-18, 2011) described concomitant changes in mRNA and protein expression. Using the same model, here we report neuroanatomical changes that include smaller brain size and abnormal inter-hemispheric asymmetry, decreases in cortical thickness, abnormalities in subcortical structures, and white matter disorganization and atrophy most severely affecting the left hemisphere. Because of the similarities between the neuroanatomical changes observed in our mouse model and those described in human survivors of ENA, this novel animal model is potentially useful for studies of human ENA too costly or cumbersome to be carried out in primates. Moreover, our current knowledge of the mouse genome makes this model particularly suited for targeted anatomical, molecular, and pharmacological experimentation not yet possible in other species.

Functional genomic and proteomic analysis reveals disruption of myelin-related genes and translation in a mouse model of early life neglect.

Early life neglect is an important public health problem which can lead to lasting psychological dysfunction. Good animal models are necessary to understand the mechanisms responsible for the behavioral and anatomical pathology that results. We recently described a novel model of early life neglect, maternal separation with early weaning (MSEW), that produces behavioral changes in the mouse that persist into adulthood. To begin to understand the mechanism by which MSEW leads to these changes we applied cDNA microarray, next-generation RNA-sequencing (RNA-seq), label-free proteomics, multiple reaction monitoring (MRM) proteomics, and methylation analysis to tissue samples obtained from medial prefrontal cortex to determine the molecular changes induced by MSEW that persist into adulthood. The results show that MSEW leads to dysregulation of markers of mature oligodendrocytes and genes involved in protein translation and other categories, an apparent downward biasing of translation, and methylation changes in the promoter regions of selected dysregulated genes. These findings are likely to prove useful in understanding the mechanism by which early life neglect affects brain structure, cognition, and behavior.

Parallel declines in cognition, motivation, and locomotion in aging mice: association with immune gene upregulation in the medial prefrontal cortex.

Aging in humans is associated with parallel changes in cognition, motivation, and motoric performance. Based on the human aging literature, we hypothesized that this constellation of age-related changes is mediated by the medial prefrontal cortex and that it would be observed in aging mice. Toward this end, we performed detailed assessments of cognition, motivation, and motoric behavior in aging mice. We assessed behavioral and cognitive performance in C57Bl/6 mice aged 6, 18, and 24 months, and followed this with microarray analysis of tissue from the medial prefrontal cortex and analysis of serum cytokine levels. Multivariate modeling of these data suggested that the age-related changes in cognition, motivation, motor performance, and prefrontal immune gene expression were highly correlated. Peripheral cytokine levels were also correlated with these variables, but less strongly than measures of prefrontal immune gene upregulation. To determine whether the observed immune gene expression changes were due to prefrontal microglial cells, we isolated CD11b-positive cells from the prefrontal cortex and subject them to next-generation RNA sequencing. Many of the immune changes present in whole medial prefrontal cortex were enriched in this cell population. These data suggest that, as in humans, cognition, motivation, and motoric performance in the mouse change together with age and are strongly associated with CNS immune gene upregulation.

Cognitive dysfunction with aging and the role of inflammation.

As the average lifespan continues to climb because of advances in medical care, there is a greater need to understand the factors that contribute to quality of life in the elderly. The capacity to live independently is highly significant in this regard, but is compromised by cognitive dysfunction. Aging is associated with decreases in cognitive function, including impairments in episodic memory and executive functioning. The prefrontal cortex appears to be particularly vulnerable to the effects of advancing age. Although the mechanism of age-related cognitive decline is not yet known, age-related inflammatory changes are likely to play a role. New insights from preclinical and clinical research may give rise to novel therapeutics which may have efficacy in slowing or preventing cognitive decline with advancing age.

Maternal separation with early weaning: a novel mouse model of early life neglect.

BACKGROUND: Childhood adversity is associated with increased risk for mood, anxiety, impulse control, and substance disorders. Although genetic and environmental factors contribute to the development of such disorders, the neurobiological mechanisms involved are poorly understood. A reliable mouse model of early life adversity leading to lasting behavioral changes would facilitate progress in elucidating the molecular mechanisms underlying these adverse effects. Maternal separation is a commonly used model of early life neglect, but has led to inconsistent results in the mouse. RESULTS: In an effort to develop a mouse model of early life neglect with long-lasting behavioral effects in C57BL/6 mice, we designed a new maternal separation paradigm that we call Maternal Separation with Early Weaning (MSEW). We tested the effects of MSEW on C57BL/6 mice as well as the genetically distinct DBA/2 strain and found significant MSEW effects on several behavioral tasks (i.e., the open field, elevated plus maze, and forced swim test) when assessed more than two months following the MSEW procedure. Our findings are consistent with MSEW causing effects within multiple behavioral domains in both strains, and suggest increased anxiety, hyperactivity, and behavioral despair in the MSEW offspring. Analysis of pup weights and metabolic parameters showed no evidence for malnutrition in the MSEW pups. Additionally, strain differences in many of the behavioral tests suggest a role for genetic factors in the response to early life neglect. CONCLUSIONS: These results suggest that MSEW may serve as a useful model to examine the complex behavioral abnormalities often apparent in individuals with histories of early life neglect, and may lead to greater understanding of these later life outcomes and offer insight into novel therapeutic strategies.

Analysis of ethanol reinforcement in 1-day-old rats: assessment through a brief and novel operant procedure.

BACKGROUND: An accumulating body of experimental evidence supports the notion that, early in development, heterogeneous rats exhibit heightened affinity for ethanol ingestion and are sensitive to the drug's postabsorptive reinforcing effects. The brevity of this ontogenetic period and the limited behavioral repertoire of the newborn represent obstacles in the examination of these phenomena. In the present study, we developed a novel operant technique aimed at examining the neonatal predisposition to gain access to intraoral infusions of different ethanol solutions and other potential reinforcers. METHODS: In all experiments, 1-day-old rats were placed in a supine position that allowed access to a touch-sensitive sensor. In Paired pups, reinforcers were delivered through an intraoral cannula in a fixed-ratio-1 schedule, based on their physical contact with the sensor. Yoked controls were included to account for overall magnitude of behavioral responding and were given infusions in accord with behavior of the corresponding Paired pup. The reinforcement effect of milk, water and different ethanol solutions (0.75 to 10% v/v) was assessed using a single 15-minute conditioning session. Additional pharmacokinetic studies were conducted to determine blood ethanol concentrations attained during the course of the training session. RESULTS: Within-subjects analysis revealed that Paired pups rapidly learned to increase the probability of behavioral execution to gain access to a biological reinforcer such as milk (Experiment 1). Follow-up experiments indicated that relatively low ethanol concentrations are equally likely to support operant performance (Experiments 2a and 3a). It was also observed that Paired pups exhibited surprisingly high levels of responding during an extinction session, particularly when initially trained with milk or 3% v/v ethanol as reinforcers (Experiment 4). The pharmacokinetic studies indicated that, within a short period of time, ethanol was detectable in blood. Levels attained during conditioning varied as a function of the ethanol concentration utilized as a reinforcer (Experiments 2b and 3b). CONCLUSIONS: The present technique appears to represent a valuable tool for examining ethanol self-administration and seeking behavior of the drug during early ontogeny. The results indicate that newborn rats, subjected to the explicit contingency comprising suckling-related behaviors and intraoral ethanol delivery (Paired pups), rapidly learn to gain access to the drug. These results are not explainable through psychomotor effects of ethanol as Yoked pups consumed similar amounts of ethanol and yet exhibited relatively low and consistent levels of responding. The overall pattern of results extends and validates previous observations of substantial affinity for ethanol during early stages of development, a phenomenon rarely encountered in genetically heterogeneous adult rats.

Olfactory learning in the one-day old rat: reinforcing effects of isoproterenol.

Within 24 h of their birth-induced norepinephrine surge, rat pups were tested for effects of a beta-receptor agonist, isoproterenol, on olfactory learning. Experiment 1 found no effect of isoproterenol on conditioning by pairing an odor (CS) with intraoral saccharin infusions. There was, however, unexpectedly strong responding in the unpaired control condition, which had the same contingency between the CS and isoproterenol as the paired condition. Experiment 2 found that pairings of odor and isoproterenol alone were sufficient for enhancing responding to the odor. Experiment 3 determined that isoproterenol had acted independently as a US for associative conditioning rather than facilitating nonassociative learning by mere exposure to the odor. These effects of isoproterenol as a US are consistent with the results of previous studies with older rats.

Trace conditioning in 1-day-old rats.

A recent test of 3-hr-old rats indicated surprisingly effective trace conditioning with a 60-s trace interval. The present study tested similar trace conditioning in pups 24-hr-old, in the absence of circumstances that immediately follow birth and might promote cognition. In Experiment 1 pairing an olfactory CS with a gustatory US yielded conditioning despite a 120-s trace. Experiment 2 determined that ambient CS odor remaining in the vicinity of the conditioning surface was not sufficient to result in the olfactory-gustatory association. Experiment 3 applied conditioning procedures more like those previously used for the 3-hr-old infant and more stringent than those applied in Experiment 1. With these procedures the 24-hr-old pups nevertheless acquired the CS-US contingency with a 60-s trace. Perhaps perinatal tolerance for long trace intervals is linked to their substantial experience in dealing with odors and tastes prenatally and postnatally.

Protracted increases in core body temperature and interleukin-1 following acute administration of lipopolysaccharide: implications for the stress response.

Administration of lipopolysaccharide (LPS) produces a fever response often precipitated by the production of pro-inflammatory cytokines in the CNS. This pro-inflammatory cascade has traditionally been regarded as a transitory event that, with a non-replicating antigen such as LPS, would subside within a few hours. We present data showing that central and peripheral levels of IL-1 were substantially elevated as much as 48 h after LPS in some structures. In order to explore other aspects of the sickness response that might follow a similarly protracted time course, rats were implanted with telemetry probes and injected (i.p.) with 0, 10 or 100 mug/kg of LPS and left undisturbed for 96 h. Rats injected with LPS evinced a polyphasic fever with intermediate temperature peaks at approximately 5 and 8 h. Although the fever appeared to subside during the first night cycle, more detailed analysis confirmed that it was masked by the circadian rise in core temperature during the dark cycle and actually persisted for approximately 36 h following LPS. In contrast, LPS produced a transient suppression of social interaction that was no longer evident 24 h after LPS. Finally, we report that prior LPS produced a sensitized fever response to social conflict 48 h later. Taken together, these results suggest that acute administration of LPS results in a protracted fever response and increased IL-1 that persist for at least 24-48 h, and that LPS may render certain aspects of the stress response to a sensitized state.

Behavioral responses during the forced swim test are not affected by anti-inflammatory agents or acute illness induced by lipopolysaccharide.

Pro-inflammatory cytokines and other molecules traditionally associated with immune function have been implicated in mediating behavioral and physiological consequences of stressor exposure. There is also evidence that cytokines are aberrantly expressed in depressive populations, suggesting they may play an etiological role in the development of depression/despair-related processes. Thus, we conducted a series of experiments to determine whether agents known to suppress cytokine activity or inflammatory responses in the CNS would alter the normal progression of behavioral responses during the forced swim test (FST, an animal model of depression/behavioral despair). Adult male Sprague-Dawley rats were injected with indomethacin (1 or 10 mg/kg intraperitoneally (i.p.)), alpha-MSH (0.25 or 0.5 microg icv), or minocycline (20 or 40 mg/kg i.p.) prior to each day of the FST and behavioral assessments were performed. Injection of indomethacin, alpha-MSH, or minocycline had no effect on the development of the immobility response during the FST on either day of testing. In a second series of experiments, we examined whether behavioral responses during forced swim would be affected by acute illness induced by a single injection of lipopolysaccharide (LPS). Acute injection of LPS (10 or 100 microg/kg i.p.) had no effect on behavioral responding during the FST irrespective of when it was injected, despite pronounced reductions in social behavior following these same doses of LPS. From these studies, we conclude that (a) endogenous inflammatory mediators do not appear to be involved in the normal progression of behavioral responses during the FST, and (b) behavioral responses during the FST are not affected by acute systemic injection of LPS.

Stress-induced increases in hypothalamic IL-1: a systematic analysis of multiple stressor paradigms.

Exposure to stressors such as footshock, tailshock, and immobilization have been shown to induce hypothalamic IL-1 production, while other stressors such as restraint, maternal separation, social isolation, and predator exposure have no effect on hypothalamic IL-1 levels. This disparity of findings has led to considerable controversy regarding the ability of stressors to induce hypothalamic IL-1 expression. Thus, the goal of the following experiments was to examine hypothalamic IL-1 responses in adult male Sprague-Dawley rats following exposure to a diverse set of stressors. Our data indicate that exposure to 2h of restraint in a Plexiglas tube, glucoprivic challenge induced by administration of 2-deoxyglucose (2-DG), or insulin-induced hypoglycemia all fail to alter hypothalamic IL-1 levels despite robust activation of the pituitary-adrenal response. However, when restraint was administered on an orbital shaker or in combination with insulin-induced hypoglycemia, robust increases in hypothalamic IL-1 were observed. No effects of glucoprivic (2-DG) challenge were observed when combined with restraint, indicating some specificity in the hypothalamic IL-1 response to stress. We also provide a preliminary validation of the ELISA detection method for IL-1, showing that (a) Western blot analyses confirmed strong immunopositive banding at the apparent molecular weight of both mature IL-1beta and the IL-1beta prohormone, and (b) footshock led to a two-fold increase in mRNA for IL-1 in the hypothalamus as detected by RT-PCR. These data provide novel insight into the characteristics of a stressor that may be necessary for the observation of stress-induced increases in hypothalamic IL-1.