Beneficial effects of postnatal choline supplementation on long-Term neurocognitive deficit resulting from fetal-Neonatal iron deficiency.

Early-life iron deficiency is a common nutrient condition worldwide and can result in cognitive impairment in adulthood despite iron treatment. In rodents, prenatal choline supplementation can diminish long-term hippocampal gene dysregulation and neurocognitive deficits caused by iron deficiency. Since fetal iron status is generally unknown in humans, we determined whether postnatal choline supplementation exerts similar beneficial effects. Male rat pups were made iron deficient (ID) by providing pregnant and nursing dams an ID diet (3-6ppm Fe) from gestational day (G) 3 through postnatal day (P) 7, and an iron-sufficient (IS) diet (200ppm Fe) thereafter. Control pups were provided IS diet throughout. Choline (5ppm) was given to half the nursing dams and weanlings in each group from P11-P30. P65 rat cognitive performance was assessed by novel object recognition (NOR). Real-time PCR was performed to validate expression levels of synaptic plasticity genes known to be dysregulated by early-life iron deficiency. Postnatal choline supplementation prevented impairment of NOR memory in formerly iron-deficient (FID) adult rats but impaired NOR memory in IS controls. Gene expression analysis revealed a recovery of 4 out of 10 dysregulated genes compared to 8 of the same 10 genes that we previously demonstrated to recover following prenatal choline supplementation. Recognition memory deficits induced by early-life iron deficiency can be prevented by postnatal choline supplementation and disrupted expression of a subset of synaptic plasticity genes can be ameliorated. The positive response to postnatal choline represents a potential adjunctive therapeutic supplement to treat iron-deficient anemic children in order to spare long-term neurodevelopmental deficits.

Conditioned object preference: an alternative approach to measuring reward learning in rats.

Pavlovian conditioned approach behavior can be directed as much toward discrete cues as it is toward the environmental contexts in which those cues are encountered. The current experiments characterized a tendency of rats to approach object cues whose prior exposure had been paired with reward (conditioned object preference, COP). To demonstrate the phenomenon, rats were conditioned to associate cocaine or saline with two different objects. Rats acquired a preference, assessed using investigation times directed toward each object, for the cocaine-paired object following conditioning. Furthermore, high levels of object investigation during cocaine conditioning predicted stronger preferences for the cocaine-paired object in the test phase. Conditioned approach diminished across extinction but was reinstated through a priming injection of cocaine. To determine whether preferences are affected by reward value, rats were conditioned using three objects paired with 0, 5, or 20 mg/kg of cocaine. This produced object preferences in the post-test that scaled with cocaine dose used for conditioning. Finally, we explored whether contextual cues modulate expression of COP by testing rats for renewal of cocaine seeking. When conditioning was conducted in one context and extinction training in a second context, COP was renewed when the rats were retested in the original context. Thus, conditioned object preferences are readily acquired, easily measured, and amenable to a number of standard Pavlovian conditioning manipulations. This task promises to become a valuable addition to the panoply of behavioral tools available to test mechanisms underlying adaptive and maladaptive reward processing.

Deletion of novel protein TMEM35 alters stress-related functions and impairs long-term memory in mice.

The mounting of appropriate emotional and neuroendocrine responses to environmental stressors critically depends on the hypothalamic-pituitary-adrenal (HPA) axis and associated limbic circuitry. Although its function is currently unknown, the highly evolutionarily conserved transmembrane protein 35 (TMEM35) is prominently expressed in HPA circuitry and limbic areas, including the hippocampus and amygdala. To investigate the possible involvement of this protein in neuroendocrine function, we generated tmem35 knockout (KO) mice to characterize the endocrine, behavioral, electrophysiological, and proteomic alterations caused by deletion of the tmem35 gene. While capable of mounting a normal corticosterone response to restraint stress, KO mice showed elevated basal corticosterone accompanied by increased anxiety-like behavior. The KO mice also displayed impairment of hippocampus-dependent fear and spatial memories. Given the intact memory acquisition but a deficit in memory retention in the KO mice, TMEM35 is likely required for long-term memory consolidation. This conclusion is further supported by a loss of long-term potentiation in the Schaffer collateral-CA1 pathway in the KO mice. To identify putative molecular pathways underlying alterations in plasticity, proteomic analysis of synaptosomal proteins revealed lower levels of postsynaptic molecules important for synaptic plasticity in the KO hippocampus, including PSD95 and N-methyl-d-aspartate receptors. Pathway analysis (Ingenuity Pathway Analysis) of differentially expressed synaptic proteins in tmem35 KO hippocampus implicated molecular networks associated with specific cellular and behavioral functions, including decreased long-term potentiation, and increased startle reactivity and locomotion. Collectively, these data suggest that TMEM35 is a novel factor required for normal activity of the HPA axis and limbic circuitry.

Prenatal Choline Supplementation Diminishes Early-Life Iron Deficiency-Induced Reprogramming of Molecular Networks Associated with Behavioral Abnormalities in the Adult Rat Hippocampus.

BACKGROUND: Early-life iron deficiency is a common nutrient deficiency worldwide. Maternal iron deficiency increases the risk of schizophrenia and autism in the offspring. Postnatal iron deficiency in young children results in cognitive and socioemotional abnormalities in adulthood despite iron treatment. The rat model of diet-induced fetal-neonatal iron deficiency recapitulates the observed neurobehavioral deficits. OBJECTIVES: We sought to establish molecular underpinnings for the persistent psychopathologic effects of early-life iron deficiency by determining whether it permanently reprograms the hippocampal transcriptome. We also assessed the effects of maternal dietary choline supplementation on the offspring's hippocampal transcriptome to identify pathways through which choline mitigates the emergence of long-term cognitive deficits. METHODS: Male rat pups were made iron deficient (ID) by providing pregnant and nursing dams an ID diet (4 g Fe/kg) from gestational day (G) 2 through postnatal day (PND) 7 and an iron-sufficient (IS) diet (200 g Fe/kg) thereafter. Control pups were provided IS diet throughout. Choline (5 g/kg) was given to half the pregnant dams in each group from G11 to G18. PND65 hippocampal transcriptomes were assayed by next generation sequencing (NGS) and analyzed with the use of knowledge-based Ingenuity Pathway Analysis. Real-time polymerase chain reaction was performed to validate a subset of altered genes. RESULTS: Formerly ID rats had altered hippocampal expression of 619 from >10,000 gene loci sequenced by NGS, many of which map onto molecular networks implicated in psychological disorders, including anxiety, autism, and schizophrenia. There were significant interactions between iron status and prenatal choline treatment in influencing gene expression. Choline supplementation reduced the effects of iron deficiency, including those on gene networks associated with autism and schizophrenia. CONCLUSIONS: Fetal-neonatal iron deficiency reprograms molecular networks associated with the pathogenesis of neurologic and psychological disorders in adult rats. The positive response to prenatal choline represents a potential adjunctive therapeutic supplement to the high-risk group.

Metabotropic Glutamate Receptor and Fragile X Signaling in a Female Model of Escalated Aggression.

BACKGROUND: Escalated aggression is a behavioral sign of numerous psychiatric disorders characterized by a loss of control. The neurobiology underlying escalated aggression is unknown and is particularly understudied in females. Research in our laboratory demonstrated that repeated aggressive experience in female hamsters resulted in an escalated response to future aggressive encounters and an increase in dendritic spine density on nucleus accumbens (NAc) neurons. We hypothesized that the activation of group I metabotropic glutamate receptor signaling though the fragile X mental retardation protein (FMRP) pathway may underlie synaptic plasticity associated with aggression escalation. METHODS: Female hamsters were given five daily aggression tests with or without prior treatment with the metabotropic glutamate receptor 5 (mGluR5) antagonist 2-methyl-6-(phenylethynyl)-pyridine. Following aggression testing, messenger RNA expression and protein levels were measured in the nucleus accumbens for postsynaptic density protein 95 (PSD-95) and SAP90/PSD-95-associated protein 3, as well as the levels of phosphorylated FMRP. RESULTS: Experience-dependent escalation of aggression in female hamsters depends on activation of mGluR5 receptors. Furthermore, aggressive experience decreases phosphorylation of FMRP in the NAc, which is coupled to a long-term increase in the expression of the synaptic scaffolding proteins PSD-95 and SAP90/PSD-95-associated protein 3. Finally, the experience-dependent increase in PSD-95 is prevented by antagonism of the mGluR5 receptor. CONCLUSIONS: Activation of the FMRP pathway by group I metabotropic glutamate receptors is involved in regulating synaptic plasticity following aggressive experience. The NAc is a novel target for preclinical studies of the treatment of escalated aggression, with the added benefit that emerging therapeutic approaches are likely to be effective in treating pathologic aggression in both female and male subjects.

Social conditioned place preference in the captive ground squirrel (Ictidomys tridecemlineatus): Social reward as a natural phenotype.

Social behaviors of wild animals are often considered within an ultimate framework of adaptive benefits versus survival risks. By contrast, studies of laboratory animals more typically focus on affective aspects of behavioral decisions, whether a rodent derives a rewarding experience from social encounter, and how this experience might be initiated and maintained by neural circuits. Artificial selection and inbreeding have rendered laboratory animals more affiliative and less aggressive than their wild conspecifics, leaving open the possibility that social reward is an artifact of domestication. We compared social behaviors of wild and captive population of juvenile 13-lined ground squirrels (Ictidomys tridecemlineatus), the latter being 2nd- and 3rd-generation descendants of wild individuals. At an age corresponding to emergence from the burrow, postnatal day (PD) 38, captive squirrels engaged in vigorous social approach and play and these juvenile behaviors declined significantly by PD 56. Similarly, young wild squirrels expressed social proximity and play; affiliative interactions declined with summer's progression and were replaced by agonistic chasing behaviors. Social conditioned place preference testing (conditioned PDs 40-50) indicated that adolescent squirrels derived a rewarding experience from social reunion. Our results support the contention that undomesticated rodents have the capacity for social reward and more generally suggest the possibility that positive affective experiences may support group cohesion, social cooperation, and altruism in the wild.

Fetal iron deficiency induces chromatin remodeling at the Bdnf locus in adult rat hippocampus.

Fetal and subsequent early postnatal iron deficiency causes persistent impairments in cognitive and affective behaviors despite prompt postnatal iron repletion. The long-term cognitive impacts are accompanied by persistent downregulation of brain-derived neurotrophic factor (BDNF), a factor critical for hippocampal plasticity across the life span. This study determined whether early-life iron deficiency epigenetically modifies the Bdnf locus and whether dietary choline supplementation during late gestation reverses these modifications. DNA methylation and histone modifications were assessed at the Bdnf-IV promoter in the hippocampus of rats [at postnatal day (PND) 65] that were iron-deficient (ID) during the fetal-neonatal period. Iron deficiency was induced in rat pups by providing pregnant and nursing dams an ID diet (4 mg/kg Fe) from gestational day (G) 2 through PND7, after which iron deficiency was treated with an iron-sufficient (IS) diet (200 mg/kg Fe). This paradigm resulted in about 60% hippocampal iron loss on PND15 with complete recovery by PND65. For choline supplementation, pregnant rat dams were given dietary choline (5 g/kg) from G11 through G18. DNA methylation was determined by quantitative sequencing of bisulfite-treated DNA, revealing a small alteration at the Bdnf-IV promoter. Chromatin immunoprecipitation analysis showed increased HDAC1 binding accompanied by reduced binding of RNA polymerase II and USF1 at the Bdnf-IV promoter in formerly ID rats. These changes were correlated with altered histone methylations. Prenatal choline supplementation reverses these epigenetic modifications. Collectively, the findings identify epigenetic modifications as a potential mechanism to explicate the long-term repression of Bdnf following fetal and early postnatal iron deficiency.

Prenatal choline supplementation ameliorates the long-term neurobehavioral effects of fetal-neonatal iron deficiency in rats.

BACKGROUND: Gestational iron deficiency in humans and rodents produces long-term deficits in cognitive and socioemotional function and alters expression of plasticity genes in the hippocampus that persist despite iron treatment. Prenatal choline supplementation improves cognitive function in other rodent models of developmental insults. OBJECTIVE: The objective of this study was to determine whether prenatal choline supplementation prevents the long-term effects of fetal-neonatal iron deficiency on cognitive and social behaviors and hippocampal gene expression. METHODS: Pregnant rat dams were administered an iron-deficient (2-6 g/kg iron) or iron-sufficient (IS) (200 g/kg iron) diet from embryonic day (E) 3 to postnatal day (P) 7 with or without choline supplementation (5 g/kg choline chloride, E11-18). Novel object recognition (NOR) in the test vs. acquisition phase, social approach (SA), and hippocampal mRNA expression were compared at P65 in 4 male adult offspring groups: formerly iron deficient (FID), FID with choline supplementation (FID-C), IS, and IS with choline supplementation. RESULTS: Relative to the intact NOR in IS rats (acquisition: 47.9%, test: 60.2%, P < 0.005), FID adult rats had impaired recognition memory at the 6-h delay (acquisition: 51.4%, test: 55.1%, NS), accompanied by a 15% reduction in hippocampal expression of brain-derived neurotrophic factor (Bdnf) (P < 0.05) and myelin basic protein (Mbp) (P < 0.05). Prenatal choline supplementation in FID rats restored NOR (acquisition: 48.8%, test: 64.4%, P < 0.0005) and increased hippocampal gene expression (FID-C vs. FID group: Bdnf, Mbp, P < 0.01). SA was also reduced in FID rats (P < 0.05 vs. IS rats) but was only marginally improved by prenatal choline supplementation. CONCLUSIONS: Deficits in recognition memory, but not social behavior, resulting from gestational iron deficiency are attenuated by prenatal choline supplementation, potentially through preservation of hippocampal Bdnf and Mbp expression. Prenatal choline supplementation may be a promising adjunct treatment for fetal-neonatal iron deficiency.

Multigenerational effects of fetal-neonatal iron deficiency on hippocampal BDNF signaling.

Fetal-neonatal iron deficiency induces adult learning impairments concomitant with changes in expression of key genes underlying hippocampal learning and memory in spite of neonatal iron replenishment. Notably, expression of brain-derived neurotrophic factor (BDNF), a gene critical for neuronal maturation and synaptic plasticity, is lowered both acutely and in adulthood following early-life iron deficiency. Although the mechanism behind its long-term downregulation remains unclear, epigenetic modification in BDNF, as seen in other models of early-life adversity, may play a role. Given that early iron deficiency occurs during critical periods in both hippocampal and gonadal development, we hypothesized that the iron-sufficient offspring (F2 IS) of formerly iron-deficient (F1 FID) rats would show a similar suppression of the BDNF gene as their parents. We compared hippocampal mRNA levels of BDNF and functionally related genes among F1 IS, F1 ID, and F2 IS male rats at postnatal day (P) 15 and P65 using RT-qPCR. As expected, the F1 ID group showed a downregulation of BDNF and associated genes acutely at P15 and chronically at P65. However, the F2 IS group showed an upregulation of these genes at P15, returning to control levels at P65. These results demonstrate that adverse effects of early iron deficiency on hippocampal gene expression observed in the F1 are not present in the F2 generation, suggesting differential effects of nutritionally induced epigenetic programing during the critical periods of hippocampal and gonadal development.

Social influences on morphine-conditioned place preference in adolescent BALB/cJ and C57BL/6J mice.

RATIONALE: Among human adolescents, drug use is substantially influenced by the attitudes and behaviors of peers. Social factors also affect the drug-seeking behaviors of laboratory animals. Conditioned place preference (CPP) experiments indicate that social context can influence the degree to which rodents derive a rewarding experience from drugs of abuse. However, the precise manner by which social factors alter drug reward in adolescent rodents remains unknown. OBJECTIVES: We employed the relatively asocial BALB/cJ (BALB) mouse strain and the more prosocial C57BL/6J (B6) strain to explore whether "low" or "high" motivation to be with peers influences the effects of social context on morphine CPP (MCPP). METHODS: Adolescent mice were conditioned by subcutaneous injections of morphine sulfate (0.25, 1.0, or 5.0 mg/kg). During the MCPP procedure, mice were housed in either isolation (Ih) or within a social group (Sh). Similarly, following injection, mice were conditioned either alone (Ic) or within a social group (Sc). RESULTS: Adolescent B6 mice expressed a robust MCPP response except when subjected to Ih-Sc, which indicates that, following isolation, mice with high levels of social motivation are less susceptible to the rewarding properties of morphine when they are conditioned in a social group. By contrast, MCPP responses of BALB mice were most sensitive to morphine conditioning when subjects experienced a change in their social environment between housing and conditioning (Ih-Sc or Sh-Ic). CONCLUSIONS: Our findings demonstrate that susceptibility to morphine-induced reward in adolescent mice is moderated by a complex interaction between social context and heritable differences in social motivation.

Age-dependent and strain-dependent influences of morphine on mouse social investigation behavior.

Opioid-coded neural circuits play a substantial role in how individuals respond to drugs of abuse. Most individuals begin using such drugs during adolescence and within a social context. Several studies indicate that adolescent mice exhibit a heightened sensitivity to the effects of morphine, a prototypical opiate drug, but it is unclear whether these developmental differences are related to aspects of motivated behavior. Moreover, exposure to opioids within the rodent brain can alter the expression of social behavior, yet little is known about whether this relationship changes as a function of development or genetic variation. In this study, we conducted a series of experiments to characterize the relationship between genetic background, adolescent development and morphine-induced changes in mouse social investigation (SI). At two time points during adolescent development [postnatal days (PD) 25 and 45], social interactions of test mice of the gregarious C57BL/6J (B6) strain were more tolerant to the suppressive effects of morphine [effective dose 50 (ED50)=0.97 mg/kg and 2.17 mg/kg morphine, respectively] than test mice from the less social BALB/cJ (BALB) strain (ED50=0.61 mg/kg and 0.91 mg/kg morphine, respectively). By contrast, this strain-dependent difference was not evident among adult mice on PD 90 (ED50=1.07 mg/kg and 1.41 mg/kg morphine for BALB and B6 mice, respectively). An additional experiment showed that the ability of morphine to alter social responsiveness was not directly related to drug-induced changes in locomotor behavior. Finally, administration of morphine to stimulus mice on PD 25 reduced social investigation of test mice only when individuals were from the B6 genetic background. Overall, these results indicate that alterations in endogenous opioid systems are related to changes in SI that occur during adolescence, and that morphine administration may mimic rewarding aspects of social encounter.

Differential entrainment of a social rhythm in adolescent mice.

Daily routines in animal activities range from sleep-wake cycles, to foraging bouts, to social interactions. Among animals living within groups, it is unclear whether the motivations that underlie social interactions respond to daily light-dark (LD) cycles or endogenous circadian rhythms. Employing two mouse strains (BALB/cJ [BALB] and C57BL/6J [B6]) with genetically based differences in social affect and circadian rhythms, we examined how social investigation (SI) is modulated by social deprivation and circadian factors. We found a genetic influence on SI that was moderated by the preceding duration of social deprivation, requiring 3-6 h of social isolation prior to testing. Following 6h of social deprivation, the SI responses of adolescent B6 mice were greater than those of BALB mice only when the isolation period was imposed during the dark phase of the LD cycle. When B6 mice were weaned into conditions of constant darkness, a novel, endogenous social rhythm emerged, which was characterized by two pronounced peaks of social responsiveness (relative to one peak under LD entrainment) that were separated by 12-h intervals. Irrespective of the lighting conditions during social isolation, the SI responses of adolescent BALB mice did not oscillate across the day. Similar strain-dependent patterns of sociability were evident within groups of mice that were left undisturbed in their home cage under LD entrainment or constant darkness. Overall, genetic influences on the social phenotypes of adolescent mice are thus moderated by an interaction between social deprivation and oscillations of an endogenous social rhythm that entrains to the LD cycle.