Chromatin accessibility and H3K9me3 landscapes reveal long-term epigenetic effects of fetal-neonatal iron deficiency in rat hippocampus.

BACKGROUND: Iron deficiency (ID) during the fetal-neonatal period results in long-term neurodevelopmental impairments associated with pervasive hippocampal gene dysregulation. Prenatal choline supplementation partially normalizes these effects, suggesting an interaction between iron and choline in hippocampal transcriptome regulation. To understand the regulatory mechanisms, we investigated epigenetic marks of genes with altered chromatin accessibility (ATAC-seq) or poised to be repressed (H3K9me3 ChIP-seq) in iron-repleted adult rats having experienced fetal-neonatal ID exposure with or without prenatal choline supplementation. RESULTS: Fetal-neonatal ID was induced by limiting maternal iron intake from gestational day (G) 2 through postnatal day (P) 7. Half of the pregnant dams were given supplemental choline (5.0 g/kg) from G11-18. This resulted in 4 groups at P65 (Iron-sufficient [IS], Formerly Iron-deficient [FID], IS with choline [ISch], and FID with choline [FIDch]). Hippocampi were collected from P65 iron-repleted male offspring and analyzed for chromatin accessibility and H3K9me3 enrichment. 22% and 24% of differentially transcribed genes in FID- and FIDch-groups, respectively, exhibited significant differences in chromatin accessibility, whereas 1.7% and 13% exhibited significant differences in H3K9me3 enrichment. These changes mapped onto gene networks regulating synaptic plasticity, neuroinflammation, and reward circuits. Motif analysis of differentially modified genomic sites revealed significantly stronger choline effects than early-life ID and identified multiple epigenetically modified transcription factor binding sites. CONCLUSIONS: This study reveals genome-wide, stable epigenetic changes and epigenetically modifiable gene networks associated with specific chromatin marks in the hippocampus, and lays a foundation to further elucidate iron-dependent epigenetic mechanisms that underlie the long-term effects of fetal-neonatal ID, choline, and their interactions.

Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition.

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes (Ntng2, Mfrp, Nr4a2, Thbs1, Atxn1, and Atxn3) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD.

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.

Recurrent Moderate Hypoglycemia Suppresses Brain-Derived Neurotrophic Factor Expression in the Prefrontal Cortex and Impairs Sensorimotor Gating in the Posthypoglycemic Period in Young Rats.

Recurrent hypoglycemia is common in infants and children. In developing rat models, recurrent moderate hypoglycemia leads to neuronal injury in the medial prefrontal cortex. To understand the effects beyond neuronal injury, 3-week-old male rats were subjected to 5 episodes of moderate hypoglycemia (blood glucose concentration, approx. 30 mg/dl for 90 min) once daily from postnatal day 24 to 28. Neuronal injury was determined using Fluoro-Jade B histochemistry on postnatal day 29. The effects on brain-derived neurotrophic factor (BDNF) and its cognate receptor, tyrosine kinase receptor B (TrkB) expression, which is critical for prefrontal cortex development, were determined on postnatal day 29 and at adulthood. The effects on prefrontal cortex-mediated function were determined by assessing the prepulse inhibition of the acoustic startle reflex on postnatal day 29 and 2 weeks later, and by testing for fear-potentiated startle at adulthood. Recurrent hypoglycemia led to neuronal injury confined primarily to the medial prefrontal cortex. BDNF/TrkB expression in the prefrontal cortex was suppressed on postnatal day 29 and was accompanied by lower prepulse inhibition, suggesting impaired sensorimotor gating. Following the cessation of recurrent hypoglycemia, the prepulse inhibition had recovered at 2 weeks. BDNF/TrkB expression in the prefrontal cortex had normalized and fear-potentiated startle was intact at adulthood. Recurrent moderate hypoglycemia during development has significant adverse effects on the prefrontal cortex in the posthypoglycemic period.

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.

Effects of age, but not sex, on elevated startle during withdrawal from acute morphine in adolescent and adult rats.

Investigations into animal models of drug withdrawal have largely found that emotional signs of withdrawal (e.g. anxiety, anhedonia, and aversion) in adolescents are experienced earlier and less severely than in their adult counterparts. The majority of these reports have examined withdrawal from ethanol or nicotine. To expand our knowledge about the emotional withdrawal state in adolescent rats, we used potentiation of the acoustic startle reflex after an acute dose of morphine (10 mg/kg, subcutaneously) as a measure of opiate withdrawal. Startle was measured at four time points after morphine injection (2, 3, 4, and 5 h) in 28-day-old and 90-day-old male and female rats. The results of this experiment revealed that peak potentiation of the startle reflex occurred at 3 h in the adolescent rats and at 5 h in the adult rats, and that the magnitude of withdrawal was larger in the adults. No sex differences were observed. Overall, these results affirm that, similar to withdrawal from ethanol and nicotine, opiate withdrawal signs are less severe in adolescent than in adult rats.

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.

Iron deficiency with or without anemia impairs prepulse inhibition of the startle reflex.

Iron deficiency (ID) during early life causes long-lasting detrimental cognitive sequelae, many of which are linked to alterations in hippocampus function, dopamine synthesis, and the modulation of dopaminergic circuitry by the hippocampus. These same features have been implicated in the origins of schizophrenia, a neuropsychiatric disorder with significant cognitive impairments. Deficits in sensorimotor gating represent a reliable endophenotype of schizophrenia that can be measured by prepulse inhibition (PPI) of the acoustic startle reflex. Using two rodent model systems, we investigated the influence of early-life ID on PPI in adulthood. To isolate the role of hippocampal iron in PPI, our mouse model utilized a timed (embryonic day 18.5), hippocampus-specific knockout of Slc11a2, a gene coding an important regulator of cellular iron uptake, the divalent metal transport type 1 protein (DMT-1). Our second model used a classic rat dietary-based global ID during gestation, a condition that closely mimics human gestational ID anemia (IDA). Both models exhibited impaired PPI in adulthood. Furthermore, our DMT-1 knockout model displayed reduced long-term potentiation (LTP) and elevated paired-pulse facilitation (PPF), electrophysiological results consistent with previous findings in the IDA rat model. These results, in combination with previous findings demonstrating impaired hippocampus functioning and altered dopaminergic and glutamatergic neurotransmission, suggest that iron availability within the hippocampus is critical for the neurodevelopmental processes underlying sensorimotor gating. Ultimately, evidence of reduced PPI in both of our models may offer insights into the roles of fetal ID and the hippocampus in the pathophysiology of schizophrenia.

Causally mapping human threat extinction relevant circuits with depolarizing brain stimulation methods.

Laboratory threat extinction paradigms and exposure-based therapy both involve repeated, safe confrontation with stimuli previously experienced as threatening. This fundamental procedural overlap supports laboratory threat extinction as a compelling analogue of exposure-based therapy. Threat extinction impairments have been detected in clinical anxiety and may contribute to exposure-based therapy non-response and relapse. However, efforts to improve exposure outcomes using techniques that boost extinction - primarily rodent extinction - have largely failed to date, potentially due to fundamental differences between rodent and human neurobiology. In this review, we articulate a comprehensive pre-clinical human research agenda designed to overcome these failures. We describe how connectivity guided depolarizing brain stimulation methods (i.e., TMS and DBS) can be applied concurrently with threat extinction and dual threat reconsolidation-extinction paradigms to causally map human extinction relevant circuits and inform the optimal integration of these methods with exposure-based therapy. We highlight candidate targets including the amygdala, hippocampus, ventromedial prefrontal cortex, dorsal anterior cingulate cortex, and mesolimbic structures, and propose hypotheses about how stimulation delivered at specific learning phases could strengthen threat extinction.

An anatomical basis for opponent process mechanisms of opiate withdrawal.

Opponent process theory predicts that the first step in the induction of drug withdrawal is the activation of reward-related circuitry. Using the acoustic startle reflex as a model of anxiety-like behavior in rats, we show the emergence of a negative affective state during withdrawal after direct infusion of morphine into the ventral tegmental area (VTA), the origin of the mesolimbic dopamine system. Potentiation of startle during withdrawal from systemic morphine exposure requires a decrease in opiate receptor stimulation in the VTA and can be relieved by administration of the dopamine receptor agonist apomorphine. Together, our results suggest that the emergence of anxiety during withdrawal from acute opiate exposure begins with activation of VTA mesolimbic dopamine circuitry, providing a mechanism for the opponent process view of withdrawal.

Canonical TGF-beta signaling is required for the balance of excitatory/inhibitory transmission within the hippocampus and prepulse inhibition of acoustic startle.

Smad4 is a unique nuclear transducer for all TGF-beta signaling pathways and regulates gene transcription during development and tissue homeostasis. To elucidate the postnatal role of TGF-beta signaling in the mammalian brain, we generated forebrain-specific Smad4 knock-out mice. Surprisingly, the mutants showed no alteration in long-term potentiation and water maze, suggesting that Smad4 is not required for spatial learning and memory. However, these mutant mice did show enhancement of paired-pulse facilitation in excitatory synaptic transmission and stronger paired-pulse depression of GABA(A) currents in the hippocampus. The alteration of hippocampal electrophysiology correlated with mouse hyperactivity in homecage and open field tests. Mutant mice also showed overgrooming as well as deficits of prepulse inhibition, a widely used endophenotype of schizophrenia. With a specific real-time PCR array focused on TGF-beta signaling pathway, we identified a novel regulation mechanism of the pathway in the hippocampal neurons, in which Smad4-mediated signaling suppresses the level of extracellular antagonism of TGF-beta ligands through transcriptional regulation of follistatin, a selective inhibitor to activin/TGF-beta signaling in the hippocampus. In summary, we suggest that the canonical TGF-beta signaling pathway is critical for use-dependent modulation of GABA(A) synaptic transmission and dendritic homeostasis; furthermore, a disruption in the balance of the excitatory and inhibitory hippocampal network can result in psychiatric-like behavior.

Behavioral predictors of individual differences in opioid addiction vulnerability as measured using i.v. self-administration in rats.

BACKGROUND: Like other forms of psychopathology, vulnerability to opioid addiction is subject to wide individual differences. Animal behavioral models are valuable in advancing our understanding of mechanisms underlying vulnerability to the disorder's development and amenability to treatment. METHODS: This review provides an overview of preclinical work on behavioral predictors of opioid addiction vulnerability as measured using the intravenous (i.v.) self-administration (SA) model in rats. We also highlight several new approaches to studying individual differences in opioid addiction vulnerability in preclinical models that could have greater sensitivity and lead to more clinically relevant findings. RESULTS AND CONCLUSIONS: Evidence for the relationship between various behavioral traits and opioid SA in the preclinical literature is limited. With the possible exceptions of sensitivity to opioid agonist/withdrawal effects and stress reactivity, predictors of individual differences in SA of other drugs of abuse (e.g. sensation-seeking, impulsivity) do not predict vulnerability to opioid SA in rats. Refinement of SA measures and the use of multivariate designs and statistics could help identify predictors of opioid SA and lead to more clinically relevant studies on opioid addiction vulnerability.

Individual Differences in Different Measures of Opioid Self-Administration in Rats Are Accounted for by a Single Latent Variable.

Individual differences in vulnerability to addiction have been widely studied through factor analysis (FA) in humans, a statistical method that identifies "latent" variables (variables that are not measured directly) that reflect the common variance among a larger number of observed measures. Despite its widespread application in behavioral genetics, FA has not been used in preclinical opioid addiction research. The current study used FA to examine the latent factor structure of four measures of i.v. morphine self-administration (MSA) in rats (i.e., acquisition, demand elasticity, morphine/cue- and stress/cue-induced reinstatement). All four MSA measures are generally assumed in the preclinical literature to reflect "addiction vulnerability," and individual differences in multiple measures of abuse liability are best accounted for by a single latent factor in some human studies. A one-factor model was therefore fitted to the data. Two different regularized FAs indicated that a one-factor model fit our data well. Acquisition, elasticity of demand and morphine/cue-induced reinstatement loaded significantly onto a single latent factor while stress/cue-induced reinstatement did not. Consistent with findings from some human studies, our results indicated a common drug "addiction" factor underlying several measures of opioid SA. However, stress/cue-induced reinstatement loaded poorly onto this factor, suggesting that unique mechanisms mediate individual differences in this vs. other MSA measures. Further establishing FA approaches in drug SA and in preclinical neuropsychopathology more broadly will provide more reliable, clinically relevant core factors underlying disease vulnerability in animal models for further genetic analyses.

Sex differences in adult social, cognitive, and affective behavioral deficits following neonatal phlebotomy-induced anemia in mice.

INTRODUCTION: Anemia is common in prematurely born infants due to blood loss resulting from frequent phlebotomies and may contribute to their neurobehavioral deficits. Preclinical models of phlebotomy-induced anemia (PIA) have revealed metabolic and genomic changes in multiple brain structures of young mice, yet the impact of neonatal PIA on early-life and adult behavior has not been assessed. METHODS: The present study employed a range of behavioral measures in phlebotomized anemic neonatal mice to investigate short- and long-term neurodevelopmental effects. PIA from postnatal (P) days 3 to 14 caused sex-specific changes in social behavior, novelty preference, and anxiety at P17 that persisted into adulthood. RESULTS: Our preclinical model suggests that PIA may contribute to acute and long-term behavioral and affective deficits and warrants further substantiation of the observed behavioral phenomena in larger samples. CONCLUSIONS: We conclude that this model is a useful tool for beginning to better understand the lasting effect that early-life PIA might have on the developing brain. The differential impact of PIA on male and female subjects warrants further exploration for the development of appropriately targeted interventions.

Prenatal Iron Deficiency and Choline Supplementation Interact to Epigenetically Regulate Jarid1b and Bdnf in the Rat Hippocampus into Adulthood.

Early-life iron deficiency (ID) causes long-term neurocognitive impairments and gene dysregulation that can be partially mitigated by prenatal choline supplementation. The long-term gene dysregulation is hypothesized to underlie cognitive dysfunction. However, mechanisms by which iron and choline mediate long-term gene dysregulation remain unknown. In the present study, using a well-established rat model of fetal-neonatal ID, we demonstrated that ID downregulated hippocampal expression of the gene encoding JmjC-ARID domain-containing protein 1B (JARID1B), an iron-dependent histone H3K4 demethylase, associated with a higher histone deacetylase 1 (HDAC1) enrichment and a lower enrichment of acetylated histone H3K9 (H3K9ac) and phosphorylated cAMP response element-binding protein (pCREB). Likewise, ID reduced transcriptional capacity of the gene encoding brain-derived neurotrophic factor (BDNF), a target of JARID1B, associated with repressive histone modifications such as lower H3K9ac and pCREB enrichments at the Bdnf promoters in the adult rat hippocampus. Prenatal choline supplementation did not prevent the ID-induced chromatin modifications at these loci but induced long-lasting repressive chromatin modifications in the iron-sufficient adult rats. Collectively, these findings demonstrated that the iron-dependent epigenetic mechanism mediated by JARID1B accounted for long-term Bdnf dysregulation by early-life ID. Choline supplementation utilized a separate mechanism to rescue the effect of ID on neural gene regulation. The negative epigenetic effects of choline supplementation in the iron-sufficient rat hippocampus necessitate additional investigations prior to its use as an adjunctive therapeutic agent.

Repeated morphine exposure activates synaptogenesis and other neuroplasticity-related gene networks in the dorsomedial prefrontal cortex of male and female rats.

BACKGROUND: Opioid abuse is a chronic disorder likely involving stable neuroplastic modifications. While a number of molecules contributing to these changes have been identified, the broader spectrum of genes and gene networks that are affected by repeated opioid administration remain understudied. METHODS: We employed Next-Generation RNA-sequencing (RNA-seq) followed by quantitative chromatin immunoprecipitation to investigate changes in gene expression and their regulation in adult male and female rats' dorsomedial prefrontal cortex (dmPFC) after a regimen of daily injection of morphine (5.0 mg/kg; 10 days). Ingenuity Pathway Analysis (IPA) was used to analyze affected molecular pathways, gene networks, and associated regulatory factors. A complementary behavioral study evaluated the effects of the same morphine injection regimen on locomotor activity, pain sensitivity, and somatic withdrawal signs. RESULTS: Behaviorally, repeated morphine injection induced locomotor hyperactivity and hyperalgesia in both sexes. 90 % of differentially expressed genes (DEGs) in morphine-treated rats were upregulated in both males and females, with a 35 % overlap between sexes. A substantial number of DEGs play roles in synaptic signaling and neuroplasticity. Chromatin immunoprecipitation revealed enrichment of H3 acetylation, a transcriptionally activating chromatin mark. Although broadly similar, some differences were revealed in the gene ontology networks enriched in females and males. CONCLUSIONS: Our results cohere with findings from previous studies based on a priori gene selection. Our results also reveal novel genes and molecular pathways that are upregulated by repeated morphine exposure, with some common to males and females and others that are sex-specific.

Higher anhedonia during withdrawal from initial opioid exposure is protective against subsequent opioid self-administration in rats.

RATIONALE: Understanding factors contributing to individual differences in vulnerability to opioid addiction is essential for developing more effective preventions and treatments, yet few reliable behavioral predictors of subsequent opioid self-administration have been identified in rodents. Sensitivity to the acute effects of initial drug exposure predicts later addiction vulnerability in both humans and animals, but the relationship between sensitivity to withdrawal from initial drug exposure and later drug use vulnerability is unclear. OBJECTIVE: The goal of the current study was to evaluate whether the degree of anhedonia experienced during withdrawal from early opioid exposure predicts subsequent vulnerability to opioid self-administration. METHODS: Rats were first tested for withdrawal sensitivity following acute injections of morphine (i.e., "acute dependence"), measured as elevations in intracranial self-stimulation (ICSS) thresholds (anhedonia-like behavior) during naloxone-precipitated and spontaneous withdrawal. Rats were then tested for addiction-like behavior using various measures of i.v. morphine self-administration (MSA) including acquisition, demand, extinction, and reinstatement induced by morphine, stress, and/or drug-associated cues. RESULTS: Greater naloxone-precipitated withdrawal across repeated morphine injections and greater peak spontaneous withdrawal severity following a single morphine injection were associated with lower addiction-like behavior on multiple MSA measures. Withdrawal-induced anhedonia predicted a wider range of MSA measures than did any individual measure of MSA itself. CONCLUSIONS: Our data establish WIA as one of the first behavioral measures to predict individual differences in opioid SA in rodents. This model promises to be useful for furthering our understanding of behavioral and neurobiological mechanisms underlying vulnerability to opioid addiction.

Sociality deficits in serine racemase knockout mice.

BACKGROUND: Studies of schizophrenia have pointed to the role of glutamate in its pathophysiology. Mice lacking D-serine show impairments in neurotransmission through NMDA receptors and display behaviors consistent with features of schizophrenia. Yet, socio-communicative deficits, a characteristic of schizophrenia, have not been reported in serine racemase knockout mice. METHODS: We use behavioral testing (the three-chambered social approach task, the dyadic interaction task, and the novel object recognition task) to examine socio-communicative behaviors in these mice. RESULTS: Serine racemase mice show abnormal social investigation and approach behavior, and differ from wild-type controls in the duration and number of vocalizations they emit in the presence of a conspecific. Serine racemase knockout mice were not impaired in a cognitive test (novel object recognition), although they displayed abnormal behavior in the acquisition phase of the task. CONCLUSIONS: Serine racemase knockout mice demonstrate abnormalities in socio-communicative behaviors consistent with an impairment in sociality, a negative symptom of schizophrenia.

Effects of the NMDA receptor antagonist memantine on the expression and development of acute opiate dependence as assessed by withdrawal-potentiated startle and hyperalgesia.

RATIONALE: While the N-methyl-D: -aspartate (NMDA) glutamate receptor has been strongly implicated in chronic opiate dependence, relatively few studies have examined the effects of NMDA receptor antagonists on withdrawal from acute opiate exposure. OBJECTIVES: The current study examined the effects of memantine, a well-tolerated NMDA receptor antagonist, on acute opiate dependence as assessed by elevations in rodent startle responding (i.e., "withdrawal-potentiated startle") and increased pain sensitivity (i.e., hyperalgesia). RESULTS: Administration of memantine either attenuated (5 mg/kg) or blocked (10 mg/kg) the expression of withdrawal-potentiated startle during naloxone (2.5 mg/kg)-precipitated withdrawal from a single dose of morphine sulfate (10 mg/kg). Pre-treatment with the NMDA receptor antagonist also inhibited the exacerbation of withdrawal-potentiated startle across repeated acute opiate exposures. Memantine blocked the expression of acute dependence, but was less effective in inhibiting its escalation, when hyperalgesia was used as a measure of withdrawal. These doses of memantine did not affect startle responding or nociception in otherwise drug-free animals. Data from additional control groups indicated that the effects of memantine on the expression of withdrawal were not influenced by nonspecific interactions between the NMDA antagonist and either morphine or naloxone. CONCLUSIONS: These findings suggest that the NMDA receptor may play a key role in the earliest stages of opiate dependence and provide further evidence that memantine may be useful for the treatment of opiate withdrawal.