Anxiety-like Behavior In Elevated Research Articles

Changes in Corticotropin-Releasing Factor Receptor Type 1, Co-Expression with Tyrosine Hydroxylase and Oxytocin Neurons, and Anxiety-Like Behaviors across the Postpartum Period in Mice

Introduction: Corticotropin-releasing factor and its primary receptor (CRFR1) are critical regulators of behavioral and neuroendocrine stress responses. CRFR1 has also been associated with stress-related behavioral changes in postpartum mice. Our previous studies indicate dynamic changes in CRFR1 levels and coupling of CRFR1 with tyrosine hydroxylase (TH) and oxytocin (OT) neurons in postpartum mice. In this study, we aimed to determine the time course of these changes during the postpartum period. Methods: Using a CRFR1-GFP reporter mouse line, we compared postpartum mice at five time points with nulliparous mice. We performed immunohistochemistry to assess changes in CRFR1 levels and changes in co-expression of TH/CRFR1-GFP and OT/CRFR1-GFP across the postpartum period. Mice were also assessed for behavioral stress responses in the open field test. Results: Relative to nulliparous mice, CRFR1 levels were elevated in the anteroventral periventricular nucleus (AVPV/PeN) but were decreased in the medial preoptic area from postpartum day 1 (P1) through P28. In the paraventricular hypothalamus (PVN), there is a transient decline in CRFR1 mid-postpartum with a nadir at P7. Co-localization of CRFR1 with TH-expressing neurons was also altered with a transient decrease found in the AVPV/PeN at P7 and P14. Co-expression of CRFR1 and OT neurons of the PVN and supraoptic nucleus was dramatically altered with virtually no co-expression found in nulliparous mice, but levels increased shortly after parturition and peaked near P21. A transient decrease in open field center time was found at P7, indicating elevated anxiety-like behavior. Conclusion: This study revealed various changes in CRFR1 across the postpartum period, which may contribute to stress-related behavior changes in postpartum mice.

G protein–biased GPR3 signaling ameliorates amyloid pathology in a preclinical Alzheimer’s disease mouse model

Biased G protein-coupled receptor (GPCR) ligands, which preferentially activate G protein or β-arrestin signaling pathways, are leading to the development of drugs with superior efficacy and reduced side effects in heart disease, pain management, and neuropsychiatric disorders. Although GPCRs are implicated in the pathophysiology of Alzheimer's disease (AD), biased GPCR signaling is a largely unexplored area of investigation in AD. Our previous work demonstrated that GPR3-mediated β-arrestin signaling modulates amyloid-β (Aβ) generation invitro and that Gpr3 deficiency ameliorates Aβ pathology invivo. However, Gpr3-deficient mice display several adverse phenotypes, including elevated anxiety-like behavior, reduced fertility, and memory impairment, which are potentially associated with impaired G protein signaling. Here, we generated a G protein-biased GPR3 mouse model to investigate the physiological and pathophysiological consequences of selective elimination of GPR3-mediated β-arrestin signaling invivo. In contrast to Gpr3-deficient mice, G protein-biased GPR3 mice do not display elevated anxiety levels, reduced fertility, or cognitive impairment. We further determined that G protein-biased signaling reduces soluble Aβ levels and leads to a decrease in the area and compaction of amyloid plaques in the preclinical AppNL-G-F AD mouse model. The changes in amyloid pathology are accompanied by robust microglial and astrocytic hypertrophy, which suggest a protective glial response that may limit amyloid plaque development in G protein-biased GPR3 AD mice. Collectively, these studies indicate that GPR3-mediated G protein and β-arrestin signaling produce discrete and separable effects and provide proof of concept for the development of safer GPCR-targeting therapeutics with more directed pharmacological action for AD.

Effects of Septin-14 Gene Deletion on Adult Cognitive/Emotional Behavior.

While various septin GTPases have been reported for their physiological functions, their roles in orchestrating complex cognitive/emotional functions in adult mammals remained scarcely explored. A comprehensive behavioral test battery was administered to two sexes of 12-week-old Septin-14 (SEPT14) knockout (KO) and wild-type (WT) mice. The sexually dimorphic effects of brain SEPT14 KO on inhibitory avoidance (IA) and hippocampal mGluR5 expression were noticed with greater IA latency and elevated mGluR5 level exclusively in male KO mice. Moreover, SEPT14 KO appeared to be associated with stress-provoked anxiety increase in a stress-related navigation task regardless of animals’ sexes. While male and female WT mice demonstrated comparable cell proliferation in the dorsal and ventral hippocampal dentate gyrus (DG), both sexes of SEPT14 KO mice had increased cell proliferation in the ventral DG. Finally, male and female SEPT14 KO mice displayed dampened observational fear conditioning magnitude and learning-provoked corticosterone secretion as compared to their same-sex WT mice. These results, taken together, prompt us to conclude that male, but not female, mice lacking the Septin-14 gene may exhibit increased aversive emotion-related learning and dorsal/ventral hippocampal mGluR5 expressions. Moreover, deletion of SEPT14 may be associated with elevated ventral hippocampal DG cell proliferation and stress-provoked anxiety-like behavior, while dampening vicarious fear conditioning magnitudes.

7,8-DHF enhances SHH in the hippocampus and striatum during early abstinence but has minor effects on alcohol intake in IA2BC paradigm and abstinence-related anxiety-like behavior in rats

A mimetic of brain-derived neurotrophic factor (BDNF), 7,8-dihydroxyflavone (7,8-DHF), alleviates some aspects of alcohol abstinence after voluntary alcohol intake in rodents. The interaction between BDNF and sonic hedgehog (SHH) was demonstrated in adult brain in some situations, though relationship between BDNF and SHH during alcohol abstinence remains obscure. We aimed to study effect of 7,8-DHF on drinking pattern, anxiety-like behavior and expression of SHH and a downstream transcription factor, GLI, in the limbic brain structures during early abstinence after voluntary ethanol intake.Male Wistar rats were subjected to intermittent access to 20% ethanol in a two-bottle choice procedure (IA2BC). The animals experienced twenty 24-h sessions of free access to two-bottle choice (water or 20% ethanol) with 24-h withdrawal periods (water only); 7,8-DHF (5 mg/kg, i.p.) was administered one hour prior to each alcohol access session. Anxiety-like behavior was estimated in the open-field (OFT) and elevated plus-maze (EPM) tests on the first and second days of abstinence, respectively. The expression of SHH and GLI was analyzed on the third day after withdrawal in the frontal cortex, hippocampus and striatum.Repeated measures ANOVA did not show significant main effect of 7,8-DHF injections during IA2BC on ethanol intake and ethanol preference over water. As expected, pair-wise comparisons of OFT and EPM data by Mann-Whitney U test revealed elevated anxiety-like behavior during early abstinence in IA2BC paradigm as compared with control group. When all groups were included in the analysis, Kruskal-Wallis ANOVA did not show significant differences in time spent in the center zone of the OFT and number of entries. However, time spent in the open arms of the EPM but not number of entries differed significantly between the groups studied according to Kruskal-Wallis ANOVA. Factorial ANOVA followed by Tukey’s HSD post hoc test demonstrated significant elevation of SHH protein levels in the hippocampus and striatum but not in the frontal cortex of animals with access to ethanol and administered with 7,8-DHF as compared with respective animals without 7,8-DHF. GLI expression changed only in the hippocampus; its protein level increased in control animals administered 7,8-DHF.Thus, 7,8-DHF administration during IA2BC procedure induces region-specific levels elevation of SHH in the brain, but does not significantly change drinking pattern and anxiety-like behavior during early abstinence.

Acute serotonin 1B/1A receptor activation impairs behavioral flexibility in C57BL/6J mice

Pharmacological activation of the serotonin (5-HT) 1B and 5-HT1A receptors has been shown to induce OCD-like perseverative circling and locomotor stereotypy in rodents. Although, several studies have examined how activation of these receptors facilitates these motor-associated OCD-like behaviors, it is not known how acute 5-HT1B and 5-HT1A activation impacts behavioral inflexibility, a common trait related to OCD. The current study examined how acute 5-HT1B/1A receptor agonist RU24969 treatment at 0.01, 0.1, and 1.0 mg/kg impacted behavioral flexibility in both female and male C57BL/6J mice. Behavioral flexibility was tested using a spatial reversal learning task, with probabilistic reward contingencies. In addition, locomotor activity and anxiety-like behaviors were also measured. RU24969 at 0.1 and 1.0 mg/kg impaired behavioral flexibility in both female and male C57BL/6J mice. RU24969 treatment at 1.0 mg/kg reduced locomotor activity in male mice, although RU24969 treatment did not significantly reduce locomotor activity in female mice. In the open field, 1.0 mg/kg elevated anxiety-like behavior in male mice only. Overall, these results demonstrate that acute 5-HT1B and 5-HT1A receptor activation leads to impairments in behavioral flexibility, a common trait associated with OCD.

Prenatal Poly I:C Challenge Affects Behaviors and Neurotransmission via Elevated Neuroinflammation Responses in Female Juvenile Rats.

BackgroundExposure to polyriboinosinic-polyribocytidylic acid (Poly I:C) in pregnant rats has been reported to cause schizophrenia-like behaviors and abnormal neurotransmissions in adult, particularly male, offspring. However, what is less well understood are the effects of maternal Poly I:C exposure on adolescent behaviors and neurotransmission in female juvenile rats.MethodsFemale adolescent Poly I:C offspring were constructed by treating with 5 mg/kg Poly I:C on timed pregnant rats (gestation day 15). A battery of behavioral tests was conducted during postnatal day 35–60. Neurotransmitter receptors and inflammation markers in brain regions were evaluated by RT-qPCR on postnatal day 60.ResultsOpen field, elevated plus maze, and forced swimming tests revealed that prenatal Poly I:C exposure led to elevated anxiety-like and depression-like behaviors in female adolescent offspring. Deficits in pre-pulse inhibition and social interaction were also observed. However, the Poly I:C rats had better performance than the controls in the novel object recognition memory test, which demonstrated a behavioral phenotype with improved cognitive function. Prenatal Poly I:C exposure caused brain region–specific elevation of the P2X7 receptor- and NF-κB-NLRP3-IL-1β inflammatory signaling in female juvenile rats. Prenatal Poly I:C exposure decreased expression of GABAA receptor subunits Gabrb3 in the prefrontal cortex and Gabrb1 and dopamine D2 receptor in the hippocampus, but increased NMDA receptor subunit Grin2a in the prefrontal cortex, 5-HT2A in the hippocampus, and Gabrb3 and D2 receptor in the nucleus accumben.ConclusionsPrenatal Poly I:C challenge causes behavioral deficits and brain-specific neurotransmission changes via elevated neuroinflammation responses in female adolescent offspring rats.

Early developmental exposure to bisphenol A and bisphenol S disrupts socio-cognitive function, isotocin equilibrium, and excitation-inhibition balance in developing zebrafish

Dysregulation of the oxytocinergic system and excitation/inhibition (E/I) balance in synaptic transmission and neural circuits are common hallmarks of various neurodevelopmental disorders. Several experimental and epidemiological studies have shown that perinatal exposure to endocrine-disrupting chemicals bisphenol A (BPA) and bisphenol S (BPS) may contribute to a range of childhood neurodevelopmental disorders. However, the effects of BPA and BPS on social-cognitive development and the associated mechanisms remain largely unknown. In this study, we explored the impacts of early developmental exposure (2hpf-5dpf) to environmentally relevant concentrations of BPA, and its analog BPS (0.001, 0.01, and 0.1 μM), on anxiety, social behaviors, and memory performance in 21 dpf zebrafish larvae. Our results revealed that early-life exposure to low concentrations of BPA and BPS elevated anxiety-like behavior, while fish exposed to higher concentrations of these chemicals displayed social deficits and impaired object recognition memory. Additionally, we found that co-exposure with an aromatase inhibitor antagonized BPA- and BPS-induced effects on anxiety levels and social behaviors, while the co-exposure to an estrogen receptor antagonist restored recognition memory in zebrafish larvae. These results indicate that BPA and BPS may affect social-cognitive function through distinct mechanisms. On the other hand, exposure to low BPA/BPS concentrations increased both the mRNA and protein levels of isotocin (zebrafish oxytocin) in the zebrafish brain, whereas a reduction in its mRNA level was observed at higher concentrations. Further, alterations in the transcript abundance of chloride transporters, and molecular markers of gamma-aminobutyric acid (GABA) and glutamatergic systems, were observed in the zebrafish brain, suggesting possible E/I imbalance following BPA or BPS exposure. Collectively, the results of this study demonstrate that early-life exposure to low concentrations of the environmental contaminants BPA and BPS can interfere with the isotocinergic signaling pathway and disrupts the establishment of E/I balance in the developing brain, subsequently leading to the onset of a suite of behavioral deficits and neurodevelopmental disorders.

Impaired hypothalamic feedback dysregulates brain glucocorticoid signaling in genetically-selected Marchigian Sardinian alcohol-preferring rats.

Genetically-selected Marchigian Sardinian alcohol-preferring (msP) rats display comorbid symptoms of increased alcohol preference and elevated anxiety-like behavior. Heightened stress sensitivity in msPs is influenced by genetic polymorphisms of the corticotropin-releasing factor receptor in the central nucleus of the amygdala (CeA), as well as reduced influence of anti-stress mechanisms that normally constrain the stress response. Given this propensity for stress dysregulation, in this study, we expand on the possibility that msPs may display differences in neuroendocrine processes that normally terminate the stress response. We utilized behavioral, biochemical, and molecular assays to compare basal and restraint stress-induced changes in the hypothalamic-pituitary-adrenal (HPA) axis of male and female msPs relative to their nonselected Wistar counterparts. The results showed that msPs display deficits in marble-burying behavior influenced by environmental factors and procedures that modulate arousal states in a sex-dependent manner. Whereas male msPs display evidence of dysregulated neuroendocrine function (higher adrenocorticotropic hormone levels and subthreshold reductions in corticosterone), females display restraint-induced elevations in corticosterone levels that were persistently higher in msPs. A dexamethasone challenge reduced the circulation of these stress hormones, although the reduction in corticosterone was generally attenuated in msP versus Wistar rats. Finally, we found evidence of diminished stress-induced glucocorticoid receptor (GR) phosphorylation in the hypothalamic paraventricular nucleus of msPs, as well as innate increases in phosphorylated GR levels in the CeA of male msPs. Collectively, these findings suggest that negative feedback processes regulating HPA responsiveness are diminished in msP rats, possibly underlying differences in the expression of anxiety-like behaviors.

Inactivation of the GATA Cofactor ZFPM1 Results in Abnormal Development of Dorsal Raphe Serotonergic Neuron Subtypes and Increased Anxiety-Like Behavior.

Serotonergic neurons in the dorsal raphe (DR) nucleus are associated with several psychiatric disorders including depression and anxiety disorders, which often have a neurodevelopmental component. During embryonic development, GATA transcription factors GATA2 and GATA3 operate as serotonergic neuron fate selectors and regulate the differentiation of serotonergic neuron subtypes of DR. Here, we analyzed the requirement of GATA cofactor ZFPM1 in the development of serotonergic neurons using Zfpm1 conditional mouse mutants. Our results demonstrated that, unlike the GATA factors, ZFPM1 is not essential for the early differentiation of serotonergic precursors in the embryonic rhombomere 1. In contrast, in perinatal and adult male and female Zfpm1 mutants, a lateral subpopulation of DR neurons (ventrolateral part of the DR) was lost, whereas the number of serotonergic neurons in a medial subpopulation (dorsal region of the medial DR) had increased. Additionally, adult male and female Zfpm1 mutants had reduced serotonin concentration in rostral brain areas and displayed increased anxiety-like behavior. Interestingly, female Zfpm1 mutant mice showed elevated contextual fear memory that was abolished with chronic fluoxetine treatment. Altogether, these results demonstrate the importance of ZFPM1 for the development of DR serotonergic neuron subtypes involved in mood regulation. It also suggests that the neuronal fate selector function of GATAs is modulated by their cofactors to refine the differentiation of neuronal subtypes.SIGNIFICANCE STATEMENT Predisposition to anxiety disorders has both a neurodevelopmental and a genetic basis. One of the brainstem nuclei involved in the regulation of anxiety is the dorsal raphe, which contains different subtypes of serotonergic neurons. We show that inactivation of a transcriptional cofactor ZFPM1 in mice results in a developmental failure of laterally located dorsal raphe serotonergic neurons and changes in serotonergic innervation of rostral brain regions. This leads to elevated anxiety-like behavior and contextual fear memory, alleviated by chronic fluoxetine treatment. Our work contributes to understanding the neurodevelopmental mechanisms that may be disturbed in the anxiety disorder.

Intergenerational effects of paternal predator cue exposure on behavior, stress reactivity, and neural gene expression

Predation threat impacts prey behavior, physiology, and fitness. Stress-mediated alterations to the paternal epigenome can be transmitted to offspring via the germline, conferring a potential advantage to offspring in predator-rich environments. While intergenerational epigenetic transmission of paternal experience has been demonstrated in mammals, how paternal predator exposure might alter offspring phenotypes across development is unstudied. We exposed male mice to a predator odor (2,4,5-trimethylthiazoline, TMT) or a neutral odor (banana extract) prior to mating and measured offspring behavioral phenotypes throughout development, together with adult stress reactivity and candidate gene expression in the prefrontal cortex, hippocampus, amygdala, and hypothalamus. We predicted that offspring of TMT-exposed males would be less active, would display elevated anxiety-like behaviors, and would have a more efficient stress response relative to controls, phenotypes that should enhance predator avoidance in a high predation risk environment. Unexpectedly, we found that offspring of TMT-exposed males are more active, exhibit less anxiety-like behavior, and have decreased baseline plasma corticosterone relative to controls. Effects of paternal treatment on neural gene expression were limited to the prefrontal cortex, with increased mineralocorticoid receptor expression and a trend towards increased Bdnf expression in offspring of TMT-exposed males. These results suggest that fathers exposed to predation threat produce offspring that are buffered against non-acute stressors and, potentially, better adapted to a predator-dense environment because they avoid trade-offs between predator avoidance and foraging and reproduction. This study provides evidence that ecologically relevant paternal experience can be transmitted through the germline, and can impact offspring phenotypes throughout development.

Dopamine D1R-neuron cacna1c deficiency: a new model of extinction therapy-resistant post-traumatic stress.

Post-traumatic stress disorder (PTSD) is characterized by persistent fear memory of remote traumatic events, mental re-experiencing of the trauma, long-term cognitive deficits, and PTSD-associated hippocampal dysfunction. Extinction-based therapeutic approaches acutely reduce fear. However, many patients eventually relapse to the original conditioned fear response. Thus, understanding the underlying molecular mechanisms of this condition is critical to developing new treatments for patients. Mutations in the neuropsychiatric risk gene CACNA1C, which encodes the Cav1.2 isoform of the L-type calcium channel, have been implicated in both PTSD and highly comorbid neuropsychiatric conditions, such as anxiety and depression. Here, we report that male mice with global heterozygous loss of cacna1c exhibit exacerbated contextual fear that persists at remote time points (up to 180 days after shock), despite successful acute extinction training, reminiscent of PTSD patients. Because dopamine has been implicated in contextual fear memory, and Cav1.2 is a downstream target of dopamine D1-receptor (D1R) signaling, we next generated mice with specific deletion of cacna1c from D1R-expressing neurons (D1-cacna1cKO mice). Notably, D1-cacna1cKO mice also show the same exaggerated remote contextual fear, as well as persistently elevated anxiety-like behavior and impaired spatial memory at remote time points, reminiscent of chronic anxiety in treatment-resistant PTSD. We also show that D1-cacna1cKO mice exhibit elevated death of young hippocampal neurons, and that treatment with the neuroprotective agent P7C3-A20 eradicates persistent remote fear. Augmenting survival of young hippocampal neurons may thus provide an effective therapeutic approach for promoting durable remission of PTSD, particularly in patients with CACNA1C mutations or other genetic aberrations that impair calcium signaling or disrupt the survival of young hippocampal neurons.

Enduring consequences of perinatal fentanyl exposure in mice.

Opioid use by pregnant women is an understudied consequence associated with the opioid epidemic, resulting in a rise in the incidence of neonatal opioid withdrawal syndrome (NOWS) and lifelong neurobehavioral deficits that result from perinatal opioid exposure. There are few preclinical models that accurately recapitulate human perinatal drug exposure and few focus on fentanyl, a potent synthetic opioid that is a leading driver of the opioid epidemic. To investigate the consequences of perinatal opioid exposure, we administered fentanyl to mouse dams in their drinking water throughout gestation and until litters were weaned at postnatal day (PD) 21. Fentanyl-exposed dams delivered smaller litters and had higher litter mortality rates compared with controls. Metrics of maternal care behavior were not affected by the treatment, nor were there differences in dams' weight or liquid consumption throughout gestation and 21 days postpartum. Twenty-four hours after weaning and drug cessation, perinatal fentanyl-exposed mice exhibited signs of spontaneous somatic withdrawal behavior and sex-specific weight fluctuations that normalized in adulthood. At adolescence (PD 35), they displayed elevated anxiety-like behaviors and decreased grooming, assayed in the elevated plus maze and sucrose splash tests. Finally, by adulthood (PD 55), they displayed impaired performance in a two-tone auditory discrimination task. Collectively, our findings suggest that perinatal fentanyl-exposed mice exhibit somatic withdrawal behavior and change into early adulthood reminiscent of humans born with NOWS.

Excess Folic Acid Supplementation Before and During Pregnancy and Lactation Activates Fos Gene Expression and Alters Behaviors in Male Mouse Offspring.

Periconceptional folic acid (FA) supplementation is recommended to prevent neural tube defects and other birth defects. After 20 years mandate food fortification with FA, serum concentration of folate and unmetabolized FA increased significantly in the North American population. But whether excess FA intake impairs neurodevelopment and behavior is still controversial. Here, we treated mice with approximately 2.5-fold (moderate dose of FA, MFA) or 10-fold (high dose of FA, HFA) the dietary requirement of FA 1 week before mating and throughout pregnancy and lactation, and examined behaviors in adult male offspring using open field test, three-chamber sociability and social novelty test, elevated plus maze, rotarod and Morris water maze. We found that early life MFA supplementation increased long-term body weight gain in adults, elevated anxiety-like behavior, and impaired social preference, motor learning and spatial learning ability without modifying motor ability and spatial memory. In contrast, HFA supplementation only induced mild behavioral abnormality. RNA sequencing revealed that FA supplementation altered the expression of brain genes at weaning, among which Fos and related genes were significantly up-regulated in MFA mice compared with control and HFA mice. Quantitative real time-PCR (qRT-PCR) and western blots confirmed the increase of these genes. Our results suggested that FA supplementation during early life stage affected gene expression in weaning mice, and exhibited long-term impairments in adult behaviors in a dose-sensitive manner.

New neurons restore structural and behavioral abnormalities in a rat model of PTSD

Post-traumatic stress disorder (PTSD) has been associated with anxiety, memory impairments, enhanced fear, and hippocampal volume loss, although the relationship between these changes remain unknown. Single-prolonged stress (SPS) is a model for PTSD combining three forms of stress (restraint, swim, and anesthesia) in a single session that results in prolonged behavioral effects. Using pharmacogenetic ablation of adult neurogenesis in rats, we investigated the role of new neurons in the hippocampus in the long-lasting structural and behavioral effects of SPS. Two weeks after SPS, stressed rats displayed increased anxiety-like behavior and decreased preference for objects in novel locations regardless of the presence or absence of new neurons. Chronic stress produced by daily restraint for 2 or 6 hr produced similar behavioral effects that were also independent of ongoing neurogenesis. At a longer recovery time point, 1 month after SPS, rats with intact neurogenesis had normalized, showing control levels of anxiety-like behavior. However, GFAP-TK rats, which lacked new neurons, continued to show elevated anxiety-like behavior and enhanced serum corticosterone response to anxiogenic experience. Volume loss in ventral CA1 region of the hippocampus paralleled increases in anxiety-like behavior, occurring in all rats exposed to SPS at the early time point and only rats lacking adult neurogenesis at the later time point. In chronic stress experiments, volume loss occurred broadly throughout the dentate gyrus and CA1 after 6-hr daily stress but was not apparent in any hippocampal subregion after 2-hr daily stress. No effect of SPS was seen on cell proliferation in the dentate gyrus, but the survival of young neurons born a week after stress was decreased. Together, these data suggest that new neurons are important for recovery of normal behavior and hippocampal structure following a strong acute stress and point to the ventral CA1 region as a potential key mediator of stress-induced anxiety-like behavior.

Adolescent Stress Disrupts the Maturation of Anxiety-related Behaviors and Alters the Developmental Trajectory of the Prefrontal Cortex in a Sex- and Age-specific Manner

Adolescence is a window of vulnerability to environmental factors such as chronic stress that can disrupt brain development and cause long-lasting behavioral dysfunction, as seen in disorders like depression, anxiety, and schizophrenia. There are also sex differences in the prevalence of these disorders across the lifespan. However, the mechanisms of how adolescent stress contributes to neuropsychiatric phenotypes are not well understood, nor are the mediating effects of sex. We hypothesize that adolescent stress disrupts the γ-aminobutyric acid (GABA) system in the prefrontal cortex (PFC) in a sex-specific manner, as this system matures during adolescence and plays an important role in cognitive and emotional functioning. We exposed male and female mice to unpredictable chronic mild stress (UCMS) during adolescence (post-natal day [PND] 28–42). One cohort underwent testing for PFC-related behavioral and molecular changes 24 h following the cessation of stress (late adolescence); a separate cohort was tested approximately 2.5 weeks after the end of UCMS (adulthood). We observed an age-related decline in anxiety-like behaviors in control mice, while mice stressed in adolescence showed elevated anxiety-like behaviors in both adolescence and adulthood. PFC-dependent cognitive functioning was also impaired in adult males stressed in adolescence. Adolescent stress disrupted expression patterns of parvalbumin (PV) and perineuronal nets (PNNs) in the PFC, as well as NMDA receptor subunit composition, in a sex- and age-specific manner. The findings presented here contribute to understanding how adolescent stress may lead to neuropsychiatric disorders such as anxiety by disrupting the development of the PFC and emotional behaviors.

Corrigendum: Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits

[This corrects the article on p. 42 in vol. 9, PMID: 29487522.].

MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors

Abnormalities in limbic neural circuits have been implicated in the onset of anxiety disorders. However, the molecular pathogenesis underlying anxiety disorders remains poorly elucidated. Here, we demonstrate that myristoylated alanine-rich C-kinase substrate like 1 (MARCKSL1) regulates amygdala circuitry to control the activity of the hypothalamic-pituitary-adrenal (HPA) axis, as well as induces anxiety-like behaviors in mice. MARCKSL1 expression was predominantly localized in the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala of the adult mouse brain. MARCKSL1 transgenic (Tg) mice exhibited anxiety-like behaviors dependent on corticotropin-releasing hormone. MARCKSL1 increased spine formation in the central amygdala, and downregulation of MARCKSL1 in the amygdala normalized both increased HPA axis activity and elevated anxiety-like behaviors in Tg mice. Furthermore, MARCKSL1 expression was increased in the PFC and amygdala in a brain injury model associated with anxiety-like behaviors. Our findings suggest that MARCKSL1 expression in the amygdala plays an important role in anxiety-like behaviors.

Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits

The role of NMDA receptors in learning, memory and hippocampal function has long been recognized. Post-mortem studies have indicated that the expression or subunit composition of the NMDA glutamate receptor subtype might be related to the impaired cognitive functions found in schizophrenia patients. NMDA receptor antagonists have been used to develop animal models of this disorder. There is accumulating evidence showing that not only the acute but also the chronic application of NMDA receptor antagonists may induce schizophrenia-like alterations in behavior and brain functions. However, limited evidence is available regarding the consequences of NMDA receptor blockage during periods of adolescence and early adulthood. This study tested the hypothesis that a 2-week treatment of male Long-Evans and Wistar rats with dizocilpine (MK-801; 0.5 mg/kg daily) starting at postnatal days (PD) 30 and 60 would cause a long-term cognitive deficit and changes in the levels of NMDA receptor subunits. The working memory version of the Morris water maze (MWM) and active place avoidance with reversal on a rotating arena (Carousel) requiring cognitive coordination and flexibility probed cognitive functions and an elevated-plus maze (EPM) was used to measure anxiety-like behavior. The western blot method was used to determine changes in NMDA receptor subunit levels in the hippocampus. Our results showed no significant changes in behaviors in Wistar rats. Slightly elevated anxiety-like behavior was observed in the EPM in Long-Evans rats with the onset of treatment on PD 30. Furthermore, Long-Evans rats treated from PD 60 displayed impaired working memory in the MWM. There were; however, no significant changes in the levels of NMDA receptor subunits because of MK-801 administration. These findings suggest that a 2-week treatment starting on PD 60 in Long-Evans rats leads to long-term changes in working memory, but this deficit is not paralleled by changes in NMDA receptor subunits. These results support the face validity, but not construct validity of this model. We suggest that chronic treatment of adolescent and adult rats does not constitute a plausible animal model of schizophrenia.

Plastic change of prefrontal cortex mediates anxiety-like behaviors associated with chronic pain in neuropathic rats.

Clinical studies show that anxiety and chronic pain are concomitant. The neural basis for the comorbidity is unclear. The prefrontal cortex (PFC) has been recognized as a critical area for affective disorders and chronic pain modulation. In this study, we examined the role of the PFC in the pathogenesis of anxiety associated with chronic pain in a rat model of neuropathic pain with spare nerve injury (SNI). The SNI rats showed apparent anxiety-like behaviors in both open field (OF) test and elevated-plus maze (EPM) test eight weeks after surgery. Thus, the number of entries to the central area in the OF decreased to 45% (±5%, n = 15) of sham control (n = 17), while the overall motor activity (i.e., total distance) was unaffected. In the EPM, the percentage of entries into the open arms significantly (p < 0.001) decreased in SNI rats (SNI: 12.58 ± 2.7%, n = 15; sham: 30.75 ± 2.82%, n = 17), so did the time spent in the open arms (SNI: 4.35 ± 1.45%, n = 15; Sham: 11.65 ± 2.18%, n = 17). To explore the neural basis for the association between anxiety and chronic pain, local field potentials (LFPs) were recorded from the medial PFC (mPFC) and ventral hippocampus. In SNI rats, there were significantly greater increases in both theta-frequency power in the mPFC and theta-frequency synchronization between the mPFC and ventral hippocampus, when animals were displaying elevated anxiety-like behaviors in avoiding anxiogenic regions in EPM and OF chamber. Western blot analyses showed a significant elevation of serotonin transporter expression in the anxious SNI rats. Inhibition of serotonin transporter effectively alleviated anxiety-like behaviors following sub-chronic (15 days) treatment with systemic citalopram (10 mg/kg/day, intraperitoneally). Moreover, the anxiety-like behaviors in the SNI rats were also suppressed by direct mPFC application of serotonin. Taken together, we conclude that the plasticity of serotonin transmission in the mPFC likely contribute to the promotion of anxiety state associated with neuropathic pain.

Effect of Early-Life Fluoxetine on Anxiety-Like Behaviors in BDNF Val66Met Mice.

Adolescence is a developmental stage in which the incidence of psychiatric disorders, such as anxiety disorders, peaks. Selective serotonin reuptake inhibitors (SSRIs) are the main class of agents used to treat anxiety disorders. However, the impact of SSRIs on the developing brain during adolescence remains unknown. The authors assessed the impact of developmentally timed SSRI administration in a genetic mouse model displaying elevated anxiety-like behaviors. Knock-in mice containing a common human single-nucleotide polymorphism (Val66Met; rs6265) in brain-derived neurotrophic factor (BDNF), a growth factor implicated in the mechanism of action of SSRIs, were studied based on their established phenotype of increased anxiety-like behavior. Timed administration of fluoxetine was delivered during one of three developmental periods (postnatal days 21-42, 40-61, or 60-81), spanning the transition from childhood to adulthood. Neurochemical and anxiety-like behavioral analyses were performed. We identified a "sensitive period" during periadolescence (postnatal days 21-42) in which developmentally timed fluoxetine administration rescued anxiety-like phenotypes in BDNF Val66Met mice in adulthood. Compared with littermate controls, BDNFMet/Met mice exhibited diminished maturation of serotonergic fibers projecting particularly to the prefrontal cortex, as well as decreased expression of the serotonergic trophic factor S100B in the dorsal raphe. Interestingly, deficient serotonergic innervation, as well as S100B levels, were rescued with fluoxetine administration during periadolescence. These findings suggest that SSRI administration during a "sensitive period" during periadolescence leads to long-lasting anxiolytic effects in a genetic mouse model of elevated anxiety-like behaviors. These persistent effects highlight the role of BDNF in the maturation of the serotonin system and the capacity to enhance its development through a pharmacological intervention.