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The retrieval of fear memory induces two opposite memory process, i.e., reconsolidation and extinction. Brief retrieval induces reconsolidation to maintain or enhance fear memory, while prolonged retrieval extinguishes this memory. Although the mechanisms of reconsolidation and extinction have been investigated, it remains unknown how fear memory phases are switched from reconsolidation to extinction during memory retrieval. Here, we show that an extracellular signal-regulated kinase (ERK)-dependent memory transition process after retrieval regulates the switch of memory phases from reconsolidation to extinction by preventing induction of reconsolidation in an inhibitory avoidance (IA) task in male mice. First, the transition memory phase, which cancels the induction of reconsolidation, but is insufficient for the acquisition of extinction, was identified after reconsolidation, but before extinction phases. Second, the reconsolidation, transition, and extinction phases after memory retrieval showed distinct molecular and cellular signatures through cAMP responsive element binding protein (CREB) and ERK phosphorylation in the amygdala, hippocampus, and medial prefrontal cortex (mPFC). The reconsolidation phase showed increased CREB phosphorylation, while the extinction phase displayed several neural populations with various combinations of CREB and/or ERK phosphorylation, in these brain regions. Interestingly, the three memory phases, including the transition phase, showed transient ERK activation immediately after retrieval. Most importantly, the blockade of ERK in the amygdala, hippocampus, or mPFC at the transition memory phase disinhibited reconsolidation-induced enhancement of IA memory. These observations suggest that the ERK-signaling pathway actively regulates the transition of memory phase from reconsolidation to extinction and this process functions as a switch that cancels reconsolidation of fear memory. SIGNIFICANCE STATEMENT Retrieval of fear memory induces two opposite memory process; reconsolidation and extinction. Reconsolidation maintains/enhances fear memory, while extinction weakens fear memory. It remains unknown how memory phases are switched from reconsolidation to extinction during retrieval. Here, we identified an active memory transition process functioning as a switch that inhibits reconsolidation. This memory transition phase showed a transient increase of extracellular signal-regulated kinase (ERK) phosphorylation in the amygdala, hippocampus and medial prefrontal cortex (mPFC). Interestingly, inhibition of ERK in these regions at the transition phase disinhibited the reconsolidation-mediated enhancement of inhibitory avoidance (IA) memory. These findings suggest that the transition memory process actively regulates the switch of fear memory phases of fear memory by preventing induction of reconsolidation through the activation of the ERK-signaling pathway., (Copyright © 2021 the authors.)

Alcohol use-associated disorders are highly comorbid with anxiety disorders; however, their mechanism remains unknown. The amygdala plays a central role in anxiety. We recently found that 7,8-dihydroxyflavone (7,8-DHF) significantly reduces withdrawal symptoms in a rat model of chronic intermittent alcohol (ethanol) exposure. This study aimed to determine the role of 7,8-DHF in regulating anxiety induced by chronic alcohol exposure and its associated underlying mechanism. Male C57BL/6J mice were exposed to chronic intermittent alcohol for 3 weeks followed by alcohol withdrawal for 12 h with or without 7,8-DHF administered intraperitoneally. All mice were tested using an open field test and elevated plus maze to assess anxiety-like behaviors. Synaptic activity and intrinsic excitability in basal and lateral amygdala (BLA) neurons were assessed using electrophysiological recordings. 7,8-DHF alleviated alcohol-induced anxiety-like behavior and attenuated alcohol-induced enhancement of activities in BLA pyramidal neurons. Furthermore, 7,8-DHF prevented alcohol withdrawal-evoked augmentation of glutamatergic transmission in the amygdala and had no effect on GABAergic transmission in the amygdala, as demonstrated by unaltered frequency and amplitude of spontaneous inhibitory postsynaptic currents. Microinjection of K252a, a tropomyosin-related kinase B (TrkB) antagonist, into the BLA blocked the effects of 7,8-DHF on anxiety-like behavior and neuronal activity in the BLA. Our findings suggest that 7,8-DHF alleviates alcohol-induced anxiety-like behavior induced by chronic alcohol exposure through regulation of glutamate transmission involving TrKB in the BLA.

The neurosteroid allopregnanolone (AL) has many beneficial functions in the brain. This study tested the hypothesis that AL administered for three days into the third brain ventricle would affect the enzymatic activity of the DNA base excision repair (BER) pathway in the hippocampal CA1 and CA3 fields and the central amygdala in luteal-phase sheep under both natural and stressful conditions. Acute stressful stimuli, including isolation and partial movement restriction, were used on the last day of infusion. The results showed that stressful stimuli increased N-methylpurine DNA glycosylase (MPG), thymine DNA glycosylase (TDG), 8-oxoguanine glycosylase (OGG1), and AP-endonuclease 1 (APE1) mRNA expression, as well as repair activities for 1, N 6 -ethenoadenine (εA), 3, N 4 -ethenocytosine (εC), and 8-oxoguanine (8-oxoG) compared to controls. The stimulated events were lower in stressed and AL-treated sheep compared to sheep that were only stressed (except MPG mRNA expression in the CA1 and amygdala, as well as TDG mRNA expression in the CA1). AL alone reduced mRNA expression of all DNA repair enzymes (except TDG in the amygdala) relative to controls and other groups. DNA repair activities varied depending on the tissue-AL alone stimulated the excision of εA in the amygdala, εC in the CA3 and amygdala, and 8-oxoG in all tissues studied compared to controls. However, the excision efficiency of lesioned bases in the AL group was lower than in the stressed and stressed and AL-treated groups, with the exception of εA in the amygdala. In conclusion, the presented modulating effect of AL on the synthesis of BER pathway enzymes and their repair capacity, both under natural and stressful conditions, indicates another functional role of this neurosteroid in brain structures.

Epidemiological studies show that maternal diabetes is associated with an increased risk of autism spectrum disorders (ASDs), although the detailed mechanisms remain unclear. The present study aims to investigate the potential effect of maternal diabetes on autism-like behavior in offspring. The results of in vitro study showed that transient hyperglycemia induces persistent reactive oxygen species (ROS) generation with suppressed superoxide dismutase 2 (SOD2) expression. Additionally, we found that SOD2 suppression is due to oxidative stress-mediated histone methylation and the subsequent dissociation of early growth response 1 (Egr1) on the SOD2 promoter. Furthermore, in vivo rat experiments showed that maternal diabetes induces SOD2 suppression in the amygdala, resulting in autism-like behavior in offspring. SOD2 overexpression restores, while SOD2 knockdown mimics, this effect, indicating that oxidative stress and SOD2 expression play important roles in maternal diabetes-induced autism-like behavior in offspring, while prenatal and postnatal treatment using antioxidants permeable to the blood-brain barrier partly ameliorated this effect. We conclude that maternal diabetes induces autism-like behavior through hyperglycemia-mediated persistent oxidative stress and SOD2 suppression. Here we report a potential mechanism for maternal diabetes-induced ASD., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Na + , K + -ATPase is an essential membrane transporter. In the brain, the α3 isoform of Na + , K + -ATPase is vital for neuronal function. The enzyme and its regulators, endogenous cardiac steroids (ECS), were implicated in neuropsychiatric disorders. GABAergic neurotransmission was also studied extensively in diseases such as schizophrenia and bipolar disorder (BD). Post mortem brain samples from subjects with depression, schizophrenia or BD and non-psychiatric controls were provided by the Stanley Medical Research Institute. ECS levels were determined by ELISA. Expression levels of the three Na + , K + -ATPase-α isoforms, α1, α2 and α3, were determined by Western blot analysis. The α3 levels in GABAergic neurons in different regions of the brain were quantified by fluorescence immunohistochemistry. The results show that Na + , K + -ATPase α3 isoform levels were lower in GABAergic neurons in the frontal cortex in BD and schizophrenia as compared with the controls (n = 15 subjects per group). A study on a 'mini-cohort' (n = 3 subjects per group) showed that the α3 isoform levels were also lower in GABAergic neurons in the hippocampus, but not amygdala, of bipolar and schizophrenic subjects. In the temporal cortex, higher Na + , K + -ATPase α3 protein levels were found in the three psychiatric groups. No significant differences in ECS levels were found in this brain area. This is the first report on the distribution of α3 in specific neurons in the human brain in association with mental illness. These results strengthen the hypothesis for the involvement of Na + , K + -ATPase in neuropsychiatric diseases., (Copyright © 2019. Published by Elsevier Ltd.)

Homocysteine (HCY) has been linked to oxidative stress and varied metabolic changes that are dependent on its concentration and affected tissues. In the present study we evaluate parameters of energy metabolism [succinate dehydrogenase (SDH), complex II and IV (cytochrome c oxidase), and ATP levels] and oxidative stress [DCFH oxidation, nitrite levels, antioxidant enzymes and lipid, protein and DNA damages, as well as nuclear factor erythroid 2-related (Nrf2) protein abundance] in amygdala and prefrontal cortex of HCY-treated rats. Wistar male rats were treated with a subcutaneous injection of HCY (0.03 µmol/g of body weight) from the 30th to 60th post-natal day, twice a day, to induce mild hyperhomocysteinemia (HHCY). The rats were euthanatized without anesthesia at 12 h after the last injection, and amygdala and prefrontal cortex were dissected for biochemical analyses. In the amygdala, mild HHCY increased activities of SDH and complex II and decreased complex IV and ATP level, as well as increased antioxidant enzymes activities (glutathione peroxidase and superoxide dismutase), nitrite levels, DNA damage, and Nrf 2 protein abundance. In the prefrontal cortex, mild HHCY did not alter energy metabolism, but increased glutathione peroxidase, catalase and DNA damage. Other analyzed parameters were not altered by HCY-treatment. Our findings suggested that chronic mild HHCY changes each brain structure, particularly and specifically. These changes may be associated with the mechanisms by which chronic mild HHCY has been linked to the risk factor of fear, mood disorders and depression, as well as in neurodegenerative diseases.

UBA6 is an alternative enzyme for ubiquitin activation in vertebrates that plays a pivotal role in early mouse development. Previously, we reported that the Uba6 brain-specific knockout (NKO) mouse is a novel autism spectrum disorder (ASD) mouse model that displays decreased social behavior and communication. To determine the therapeutic impact of environmental stimulation in ASDs, we investigated the behavioral and molecular changes of the NKO and control mice after exposure to environmental enrichment and paired housing in different developmental phases. Our results demonstrated that early paired housing could diminish the ASD phenotypes of NKO mice such as impaired nest building and social interaction and anxiety. Additionally, increased histone acetylation in the amygdala was observed in NKO mice after paired housing without a change in Ube3a levels. Our data suggest that paired housing at an early time point can play a crucial role in ameliorating ASD behavior and can be applied in other ASD animal models or clinical settings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase and NH 2 -terminal lipid phosphatase activities, is expressed in regions of the brain such as the cortex, white matter, hippocampus, substantia nigra, and striatum. sEH is involved in the regulation of cerebrovascular and neuronal function upon pathological insults. However, the physiological significance of sEH and its underlying mechanism in modulating brain function are not fully understood. In this study, we investigated the role of sEH in anxiety and potential underlying mechanisms in mice. Western blot for protein phosphorylation and expression was performed. Immunohistochemical analyses and Nissl and Golgi staining were performed for histological examination. Mouse behaviors were evaluated by open field activity, elevated plus maze, classical fear conditioning, social preference test, and Morris water maze. Our results demonstrated that the expression of sEH was upregulated during postnatal development in wild-type (WT) mice. Genetic deletion of sEH (sEH -/- ) in mice resulted in anxiety-like behavior and disrupted social preference. Increased olfactory bulb (OB) size and altered integrity of neurites were observed in sEH -/- mice. In addition, ablation of sEH in mice decreased protein expression of tyrosine hydroxylase and reduced dopamine production in the brain. Moreover, the level of phosphorylated calmodulin kinase II (CaMKII) and glycogen synthase kinase 3 α/β (GSK3α/β) was higher in sEH -/- mice than in WT mice. Collectively, these findings suggest that sEH is a key player in neurite outgrowth of neurons, OB development in the brain, and the development of anxiety-like behavior, by regulating the CaMKII-GSK3α/β signaling pathway.

In mammals, the extended amygdala is a neural hub for social and emotional information processing. In the rat, the extended amygdala receives inhibitory GABAergic projections from the nucleus incertus (NI) in the pontine tegmentum. NI neurons produce the neuropeptide relaxin-3, which acts via the G i/o -protein-coupled receptor, RXFP3. A putative role for RXFP3 signalling in regulating social interaction was investigated by assessing the effect of intracerebroventricular infusion of the RXFP3 agonist, RXFP3-A2, on performance in the 3-chamber social interaction paradigm. Central RXFP3-A2, but not vehicle, infusion, disrupted the capacity to discriminate between a familiar and novel conspecific subject, but did not alter differentiation between a conspecific and an inanimate object. Subsequent studies revealed that agonist-infused rats displayed increased phosphoERK(pERK)-immunoreactivity in specific amygdaloid nuclei at 20 min post-infusion, with levels similar to control again after 90 min. In parallel, we used immunoblotting to profile ERK phosphorylation dynamics in whole amygdala after RXFP3-A2 treatment; and multiplex histochemical labelling techniques to reveal that after RXFP3-A2 infusion and social interaction, pERK-immunopositive neurons in amygdala expressed vesicular GABA-transporter mRNA and displayed differential profiles of RXFP3 and oxytocin receptor mRNA. Overall, these findings demonstrate that central relaxin-3/RXFP3 signalling can modulate social recognition in rats via effects within the amygdala and likely interactions with GABA and oxytocin signalling.

Although infants learn and remember, they rapidly forget, a phenomenon known as infantile amnesia. While myriad mechanisms impact this rapid forgetting, the molecular events supporting memory maintenance have yet to be explored. To explore memory mechanisms across development, we used amygdala-dependent odor-shock conditioning and focused on mechanisms important in adult memory, the AMPA receptor subunits GluA1/2 and upstream protein kinases important for trafficking AMPAR, protein kinase M zeta (PKMζ) and iota/lambda (PKCι/λ). We use odor-shock conditioning in infant rats because it is late-developing (postnatal day, PN10) and can be modulated by corticosterone during a sensitive period in early life. Our results show that memory-related molecules did not change in pups too young to learn threat (PN8) but were activated in pups old enough to learn (PN12), with increased PKMζ-PKCι/λ and GluA2 similar to that observed in adult memory, but with an uncharacteristic decrease in GluA1. This molecular signature and behavioral avoidance of the conditioned odor was recapitulated in PN8 pups injected with CORT before conditioning to precociously induce learning. Blocking learning via CORT inhibition in older pups (PN12) blocked the expression of these molecules. PN16 pups showed a more adult-like molecular cascade of increased PKMζ-PKCι/λ and GluA1-2. Finally, at all ages, zeta inhibitory peptide (ZIP) infusions into the amygdala 24 hr after conditioning blocked memory. Together, these results identify unique features of memory processes across early development: AMPAR subunits GluA1/2 and PKC isoform expression are differentially used, which may contribute to mechanisms of early life forgetting.

Neuropeptide S (NPS) is an important anxiolytic substance of the brain. However, the signaling pathways downstream of NPS receptor (NPSR) activation, underlying the behavioral effect of NPS, remain largely unknown. Here, we show that bilateral microinfusion of NPS (0.2 nmol/0.5 μl) into the medial amygdala (MeA) of male adult Wistar rats reduced anxiety-related behavior on both the elevated plus-maze and the open field. Moreover, as shown in amygdala tissue micropunches intracerebroventricular infusion of NPS (1 nmol/5 μl) (1) evoked phosphorylation and synthesis of CaMKIIα in relation to reference protein β-tubulin representing Ca 2+ influx, and (2) induced phosphorylation of mitogen-activated protein kinase ERK1/2. The NPS-induced anxiolysis was prevented by local inhibition of phospholipase C signaling using U73122 (0.5 nmol/0.5 μl) in the MeA, indicating the behavioral relevance of this pathway. Conversely, local pharmacological blockade of adenylyl cyclase signaling using 2',5'-dideoxyadenosine (12.5 nmol/0.5 μl) failed to inhibit the anxiolytic effect of NPS infused into the MeA. Hence, NPS promotes acute anxiolysis within the MeA dependent on NPSR-mediated phospholipase C signaling. Taken together, our study extends the knowledge about the intracellular signaling mechanisms underlying the potent anxiolytic profile of NPS.

The hippocampus and amygdala are essential brain regions responsible for contextual fear conditioning (CFC). The autophosphorylation of alpha calcium-calmodulin kinase II (αCaMKII) at threonine-286 (T286) is a critical step implicated in long-term potentiation (LTP), learning and memory. However, the changes in αCaMKII levels with aging and training in associated brain regions are not fully understood. Here, we studied how aging and training affect the levels of phosphorylated (T286) and proportion of phosphorylated:total αCaMKII in the hippocampus and amygdala. Young and aged mice, naïve (untrained) and trained in CFC, were analysed by immunohistochemistry for the levels of total and phosphorylated αCaMKII in the hippocampus and amygdala. We found that two hours after CFC training, young mice exhibited a higher level of phosphorylated and increased ratio of phosphorylated:total αCaMKII in hippocampal CA3 stratum radiatum. Furthermore, aged untrained mice showed a higher ratio of phosphorylated:total αCaMKII in the CA3 region of the hippocampus when compared to the young untrained group. No effect of training or aging were seen in the central, lateral and basolateral amygdala regions, for both phosphorylated and ratio of phosphorylated:total αCaMKII. These results show that aging impairs the training-induced upregulation of autophosphorylated (T286) αCaMKII in the CA3 stratum radiatum of the hippocampus. This indicates that distinct age-related mechanisms underlie CFC that may rely more heavily on NMDA receptor-dependent plasticity in young age., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

During development sex differences in aromatase expression in limbic regions of mouse brain depend on sex chromosome factors. Genes on the sex chromosomes may affect the hormonal regulation of aromatase expression and this study was undertaken to explore that possibility. Male E15 anterior amygdala neuronal cultures expressed higher levels of aromatase (mRNA and protein) than female cultures. Furthermore, treatment with oestradiol (E2) or dihydrotestosterone (DHT) increased Cyp19a1 expression and aromatase protein levels only in female neuronal cultures. The effect of E2 on aromatase expression was not imitated by oestrogen receptor (ER) α agonist PPT or the GPER agonist G1, but it was fully reproduced by DPN, a specific ligand of ERβ. By contrast, the effect of DHT on aromatase expression was not blocked by the anti-androgen flutamide, but completely abrogated by the ERβ antagonist PHTPP. Experiments using the four core genotype model showed a sex chromosome effect in ERβ expression (XY > XX) and regulation by E2 or DHT (only XX respond) in amygdala neurons. In conclusion, sex chromosome complement governs the hormonal regulation of aromatase expression through activation of ERβ in developing mouse brain.

Chemical and psychological stressors can exert long lasting changes in brain function and behaviour. Changes in DNA methylation have been shown to be an important mechanism mediating long lasting changes in neural function and behaviour, especially for anxiety-like or stress responses. In the present study, we examined the effects of either a social or chemical stressor on DNA methyltransferase (DNMT) gene expression in the amygdala, an important brain region modulating stress responses and anxiety. In adult California mice (Peromyscus californicus) that were naïve to social defeat, females had higher levels of Dnmt1 expression in punch samples of the central amygdala (CeA) than males. In addition, mice that underwent social defeat stress showed reduced Dnmt1 and Dnmt3a expression in the CeA of females but not males. A second study using more anatomically specific punch samples replicated these effects for Dnmt1. Perinatal exposure (spanning from periconception through lactation) to bisphenol A or ethinyl oestradiol (oestrogens in birth control pills) also abolished sex differences in Dnmt1 expression in the CeA but not the basolateral amygdala. These findings identify a robust sex difference in Dnmt1 expression in the CeA that is sensitive to both psychological and chemical stressors. Future studies should aim to examine the impact of psychological and chemical stressors on DNA methylation in the CeA and also investigate whether Dnmt1 may have an underappreciated role in plasticity in behaviour., (© 2017 British Society for Neuroendocrinology.)

Objective: Protein tyrosine phosphatase 1B (PTP1B) has been extensively implicated in the regulation of body weight, food intake, and energy expenditure. The role of PTP1B appears to be cell and brain region dependent., Results: Herein, we demonstrated that chronic high-fat feeding enhanced PTP1B expression in the central nucleus of the amygdala (CeA) of rats compared to rats on chow. Knocking down PTP1B with oligonucleotide antisense (ASO) decreased its expression and was sufficient to improve the anorexigenic effect of insulin through IR/Akt signaling in the CeA. ASO treatment reduces body weight, fat mass, serum leptin levels, and food intake and also increases energy expenditure, without altering ambulatory activity. These changes were explained, at least in part, by the improvement of insulin sensitivity in the CeA, decreasing NPY and enhancing oxytocin expression. There was a slight decline in fasting blood glucose and serum insulin levels possibly due to leanness in rats treated with ASO. Surprisingly, the elevated plus maze test revealed an anxiolytic behavior after reduction of PTP1B in the CeA., Conclusions: Thus, the present study highlights the deleterious role that the amygdalar PTP1B has on energy homeostasis in obesity states. The reduction of PTP1B in the CeA may be a strategy for the treatment of obesity, insulin resistance and anxiety disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The present study examined the involvement of extracellular signal-regulated kinase 1/2 (ERK) signaling on the serotonin (5-HT) 7 receptor-mediated fear conditioning. Conditioning was performed in a trial in which a tone was followed by an electrical foot-shock. Context- and tone-dependent fear were examined in tests conducted 24 and 48h after conditioning, respectively. The selective 5-HT 7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,-tetrahydrobenzo(c,d)indol-2-(1H)-one (DR4004) (5mg/kg), when administered intraperitoneally (i.p.) immediately after conditioning, caused a significant decrease in both context- and tone-dependent fear responses (freezing behavior). A significant increase in ERK activity was observed in the amygdala of mice that displayed context- or tone-dependent fear responses, and these changes were also inhibited by the administration of DR4004 (5mg/kg, i.p.) immediately after conditioning. In contrast, the increase in hippocampal ERK activity in mice that displayed context-dependent fear responses was further enhanced by the administration of DR4004 (5mg/kg, i.p.). These results suggest that 5-HT 7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Human genetic studies have indicated that mutations in calcium/calmodulin-dependent serine protein kinase ( CASK ) result in X-linked mental retardation and autism-spectrum disorders. We aimed to establish a mouse model to study how Cask regulates mental ability., Methods: Because Cask encodes a multidomain scaffold protein, a possible strategy to dissect how CASK regulates mental ability and cognition is to disrupt specific protein-protein interactions of CASK in vivo and then investigate the impact of individual specific protein interactions. Previous in vitro analyses indicated that a rat CASK T724A mutation reduces the interaction between CASK and T-brain-1 (TBR1) in transfected COS cells. Because TBR1 is critical for glutamate receptor, ionotropic, N -methyl-D-aspartate receptor subunit 2B ( Grin2b ) expression and is a causative gene for autism and intellectual disability, we then generated CASK T740A (corresponding to rat CASK T724A) mutant mice using a gene-targeting approach. Immunoblotting, coimmunoprecipitation, histological methods and behavioural assays (including home cage, open field, auditory and contextual fear conditioning and conditioned taste aversion) were applied to investigate expression of CASK and its related proteins, the protein-protein interactions of CASK, and anatomic and behavioural features of CASK T740A mice., Results: The CASK T740A mutation attenuated the interaction between CASK and TBR1 in the brain. However, CASK T740A mice were generally healthy, without obvious defects in brain morphology. The most dramatic defect among the mutant mice was in extinction of associative memory, though acquisition was normal., Limitations: The functions of other CASK protein interactions cannot be addressed using CASK T740A mice., Conclusion: Disruption of the CASK and TBR1 interaction impairs extinction, suggesting the involvement of CASK in cognitive flexibility.

We examined the neural substrates of fear memory formation and maintenance when repeated recall was used to prevent forgetting in young animals. In contrast to adult rats, juveniles failed to show contextual fear responses at 4 d post-fear conditioning. Reconsolidation sessions 3 and 6 d after conditioning restored contextual fear responses in juveniles 7 d after initial training. In juveniles that received reconsolidation sessions, protein kinase M zeta (PKMζ) increased in the amygdala, but not in the hippocampus. These data suggest that repeated reminders and increased PKMζ maintain fear responses in juvenile animals that otherwise would not exhibit this behavior., (© 2016 Oliver et al.; Published by Cold Spring Harbor Laboratory Press.)

Aromatase (ARO) is a cytochrome P450 enzyme that accounts for local estrogen production in the brain. The goal of this study was to develop a microsomal based assay to sensitively and reliably detect the low levels of ARO activity in different brain regions. Enzyme activity was detected based on the conversion of testosterone to estradiol. Quantity of estradiol was measured using ultra performance liquid chromatography-mass spectrometry. Detection was linear over a range of 2.5-200pg/ml estradiol, and was reproducible with intra- and inter-assay coefficients of variation (CV) <15%. Estradiol production using isolated microsomes was linear with time up to 30min as well as linearly related to amount of microsome. Substrate concentration curves revealed enzymatic kinetics (hippocampus: Vmax and Km: 0.57pmol estradiol/h per mg microsome and 48.58nM; amygdala: Vmax and Km: 1.69pmol estradiol/h per mg microsome and 48.4nM; preoptic area: Vmax and Km: 0.96pmol estradiol/h per mg microsome and 44.31nM) with testosterone used at a saturating concentration of 400nM. Anastrozole treatment blocked ARO activity in hippocampal and ovarian microsomes, indicating that the assay is specific for ARO. Also, we showed that the distribution of the long form ARO mRNA (CYP19A1) in different regions of the brain is correlated with ARO activity, with highest levels in the amygdala, followed by preoptic area and hippocampus. In the frontal cortex, very little long form ARO mRNA, and little to no ARO activity, were detected. These findings demonstrate that the microsomal incubation (MIB) assay is a sensitive and reliable method for quantifying ARO activity in discrete brain regions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Destabilization refers to a memory that becomes unstable when reactivated and is susceptible to disruption by amnestic agents. Here we delineated the cellular mechanism underlying the destabilization of drug memory. Mice were conditioned with methamphetamine (MeAM) for 3 d, and drug memory was assessed with a conditioned place preference (CPP) protocol. Anisomycin (ANI) was administered 60 min after the CPP retrieval to disrupt reconsolidation. We found that destabilization of MeAM CPP after the application of ANI was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 and the NR2B antagonist ifenprodil (IFN) but not by the NR2A antagonist NVP-AAM077 (NVP). In addition, decrease in the phosphorylation of GluR1 at Serine845 (p-GluR1-Ser845), decrease in spine density, and a reduction in the AMPAR/NMDAR ratio in the basolateral amygdala (BLA) were reversed after the MK-801 treatment. The effect of ANI on destabilization was prevented by the protein phosphatase 2B (calcineurin, CaN) inhibitors cyclosporine A (CsA) and FK-506 and the protein phosphatase 1 (PP1) inhibitors calyculin A (CA) and okadaic acid (OA). These results suggest that memory destabilization involves the activation of NR2B-containing NMDARs, which in turn allows the influx of Ca(2+) Increased intracellular Ca(2+) stimulates CaN, leading to the dephosphorylation and inactivation of inhibitor 1 and the activation of PP1. PP1 then dephosphorylates p-GluR1-Ser845 to elicit AMPA receptor (AMPAR) endocytosis and destabilization of the drug memory., (© 2016 Yu et al.; Published by Cold Spring Harbor Laboratory Press.)

Ropivacaine (Ropi), one of the newest and safest amino amide local anesthetics, is linked to toxicity, including the potential for seizures, changes in behavior, and even cardiovascular collapse. Dexmedetomidine (Dex), an α2-adrenergic receptor agonist, has been widely used in anesthesia and critical care practice. To date, the underlying mechanisms of the effects of Dex premedication on Ropi-induced toxicity have not been clearly identified. In the current study, we investigated the effects of increasing doses of Dex premedication on 50% convulsive dose (CD50) of Ropi. With increasing doses of intraperitoneal (i.p.) Dex 10 min prior to each i.p. RopiCD50, the latency and duration of seizure activity were recorded. Open-field (OF) and elevated plus maze (EPM) test were used to measure negative behavioral emotions such as depression and anxiety. Immunohistochemistry and Western blot were utilized to investigate phosphorylation-extracellular regulated protein kinases (p-ERK) expression in the basolateral amygdala (BLA) on 2 h and in the central amygdala (CeA) on 24 h after convulsion in mice. The results of our investigation demonstrated that Dex dose-dependently increased RopiCD50, prolonged the latency and shortened the duration of each RopiCD50-induced seizure, improved the negative emotions revealed by both OF and EPM test, and inhibited p-ERK expression in the BLA and the CeA.

κ opioid receptor agonists produce aversive effects in rodents, however the underlying mechanisms remain unclear. Activation of p38 mitogen-activated protein kinase (MAPK) has been discovered to play a critical role in the modulation of affective behaviors. The present study was undertaken to detect the possible involvement of p38 MAPK in the aversive effects induced by κ opioid receptor activation. We found that the κ opioid receptor agonist trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzenacetamide methanesulfonate salt (U50,488H) produced significant place aversion in mice as measured by the conditioned place preference procedure, accompanied with significant p38 MAPK activation in the amygdala, but not in the nucleus accumbens and hippocampus. Stereotaxic microinjection of the p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridy-l)-1H-imidazole (SB203580) into amygdala significantly inhibited p38 MAPK activation and completely blocked the conditioned place aversion in mice. Thus, these results suggested that activation of p38 MAPK in the amygdala was required to mediate κ opioid receptor-induced aversive behavior., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and β-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Long-term memory (LTM) of fear stores activity dependent modifications that include changes in amygdala signaling. Previously, we identified an enhanced probability of release of glutamate mediated signaling to be important in rat fear potentiated startle (FPS), a well-established translational behavioral measure of fear. Here, we investigated short- and long-term synaptic plasticity in FPS involving metabotropic glutamate receptors (mGluRs) and associated downstream proteomic changes in the thalamic-lateral amygdala pathway (Th-LA). Aldolase A, an inhibitor of phospholipase D (PLD), expression was reduced, concurrent with significantly elevated PLD protein expression. Blocking the PLD-mGluR signaling significantly reduced PLD activity. While transmitter release probability increased in FPS, PLD-mGluR agonist and antagonist actions were occluded. In the unpaired group (UNP), blocking the PLD-mGluR increased while activating the receptor decreased transmitter release probability, consistent with decreased synaptic potentials during tetanic stimulation. FPS Post-tetanic potentiation (PTP) immediately following long-term potentiation (LTP) induction was significantly increased. Blocking PLD-mGluR signaling prevented PTP and reduced cumulative PTP probability but not LTP maintenance in both groups. These effects are similar to those mediated through mGluR7, which is co-immunoprecipitated with PLD in FPS. Lastly, blocking mGluR-PLD in the rat amygdala was sufficient to prevent behavioral expression of fear memory. Thus, our study in the Th-LA pathway provides the first evidence for PLD as an important target of mGluR signaling in amygdala fear-associated memory. Importantly, the PLD-mGluR provides a novel therapeutic target for treating maladaptive fear memories in posttraumatic stress and anxiety disorders., (Published by Elsevier Inc.)

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala - peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 °C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Background: Social defeat (SD) stress induces social avoidance and anxiety-like phenotypes. Amygdala is recognized as an emotion-related brain region such as fear, aversion and anxiety. It is conceivable to hypothesize that activation of amygdala is involved in SD-dependent behavioral defects., Results: SD model was established using C57BL/6J mice that were physically defeated by different CD-1 mice for 10 days. Stressed mice exhibited decreased social interaction level in social interaction test and significant anxiety-like behaviors in elevated plus maze and open field tests. Meanwhile, a higher phosphorylation of PKA and CREB with a mutually linear correlation, and increased Fos labeled cells in the basolateral amygdala (BLA) were observed. Activation of PKA in the BLA by 8-Br-cAMP, a PKA activitor, significantly upregulated pCREB and Fos expression. To address the role of PKA activation on SD stress-induced social avoidance and anxiety-like behaviors, 8-Br-cAMP or H-89, a PKA inhibitor, was continuously administered into the bilateral BLA by a micro-osmotic pump system during the 10-day SD period. Neither H-89 nor 8-Br-cAMP affected the social behavior. Differently, 8-Br-cAMP significantly relieved anxiety-like behaviors in both general and moderate SD protocols. H-89 per se did not have anxiogenic effect in naïve mice, but aggravated moderate SD stress-induced anxiety-like behaviors. The antidepressant clomipramine reduced SD-induced anxiety and up-regulated pPKA level in the BLA., Conclusions: These results suggest that SD-driven PKA activation in the basolateral amygdala is actually a compensatory rather than pathogenic response in the homeostasis, and modulating amygdaloid PKA may exhibit potency in the therapy of social derived disorders.

Background: While the Albizia adianthifolia (Schumach.) W. Wright (Fabaceae) is a traditional herb largely used in the African traditional medicine as analgesic, purgative, antiinflammatory, antioxidant, antimicrobial, memory-enhancer, anxiolytic and antidepressant drug, there are no scientific data that clarify the anxiolytic and antidepressant-like effects in 6-hydroxydopamine (6-OHDA)-lesioned animal model of Parkinson's disease. This study was undertaken in order to identify the effects of aqueous extract of A. adianthifolia leaves on 6-hydroxydopamine-induced anxiety, depression and oxidative stress in the rat amygdala., Methods: The effect of the aqueous extract of A. adianthifolia leaves (150 and 300 mg/kg, orally, daily, for 21 days) on anxiety and depression was assessed using elevated plus-maze and forced swimming tests, as animal models of anxiety and depression. Also, the antioxidant activity in the rat amygdala was assessed using assessed using superoxide dismutase, glutathione peroxidase and catalase specific activities, the total content of the reduced glutathione, protein carbonyl and malondialdehyde levels. Statistical analyses were performed using by one-way analysis of variance (ANOVA). Significant differences were determined by Tukey's post hoc test. F values for which p < 0.05 were regarded as statistically significant. Pearson's correlation coefficient and regression analysis were used in order to evaluate the connection between behavioral measures, the antioxidant defence and lipid peroxidation., Results: 6-OHDA-lesioned rats exhibited the following: decrease of the exploratory activity, the percentage of the time spent and the number of entries in the open arm within elevated plus-maze test and decrease of swimming time and increase of immobility time within forced swimming test. Administration of the aqueous extract significantly exhibited anxiolytic- and antidepressant-like effects and also antioxidant potential in the rat amygdala., Conclusions: Our results suggest that the aqueous extract ameliorates 6-OHDA-induced anxiety and depression by attenuation of the oxidative stress in the rat amygdala. These pieces of evidence accentuate its use in traditional medicine.

The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p<0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Depression is one of the most common mental disorders, but its etiology is not completely understood. It is assumed that peptidergic system components are involved in the formation of this pathology. Neuropeptides play an important role in the regulation of mental and emotional states. Сarboxypeptidase E is a key enzyme of peptide processing; it regulates neuropeptide levels in the various structures of the nervous system. We have studied effects of a single dose of reboxetine on the activity of carboxypeptidase E in various brain regions and the adrenal glands of rats. The reboxetine injection decreased carboxypeptidase E activity in the pituitary gland (12 h after injection), in the pituitary gland, the quadrigeminal bodies, the medulla oblongata, the hypothalamus, the hippocampus and the amygdala (24 h after injection), in the pituitary gland and striatum (72 h after injection). The enzyme activity in adrenal glands remained basically unchanged. Apparently, the decrease of carboxypeptidase E activity may influence the level of regulatory peptides involved in the pathogenesis of depression.

The incidence of anxiety-related diseases is increasing these days, hence there is a need to understand the mechanisms that underlie its nature and consequences. It is known that limbic structures, mainly the prefrontal cortex and amygdala, are involved in the processing of anxiety, and that projections from prefrontal cortex and amygdala can induce activity of the hypothalamic-pituitary-adrenal axis with consequent cardiovascular changes, increase in oxygen consumption, and ROS production. The compensatory reaction can include increased antioxidant enzymes activities, overexpression of antioxidant enzymes, and genetic shifts that could include the activation of antioxidant genes. The main objective of this study was to evaluate the oxidant/antioxidant effect that chronic anxiogenic stress exposure can have in prefrontal cortex, amygdala, and hypothalamus by exposition to predator odor. Results showed (a) sensitization of the HPA axis response, (b) an enzymatic phase 1 and 2 antioxidant response to oxidative stress in amygdala, (c) an antioxidant stability without elevation of oxidative markers in prefrontal cortex, (d) an elevation in phase 1 antioxidant response in hypothalamus. Chronic exposure to predator odor has an impact in the metabolic REDOX state in amygdala, prefrontal cortex, and hypothalamus, with oxidative stress being prevalent in amygdala as this is the principal structure responsible for the management of anxiety.

Phyto-oestrogens are biologically active components of many human and laboratory animal diets. In the present study, we investigated, in adult male mice with C57BL/6 genetic background, the effects of a reduced phyto-oestrogens intake on anxiety-related behaviour and associated gene expression in the amygdala. After 6 weeks on a low-phyto-oestrogen diet (< 20 μg/g cumulative phyto-oestrogen content), animals showed reduced centre exploration in an open-field task compared to their littermates on a soybean-based standard diet (300 μg/g). Freezing behaviour in an auditory fear memory task, in contrast, was not affected. We hypothesised that this mildly increased anxiety may involve changes in the function of GABAergic local circuit neurones in the amygdala. Using GAD67(+/GFP) mice, we could demonstrate reduced transcription of the GAD67 gene in the lateral and basolateral amygdala under the low-phyto-oestrogen diet. Analysis of mRNA levels in microdissected samples confirmed this regulation and demonstrated concomitant changes in expression of the second glutamic acid decarboxylase (GAD) isoform, GAD65, as well as the anxiolytic neuropeptide Y. These molecular and behavioural alterations occurred without apparent changes in circulating oestrogens or testosterone levels. Our data suggest that expression regulation of interneurone-specific gene products in the amygdala may provide a mechanism for the control of anxiety-related behaviour through dietary phyto-oestrogens., (© 2015 British Society for Neuroendocrinology.)

Brain enkephalin, vasopressin and oxytocin are anxiolytic agents involved in the stress response. Acute restraint stress influences certain neuropeptidase activities, such as some enkephalin-degrading peptidases and vasopressinase/oxytocinase, in the medial prefrontal cortex (mPFC), amygdala (AM) or hippocampus (HC), which are involved in this response. Because these regions form a unified circuit and cooperate in their response to stress, it is important to analyze the profile of the regional distribution of these activities as well as their inter-regional model of interaction in this circuit. Regarding the regional study, although most activities showed a marked predominance of the AM over the HC and mPFC, both in control and stressed animals, enkephalin-degrading activity, assayed as membrane-bound alanyl aminopeptidase activity, showed a change after stress, increasing in the HC and decreasing in the AM. The correlational study in controls indicated essentially a positive interaction between the mPFC and AM. In marked contrast, there was a highly significant change in the functional status of this circuit after stress, showing mainly a positive correlation between the mPFC and HC and between the AM and HC. The existence of correlations does not demonstrate a direct relationship between regions. However, reasons for such strong associations after restraint stress should be examined. The present study may indicate a connection between neuropeptidase activities and their corresponding neuropeptidergic substrates due to significant changes in the functional status of the cortico-limbic circuit after restraint stress., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.

An imbalance of the gamma-aminobutyric acid (GABA) system is considered a major neurobiological pathomechanism of anxiety, and the amygdala is a key brain region involved. Reduced GABA levels have been found in anxiety patients, and genetic variations of glutamic acid decarboxylase (GAD), the rate-limiting enzyme of GABA synthesis, have been associated with anxiety phenotypes in both humans and mice. These findings prompted us to hypothesize that a deficiency of GAD65, the GAD isoform controlling the availability of GABA as a transmitter, affects synaptic transmission and plasticity in the lateral amygdala (LA), and thereby interferes with fear responsiveness. Results indicate that genetically determined GAD65 deficiency in mice is associated with (1) increased synaptic length and release at GABAergic connections, (2) impaired efficacy of GABAergic synaptic transmission and plasticity, and (3) reduced spillover of GABA to presynaptic GABAB receptors, resulting in a loss of the associative nature of long-term synaptic plasticity at cortical inputs to LA principal neurons. (4) In addition, training with high shock intensities in wild-type mice mimicked the phenotype of GAD65 deficiency at both the behavioral and synaptic level, indicated by generalization of conditioned fear and a loss of the associative nature of synaptic plasticity in the LA. In conclusion, GAD65 is required for efficient GABAergic synaptic transmission and plasticity, and for maintaining extracellular GABA at a level needed for associative plasticity at cortical inputs in the LA, which, if disturbed, results in an impairment of the cue specificity of conditioned fear responses typifying anxiety disorders.

Genistein is a phytoestrogen, particularly abundant in soybeans, that is able to bind estrogen receptors exerting both estrogenic and antiestrogenic activities. Genistein is largely present in the human diet even during pregnancy. Embryos and fetuses are therefore, commonly exposed to genistein during the development and after birth. In the present study, we used a murine model as a test end-point to investigate the effects of early exposure to genistein on adult male behavior and related neural circuits. Daily exposure of dams to genistein (100 μg/g of body weight) during late pregnancy and early lactation, produced in male offspring, when adults, significant changes in anxiety and aggressive behaviors. Moreover, we found statistically significant variations in the number of neuronal nitric-oxide synthase positive cells in the amygdala. In conclusions, these data indicate that early exposure to phytoestrogens may induce life-long effects on the differentiation of brain structures and behaviors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Copper (Cu) is an essential element with many biological roles, but its roles in the mammalian nervous system are poorly understood. Mice deficient in the cuproenzyme peptidylglycine α-amidating monooxygenase (Pam(+/-) mice) were initially generated to study neuropeptide amidation. Pam(+/-) mice exhibit profound deficits in a few behavioral tasks, including enhancements in innate fear along with deficits in acquired fear. Interestingly, several Pam(+/-) phenotypes were recapitulated in Cu-restricted wild-type mice and rescued in Cu-supplemented Pam(+/-) mice. These behaviors correspond to enhanced excitability and deficient synaptic plasticity in the amygdala of Pam(+/-) mice, which are also rescued by Cu supplementation. Cu and ATP7A are present at synapses, in key positions to respond to and influence synaptic activity. Further study demonstrated that extracellular Cu is necessary for wild-type synaptic plasticity and sufficient to induce long-term potentiation. These experiments support roles for PAM in Cu homeostasis and for synaptic Cu in amygdalar function., (© 2014 New York Academy of Sciences.)

Emotional and motivational dysfunctions observed in Parkinson's disease, schizophrenia, and drug addiction are associated to an alteration of the mesocortical and mesolimbic dopaminergic pathways, which include axons projecting to the prefrontal cortex, the ventral striatum, and the amygdala. Subpopulations of catecholaminergic neurons have been described in the cortex and striatum of several mammals, but the presence of such cells in the adult amygdala is unclear in murine rodents, and in other rodents appears to show variations depending on the species. Moreover, the embryonic origin of telencephalic tyrosine hydroxylase (TH) cells is unknown, which is essential for trying to understand aspects of their evolution, distribution and function. Herein we investigated the expression of TH mRNA and protein in cells of the striatum and amygdala of developing and adult mice, and analyzed the embryonic origin of such cells using in vitro migration assays. Our results showed the presence of TH mRNA and protein expressing cells in the striatum (including nucleus accumbens), central and medial extended amygdala during development, which are persistent in adulthood although they are less numerous, generally show weak mRNA expression, and some appear to lack the protein. Fate mapping analysis showed that these cells include at least two subpopulations with different embryonic origin in either the commissural preoptic area of the subpallium or the supraopto-paraventricular domain of the alar hypothalamus. These data are important for future studies trying to understand the role of catecholamines in modulation of emotion, motivation, and reward.

The first 2 weeks of life are a critical period for neural development in rats. Repeated long-term separation from the dam is considered to be one of the most potent stressors to which rat pups can be exposed, and permanently modifies neurobiological and behavioral parameters. Prolonged periods of maternal separation (MS) usually increase stress reactivity during adulthood, and enhance anxiety-like behavior. The aim of this study was to verify the effects of maternal separation during the neonatal period on memory as well as on biochemical parameters (Na(+), K(+)-ATPase and antioxidant enzymes activities) in the amygdala of adult rats. Females and male Wistar rats were subjected to repeated maternal separation (incubator at 32 °C, 3 h/day) during postnatal days 1-10. At 60 days of age, the subjects were exposed to a Contextual fear conditioning task. One week after the behavioral task, animals were sacrificed and the amygdala was dissected for evaluation of Na(+), K(+)-ATPase and antioxidant enzymes activities. Student-t test showed significant MS effect, causing an increase of freezing time in the three exposures to the aversive context in both sexes. Considering biochemical parameters Student-t test showed significant MS effect causing an increase of Na(+), K(+)-ATPase activity in both sexes. On the other hand, no differences were found among the groups on the antioxidant enzymes activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] in male rats, but in females, we found a significant MS effect, causing an increase of CAT activity and no differences were found among the groups on SOD and GPx activities. Our results suggest a role of early rearing environment in programming fear learning and memory in adulthood. An early stress experience such as maternal separation may increase activity in the amygdala (as pointed by the increased activity of Na(+), K(+)-ATPase), affecting behaviors related to fear in adulthood, and this effect could be task-specific.

Objective: The aim of this study was to evaluate if nitric oxide (NO) in the central amygdala (CeA) is involved in the expression of withdrawal aspects induced by morphine., Materials and Methods: Male Wistar rats (weighing 200-250 g) were bilaterally cannulated in the CeA and conditioned to morphine using an unbiased paradigm. Morphine (2.5-10 mg/kg) was subcutaneously injected once a day throughout the conditioning phase of the procedure. This phase also included 3-saline paired sessions. Naloxone (0.1-0.4 mg/kg, intraperitoneally [i.p.]), an antagonist of opioid receptors, was administered i.p. 10 min prior to testing of morphine-induced withdrawal features. The NO precursor, L-arginine (0.3-3 μg/rat) was intra-CeA injected prior to testing of naloxone response. To evaluate the involvement of NO system an inhibitor of NO synthase (NOS), N(G)-nitro-L-arginine methyl ester (L-NAME) (0.3-3 μg/rat), was injected ahead of L-arginine. Control group received saline solely instead of drug. As a complementary study, the activation of NOS was studied by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)., Results: Morphine induced a significant increase in wet dog shaking and grooming behaviors compared with controls. Injection of naloxone pre-testing of morphine response significantly reversed the response to morphine. However, pre-microinjection of L-arginine intra-CeA recovered the response to morphine. Injection of L-NAME intra-CeA ahead of L-arginine though had no effect behaviorally, but, inhibited the NOS which has been evidenced by NADPH-d., Conclusion: The present study shows that NO in the CeA potentiates the expression of conditioned withdrawal induced by morphine paired with naloxone.

We have previously shown that auditory Pavlovian fear conditioning is associated with an increase in DNA methyltransferase (DNMT) expression in the lateral amygdala (LA) and that intra-LA infusion or bath application of an inhibitor of DNMT activity impairs the consolidation of an auditory fear memory and long-term potentiation (LTP) at thalamic and cortical inputs to the LA, in vitro. In the present study, we use awake behaving neurophysiological techniques to examine the role of DNMT activity in memory-related neurophysiological changes accompanying fear memory consolidation and reconsolidation in the LA, in vivo. We show that auditory fear conditioning results in a training-related enhancement in the amplitude of short-latency auditory-evoked field potentials (AEFPs) in the LA. Intra-LA infusion of a DNMT inhibitor impairs both fear memory consolidation and, in parallel, the consolidation of training-related neural plasticity in the LA; that is, short-term memory (STM) and short-term training-related increases in AEFP amplitude in the LA are intact, while long-term memory (LTM) and long-term retention of training-related increases in AEFP amplitudes are impaired. In separate experiments, we show that intra-LA infusion of a DNMT inhibitor following retrieval of an auditory fear memory has no effect on post-retrieval STM or short-term retention of training-related changes in AEFP amplitude in the LA, but significantly impairs both post-retrieval LTM and long-term retention of AEFP amplitude changes in the LA. These findings are the first to demonstrate the necessity of DNMT activity in the consolidation and reconsolidation of memory-associated neural plasticity, in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Rationale: Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response-cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon., Objectives: The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response-cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior., Methods: Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context., Results: Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion., Conclusions: PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental cocaine-seeking behavior.

Stress during gestation increases vulnerability to disease and changes behavior in offspring. We previously reported that hypoxia and restraint during pregnancy sensitized the hypothalamic-pituitary-adrenal (HPA) axis and induced anxiety-like behavior in the adult offspring. Here, we report that gestational intermittent hypoxia (GIH) elicited a sex-dependent anxiety-like behavior in male P90 offspring and activation of corticotropin-releasing hormone (CRH) and CRH type-1 receptor (CRHR1) mRNA in the hypothalamic paraventricular nucleus (PVN) and in male E19 hypothalamus. These linked to demethylation at several specific sites of CpG island of Crhr1 promoter in P90 PVN and E19 embryo hypothalamus in GIH groups. Crhr1 DNA demethylation is more crucial in CpG island 1 than island 2 for activation of CRHR1 mRNA. DNMT3b is required for the Crhr1 DNA methylation than DNMT1 and DNMT3a in increased CRHR1 mRNA. We first address a novel hypothesis that GIH-induced male-sex-dependent demethylation at CpG sites of Crhr1 DNA in promoter triggers elevation of CRHR1 mRNA in PVN and anxiety-like behavior in adult offspring.

We studied the effect of IL-4 on antioxidant enzyme activity in brain structures (hypothalamus, sensorimotor cortex, and amygdala) in behaviorally passive and active rats. One-hour immobilization of animals with simultaneous delivery of subthreshold electrocutaneous stimulation was used as the model of acute stress. Intraperitoneal injection of IL-4 (5 μg/kg) was followed by an increase in activities of glutathione peroxidase and Cu/Zn SOD in the hypothalamus of non-stressed rats. Activities of glutathione peroxidase and Cu/Zn SOD in the amygdala were shown to decrease. Administration of IL-4 was accompanied by activation of glutathione peroxidase (active and passive rats), glutathione reductase (passive rats), and Cu/Zn SOD (active rats) in the sensorimotor cortex. These data indicate that the efficiency of antioxidant protection increases in the hypothalamus and sensorimotor cortex, but decreases in the amygdala of rats receiving IL-4. Pretreatment with IL-4 abolished a poststress increase in glutathione peroxidase activity in the sensorimotor cortex of passive animals.

A long-standing literature linking endocannabinoids (ECBs) to stress, fear, and anxiety has led to growing interest in developing novel anxiolytics targeting the ECB system. Following rapid on-demand biosynthesis and degradation upon neuronal activation, the ECB N-arachidonoylethanolamide (anandamide, AEA) is actively degraded by the serine hydrolase enzyme, fatty acid amide hydrolase (FAAH). Exposure to stress rapidly mobilizes FAAH to deplete the signaling pool of AEA and increase neuronal excitability in a key anxiety-mediating region--the basolateral amygdala (BLA). Gene deletion or pharmacological inhibition of FAAH prevents stress-induced reductions in AEA and associated increases in BLA dendritic hypertrophy and anxiety-like behavior. Additionally, inhibition of FAAH facilitates long-term fear extinction and rescues deficient fear extinction in rodent models by enhancing AEA-CB1 (cannabinoid type 1) receptor signaling and synaptic plasticity in the BLA. These preclinical findings propose restoring deficient BLA AEA levels by pharmacologically inhibiting FAAH as a mechanism to therapeutically mitigate the effects of traumatic stress., (Copyright © 2013. Published by Elsevier Ltd.)

Substantial evidence indicates that predisposition to diseases can be acquired during early stages of development and interactions between environmental and genetic factors may be implicated in the onset of many pathological conditions. We have shown that perinatal exposure to bisphenol A (BPA) at environmental dose level causes long-term anxiety-like behaviors in rats. The aim of this study was to examine epigenetic reprogramming effect of BPA on anxiety-related neurobehavior in the rat offspring. The results of real-time RT-PCR displayed that the overexpression of DNA methyltransferase 1 (DNMT1) mRNA was accompanied by the reduction of glutamic acid decarboxylase 67 (GAD67) mRNA level in the basolateral amygdala (BLA) of postnatal day 45 BPA-exposed female rats. Chronic intro-BLA injection with 5-ada-CdR could rectify the GAD67 mRNA expression. Behavioral data showed that the anxiety-like behaviors in BPA-exposed rats were reversed by intro-BLA treatment with 5-ada-CdR which could be further blocked by PTX. Electrophysiological study revealed behavioral alterations were associated with the increase of postsynaptic neuronal excitability in the cortical-BLA pathway which appeared as multispike responses, paired-pulse facilitation instead of paired-pulse inhibition and long-term potentiation and 5-aza-CdR treatment restored the increased synaptic transmission in the BLA via improving GABAergic system. The above results suggest that the overexpression of DNMT1 in the BLA is responsible for the etiology of anxiety associated with BPA exposure via GABAergic disinhibition. In addition, we also find these long-term neurobehavioral effects of developmental BPA exposure are reversible in adolescent period., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Exposure to nerve agents induces intense seizures (status epilepticus, SE), which cause brain damage or death. Identification of the brain regions that are critical for seizure initiation after nerve agent exposure, along with knowledge of the physiology of these regions, can facilitate the development of pretreatments and treatments that will successfully prevent or limit the development of seizures and brain damage. It is well-established that seizure initiation is due to excessive cholinergic activity triggered by the nerve agent-induced irreversible inhibition of acetylcholinesterase (AChE). Therefore, the reason that when animals are exposed to lethal doses of a nerve agent, a small proportion of these animals do not develop seizures, may have to do with failure of the nerve agent to inhibit AChE in brain areas that play a key role in seizure initiation and propagation. In the present study, we compared AChE activity in the basolateral amygdala (BLA), hippocampus, and piriform cortex of rats that developed SE (SE rats) after administration of the nerve agent soman (154μg/kg) to AChE activity in these brain regions of rats that received the same dose of soman but did not develop SE (no-SE rats). The levels of AChE activity were measured at the onset of SE in SE rats, 30min after soman administration in no-SE rats, as well as in controls which received saline in place of soman. In the control group, AChE activity was significantly higher in the BLA compared to the hippocampus and piriform cortex. Compared to controls, AChE activity was dramatically lower in the hippocampus and the piriform cortex of both the SE rats and the no-SE rats, but AChE activity in the BLA was reduced only in the SE rats. Consistent with the notion that soman-induced neuropathology is due to intense seizures, rather than due to a direct neurotoxic effect of soman, no-SE rats did not present any neuronal loss or degeneration, 7 days after exposure. The results suggest that inhibition of AChE activity in the BLA is necessary for the generation of seizures after nerve agent exposure, and provide strong support to the view that the amygdala is a key brain region for the induction of seizures by nerve agents., (Published by Elsevier B.V.)

The Uba6 (E1)-Use1 (E2) ubiquitin transfer cascade is a poorly understood alternative arm of the ubiquitin proteasome system (UPS) and is required for mouse embryonic development, independent of the canonical Uba1-E2-E3 pathway. Loss of neuronal Uba6 during embryonic development results in altered patterning of neurons in the hippocampus and the amygdala, decreased dendritic spine density, and numerous behavioral disorders. The levels of the E3 ubiquitin ligase Ube3a (E6-AP) and Shank3, both linked with dendritic spine function, are elevated in the amygdala of Uba6-deficient mice, while levels of the Ube3a substrate Arc are reduced. Uba6 and Use1 promote proteasomal turnover of Ube3a in mouse embryo fibroblasts (MEFs) and catalyze Ube3a ubiquitylation in vitro. These activities occur in parallel with an independent pathway involving Uba1-UbcH7, but in a spatially distinct manner in MEFs. These data reveal an unanticipated role for Uba6 in neuronal development, spine architecture, mouse behavior, and turnover of Ube3a., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The reconsolidation of cocaine memories following retrieval is necessary for the sustained ability of a cocaine-paired environmental context to elicit cocaine seeking. Extracellular signal-regulated kinase (ERK) is an intracellular signaling molecule involved in nucleus accumbens core (NACc)-mediated reconsolidation of Pavlovian cocaine memories. Here, we used a rodent model of drug context-elicited relapse to test the hypothesis that ERK would be similarly required for the reconsolidation of context-response-cocaine memories that underlie drug context-induced reinstatement of instrumental cocaine-seeking behavior, with a focus on the NACc and on the basolateral amygdala (BLA), another important locus for the reconsolidation of cocaine memories. We show that the mitogen-activated protein kinase (MEK)/ERK1/2 inhibitor, U0126 (1.0 μg/0.5 μl/hemisphere), microinfused bilaterally into the BLA--but not the NACc--immediately after brief re-exposure to a previously cocaine-paired context (that is, cocaine-memory reactivation), significantly attenuated subsequent drug context-induced cocaine seeking relative to vehicle (VEH). This effect in the BLA was associated with a transient inhibition of ERK1/2 phosphorylation, and it depended on memory reactivation given that U0126 administered following exposure to a novel context did not alter subsequent cocaine seeking. Furthermore, similar to U0126, baclofen+muscimol-induced (B+M; 106.8/5.7 ng/0.5 μl/hemisphere) neural inactivation of the NACc, following cocaine-memory reactivation, failed to alter subsequent cocaine seeking. These findings demonstrate that ERK activation in the BLA, but not the NACc, is required for the reconsolidation of context-response-cocaine associative memories. Together with prior research, these results suggest that contextual drug-memory reconsolidation in Pavlovian and instrumental settings involves distinct neuroanatomical mechanisms.

Chronic exposure to low doses of organophosphates during brain development can induce persistent neurochemical and behavioral effects. This study sought to determine the long-lasting effects of developmental exposure to chlorpyrifos (CPF) and diazinon (DZN) on passive avoidance (PA) performance and neuronal nitric oxide synthase (nNOS)-containing neurons in the subnuclei within basolateral complex of amygdala (BLC). Developing rats were exposed to daily dose (1mg/kg) of CPF or DZN during gestational days 15-18 and postnatal days (PND) 1-4. PA performance was assessed in young adulthood (PND 60). Brain sections were also processed by NADPH-diaphorase (NADPH-d) and nNOS immunohistochemistry. Gestational exposure to CPF increased NADPH-d(+)/nNOS-immunoreactive (IR) neurons within the basolateral nucleus (BL) and medial paracapsular intercalated cluster, which was along with PA retention impairment in both male and female rats. Prenatal exposure to DZN did not significantly change the number of NADPH-d(+)/nNOS-IR neurons in the BLC while impaired PA retention in females. Postnatal exposure to CPF decreased NADPH-d(+)/NOS-IR neurons in the BL without affecting PA performance. Exposure to DZN during early postnatal period impaired PA retention in both sexes, albeit to a lesser extent in females, and was along with a considerable sex independent reduction of NADPH-d(+)/NOS-IR neurons in all BLC subnuclei. Our data suggest that developmental exposure to apparently subtoxic dose of CPF and DZN elicit long-lasting impairment in PA retention that are associated, but not necessarily correlated with effects on NADPH-d(+)/NOS-IR neurons in BLC of the amygdala., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Recent findings implicate the central lateral amygdala (CeL) in conditioned fear. Indeed, CeL contains neurons exhibiting positive (CeL-On) or negative (CeL-Off) responses to fear-inducing conditioned stimuli (CSs). In mice, these cells differ in their expression of protein kinase Cδ (PKCδ) and physiological properties. CeL-Off cells are PKCδ(+) and late firing (LF), whereas CeL-On cells are PKCδ(-) and express a regular-spiking (RS) or low-threshold bursting (LTB) phenotype. However, the scarcity of LF cells in rats raises questions about the correspondence between the organization of CeL in mice and rats. Therefore, we studied the PKCδ expression, morphological properties, synaptic responsiveness, and fear conditioning-induced plasticity of rat CeL neurons. No PKCδ(+) LF cells were encountered, but ≈20-25% of RS and LTB neurons were PKCδ(+). Compared with RS neurons, a higher proportion of LTB cells projected to central medial amygdala (CeM) and they had fewer primary dendritic branches, yet the amplitude of excitatory postsynaptic potentials (EPSPs) evoked by lateral amygdala (LA) stimulation was similar in RS and LTB cells. In contrast, LA-evoked inhibitory postsynaptic potentials (IPSPs) had a higher amplitude in LTB than RS neurons. Finally, fear conditioning did not induce plasticity at LA inputs to RS or LTB neurons. These findings point to major species differences in the organization of CeL. Since rat LTB cells are subjected to stronger feedforward inhibition, they are more likely to exhibit inhibitory CS responses than RS cells. This is expected to cause a disinhibition of CeM fear output neurons and therefore an increase in fear expression.