7,8-Dihydroxyflavone Alleviates Anxiety-Like Behavior Induced by Chronic Alcohol Exposure in Mice Involving Tropomyosin-Related Kinase B in the Amygdala.

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.

Active Transition of Fear Memory Phase from Reconsolidation to Extinction through ERK-Mediated Prevention of Reconsolidation.

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.

The Effect of Allopregnanolone on Enzymatic Activity of the DNA Base Excision Repair Pathway in the Sheep Hippocampus and Amygdala under Natural and Stressful Conditions.

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,N6-ethenoadenine (εA), 3,N4-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.

Maternal diabetes induces autism-like behavior by hyperglycemia-mediated persistent oxidative stress and suppression of superoxide dismutase 2.

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.

Na+, K+-ATPase α3 isoform in frontal cortex GABAergic neurons in psychiatric diseases.

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.

Early-stage paired housing improves social interaction in neuronal Uba6-deficient mice.

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.

Chronic mild Hyperhomocysteinemia impairs energy metabolism, promotes DNA damage and induces a Nrf2 response to oxidative stress in rats brain.

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.

Central relaxin-3 receptor (RXFP3) activation impairs social recognition and modulates ERK-phosphorylation in specific GABAergic amygdala neurons.

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 Gi/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.

Genetic Deletion of Soluble Epoxide Hydroxylase Causes Anxiety-Like Behaviors in Mice.

Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase and NH2-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/ß (GSK3α/ß) 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/ß signaling pathway.

Developmental transitions in amygdala PKC isoforms and AMPA receptor expression associated with threat memory in infant rats.

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 Induces Acute Anxiolysis by Phospholipase C-Dependent Signaling within the Medial Amygdala.

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 ß-tubulin representing Ca2+ 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.

Regulation of aromatase expression in the anterior amygdala of the developing mouse brain depends on ERß and sex chromosome complement.

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 ERß. By contrast, the effect of DHT on aromatase expression was not blocked by the anti-androgen flutamide, but completely abrogated by the ERß antagonist PHTPP. Experiments using the four core genotype model showed a sex chromosome effect in ERß 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 ERß in developing mouse brain.

Age-dependent changes in autophosphorylation of alpha calcium/calmodulin dependent kinase II in hippocampus and amygdala after contextual fear conditioning.

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.

Knocking down amygdalar PTP1B in diet-induced obese rats improves insulin signaling/action, decreases adiposity and may alter anxiety behavior.

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.

Exposure to extrinsic stressors, social defeat or bisphenol A, eliminates sex differences in DNA methyltransferase expression in the amygdala.

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.

Calcium/calmodulin-dependent serine protein kinase (CASK), a protein implicated in mental retardation and autism-spectrum disorders, interacts with T-Brain-1 (TBR1) to control extinction of associative memory in male mice.

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.

5-HT7 receptor-mediated fear conditioning and possible involvement of extracellular signal-regulated kinase.

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-HT7 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-HT7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory.

Repeated recall and PKMζ maintain fear memories in juvenile rats.

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.

Involvement of protein phosphatases in the destabilization of methamphetamine-associated contextual memory.

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.

A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography-mass spectrometry.

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.