Development of the fear regulation system from early adolescence to young adulthood in female mice.

Onset of fear-related disorders such as post-traumatic stress disorder is enhanced from adolescence until adulthood. However, the biological mechanisms underlying this vulnerability remain unclear. Therefore, we investigated contextual fear memory and extinction in 4-, 6-, 8-, 10-, and 15-week-old female mice. We also measured phosphorylation of ERK2 in the medial prefrontal cortex (mPFC) and the dorsal hippocampus following fear conditioning or extinction in 6- and 15-week-old mice. We found that 10- and 15-week-old mice showed stronger fear memory and more resistance to fear extinction than 6-week-old mice. Moreover, 15-week-old mice showed lower ERK2 phosphorylation levels following fear extinction in the mPFC than those 6 weeks old. Our results suggest that female mice acquire strong fear memory and resistance to fear extinction throughout adulthood, which may be related to alteration in ERK2 activation in the mPFC.

Intermittent Ibandronate Maintains Bone Mass, Bone Structure, and Biomechanical Strength of Trabecular and Cortical Bone After Discontinuation of Parathyroid Hormone Treatment in Ovariectomized Rats.

Although parathyroid hormone (PTH) expresses an anabolic effect on bone mass, the increased bone mass disappears once PTH treatment is withdrawn. Therefore, sequential treatment with anti-bone-resorptive agents is required to maintain bone mass after PTH treatment. We examined the effect of sequential treatment with ibandronate (IBN), a nitrogen-containing bisphosphonate, following PTH in ovariectomized (OVX) rats. Wistar-Imamichi rats (27 weeks old) were ovariectomized and treated with PTH (10 µg/kg, s.c.; 5 times/week; PTH group) for 8 weeks from 8 weeks after OVX. Thereafter, PTH was withdrawn and rats were administered IBN (10 µg/kg, s.c.; every 4 weeks; PTH-IBN group) or vehicle (PTH-Veh group) for another 8 weeks. PTH increased bone mineral density (BMD) measured by dual-energy X-ray absorptiometry and biomechanical strength in the lumbar spine and femur as compared to the disease control rats. BMD and biomechanical strength in the PTH-Veh group were lower than in the PTH group, whereas in the PTH-IBN group they were maintained at the level of the PTH group. Microstructure of the trabecular and cortical bone in the PTH-IBN group was not significantly different from that in the PTH group. In histomorphometric analysis of the lumbar vertebra, eroded surface and osteoclast surface in the PTH-Veh group were no different from those in the PTH group, whereas they were lower in the PTH-IBN group. Osteoid surface, osteoblast surface, and mineralize surface decreased in both PTH-IBN and PTH-Veh groups compared to the PTH group, and these parameters in the PTH-IBN group were lower than in the PTH-Veh group. These results indicated that intermittent IBN after PTH treatment suppressed bone turnover and maintained BMD, biomechanical strength, and microstructure in the lumbar spine and femur of OVX rats.

Sex differences in fear extinction and involvements of extracellular signal-regulated kinase (ERK).

Stress-related disorders, such as post-traumatic stress disorder (PTSD) and panic disorders, are disproportionately prevalent in females. However, the biological mechanism underlying these sex differences in the prevalence rate remains unclear. In the present study, we examined sex differences in fear memory, fear extinction, and spontaneous recovery of fear. We investigated the presence of sex differences in recent and remote fear memory in mice using contextual fear conditioning, as well as sex differences in spontaneous recovery of fear memory using a consecutive fear extinction paradigm. We examined the number of fear extinction days required to prevent spontaneous recovery of fear in either sex. We investigated whether ovariectomy affected fear extinction and spontaneous recovery. We also measured the activation of extracellular signal-regulated kinase (ERK) 1 and 2 in the dorsal hippocampus and the medial prefrontal cortex following fear extinction sessions. In our results, we found no sex difference in recent or remote fear memory. However, females required more fear extinction sessions compared to males to prevent spontaneous recovery. Within-extinction freezing also differed between males and females. Moreover, females required more extinction sessions than males to increase ERK2 phosphorylation in the dorsal hippocampus. Our data suggest that contextual fear extinction was unstable in females compared to males and that such sex differences may be related to the ERK2 phosphorylation in the hippocampus.

Perinatal exposure to bisphenol A enhances contextual fear memory and affects the serotoninergic system in juvenile female mice.

Perinatal exposure to bisphenol A (BPA), an endocrine-disrupting chemical, affects the central nervous system, including effects on emotional responses and neurotransmitter release. In this study, we investigated the effects of BPA (250 ng/kg/day, from gestational day 10 to postnatal day 20) on fear memory and serotonin (5-HT) metabolites in the brain using contextual fear conditioning (FC) and high-performance liquid chromatography (HPLC), respectively, in adult and juvenile mice of both sexes. Furthermore, we studied the effects of BPA on the gene expression of 5-HT metabolite-related enzymes and 5-HT receptors using quantitative real-time RT PCR in the brains of juvenile females. BPA enhanced fear memory and increased serotonin metabolite (5-HIAA) levels and 5-HIAA/5-HT in the hippocampus, the striatum, the midbrain, the pons, and the medulla oblongata of juvenile female mice. In contrast, alterations in those areas were much smaller in adult females and in both juvenile and adult males. Furthermore, BPA induced increases in the expression levels of Tph2, Slc6a4, and Maoa mRNA in the hippocampus of juvenile females, indicating that BPA induces hyper 5-HT turnover in the hippocampus. Our results suggest that perinatal exposure to a low dose of BPA enhances fear memory and the 5-HTergic system in juvenile mice.

An isolated retrieval trial before extinction session does not prevent the return of fear.

Several studies have shown that an isolated retrieval trial before the extinction session (retrieval-extinction) prevents the return of fear memory by inhibition of reconsolidation. Other studies have reported that retrieval-extinction did not prevent the return of the fear. To date, it is still unclear whether retrieval-extinction prevents the return of the original fear memory. A previous study revealed that reconsolidation of conditioned fear memory was not induced by the brevity of the retrieval session. Thus, we examined whether the number of retrievals in the retrieval-extinction paradigm was involved in the prevention of return of fear (Experiment 1). Furthermore, studies with different-age experimental subjects have shown conflicting results. We investigated the potential impact of age on the inhibitory effect of retrieval-extinction on the return of fear (Experiment 2). Our major findings were as follows: (1) Retrieval-extinction procedure did not prevent the return of fear, regardless of the intensity (number of presentations) of the stimulus inducing retrieval of fear memory. (2) The mice in both juvenile and adult age groups (4 and 8 weeks old) retrieved fear memory after retrieval-extinction. These results suggest the possibility that extinction after retrieval does not inhibit reconsolidation of previously consolidated fear memory.

Transient contribution of left posterior parietal cortex to cognitive restructuring.

Cognitive restructuring is a fundamental method within cognitive behavioural therapy of changing dysfunctional beliefs into flexible beliefs and learning to react appropriately to the reality of an anxiety-causing situation. To clarify the neural mechanisms of cognitive restructuring, we designed a unique task that replicated psychotherapy during a brain scan. The brain activities of healthy male participants were analysed using functional magnetic resonance imaging. During the brain scan, participants underwent Socratic questioning aimed at cognitive restructuring regarding the necessity of handwashing after using the restroom. The behavioural result indicated that the Socratic questioning effectively decreased the participants' degree of belief (DOB) that they must wash their hands. Alterations in the DOB showed a positive correlation with activity in the left posterior parietal cortex (PPC) while the subject thought about and rated own belief. The involvement of the left PPC not only in planning and decision-making but also in conceptualization may play a pivotal role in cognitive restructuring.

Effects of memory age and interval of fear extinction sessions on contextual fear extinction.

Fear extinction is a major task in our understanding of the biological mechanisms of exposure therapy, one of the most used treatments for stress-related disorders. It was recently reported that an extinction of 5 consecutive days prevents spontaneous recovery of fear memory. Memory age and the timing of fear extinction influence the effect of fear extinction. In this study, we used contextual fear extinction in adult male mice to examine whether memory age influences an extinction of 5 consecutive days and whether consecutiveness is necessary to prevent spontaneous recovery. Our results showed that, although fear memory was not affected by the passage of time, the old fear memory (28 days after fear conditioning) was more sensitive to fear extinction than the young fear memory (7 days after fear conditioning). Additionally, we demonstrated that consecutiveness of extinction sessions is not necessary to prevent spontaneous recovery. Instead, fear extinction sessions at spaced intervals were found to be more effective than consecutive extinction sessions for young fear memory. Our results suggest that taking memory age and the interval of fear extinction sessions into consideration would help to optimize exposure therapy.

Methyl donor-deficient diet during development can affect fear and anxiety in adulthood in C57BL/6J mice.

DNA methylation is one of the essential factors in the control of gene expression. Folic acid, methionine and choline (methyl donors)--all nutrients related to one-carbon metabolism--are known as important mediators of DNA methylation. A previous study has shown that long-term administration of a diet lacking in methyl donors caused global DNA hypermethylation in the brain (Pogribny et al., 2008). However, no study has investigated the effects of a diet lacking in methyl donors during the developmental period on emotional behaviors such as fear and anxiety-like behavior in association with gene expressions in the brain. In addition, it has not been elucidated whether a diet supplemented with methyl donors later in life can reverse these changes. Therefore, we examined the effects of methyl donor deficiency during the developmental period on fear memory acquisition/extinction and anxiety-like behavior, and the relevant gene expressions in the hippocampus in juvenile (6-wk) and adult (12-wk) mice. We found that juvenile mice fed a methyl-donor-deficient diet had impaired fear memory acquisition along with decreases in the gene expressions of Dnmt3a and Dnmt3b. In addition, reduced anxiety-like behavior with decreased gene expressions of Grin2b and Gabar2 was observed in both the methyl-donor-deficient group and the body-weight-matched food-restriction group. After being fed a diet supplemented with methyl donors ad libitum, adult mice reversed the alteration of gene expression of Dnmt3a, Dnmt3b, Grin2b and Gabar2, but anxiety-like behavior became elevated. In addition, impaired fear-memory formation was observed in the adult mice fed the methyl-donor-deficient diet during the developmental period. Our study suggested that developmental alterations in the one-carbon metabolic pathway in the brain could have effects on emotional behavior and memory formation that last into adulthood.

No erasure effect of retrieval-extinction trial on fear memory in the hippocampus-independent and dependent paradigms.

Recently, Monfils et al. [9] and Clem and Huganir [3] have shown that an isolated retrieval trial before the extinction sessions (retrieval-extinction) in mice and rats prevented the renewal and spontaneous recovery of the original fear memory by inhibiting reconsolidation in a hippocampus-independent manner. In contrast, Chan et al. [2], using the same paradigm, reported that retrieval extinction in rats augmented the renewal and reinstatement of extinguished fear. However, it remains unclear whether or not retrieval extinction in a hippocampus-independent paradigm erases the original fear memory by inhibiting reconsolidation. We therefore conducted three experiments to investigate whether or not retrieval extinction erases the original fear memory by inhibiting reconsolidation in mice. Our major findings were as follows. (1) Retrieval-extinction in mice did not suppress spontaneous recovery and fear renewal in a hippocampus-independent paradigm. (2) Fear renewal was observed when retrieval-strong extinction in a hippocampus-independent paradigm was performed. (3) Retrieval extinction in a hippocampus-dependent paradigm did not erase the original fear memory. These results suggested that fear extinction after retrieval in mice does not inhibit reconsolidation of previously consolidated fear memory in either a hippocampus-independent or -dependent paradigm.

Effects of perinatal exposure to low dose of bisphenol A on anxiety like behavior and dopamine metabolites in brain.

Bisphenol A (BPA), an endocrine-disrupting chemical, is widely present in the environment. It has been reported that perinatal exposure to low doses of BPA that are less than the tolerable daily intake level (50µg/kg/day) affects anxiety-like behavior and dopamine levels in the brain. Although the dopaminergic system in the brain is considered to be related to anxiety, no study has reported the effects of low-dose BPA exposure on the dopaminergic system in the brain and on anxiety-like behavior using the same methods of BPA exposure. To investigate the relationship between alterations in anxiety-like behavior and changes in the dopaminergic system in the brain induced by BPA, we examined the effects of BPA on anxiety-like behavior using an open field test in juvenile and adult mice and measured DA and DOPAC levels and the DOPAC/DA ratio in the dorsal hippocampus (HIP), amygdala (AMY), and medulla oblongata (MED) using high-performance liquid chromatography (HPLC) in adult mice. In males, BPA decreased the time spent in the center area of the open field in both juveniles and adults. In addition, BPA increased DA levels in the dorsal HIP and MED and decreased the DOPAC/DA ratio in the dorsal HIP, AMY, and MED in adults. The activity of monoamine oxidase (MAO)-B, the enzyme that metabolizes DA into DOPAC, was reduced in the MED. In females, those changes were not observed. These results suggest that an increase in anxiety-like behavior induced by perinatal exposure to BPA may be related to decreases in DA metabolites in the brain, and there are sex differences in those BPA effects.

d-serine enhances extinction of auditory cued fear conditioning via ERK1/2 phosphorylation in mice.

Several lines of evidence suggest that the N-methyl-D-aspartate (NMDA) receptor plays a significant role in fear conditioning and extinction. However, our knowledge of the role of D-serine, an endogenous ligand for the glycine site of the NMDA receptor, in fear extinction is quite limited compared to that of D-cycloserine, an exogenous partial agonist for the same site. In the current study, we examined the effects of D-serine on fear extinction and phosphorylation of extracellular signal-regulated kinase (ERK) in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) during the process of fear extinction. Systemic administrations of D-serine (2.7 g/kg, i.p.) with or without the ERK inhibitor SL327 (30 mg/kg, i.p.) to C57BL/6J mice were performed before fear extinction in a cued fear conditioning and extinction paradigm. Cytosolic and nuclear ERK 1/2 phosphorylation in the hippocampus, BLA, and mPFC were measured 1h after extinction (E1h), 24h after extinction (E24h), and 1h after recall (R1h) by Western blotting. We found that D-serine enhanced the extinction of fear memory, and the effects of D-serine were reduced by the ERK phosphorylation inhibitor SL327. The Western blot analyses showed that D-serine significantly increased cytosolic ERK 2 phosphorylation at E1h in the hippocampus and cytosolic ERK 1/2 phosphorylation at R1h in the BLA. The present study suggested that D-serine might enhance fear extinction through NMDA receptor-induced ERK signaling in mice, and that D-serine has potential clinical importance for the treatment of anxiety disorders.