Ya Hsin Hsiao

Extinction Training in Conjunction with a Partial Agonist of the Glycine Site on the NMDA Receptor Erases Memory Trace

Much evidence indicates that extinction training does not erase memory traces but instead forms inhibitory learning that prevents the expression of original memory. Fear conditioning induces long-term potentiation and drives synaptic insertion of AMPA receptors into the amygdala. Here we show that extinction training applied 1 h after training reversed the conditioning-induced increase in surface glutamate receptor subunit 1 (GluR1) in parallel with the inhibition of startle potentiation. However, if applied 24 h after training, extinction training reduced startle potentiation without influencing the GluR1 increase. We infused d-cycloserine (DCS), a partial agonist of the glycine site on the NMDA receptor, bilaterally into the amygdala 30 min before extinction training. This augmented the extinction training-elicited reduction in startle and reversed the conditioning-induced increase in GluR1. Delivery of five sets of tetanic stimulation (TS) to the external capsule produced a robust enhancement of synaptic responses in the lateral amygdala neurons that persisted for >2 h. Low-frequency stimulation applied 1 h after TS had no long-lasting effect on synaptic responses. The same treatments, however, induced depotentiation in the presence of DCS and reversed TS-induced increase in surface GluR1. Together, this study has two important findings: (1) whether a memory trace remains intact or is erased depends on the interval between conditioning and extinction training and (2) DCS facilitates the reversal of memory trace. DCS-induced augmentation of extinction and reversal of GluR1 surface expression are likely mediated by DCS-facilitated endocytosis of AMPA receptors.

Social interaction rescues memory deficit in an animal model of Alzheimer’s disease by increasing BDNF-Dependent Hippocampal Neurogenesis

It has been recognized that the risk of cognitive decline during aging can be reduced if one maintains strong social connections, yet the neural events underlying this beneficial effect have not been rigorously studied. Here, we show that amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic (APP/PS1) mice demonstrate improvement in memory after they are cohoused with wild-type mice. The improvement was associated with increased protein and mRNA levels of BDNF in the hippocampus. Concomitantly, the number of BrdU+/NeuN+ cells in the hippocampal dentate gyrus was significantly elevated after cohousing. Methylazoxymethanol acetate, a cell proliferation blocker, markedly reduced BrdU+ and BrdU/NeuN+ cells and abolished the effect of social interaction. Selective ablation of mitotic neurons using diphtheria toxin (DT) and a retrovirus vector encoding DT receptor abolished the beneficial effect of cohousing. Knockdown of BDNF by shRNA transfection blocked, whereas overexpression of BDNF mimicked the memory-improving effect. A tropomyosin-related kinase B agonist, 7,8-dihydroxyflavone, occluded the effect of social interaction. These results demonstrate that increased BDNF expression and neurogenesis in the hippocampus after cohousing underlie the reversal of memory deficit in APP/PS1 mice.

Amelioration of social isolation-triggered onset of early Alzheimer's disease-related cognitive deficit by N-acetylcysteine in a transgenic mouse model

Epidemiological study reveals that socially isolated persons have increased risk of developing Alzheimers disease (AD). Whether this risk arises from an oxidative stress is unclear. Here we show that N-acetylcysteine (NAC), an anti-oxidant, is capable of preventing social isolation-induced accelerated impairment of contextual fear memory and rundown of hippocampal LTP in 3-month old APP/PS1 mice. Increased hippocampal levels of γ-secretase activity, Aβ-40 and Aβ-42 seen in the isolated APP/PS1 mice were reduced by chronic treatment of NAC. In addition, social isolation-induced increase in calpain activity and p25/p35 ratio concomitant with decrease in membrane-associated p35 and p35/Cdk5 activity was normalized by NAC. NAC pretreatment also reversed isolation-induced decrease in GluR1 Ser831 phosphorylation, surface expression of AMPARs and p35-GluR1-CaMKII interactions. These results suggest that NAC decreases γ-secretase activity resulting in the attenuation of Aβ production, calpain activity and conversion of p35 to p25 which stabilized p35-GluR1-CaMKII interactions and restored GluR1 and GluR2 surface expression. Our results indicate that NAC is effective in mouse models of AD and has translation potential for the human disorder.

Social isolation-induced increase in NMDA receptors in the hippocampus exacerbates emotional dysregulation in mice.

Epidemiological studies have shown that early life adverse events have long‐term effects on the susceptibility to subsequent stress exposure in adolescence, but the precise mechanism is unclear. In the present study, mice on postnatal day 21–28 were randomly assigned to either a group or isolated cages for 8 weeks. The socially isolated (SI) mice exhibited a higher level of spontaneous locomotor activity, a longer duration of immobility in the forced swimming test (FST), significantly less prepulse inhibition (PPI) and an increase in aggressive (but not attack) behavior. However, acute stress markedly exacerbated the attack counts of the SI mice but did not affect the group housing (GH) mice. SI mice exhibited higher synaptosomal NR2A and NR2B levels in the hippocampus as compared to the GH mice. Whole‐cell patch clamp recordings of CA1 neurons in hippocampal slices showed that the SI mice exhibited a higher input‐output relationship of NMDAR‐EPSCs as compared to the GH mice. Application of the NR2B‐specific antagonist ifenprodil produced a greater attenuating effect on NMDAR‐EPSCs in slices from the SI mice. NMDAR EPSCs recorded from the SI mice had a slower deactivation kinetic. MK‐801, CPP and ifenprodil, the NMDA antagonists, reversed acute stress‐induced exaggeration of aggressive and depressive behaviors. Furthermore, acute stress‐induced exacerbation of attack behavior in the SI mice was abolished after the knockdown of NR2B expression. These results suggest that social isolation‐induced increased expression of NMDA receptors in the hippocampus involves stress exacerbation of aggressive behaviors. Amelioration of aggressive behaviors by NMDA antagonists may open a new avenue for the treatment of psychopathologies that involve outbursts of emotional aggression in neglected children.

The Involvement of Cdk5 Activator p35 in Social Isolation-Triggered Onset of Early Alzheimer's Disease-Related Cognitive Deficit in the Transgenic Mice

Epidemiological studies indicate that isolated persons have increased risk of developing Alzheimers disease (AD). This study investigated the cellular mechanisms of how social isolation influenced amyloid β peptide (Aβ) accumulation and affected the severity of AD-associated cognitive decline in a mouse model of AD. Amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic (APP/PS1) mice were placed either in isolation or in group from postnatal day 28 and tested for cognitive performance at the age of 3 months with fear-conditioning paradigms. We found that social isolation accelerated impairment of contextual fear memory in the APP/PS1 mice. The magnitude of long-term potentiation in the hippocampal CA1 neurons was significantly lower in the isolated APP/PS1 mice compared with group APP/PS1 and wild-type mice. Hippocampal level of Aβ was significantly elevated in the isolated APP/PS1 mice, which was accompanied by an increased calpain activity and p25/p35 ratio. In addition, surface expression of GluR1 subunit of AMPA receptor was decreased by social isolation. The association of p35, and α-CaMKII was significantly less in the isolated APP/PS1 mice indicating that their interaction was impaired. These results suggest that social isolation exacerbates memory deficit by increasing Aβ level, leading to the increased calpain activity, conversion of p35 to p25 and decrease in association of p35, α-CaMKII, and GluR1, resulting in the endocytosis of AMPA receptors.

N‐acetylcysteine prevents β‐amyloid toxicity by a stimulatory effect on p35/cyclin‐dependent kinase 5 activity in cultured cortical neurons

Although previous studies have indicated that the neuroprotective effect of N‐acetylcysteine (NAC) required activation of the Ras‐extracellular‐signal‐regulated kinase (ERK) pathway, the detailed mechanisms and signal cascades leading to activation ERK are not clear. In the present study, we investigated the effect of NAC on Aβ25–35‐induced neuronal death. Pretreatment of neurons with NAC 1 hr before application of Aβ prevented Aβ‐mediated cell death. NAC increased cyclin‐dependent kinase 5 (Cdk5) phosphorylation, an effect that was blocked by Cdk5 inhibitor. The neuroprotective effect of NAC was significantly attenuated by Cdk5 inhibitors or in neurons transfected with Cdk5 or p35 small interfering RNA (siRNA). Conversely, pretreatment of neurons with the calpain inhibitors calpeptin or MDL28170 enhanced the neuroprotective effect of NAC. Aβ25–35 caused a significant decrease in the level of p35, with a concomitant increase in p25, which was completely prevented by NAC. This effect of NAC was blocked by the Cdk5 inhibitors roscovitine and butyrolactone. In addition, NAC increased Cdk5/p35 kinase activity but reduced Cdk5 kinase activity. Aβ25–35 treatment decreased phosphorylated levels of ERK, which could be reversed by NAC. The effect of NAC was completely blocked by Cdk5 inhibitors. NAC reversed the Aβ25–35–induced decrease in the expression of Bcl‐2, which could be blocked by the MAPK kinase (MEK) inhibitor or Cdk5 inhibitors. These results suggest that NAC‐mediated neuroprotection against Aβ toxicity is likely mediated by the p35/Cdk5‐ERKs‐Bcl‐2 signal pathway.

Activation of mGluR2/3 underlies the effects of N-acetylcystein on amygdala-associated autism-like phenotypes in a valproate-induced rat model of autism

Autism-like phenotypes in male valproate (VPA)-exposed offspring have been linked to high glutamatergic neurotransmission in the thalamic-amygdala pathway. Glial cystine/glutamate exchange (system Xc−), which exchanges extracellular cystine for intracellular glutamate, plays a significant role in the maintenance of extracellular glutamate. N-acetylcysteine (NAC) is a cystine prodrug that restores extracellular glutamate by stimulating system Xc−. In this study, we examined the effects of NAC on autism-like phenotypes and neurotransmission in the thalamic–amygdala synapses, as well as the involvement of metabotropic glutamate receptors 2/3 (mGluR2/3). Valproate-treated rats received a single intraperitoneal injection of 500 mg/kg NaVPA on E12.5. On postnatal day 21 (P21), NAC or saline was administered once daily for 10 days. From day 8 to 10, NAC was given 1/2 h prior to behavioral testing. Chronic administration of NAC restored the duration and frequency of social interaction and ameliorated anxiety-like behaviors in VPA-exposed offspring. In amygdala slices, NAC treatment normalized the increased frequency of mEPSCs and decreased the paired pulse facilitation (PPF) induced by VPA exposure. The effects of NAC on social interaction and anxiety-like behavior in the VPA-exposed offspring were blocked after intra-amygdala infusion of mGluR2/3 antagonist LY341495. The expressions of mGluR2/3 protein and mGluR2 mRNA were significantly lower in the VPA-exposed offspring. In contrast, the mGluR3 mRNA level did not differ between the saline- and VPA-exposed offspring. These results provide the first evidence that the disruption of social interaction and enhanced presynaptic excitatory transmission in VPA-exposed offspring could be rescued by NAC, which depends on the activation of mGluR2/3.

Epigenetic regulation of BDNF in the learned helplessness-induced animal model of depression

Major depressive disorder (MDD), one of the most common mental disorders, is a significant risk factor for suicide and causes a low quality of life for many people. However, the causes and underlying mechanism of depression remain elusive. In the current work, we investigated epigenetic regulation of BDNF in the learned helplessness-induced animal model of depression. Mice were exposed to inescapable stress and divided into learned helplessness (LH) and resilient (LH-R) groups depending on the number they failed to escape. We found that the LH group had longer immobility duration in the forced swimming test (FST) and tail suspension tests (TST), which is consistent with a depression-related phenotype. Western blotting analysis and enzyme-linked immunosorbent assay (ELISA) revealed that the LH group had lower BDNF expression than that of the LH-R group. The LH group consistently had lower BDNF mRNA levels, as detected by qPCR assay. In addition, we found BDNF exon IV was down-regulated in the LH group. Intraperitoneal injection of imipramine or histone deacetylase inhibitors (HDACi) to the LH mice for 14 consecutive days ameliorated depression-like behaviors and reversed the decrease in BDNF. The expression of HDAC5 was up-regulated in the LH mice, and a ChIP assay revealed that the level of HDAC5 binding to the promoter region of BDNF exon IV was higher than that seen in other groups. Knockdown of HDAC5 reduced depression-like behaviors in the LH mice. Taken together, these results suggest that epigenetic regulation of BDNF by HDAC5 plays an important role in the learned helplessness model of depression.

Sonic hedgehog signaling regulates amygdalar neurogenesis and extinction of fear memory

It is now recognized that neurogenesis occurs throughout life predominantly in the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle. In the present study, we investigated the relationship between neurogenesis in the amygdala and extinction of fear memory. Mice received 15 tone-footshock pairings. Twenty-four hours after training, the mice were given 15 tone-alone trials (extinction training) once per day for 7 days. Two hours before extinction training, the mice were injected intraperitoneally with 5-bromo-3-deoxyuridine (BrdU). BrdU-positive and NeuN-positive cells were analyzed 52 days after the training. A group of mice that received tone-footshock pairings but no extinction training served as controls (FC+No-Ext). The number of BrdU(+)/NeuN(+) cells was significantly higher in the extinction (FC+Ext) than in the FC+No-Ext mice. Proliferation inhibitor methylazoxymethanol acetate (MAM) or DNA synthesis inhibitor cytosine arabinoside (Ara-C) reduced neurogenesis and retarded extinction. Silencing Sonic hedgehog (Shh) gene with short hairpin interfering RNA (shRNA) by means of a retrovirus expression system to knockdown Shh specifically in the mitotic neurons reduced neurogenesis and retarded extinction. By contrast, over-expression of Shh increased neurogenesis and facilitated extinction. These results suggest that amygdala neurogenesis and Shh signaling are involved in the extinction of fear memory.

Learning Induces Sonic Hedgehog Signaling in the Amygdala which Promotes Neurogenesis and Long-Term Memory Formation

Background: It is known that neurogenesis occurs throughout the life mostly in the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle. We investigated whether neurogenesis occurred in the amygdala and its function in fear memory formation. Methods: For detection of newborn neurons, mice were injected intraperitoneally with 5-bromo-2’-deoxyuridine (BrdU) 2h before receiving 15 tone–footshock pairings, and newborn neurons were analyzed 14 and 42 days after training. To determine the relationship between neurogenesis and memory formation, mice were given a proliferation inhibitor methylazoxymethanol (MAM) or a DNA synthesis inhibitor cytosine arabinoside (Ara-C). To test whether sonic hedgehog (Shh) signaling was required for neurogenesis, Shh-small hairpin–interfering RNA (shRNA) was inserted into a retroviral vector (Retro-Shh-shRNA). Results: The number of BrdU+/Neuronal nuclei (NeuN)+ cells was significantly higher in the conditioned mice, suggesting that association of tone with footshock induced neurogenesis. MAM and Ara-C markedly reduced neurogenesis and impaired fear memory formation. Shh, its receptor patched 1 (Ptc1), and transcription factor Gli1 protein levels increased at 1 day and returned to baseline at 7 days after fear conditioning. Retro-Shh-shRNA, which knocked down Shh specifically in the mitotic neurons, reduced the number of BrdU+/NeuN+ cells and decreased freezing responses. Conclusions: These results suggest that fear learning induces Shh signaling activation in the amygdala, which promotes neurogenesis and fear memory formation.