Enhancing actions at the glycine modulatory site of NMDA receptor augment fear extinction by erasing memory trace

• Main Authors: Sheng-Chun Mao, 毛盛駿
• Other Authors: Po-Wu Gean
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/72446391544072677341

博士 === 國立成功大學 === 基礎醫學研究所 === 97 === Exposure-based psychotherapy is a kind of treatment for cognitive behavior. It means that make patients expose to different kinds of stimulating situation, and make them overcome gradually. It is the most common behavioral therapy for anxiety disorders such as posttraumatic stress disorder, social panic and obsessive-compulsive disorder. The overwhelming majority of the treatment for anxiety disorders can adopt the exposure therapy. Patients are exposed to stimulation related to the emotional response step by step, and previously acquired responses will gradually decline. Much evidence indicates that extinction training does not erase memory traces but forms an inhibitory learning that prevents the expression of original memory. It still remained to be determined whether NMDA-mediated facilitation of extinction is mediated by the enhancement of inhibitory memory and/or the partial erasure of the original memory. Here we investigate the effect of glycine modulatory site on NMDA receptor using NMDA receptor partial agonist (D-cycloserine) and glycine transporter blockers (NFPS) on the conditioning and extinction of fear memory assessed by fear-potentiated startle. The thesis is divided into 3 parts. In the first part, we investigated the molecular mechanism underlying the effect of the partial agonist D-cycloserine (DCS) at the glycine modulatory site of NMDA receptor on the extinction of fear. Extinction training applied 1 h after training reversed the conditioning-induced increase in surface GluR1 in parallel with the inhibition of startle potentiation. However, if applied 24 h after training, extinction training reduced startle potentiation without influencing GluR1 increase. We infused 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 of startle and reversed the conditioning-induced increase in GluR1. Delivery of five sets of tetanic stimulation (TS) to the external capsule (EC) produced a robust enhancement of synaptic responses in the LA neurons that persisted for more than 2 h. Low-frequency stimulation (LFS) applied at 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. In the second part, we further investigated the underlying mechanism of DCS on the extinction of fear memory. In the in vitro experiments on the amygdalar slice, DCS selectively increased NMDA receptor-mediated synaptic response without affecting AMPA receptor-mediated synaptic response. Low-frequency stimulation (LFS) when applied in the presence of DCS induced GluR1 and GluR2 internalization in the amygdala slices. N-methyl-D-aspartate (NMDA) at 10 �嵱 that normally was ineffective at inducing long-term depression (LTD) and reducing surface expression of GluR1 and GluR2 succeeded in the presence of DCS. Simultaneously, LFS in combination with DCS increased the level of depotentiation and receptor internalization, which was abolished by proteasome inhibitors. Furthermore, DCS in combination with LFS reduced cellular levels of PSD-95 and synapse-associated protein 97 (SAP97) that were also blocked by proteasome inhibitors. In the in vivo animal experiments, DCS-induced further reduction of fear-potentiated startle and reversal of conditioning-induced increase in surface expression of GluR1 and GluR2 were blocked by proteasome inhibitors. Finally, DCS-treated rats exhibit partial reinstatement after US alone presentations. In the last part, we investigated the molecular and behavioral effect of glycine transporter blockers (NFPS) on the extinction of fear memory. In the in vitro experiments on the amygdalar slice, NFPS selectively increased NMDA receptor- mediated synaptic response without affecting AMPA receptor-mediated synaptic response. NMDA at a concentration that normally was without effect reduced cellular levels of PSD-95 and SAP97, and the surface expression of GluR1 and GluR2 in the presence of NFPS. In the in vivo animal experiments, Extinction training reduced startle potentiation without influencing conditioning-induced increase in surface expression of GluR1 and GluR2. However, NFPS treatment in conjunction with extinction training augmented extinction and exhibit only partial reinstatement after US alone presentations. Also, it reverse conditioning-induced increase in GluR1 and GluR2 as well as AMPA/NMDA ratio. Finally, Tat-GluR23Y, a synthetic peptide which has been shown to block AMPA receptor endocytosis, inhibited only the additional reduction caused by NFPS treatment. Taken together, these study have three important findings: 1. Whether a memory trace remains intact or is erased depends on the interval between conditioning and extinction training, and 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. 2. DCS facilitates GluR1 and GluR2 internalization in the presence of extinction training resulting in augmented reduction of startle potentiation and these effects are regulated by ubiquitin-proteasome system. 3. NFPS in combination with extinction training reverses GluR1 and GluR2 surface expression and thus augments extinction of conditioned fear, and Tat-GluR23Y specifically blocks the effect of NFPS on the extinction. These results suggest that NFPS-induced augmentation of extinction and reversal of GluR1 and GluR2 surface expression are likely mediated by NFPS-facilitated endocytosis of AMPA receptors.