Hui-Ching Lin

The Role of Prefrontal Cortex CB1 Receptors in the Modulation of Fear Memory

Understanding the mechanism of how fear memory can be extinguished could provide potential therapeutic strategies for the treatment of posttraumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) retarded cue-alone-induced reduction of fear-potentiated startle. Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated the extinction. Unexpectedly, administration of WIN without cue-alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by endocannabinoid uptake or fatty acid amide hydrolase inhibitors. Rats were trained with 10 conditioned stimulus (CS(+)) (yellow light)-shock pairings. Extinction training with CS(+) (yellow light)-alone but not CS(-) (blue light)-alone trials decreased fear-potentiated startle. Intra-IL infusion of WIN before CS(-)-alone trials decreased startle potentiation, suggesting that the cannabinoid agonist decreased conditioned fear irrespective of whether the rats underwent CS(+)- or CS(-)-alone trials. Cannabinoid agonists activated extracellular signal-regulated kinases (ERKs) in mPFC slices, and ERK inhibitor blocked the effect of cannabinoid agonists on fear-potentiated startle. These results suggest that CB1 receptors acting through the phosphorylation of ERK are involved not only in the extinction of conditioned fear but also in the adaptation to aversive situations in general.

Synaptic expression of glutamate receptor after encoding of fear memory in the rat amygdala

Fear conditioning has been ascribed to presynaptic mechanisms, particularly presynaptic facilitation of transmission at thalamo- and cortico-amygdala synapses. Here, by labeling surface receptors with biotin or using membrane fractionation approaches, we report that fear conditioning resulted in an increase in surface expression of GluR1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the amygdala, whereas total GluR1 mRNA and protein levels were unchanged. The control group that received conditioned stimulus (CS) and unconditioned stimulus in an unpaired fashion did not present any increase, indicating that GluR1 increase was specific to the learning component of the task. Conditioning-induced increase in surface expression of GluR1 depended on the activation of N-methyl-d-aspartate receptors and protein kinases and required the synthesis of new proteins. CS-alone trials applied 24 h before training attenuated fear-potentiated startle and prevented conditioning-induced increase in surface expression of GluR1. Increase in GluR1 was also observed in the amygdala slices after delivery of tetanic stimulation that elicited long-term potentiation of synaptic transmission. Proteasome inhibitor increased surface expression of GluR1 in a time- and dose-dependent manner. Furthermore, pretraining administration of proteasome inhibitor into the amygdala facilitated the fear-potentiated startle. These results suggest that long-term memory formation is correlated with the change in synaptic expression of GluR1, and trafficking of GluR1 to the synaptic sites contributes at least in part to the expression of fear memory.

Block of γ-Aminobutyric Acid-A Receptor Insertion in the Amygdala Impairs Extinction of Conditioned Fear

BACKGROUND Extinction is a complex phenomenon but generally is regarded as a new inhibitory learning that suppresses the original memory. However, how or from where the inhibition originates remains to be determined. In the present study, we examine whether increase in the expression of gamma-aminobutyric acid (GABA)(A) receptors in the amygdala is required for extinction by employing cell-permeable TAT-conjugated peptide (TAT)-GABA receptor-associated protein (GABARAP) inhibitory peptide to block GABA(A) receptor insertion. METHODS Retention of fear memory was assessed with fear-potentiated startle paradigm. Whole cell patch clamp recordings were performed to record miniature inhibitory postsynaptic current (mIPSC). Western blotting analysis was used to measure the expression of gephyrin, beta2, and gamma2 subunits of GABA(A) receptor. RESULTS Fear conditioning decreased frequency and amplitude of mIPSC and surface protein levels of beta2 and gamma2 subunits of GABA(A) receptor. Extinction training, by contrast, reversed the decreased frequency and amplitude of mIPSC and surface protein levels of gephyrin and beta2 subunit of GABA(A) receptor. Disruption of GABARAP-GABA(A) receptor interaction in the amygdala with GABARAP inhibitory peptide blocked N-methyl-D-aspartate-mediated GABA(A) receptor insertion in the amygdala. Importantly, it also blocked extinction-induced increase in the frequency and amplitude of mIPSCs, and the reduction of fear-potentiated startle. CONCLUSIONS GABA(A) receptor insertion in the amygdala contributes a significant part to the extinction of fear memory.

Augmentation of Fear Extinction by D-Cycloserine is Blocked by Proteasome Inhibitors

D-Cycloserine (DCS) has been shown to facilitate extinction of conditioned fear in rats and to improve fear reduction of social phobia and fear of heights in human studies. Here, we investigate the mechanism of DCS effect by measuring internalized GluR1 and GluR2 using cell-surface biotinylation techniques. 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. Proteasome inhibitors block DCS facilitation of LFS-induced depotentiation and a reduction in surface levels of GluR1 and GluR2. Furthermore, DCS in combination with LFS reduced cellular levels of PSD-95 and synapse-associated protein 97 (SAP97), which were also blocked by proteasome inhibitors. In the in vivo experiments, DCS-induced reduction of fear-potentiated startle and reversal of conditioning-induced increase in surface expression of GluR1 were blocked by proteasome inhibitors. DCS-treated rats fail to exhibit reinstatement after US-alone presentations. These results suggest that DCS facilitates receptor internalization in the presence of extinction training, resulting in augmented reduction of startle potentiation.

Chronic cannabinoid administration in vivo compromises extinction of fear memory

Endocannabinoids are critically involved in the extinction of fear memory. Here we examined the effects of repeated cannabinoid administration on the extinction of fear memory in rats and on inhibitory synaptic transmission in medial prefrontal cortex (mPFC) slices. Rats were treated with the CB1 receptor agonist WIN55212-2 (WIN 10 mg/kg, i.p.) once per day for 7 d. On day 8, the rats were submitted to a standard fear conditioning procedure, and retention of memory was measured with potentiated startle paradigm. We found that (1) WIN-pretreated rats exhibited much less extinction to cue alone presentations; (2) the reduction of fear-potentiated startle normally seen when the CB1 receptor agonists were infused into the mPFC was absent in the WIN-pretreated rats; (3) WIN-induced inhibition of GABAergic transmission was significantly less in slices from the WIN-pretreated rats than that from the vehicle-pretreated control; (4) WIN failed to induce extracellular signal-regulated kinases (ERKs) phosphorylation in the WIN-pretreated rats; and (5) the level of CB1 receptor in the WIN-pretreated rats was lower than that of vehicle-pretreated rats. These results suggest that endocannabinoids within the mPFC play an important role in the extinction of conditioned fear. However, long-term marijuana use may limit its clinical efficacy for the treatment of anxiety disorders.

The Amygdala Excitatory/Inhibitory Balance in a Valproate-Induced Rat Autism Model

The amygdala is an important structure contributing to socio-emotional behavior. However, the role of the amygdala in autism remains inconclusive. In this study, we used the 28–35 days valproate (VPA)-induced rat model of autism to observe the autistic phenotypes and evaluate their synaptic characteristics in the lateral nucleus (LA) of the amygdala. The VPA-treated offspring demonstrated less social interaction, increased anxiety, enhanced fear learning and impaired fear memory extinction. Slice preparation and electrophysiological recordings of the amygdala showed significantly enhanced long-term potentiation (LTP) while stimulating the thalamic-amygdala pathway of the LA. In addition, the pair pulse facilitation (PPF) at 30- and 60-ms intervals decreased significantly. Whole-cell recordings of the LA pyramidal neurons showed an increased miniature excitatory postsynaptic current (EPSC) frequency and amplitude. The relative contributions of the AMPA receptor and NMDA receptor to the EPSCs did not differ significantly between groups. These results suggested that the enhancement of the presynaptic efficiency of excitatory synaptic transmission might be associated with hyperexcitibility and enhanced LTP in LA pyramidal neurons. Disruption of the synaptic excitatory/inhibitory (E/I) balance in the LA of VPA-treated rats might play certain roles in the development of behaviors in the rat that may be relevant to autism. Further experiments to demonstrate the direct link are warranted.

Alterations of excitatory transmission in the lateral amygdala during expression and extinction of fear memory.

Understanding the neurophysiology of fear extinction has important implications for the treatment of post-traumatic stress disorders. Here we report that fear conditioning resulted in an increase in AMPA/NMDA ratio as well as depression of paired-pulse facilitation (PPF) in neurons of the lateral nucleus of amygdala. These conditioning-induced changes in synaptic transmission were not affected by extinction training. D-cycloserine (DCS), a partial agonist at the glycine-binding site of the NMDA receptor, facilitated extinction and reversed the increase in AMPA/NMDA ratio without altering the depression of PPF when administered before extinction training. Extinction training, however, significantly increased the frequency and amplitude of miniature inhibitory post-synaptic currents and these effects were unaffected by the DCS treatment. Disruption of AMPA receptor endocytosis with a synthetic peptide containing a short C-terminal sequence of GluR2 (869YKEGYNVYG877, GluR23Y) specifically blocked DCS-induced reversal of AMPA/NMDA ratio and the facilitation of extinction. These results suggest that extinction training mainly increases inhibitory transmission leaving conditioning-induced excitatory association unaltered. DCS does not affect inhibitory transmission but reverses the conditioning-induced post-synaptic memory trace when administered before extinction training.

GABAA Receptor Endocytosis in the Basolateral Amygdala Is Critical to the Reinstatement of Fear Memory Measured by Fear-Potentiated Startle

Reinstatement represents a phenomenon that may be used to model the effects of retraumatization observed in patients with posttraumatic stress disorder (PTSD). In this study, we found intraperitoneal injection of the β-adrenergic receptor antagonist propranolol (10 mg/kg) 1 h before reinstatement training attenuated reinstatement of fear memory in rats. Conversely, reinstatement was facilitated by intra-amygdalar administration of β-adrenergic receptor agonist isoproterenol (Iso; 2 μg per side) 30 min before reinstatement training. The frequency and amplitude of the miniature IPSC (mIPSC) and the surface expression of the β3 and γ2 subunits of the GABAA receptor (GABAAR) were significantly lower in reinstated than in extinction rats, whereas the AMPA/NMDA ratio and the surface expression of GluR1 and GluR2 in the amygdala did not differ between groups. In amygdala slices, Iso-induced decrease in the surface β3 subunit of GABAA receptor was blocked by a Tat-conjugated dynamin function-blocking peptide (Tat-P4) pretreatment (10 μm for 30 min). By contrast, Tat-scramble peptide had no effect. Intravenous injection (3 μmol/kg) or intra-amygdalar infusion (30 pmol per side) of Tat-P4 interfered with reinstatement. Reinstatement increased the association between protein phosphatase 2A (PP2A) and the β3 subunit of the GABAAR, which was abolished by PP1/PP2A inhibitors okadaic acid and calyculin A. These results suggest the involvement of β-adrenergic receptor activation and GABAA receptor endocytosis in the amygdala for the reinstatement in fear memory.

5-HT1A-receptor agonist modified amygdala activity and amygdala-associated social behavior in a valproate-induced rat autism model

Accumulating evidence suggests that dysfunction of the amygdala is related to abnormal fear processing, anxiety, and social behaviors noted in autistic spectrum disorders (ASDs). In addition, studies have shown that disrupted brain serotonin homeostasis is linked to ASD. With a valproate (VPA)-induced rat ASD model, we investigated the possible role of amygdala serotonin homeostasis in autistic phenotypes and further explored the underlying mechanism. We first discovered that the distribution of tryptophan hydroxylase immunoreactivity in the caudal raphe system was modulated on postnatal day (PD) 28 of the VPA-exposed offspring. Then, we found a significantly higher serotonin transporter availability in the amygdala of the VPA-exposed offspring on PD 56 by using single photon emission computed tomography and computed tomography co-registration following injection of (123)I-labeled 2-((2-(dimethylamino)methyl)phenyl)thio)-5-iodophenylamine((123)I[ADAM]). Furthermore, treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, increased social interaction and improved fear memory extinction in the VPA-exposed offspring. 8-OH-DPAT treatment also reversed the characteristics of miniature excitatory post-synaptic currents as well as paired pulse facilitation observed in lateral amygdala slices. These results provided further evidence to support the role of the amygdala in characteristic behavioral changes in the rat ASD model. The serotonergic projections that modulate the amygdala function might play a certain role in the development and treatment of behavioral symptoms exhibited in individuals with ASD.

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.