Reversible Plasticity of Fear Memory-Encoding Amygdala Synaptic Circuits Even after Fear Memory Consolidation

It is generally believed that after memory consolidation, memory-encoding synaptic circuits are persistently modified and become less plastic. This, however, may hinder the remaining capacity of information storage in a given neural circuit. Here we consider the hypothesis that memory-encoding synaptic circuits still retain reversible plasticity even after memory consolidation. To test this, we employed a protocol of auditory fear conditioning which recruited the vast majority of the thalamic input synaptic circuit to the lateral amygdala (T-LA synaptic circuit; a storage site for fear memory) with fear conditioning-induced synaptic plasticity. Subsequently the fear memory-encoding synaptic circuits were challenged with fear extinction and re-conditioning to determine whether these circuits exhibit reversible plasticity. We found that fear memory-encoding T-LA synaptic circuit exhibited dynamic efficacy changes in tight correlation with fear memory strength even after fear memory consolidation. Initial conditioning or re-conditioning brought T-LA synaptic circuit near the ceiling of their modification range (occluding LTP and enhancing depotentiation in brain slices prepared from conditioned or re-conditioned rats), while extinction reversed this change (reinstating LTP and occluding depotentiation in brain slices prepared from extinguished rats). Consistently, fear conditioning-induced synaptic potentiation at T-LA synapses was functionally reversed by extinction and reinstated by subsequent re-conditioning. These results suggest reversible plasticity of fear memory-encoding circuits even after fear memory consolidation. This reversible plasticity of memory-encoding synapses may be involved in updating the contents of original memory even after memory consolidation

Cortical Synaptic AMPA Receptor Plasticity during Motor Learning

Modulation of synaptic strength through trafficking of AMPA receptors (AMPARs) is a fundamental mechanism underlying synaptic plasticity, learning, and memory. However, the dynamics of AMPAR trafficking in vivo and its correlation with learning have not been resolved. Here, we used in vivo two-photon microscopy to visualize surface AMPARs in mouse cortex during the acquisition of a forelimb reaching task. Daily training leads to an increase in AMPAR levels at a subset of spatially clustered dendritic spines in the motor cortex. Surprisingly, we also observed increases in spine AMPAR levels in the visual cortex. There, synaptic potentiation depends on the availability of visual input during motor training, and optogenetic inhibition of visual cortex activity impairs task performance. These results indicate that motor learning induces widespread cortical synaptic potentiation by increasing the net trafficking of AMPARs into spines, including in non-motor brain regions

Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer's disease using pI shift and label-free quantification without enrichment

Posttranslational modifications modulate protein function in cells. Global analysis of multiple posttranslational modifications can provide insight into physiology and disease, but presents formidable challenges. In the present study, we used a technique that does not require target enrichment to analyze alterations in the phosphorylation and ubiquitination of proteins from patients with Alzheimer’s disease (AD). Guided by our previous findings, we applied three strategies to further our understanding of the dysregulation of posttranslationally modified proteins.We first identified phosphorylation sites by determining peptide pI shifts using OFFGEL. Second, using tandem mass spectrometry, we determined the ubiquitination status of the proteins using an assay for a trypsin digestion remnant of ubiquitination (Gly- Gly). Third, for large-scale discovery, we quantified the global differences in protein expression. Of the proteins expressed in AD tissue at levels of 2.0 or greater compared with controls, 60 were phosphorylated and 56 were ubiquitinated. Of the proteins expressed at levels of 0.5 or lower compared with controls, 81 were phosphorylated and 56 were ubiquitinated. Approximately 98 % of the phosphopeptides exhibited a pI shift.We identified 112 new phosphorylation sites (51.38 %), and 92 new ubiquitination sites (96.84 %). Taken together, our findings suggest that analysis of the alterations in posttranslationally modified proteins may contribute to understanding the pathogenesis of AD and other diseases.1451sciescopu

ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion

Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy

Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized\ua0medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their\ua0application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in\ua0vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases

The GluR1_S peptide inhibits both low-threshold potentiation and its associated enhancement in the RI.

<p><b>A.</b> Left: Inclusion of GluA1_S in the internal solution inhibited the low-threshold potentiation relative to GluA1_A (GluR1_A, 135.9±7.8% of baseline, n = 12; GluA1_S, 113.3±6.6% of baseline, n = 16). Representative traces are superimposed averages of the EPSCs before and 20 min after pairing. Scale bars, 20 ms and 50 pA. Right: The inclusion of GluA1_S or GluA1_A in the internal solution did not have a significant effect on basal synaptic transmission (GluA1s, 104.1±6.7% of baseline, n = 5, p>0.5; GluA1_A, 109.7±5.8 of baseline, n = 5, p>0.1; paired <i>t</i>-test). <b>B1</b>. The pairing protocol produced an enhancement in the RI and the AMPA EPSC amplitudes when cells were dialyzed with GluA1_A. Left: Sample traces from an individual experiment. Right: Summary data for the RI changes after the induction of low-threshold potentiation (114.9±5.9% of baseline, n = 13). NMDAR-meditated EPSCs did not change significantly after the induction of low-threshold potentiation (113.4±7.4% of baseline, p = 0.0930, paired <i>t</i>-test). <b>B2</b>. The inclusion of GluA1_S in the internal solution attenuated the RI increase associated with the low-threshold potentiation. Left, Sample traces from an individual experiment. Right, Summary data for the RI changes after the induction of low-threshold potentiation (103.9±4.5% of baseline, n = 13). NMDAR-meditated EPSCs did not change (106.9±4.6% of baseline, p = 0.1563, paired <i>t</i>-test). *, p<0.05.</p

ABA renewal-inducing stimuli produce a context-dependent enhancement of synaptic efficacy at T-LA synapses.

<p><b>A.</b> The behavioral procedure. On Day 7, brain slices were prepared immediately after a tone test (ABB-tone and ABA-tone group) or context exposure (ABC-context group). In unpaired controls, one set of rats was killed for the preparation of brain slices, while another set was monitored for conditioned freezing to a CS on Day 7. <b>B</b> and <b>C.</b> Pooled behavioral results. **, p<0.01. <b>D.</b> Input-output curves for EPSCs in unpaired controls (n = 7), ABC-context (n = 7), ABB-tone (n = 16) and ABA-tone groups (n = 9). The representative traces are the averages of four responses evoked by input stimulations of 35 µA. Scale bars, 20 ms and 100 pA.</p

The microinjection of a cell permeable form of the GluA1-derived peptides into the LA attenuates ABA renewal.

<p><b>A.</b> The behavioral procedure. The tone stimulus used for renewal was 60<b> </b>sec in duration on Day 6. In this figure, a weaker conditioning protocol was used (<i>see</i> Methods for additional details). <b>B1</b>. Upper, The microinjection of GluA1_S into the LA impaired ABA renewal relative to GluA1_A (GluA1_A, 41.43±2.68%, n = 8; GluA1_S, 21.24±5.63%, n = 9; p<0.01, unpaired <i>t</i>-test). Lower, Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100108#pone.0100108-Paxinos1" target="_blank">[50]</a> (○, GluA1_A; •, GluA1_S). <b>B2</b>. Top, GluA1_A peptide injection had no effects on ABA renewal relative to vehicle controls (vehicle, 56.09±6.78%, n = 9; GluA1_A, 53.70±11.05%, n = 10; p>0.5, unpaired <i>t</i>-test). Bottom, Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas (○, Vehicle; •, GluA1_A). <b>C.</b> Diffusion of the fluorescent dansyl-tat-GluR1_S peptide (1<b> </b>nmol) within 1<b> </b>h after the microinjection, as visualized with a multiphoton microscope (top). The white arrow indicates the end of the injector cannula. Peptide transduction in individual LA neurons at high magnification (bottom). Ce, central amygdala; BA, basal amygdala. <b>D1</b>. Top: Microinjection of GluA1_D into the LA impaired ABA renewal relative to GluA1_A (GluA1_A, 66.82±5.62%, n = 6; GluA1_D, 36.70±6.77%, n = 6; p<0.01, unpaired t-test). Bottom: Schematic representation of the injector cannula tips (○, GluA1_A; •, GluA1_D). <b>D2</b>. Top: GluA1_A injection had no effects on ABA renewal relative to vehicle controls (vehicle, 74.01±5.65%, n = 6; GluA1_A, 77.37±7.24%, n = 4; p>0.05, unpaired t-test). Bottom: Schematic representation of the injector cannula tips (○, Vehicle; •, GluA1_A).</p