Epigenetic Alterations Are Critical for Fear Memory Consolidation and Synaptic Plasticity in the Lateral Amygdala

Epigenetic mechanisms, including histone acetylation and DNA methylation, have been widely implicated in hippocampal-dependent learning paradigms. Here, we have examined the role of epigenetic alterations in amygdala-dependent auditory Pavlovian fear conditioning and associated synaptic plasticity in the lateral nucleus of the amygdala (LA) in the rat. Using Western blotting, we first show that auditory fear conditioning is associated with an increase in histone H3 acetylation and DNMT3A expression in the LA, and that training-related alterations in histone acetylation and DNMT3A expression in the LA are downstream of ERK/MAPK signaling. Next, we show that intra-LA infusion of the histone deacetylase (HDAC) inhibitor TSA increases H3 acetylation and enhances fear memory consolidation; that is, long-term memory (LTM) is enhanced, while short-term memory (STM) is unaffected. Conversely, intra-LA infusion of the DNA methyltransferase (DNMT) inhibitor 5-AZA impairs fear memory consolidation. Further, intra-LA infusion of 5-AZA was observed to impair training-related increases in H3 acetylation, and pre-treatment with TSA was observed to rescue the memory consolidation deficit induced by 5-AZA. In our final series of experiments, we show that bath application of either 5-AZA or TSA to amygdala slices results in significant impairment or enhancement, respectively, of long-term potentiation (LTP) at both thalamic and cortical inputs to the LA. Further, the deficit in LTP following treatment with 5-AZA was observed to be rescued at both inputs by co-application of TSA. Collectively, these findings provide strong support that histone acetylation and DNA methylation work in concert to regulate memory consolidation of auditory fear conditioning and associated synaptic plasticity in the LA

Using fMRI connectivity to define a treatment-resistant form of post-traumatic stress disorder.

A mechanistic understanding of the pathology of psychiatric disorders has been hampered by extensive heterogeneity in biology, symptoms, and behavior within diagnostic categories that are defined subjectively. We investigated whether leveraging individual differences in information-processing impairments in patients with post-traumatic stress disorder (PTSD) could reveal phenotypes within the disorder. We found that a subgroup of patients with PTSD from two independent cohorts displayed both aberrant functional connectivity within the ventral attention network (VAN) as revealed by functional magnetic resonance imaging (fMRI) neuroimaging and impaired verbal memory on a word list learning task. This combined phenotype was not associated with differences in symptoms or comorbidities, but nonetheless could be used to predict a poor response to psychotherapy, the best-validated treatment for PTSD. Using concurrent focal noninvasive transcranial magnetic stimulation and electroencephalography, we then identified alterations in neural signal flow in the VAN that were evoked by direct stimulation of that network. These alterations were associated with individual differences in functional fMRI connectivity within the VAN. Our findings define specific neurobiological mechanisms in a subgroup of patients with PTSD that could contribute to the poor response to psychotherapy.PEV was supported by the Medical Research Council (grant no. MR/K020706/1) and is a Fellow of MQ: Transforming Mental Health (MQF17_24)

Stereotactic Body Radiotherapy for Extracranial Oligometastatic Disease from Head and Neck Primary Cancers: A Systematic Review and Meta-Analysis

Introduction: Stereotactic body radiotherapy (SBRT) is increasingly used to treat disease in the oligometastatic (OM) setting due to mounting evidence demonstrating its efficacy and safety. Given the low population representation in prospective studies, we performed a systematic review and meta-analysis of outcomes of HNC patients with extracranial OM disease treated with SBRT. Methods: A systematic review was conducted with Cochrane, Medline, and Embase databases queried from inception to August 2022 for studies with extracranial OM HNC treated with stereotactic radiotherapy. Polymetastatic patients (>five lesions), mixed-primary cohorts failing to report HNC separately, lack of treatment to all lesions, nonquantitative endpoints, and other definitive treatments (surgery, conventional radiotherapy, and radioablation) were excluded. The meta-analysis examined the pooled effects of 12- and 24-month local control (LC) per lesion, progression-free survival (PFS), and overall survival (OS). Weighted random-effects were assessed using the DerSimonian and Laird method, with heterogeneity evaluated using the I2 statistic and Cochran Qtest. Forest plots were generated for each endpoint. Results: Fifteen studies met the inclusion criteria (639 patients, 831 lesions), with twelve eligible for quantitative synthesis with common endpoints and sufficient reporting. Fourteen studies were retrospective, with a single prospective trial. Studies were small, with a median of 32 patients (range: 6–81) and 63 lesions (range: 6–126). The OM definition varied, with a maximum of two to five metastases, mixed synchronous and metachronous lesions, and a few studies including oligoprogressive lesions. The most common site of metastasis was the lung. Radiation was delivered in 1–10 fractions (20–70 Gy). The one-year LC (LC1), reported in 12 studies, was 86.9% (95% confidence interval [CI]: 79.3–91.9%). LC2 was 77.9% (95% CI: 66.4–86.3%), with heterogeneity across studies. PFS was reported in five studies, with a PFS1 of 43.0% (95% CI: 35.0–51.4%) and PFS2 of 23.9% (95% CI: 17.8–31.2%), with homogeneity across studies. OS was analyzed in nine studies, demonstrating an OS1 of 80.1% (95% CI: 74.2–85.0%) and OS2 of 60.7% (95% CI: 51.3–69.4%). Treatment was well tolerated with no reported grade 4 or 5 toxicities. Grade 3 toxicity rates were uniformly below 5% when reported. Conclusions: SBRT offers excellent LC and promising OS, with acceptable toxicities in OM HNC. Durable PFS remains rare, highlighting the need for effective local or systemic therapies in this population. Further investigations on concurrent and adjuvant therapies are warranted

Histone acetylation and DNA methylation interact to regulate memory consolidation in the LA.

<p>(A) Schematic of the behavioral protocol. Rats were trained with 3 tone-shock pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 7) or 5-AZA (n = 7) and sacrificed 30 min after infusion. <i>(B)</i> Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from punches taken from the LA. Here, acetyl-H3 and acetyl-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle group. (*) <i>p</i><0.05 relative to vehicle group. (<i>C</i>) Mean (±SEM) total-H3 and total-H4 immunoreactivity from the samples in (B). Here, total-H3 and total-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (<i>D</i>) Representative blots for acetyl-H3/H4 and total-H3/H4, respectively. (<i>E</i>) Schematic of the behavioral protocol. Rats were trained and immediately after given intra-LA infusion of either (1 µg in 0.5 µl/side) TSA or Vehicle (0.5 µl/side) followed 60 min later by intra-LA infusion of (1 µg in 0.5 µl/side) 5-AZA or Vehicle (0.5 µl/side), creating the following groups: Veh-Veh (<i>n</i> = 6), Veh-5-AZA (<i>n</i> = 5), and TSA-5-AZA (<i>n</i> = 6). LTM was examined 24 hrs later. (<i>F</i>) Post-shock freezing scores in each group immediately after the conditioning trials. (<i>G</i>) Mean (±SEM) LTM retention test scores across each trial. (<i>H</i>) Histological verification of cannula placements for rats infused with Vehicle-Vehicle (black circles), or Vehicle-5-AZA (gray circles), or TSA-5-AZA (white circles). Panels adapted from Paxinos and Watson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019958#pone.0019958-Paxinos1" target="_blank">[40]</a>.</p

Intra-LA infusion of a DNMT inhibitor impairs auditory fear memory consolidation.

<p>(A) Schematic of behavioral protocol. Rats were conditioned with 3 tone-shock pairings, followed by intra-LA infusion of vehicle or 5-AZA 1 hr later (n = 5, each group). LTM was assessed ∼24 hr after training in a distinct context. Rats were re-conditioned drug free and re-tested for LTM ∼1 week later. (<i>B</i>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<i>C</i>) Mean (±SEM) LTM retention test scores across each trial. (<i>D</i>) Mean (±SEM) LTM retention re-test scores across each trial following re-conditioning one week later. (<i>E</i>) Schematic of behavioral protocol. Rats were conditioned with 3 tone-shock pairings, followed 1 hr later by intra-LA infusion of vehicle (n = 4) or 5-AZA (n = 4). STM was assessed 1 hr after training in a distinct context. (<i>F</i>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<i>G</i>) Mean (±SEM) STM retention test scores across each trial. (<i>H</i>) Histological verification of cannula placements for rats in LTM (left) and STM (right) experiments infused with vehicle (black circles) or 5-AZA (white circles). Panels adapted from Paxinos and Watson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019958#pone.0019958-Paxinos1" target="_blank">[40]</a>.</p

Intra-LA infusion of an HDAC inhibitor increases histone acetylation and enhances auditory fear memory consolidation.

<p>(A) Schematic of the behavioral protocol. Rats were trained with 2 tone-shock pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 8) or TSA (n = 8) and sacrificed 30 min after infusion. (<i>B</i>) Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from punches taken from the LA. Here, acetyl-H3/H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (*) <i>p</i><0.05 relative to vehicle-infused rats. (<i>C</i>) Mean (±SEM) total-H3/H4 from the samples in (B). Here, total-H3/H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle group. (<i>D</i>) Representative blots for acetyl-H3/H4 and total-H3/H4, respectively. (<i>E</i>) Schematic of behavioral protocol. Rats were conditioned with 2 tone-shock pairings, followed 1 hr later by intra-LA infusion of vehicle (n = 6) or TSA (n = 6). LTM was assessed ∼24 hr after training in a distinct context. (<i>F</i>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<i>G</i>) Mean (±SEM) LTM retention test scores across each trial. (<i>H</i>) Schematic of behavioral protocol. Rats were conditioned with 2 tone-shock pairings, followed 1 hr later by intra-LA infusion of vehicle (n = 4) or TSA (n = 4). STM was assessed 1 hr after infusion in a distinct context. (<i>I</i>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<i>J</i>) Mean (±SEM) STM retention test scores across each trial. (<i>K</i>) Histological verification of cannula placements for rats infused with vehicle (black circles) or TSA (white circles) for the LTM (top) and STM (bottom) experiments. Panels adapted from Paxinos and Watson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019958#pone.0019958-Paxinos1" target="_blank">[40]</a>.</p

Auditory fear conditioning regulates histone acetylation and DNMT expression in the LA.

<p>(A) Schematic of the behavioral protocol. Rats were habituated to handling, trained with 3 tone-shock pairings, and sacrificed at 30, 60, or 90 min later. (<i>B</i>) Representative Western blots for acetylated histone (top), total histone (middle), and DNMT expression (bottom) at each time point. (<i>C</i>) Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from LA punches taken from Naïve (n = 7) and trained rats sacrificed at 30 min (n = 8), 60 min (n = 8), or 90 min (n = 7). Here, acetyl-H3 and acetyl-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (*) <i>p</i><0.05 relative to all other time points. (<i>D</i>) Mean (±SEM) total-H3 and total-H4 immunoreactivity from LA punches taken from the samples in (B). Here, total-H3 and total-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (E) Mean (±SEM) DNMT3A/3B immunoreactivity from LA punches taken from Naïve (n = 7) and trained rats sacrificed at 30 min (n = 8), 60 min (n = 7), or 90 min (n = 8). (*) <i>p</i><0.05 relative to all other time points. Here, DNMT3A/3B protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group.</p

Regulation of histone H3 acetylation and DNMT3A expression in the LA following fear conditioning is ERK-dependent.

<p>(A) Schematic of the behavioral protocol. Rats received intra-LA infusion of either vehicle (n = 7) or U0126 (n = 8) followed 30 min later by fear conditioning consisting of 3 tone-shock pairings and were sacrificed 90 min after training. (<i>B</i>) Representative Western blots for acetylated histone (top), total histone (middle), and DNMT3A expression (bottom) at each time point. (<i>C</i>) Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from punches taken from the LA. Here, acetyl-H3 and acetyl-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (*) <i>p</i><0.05 relative to vehicle group. (<i>D</i>) Mean (±SEM) total-H3 and total-H4 immunoreactivity from the samples in (C). Here, total-H3 and total-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (<i>E</i>) Mean (±SEM) DNMT3A immunoreactivity from punches taken from the LA from vehicle (n = 7) and U0126-treated rats (n = 7). Here, DNMT3A protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (*) <i>p</i><0.05 relative to vehicle group.</p

DNMT and HDAC inhibition impairs or enhances, respectively, amygdala LTP at thalamic and cortical inputs.

<p>(A–B) Schematic of amygdala slice preparation for “thalamic” and “cortical” stimulation experiments, showing placement of stimulating and recording electrodes. (<i>C</i>) Mean (±SEM) percent field potential amplitude (relative to baseline) in slices treated with vehicle (n = 7; black circles) or 30 µM 5-AZA (n = 5; gray circles) followed by LTP induction at thalamic inputs. Traces from an individual experiment before and 30 min following tetanic stimulation and baseline transmission following 20 min of bath application of drug are shown in the <i>inset</i>. Scale, 0.2 mV by 10 msec. (<i>D</i>) Mean (±SEM) percent field potential amplitude (relative to baseline) in slices treated with vehicle (n = 6; black circles) or 30 µM 5-AZA (n = 7; gray circles) followed by LTP induction at cortical inputs. Traces from an individual experiment before and 30 min following tetanic stimulation and baseline transmission following 20 min of bath application of drug are shown in the <i>inset</i>. Scale, 0.2 mV by 10 msec. (<i>E</i>) Mean (±SEM) percent field potential amplitude (relative to baseline) in slices treated with vehicle (n = 10; black circles) or 2.5 µM TSA (n = 7; gray circles) followed by LTP at thalamic inputs. Traces from an individual experiment before and 30 min following tetanic stimulation and transmission following 20 min of bath application of drug are shown in the <i>inset</i>. Scale, 0.2 mV by 10 msec. (<i>F</i>) Mean (±SEM) percent field potential amplitude (relative to baseline) in slices treated with vehicle (n = 6; black circles) or 2.5 µM TSA (n = 7; gray circles) followed by LTP at cortical inputs. Traces from an individual experiment before and 30 min following tetanic stimulation and transmission following 20 min of bath application of drug are shown in the <i>inset</i>. Scale, 0.2 mV by 10 msec. (<i>G</i>) Mean (±SEM) percent field potential amplitude (relative to baseline) in slices treated with vehicle (n = 6; black circles) or 30 µM 5-AZA+2.5 µM TSA (n = 6; gray circles) followed by LTP at thalamic inputs. Traces from an individual experiment before and 30 min following tetanic stimulation and transmission following 20 min of bath application of drug are shown in the <i>inset</i>. Scale, 0.2 mV by 10 msec. (<i>H</i>) Mean (±SEM) percent field potential amplitude (relative to baseline) in slices treated with vehicle (n = 6; black circles) or 30 µM 5-AZA+2.5 µM TSA (n = 5; gray circles) followed by LTP at cortical inputs. Traces from an individual experiment before and 30 min following tetanic stimulation and transmission following 20 min of bath application of drug are shown in the <i>inset</i>. Scale, 0.2 mV by 10 msec.</p

Training-related regulation of histone H3 acetylation and DNMT3A expression in the LA is specific to tone-shock pairing.

<p>(A) Schematic of the behavioral protocol. In two separate experiments, rats were either given no stimulation (“Naïve”), 3 tone-alone presentations (“Tone Alone”), 3 immediate shocks (“Imm. Shock”), or 3 tone-shock pairings (“Paired”) and sacrificed 90 min later. (<i>B</i>) Representative Western blots for acetylated and total histone H3 (top) and DNMT3A/B expression (bottom) in each group. (<i>C</i>) Mean (±SEM) acetyl-H3 and total H3 immunoreactivity from LA punches taken from Naïve (n = 8), Tone Alone (n = 7), Imm. Shock (n = 8), and Paired (n = 6) rats. Here, acetyl-H3 and total H3 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (*) <i>p</i><0.05 relative to all other groups. (<i>D</i>) Mean (±SEM) DNMT3A/B immunoreactivity from LA punches taken from Naïve (n = 8), Tone Alone (n = 8), Imm. Shock (n = 8), and Paired (n = 8) rats. Here, DNMT3A/B protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (*) <i>p</i><0.05 relative to all other groups.</p