A Role for Nitric Oxide-Driven Retrograde Signaling in the Consolidation of a Fear Memory

In both invertebrate and vertebrate models of synaptic plasticity, signaling via the putative “retrograde messenger” nitric oxide (NO) has been hypothesized to serve as a critical link between functional and structural alterations at pre- and postsynaptic sites. However, while in vitro models of synaptic plasticity have consistently implicated NO signaling in linking postsynaptic induction mechanisms with accompanying presynaptic changes, a convincing role of such “retrograde signaling” in mammalian memory formation has remained elusive. Using auditory Pavlovian fear conditioning, we show that synaptic plasticity and NO signaling in the lateral nucleus of the amygdala (LA) regulate the expression of the ERK-driven immediate early gene early growth response gene I (EGR-1) in regions of the auditory thalamus that are presynaptic to the LA. Further, antisense knockdown of EGR-1 in the auditory thalamus impairs both fear memory consolidation and the training-induced elevation of two presynaptically localized proteins in the LA. These findings indicate that synaptic plasticity and NO signaling in the LA during auditory fear conditioning promote alterations in ERK-driven gene expression in auditory thalamic neurons that are required for both fear memory consolidation as well as presynaptic correlates of fear memory formation in the LA, and provide general support for a role of NO as a “retrograde signal” in mammalian memory formation

Synaptic Plasticity and NO-cGMP-PKG Signaling Regulate Pre- and Postsynaptic Alterations at Rat Lateral Amygdala Synapses Following Fear Conditioning

In vertebrate models of synaptic plasticity, signaling via the putative “retrograde messenger” nitric oxide (NO) has been hypothesized to serve as a critical link between functional and structural alterations at pre- and postsynaptic sites. In the present study, we show that auditory Pavlovian fear conditioning is associated with significant and long-lasting increases in the expression of the postsynaptically-localized protein GluR1 and the presynaptically-localized proteins synaptophysin and synapsin in the lateral amygdala (LA) within 24 hrs following training. Further, we show that rats given intra-LA infusion of either the NR2B-selective antagonist Ifenprodil, the NOS inhibitor 7-Ni, or the PKG inhibitor Rp-8-Br-PET-cGMPS exhibit significant decreases in training-induced expression of GluR1, synaptophysin, and synapsin immunoreactivity in the LA, while those rats infused with the PKG activator 8-Br-cGMP exhibit a significant increase in these proteins in the LA. In contrast, rats given intra-LA infusion of the NO scavenger c-PTIO exhibit a significant decrease in synapsin and synaptophysin expression in the LA, but no significant impairment in the expression of GluR1. Finally, we show that intra-LA infusions of the ROCK inhibitor Y-27632 or the CaMKII inhibitor KN-93 impair training-induced expression of GluR1, synapsin, and synaptophysin in the LA. These findings suggest that the NO-cGMP-PKG, Rho/ROCK, and CaMKII signaling pathways regulate fear memory consolidation, in part, by promoting both pre- and post-synaptic alterations at LA synapses. They further suggest that synaptic plasticity in the LA during auditory fear conditioning promotes alterations at presynaptic sites via NO-driven “retrograde signaling”

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

Decreased SGK1 Expression and Function Contributes to Behavioral Deficits Induced by Traumatic Stress

Exposure to extreme stress can trigger the development of major depressive disorder (MDD) as well as post-traumatic stress disorder (PTSD). The molecular mechanisms underlying the structural and functional alterations within corticolimbic brain regions, including the prefrontal cortex (PFC) and amygdala of individuals subjected to traumatic stress, remain unknown. In this study, we show that serum and glucocorticoid regulated kinase 1 (SGK1) expression is down-regulated in the postmortem PFC of PTSD subjects. Furthermore, we demonstrate that inhibition of SGK1 in the rat medial PFC results in helplessness- and anhedonic-like behaviors in rodent models. These behavioral changes are accompanied by abnormal dendritic spine morphology and synaptic dysfunction. Together, the results are consistent with the possibility that altered SGK1 signaling contributes to the behavioral and morphological phenotypes associated with traumatic stress pathophysiology

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way

The NO-cGMP-PKG signaling pathway regulates synaptic plasticity and fear memory consolidation in the lateral amygdala via activation of ERK/MAP kinase

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and long-term potentiation (LTP) at thalamic and cortical input pathways to the LA. In behavioral experiments, rats given intra-LA infusions of either the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibited dose-dependent impairments or enhancements of fear memory consolidation, respectively. In slice electrophysiology experiments, bath application of Rp-8-Br-PET-cGMPS or the guanylyl cyclase inhibitor LY83583 impaired LTP at thalamic, but not cortical inputs to the LA, while bath application of 8-Br-cGMP or the guanylyl cyclase activator YC-1 resulted in enhanced LTP at thalamic inputs to the LA. Interestingly, YC-1-induced enhancement of LTP in the LA was reversed by concurrent application of the MEK inhibitor U0126, suggesting that the NO-cGMP-PKG signaling pathway may promote synaptic plasticity and fear memory formation in the LA, in part by activating the ERK/MAPK signaling cascade. As a test of this hypothesis, we next showed that rats given intra-LA infusion of the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibit impaired or enhanced activation, respectively, of ERK/MAPK in the LA after fear conditioning. Collectively, our findings suggest that an NO-cGMP-PKG-dependent form of synaptic plasticity at thalamic input synapses to the LA may underlie memory consolidation of Pavlovian fear conditioning, in part, via activation of the ERK/MAPK signaling cascade

A dietary regimen of caloric restriction or pharmacological activation of SIRT1 to delay the onset of neurodegeneration

Caloric restriction (CR) is a dietary regimen known to promote lifespan by slowing down the occurrence of age-dependent diseases. The greatest risk factor for neurodegeneration in the brain is age, from which follows that CR might also attenuate the progressive loss of neurons that is often associated with impaired cognitive capacities. In this study, we used a transgenic mouse model that allows for a temporally and spatially controlled onset of neurodegeneration to test the potentially beneficial effects of CR. We found that in this model, CR significantly delayed the onset of neurodegeneration and synaptic loss and dysfunction, and thereby preserved cognitive capacities. Mechanistically, CR induced the expression of the known lifespan-regulating protein SIRT1, prompting us to test whether a pharmacological activation of SIRT1 might recapitulate CR. We found that oral administration of a SIRT1-activating compound essentially replicated the beneficial effects of CR. Thus, SIRT1-activating compounds might provide a pharmacological alternative to the regimen of CR against neurodegeneration and its associated ailments

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

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