Fluorescence in situ hybridization (FISH) of neural activity-regulated, immediate-early gene (IEG) expression provides a method of functional brain imaging with cellular resolution. This enables the identification, in one brain, of which specific principal neurons were active during each of two distinct behavioral epochs. The unprecedented potential of this differential method for large-scale analysis of functional neural circuits is limited, however, by the time-intensive nature of manual image analysis. A comprehensive software tool for processing three-dimensional, multi-spectral confocal image stacks is described which supports the automation of this analysis. Nuclei counterstained with conventional DNA dyes and FISH signals indicating the sub-cellular distribution of specific, IEG RNA species are imaged using different spectral channels. The DNA channel data are segmented into individual nuclei by a three-dimensional multi-step algorithm that corrects for depth-dependent attenuation, non-isotropic voxels, and imaging noise. Intra-nuclear and cytoplasmic FISH signals are associated spatially with the nuclear segmentation results to generate a detailed tabular/database and graphic representation. Here we present a comprehensive validation of data generated by the automated software against manual quantification by human experts on hippocampal and parietal cortical regions (96.5% concordance with multi-expert consensus). The high degree of reliability and accuracy suggests that the software will generalize well to multiple brain areas and eventually to large-scale brain analysis.

17β-Estradiol (E2) is a well-known neuroprotective factor in the brain. Recently, our lab demonstrated that the neuroprotective and cognitive effects of E2 require mediation by the estrogen receptor (ER) coregulator protein and proline-, glutamic acid-, and leucine-rich protein 1 (PELP1). In the current study, we examined whether E2, acting via PELP1, can exert anti-inflammatory effects in the ovariectomized rat and mouse hippocampus to regulate NLRP3 inflammasome activation after global cerebral ischemia (GCI). Activation of the NLRP3 inflammasome pathway and expression of its downstream products, cleaved caspase-1 and IL-1β, were robustly increased in the hippocampus after GCI, with peak levels observed at 6-7 days. Expression of P2X7 receptor, an upstream regulator of NLRP3, was also increased after GCI. E2 markedly inhibited NLRP3 inflammasome pathway activation, caspase-1, and proinflammatory cytokine production, as well as P2X7 receptor expression after GCI (at both the mRNA and protein level). Intriguingly, the ability of E2 to exert these anti-inflammatory effects was lost in PELP1 forebrain-specific knockout mice, indicating a key role for PELP1 in E2 anti-inflammatory signaling. Collectively, our study demonstrates that NLRP3 inflammasome activation and proinflammatory cytokine production are markedly increased in the hippocampus after GCI, and that E2 signaling via PELP1 can profoundly inhibit these proinflammatory effects.

Silicosis is the most serious occupational disease in China. The objective of this study was to screen various proteins related to mechanisms of the pathogenesis of silicosis underlying the anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) using proteomic profile analysis. We also aimed to explore a potential mechanism of acetylated α-tubulin (α-Ac-Tub) regulation by Ac-SDKP. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to assess the different protein expression profiles between control and silicosis rats treated with or without Ac-SDKP. Twenty-nine proteins were identified to be potentially involved in the progression of silicosis and the anti-fibrotic effect of Ac-SDKP. Our current study finds that 1) the lost expression of Ac-Tub-α may be a new mechanism in rat silicosis; 2) treatment of silicotic rats with N-acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits myofibroblast differentiation and collagen deposition accompanied by stabilizing the expression of α-Ac-Tub in vivo and in vitro, which is related with deacetylase family member 6 (HDAC6) and α-tubulin acetyl transferase (α-TAT1). Our data suggest that α-Ac-Tub regulation by Ac-SDKP may potentially be a new anti-fibrosis mechanism.

Myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, is a key process in organ fibrosis, and is induced by TGF-β. Here we examined whether an anti-fibrotic agent, N-acetyl-seryl-aspartyl-lysylproline (Ac-SDKP), can regulate induction of TGF-β signaling and myofibroblast differentiation as a potential key component of its anti-fibrotic mechanism in vivo and in vitro.

N‑acetyl‑seryl‑aspartyl‑lysyl‑proline (Ac‑SDKP) is a natural tetrapeptide that is released from thymosin β4 by prolyl oligopeptides. It is hydrolyzed by the key enzyme of the renin‑angiotensin system, angiotensin‑converting enzyme (ACE). The aim of the present study was to investigate the alterations in Ac‑SDKP and the ACE/angiotensin II (Ang II)/angiotensin II type 1 (AT1) receptor axis and its impact on the pathogenesis and development of silicotic fibrosis. For in vivo studies, a HOPE MED 8050 exposure control apparatus was used to establish different stages of silicosis in a rat model treated with Ac‑SDKP. For in vitro studies, cultured primary lung fibroblasts were induced to differentiate into myofibroblasts by Ang II, and were pretreated with Ac‑SDKP and valsartan. The results of the present study revealed that, during silicosis development, ACE/Ang II/AT1 expression in local lung tissues increased, whereas that of Ac‑SDKP decreased. Ac‑SDKP and the ACE/AT1/Ang II axis were inversely altered in the development of silicotic fibrosis. Ac‑SDKP treatment had an anti‑fibrotic effect in vivo. Compared with the silicosis group, the expression of α‑smooth muscle actin (α‑SMA), Collagen (Col) I, Fibronectin (Fn) and AT1 were significantly downregulated, whereas matrix metalloproteinase‑1 (MMP‑1) expression and the MMP‑1/tissue inhibitor of metalloproteinases‑1 (TIMP‑1) ratio was increased in the Ac‑SDKP treatment group. In vitro, pre‑treatment with Ac‑SDKP or valsartan attenuated the expression of α‑SMA, Col I, Fn and AT1 in Ang II‑induced fibroblasts. In addition, MMP‑1 expression and the MMP‑1/TIMP‑1 ratio were significantly higher in Ac‑SDKP and valsartan pre‑treatment groups compared with the Ang II group. In conclusion, the results of the present study suggest that an imbalance between Ac‑SDKP and ACE/Ang II/AT1 molecules promotes the development of silicosis and that Ac‑SDKP protects against silicotic fibrosis by inhibiting Ang II‑induced myofibroblast differentiation and extracellular matrix production.

Post-Traumatic Stress Disorder (PTSD) is a psychological disorder that occurs after a traumatic event in a subset of exposed individuals. This implies the existence of susceptibility factors that foster the development of PTSD. Susceptibility factors are present before trauma and can contribute to the development and maintenance of PTSD after trauma. Manipulation of susceptibility factors may decrease the probability of developing PTSD. A putative susceptibility factor is inflammation. Patients with PTSD have been documented to have a higher pro-inflammatory profile compared to non-PTSD subjects. In addition, they are more likely to develop and die from cardiovascular disease which has a strong inflammation component. It is not known, however, whether inflammation plays a role in developing PTSD or whether reducing inflammation can prevent PTSD.

G-protein-coupled estrogen receptor (GPER/GPR30) is a novel membrane-associated estrogen receptor that can induce rapid kinase signaling in various cells. Activation of GPER can prevent hippocampal neuronal cell death following transient global cerebral ischemia (GCI), although the mechanisms remain unclear. In the current study, we sought to address whether GPER activation exerts potent anti-inflammatory effects in the rat hippocampus after GCI as a potential mechanism to limit neuronal cell death.

The neurobiology of emotion and episodic memory are well-researched subjects, as is their intersection: memory of emotional events (i.e. emotional memory). We and others have previously demonstrated that the emotional valence of stimuli is encoded in the dorsal hippocampus, a structure integral to the acquisition, consolidation and retrieval of long-term episodic memories. Such findings are consistent with the idea that the emotional valence of stimuli contributes to the "what" component of episodic memories ("where" and "when" being the other components). We hypothesized that being in a heightened emotional state by itself does not contribute to the "what" component of episodic memories. We tested an inference of this hypothesis - that negative emotional state does not alter re-encoding of a spatial episodic event. Rats from the experimental group explored a novel place at their baseline emotional state (Event 1) and 20 min later re-explored the same place (Event 2) in a negative emotional state induced by a state-altering event prior to Event 2. We examined neuronal ensembles that induced expression of Arc and Homer1a, two immediate-early genes (IEGs) necessary for synaptic plasticity and consolidation of long-term memories, during both events. We found that in dorsal CA1 and dorsal CA3, Event 1 and Event 2 induced IEG expression in different neuronal ensembles. This finding was reflected in a low Fidelity score, which assesses the percentage of the Event 1 IEG-expressing ensemble re-activated during Event 2. The Fidelity score was significantly higher in a control group which was at a baseline emotional state during Event 2. Groups which were matched for non-specific disruptions from the state-altering event had intermediate Fidelity scores in dorsal CA1. The Fidelity scores of the dorsal CA3 in the latter groups were similar to those of the control group. Combined, the findings reject the tested hypothesis and suggest that a negative emotional state is encoded in the hippocampus as part of the long-term memory of episodic events that lack explicit emotion-inducing stimuli. These findings also suggest that individuals who often experience strong negative emotional states incorporate these states into ongoing non-emotional episodic memories.

Anxiety and anxiety-influenced disorders are sexually dimorphic with women being disproportionately affected compared to men. Given the increased prevalence in women and the documented differences in anxiety and trauma behavior between male and female rats this paper sought to examine the link between stress, anxiety, and fear learning and extinction in female rats. We tested the hypothesis that a mild stressor will induce short-and long-term increases in anxiety and produce long term effects on subsequent fear learning and extinction behavior.

Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs) correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration.

Active behavior, such as exploring a novel environment, induces the expression of the immediate-early gene Arc (activity-regulated cytoskeletal associated protein, or Arg 3.1) in many brain regions, including the hippocampus, neocortex, and striatum. Arc messenger ribonucleic acid and protein are localized in activated dendrites, and Arc protein is required for the maintenance of long-term potentiation and memory consolidation. Although previous evidence suggests that Arc is expressed in neurons, there is no direct demonstration that only neurons can express Arc. Furthermore, there is no characterization of the main neuronal types that express Arc. The data reported here show that behavior- or seizure-induced Arc expression in the hippocampus, primary somatosensory cortex, and dorsal striatum of rats colocalizes only with neuronal (NeuN-positive) and not with glial (GFAP-positive) cells. Furthermore, Arc was found exclusively in non-GABAergic alpha-CaMKII-positive hippocampal and neocortical neurons of rats that had explored a novel environment. Some GAD65/67-positive neurons in these regions were observed to express Arc, but only after a very strong stimulus (electroconvulsive seizure). In the dorsal striatum, spatial exploration induced Arc only in GABAergic and alpha-CaMKII-positive neurons. Combined, these results show that although a very strong stimulus (seizure) can induce Arc in a variety of neurons, behavior induces Arc in the CaMKII-positive principal neurons of the hippocampus, neocortex, and dorsal striatum. These results, coupled with recent in vitro findings of interactions between Arc and CaMKII, are consistent with the hypothesis that Arc and CaMKII act as plasticity partners to promote functional and/or structural synaptic modifications that accompany learning.

We hypothesize that dorsal hippocampal (dHC) neurons, which are critical for episodic memory, form a memory of a meal and inhibit the initiation of the next meal and the amount ingested during that meal. In support, we showed previously that (1) consuming a sucrose meal induces expression of the synaptic plasticity marker activity-regulated cytoskeleton-associated protein (Arc) in dHC neurons and (2) reversible inactivation of these neurons immediately following a sucrose meal accelerates the onset of the next meal and increases the size of that meal. These data suggest that hippocampal-dependent memory inhibits intake; therefore, the following experiments were conducted to determine whether hippocampal-dependent memory impairments are associated with increased intake. We reported recently that one episode of early life inflammatory pain impairs dHC-dependent memory in adult rats. The present study determined whether neonatal inflammatory pain also increases sucrose intake and attenuates sucrose-associated Arc expression. Male and female Sprague-Dawley rats were given an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth and sucrose intake and sucrose-associated dHC Arc expression were measured in adulthood. Neonatal inflammatory pain increased sucrose intake in adult female and male rats, decreased sucrose-associated dHC Arc expression in female rats, and tended to have a similar effect on Arc expression in male rats. Neonatal inflammatory pain significantly decreased the interval between two sucrose meals in female but not in male rats. Morphine administration at the time of insult attenuated the effects of injury on sucrose intake. Collectively, these findings indicate that one brief episode of inflammatory pain on the day of birth has a long long-lasting, sex-dependent impact on intake of a palatable food in adulthood.

Myofibroblasts play a major role in the synthesis of extracellular matrix (ECM) and the stimulation of these cells is thought to play an important role in the development of silicosis. The present study was undertaken to investigate the anti-fibrotic effects of dibutyryl-cAMP (db-cAMP) on rats induced by silica.

Emotionally traumatic experiences can lead to debilitating anxiety disorders, such as phobias and Post-Traumatic Stress Disorder (PTSD). Exposure to such experiences, however, is not sufficient to induce pathology, as only up to one quarter of people exposed to such events develop PTSD. These statistics, combined with findings that smaller hippocampal size prior to the trauma is associated with higher risk of developing PTSD, suggest that there are pre-disposing factors for such pathology. Because prospective studies in humans are limited and costly, investigating such pre-dispositions, and thus advancing understanding of the genesis of such pathologies, requires the use of animal models where predispositions are identified before the emotional trauma. Most existing animal models are retrospective: they classify subjects as those with or without a PTSD-like phenotype long after experiencing a traumatic event. Attempts to create prospective animal models have been largely unsuccessful.

VPS35, a major component of the retromer complex, is important for endosome-to-Golgi retrieval of membrane proteins. Although implicated in Alzheimer's disease (AD), how VPS35 regulates AD-associated pathology is unknown. In this paper, we show that hemizygous deletion of Vps35 in the Tg2576 mouse model of AD led to earlier-onset AD-like phenotypes, including cognitive memory deficits, defective long-term potentiation, and impaired postsynaptic glutamatergic neurotransmission in young adult age. These deficits correlated well with an increase of β-amyloid peptide (Aβ) level in the mutant hippocampus. We further demonstrate that VPS35 is predominantly expressed in pyramidal neurons of young adult hippocampus and interacts with BACE1, a protease responsible for Aβ production. Loss of VPS35 function in the mouse hippocampus increased BACE1 activity. Suppression of VPS35 expression in culture decreased BACE1 trans-Golgi localization but enriched it in endosomes. These results demonstrate an essential role for VPS35 in suppression of AD neuropathology and in inhibition of BACE1 activation and Aβ production by promoting BACE1 endosome-to-Golgi retrieval.

Rats can acquire the cognitive component of CS-US associations between sensory and aversive stimuli without a functional basolateral amygdala (BLA). Thus, other brain regions should support such associations. Some septal/dorsal CA1 (dCA1) neurons respond to both spatial stimuli and footshock, suggesting that dCA1 could be one such region. We report that, in both dorsal and ventral hippocampus, different neuronal ensembles express immediate-early genes (IEGs) when a place is experienced alone vs. when it is associated with foot shock. We assessed changes in the size and overlap of hippocampal neuronal ensembles activated by two behavioral events using a cellular imaging method, Arc/Homer1a catFISH. The control group (A-A) experienced the same place twice, while the experimental group (A-CFC) received the same training plus two foot shocks during the second event. During fear conditioning, A-CFC, compared to A-A, rats had a smaller ensemble size in dCA3, dCA1, and vCA3, but not vCA1. Additionally, A-CFC rats had a lower overlap score in dCA1 and vCA3. Locomotion did not correlate with ensemble size. Importantly, foot shocks delivered in a training paradigm that prevents establishing shock-context associations, did not induce significant Arc expression, rejecting the possibility that the observed changes in ensemble size and composition simply reflect experiencing a foot shock. Combined with data that Arc is necessary for lasting synaptic plasticity and long-term memory, the data suggests that Arc/H1a+ hippocampal neuronal ensembles encode aspects of fear conditioning beyond space and time. Rats, like humans, may use the hippocampus to create integrated episodic-like memory during fear conditioning.

A precise fear memory encoding a traumatic event enables an individual to avoid danger and identify safety. An impaired fear memory (contextual amnesia), however, puts the individual at risk of developing posttraumatic stress disorder (PTSD) due to the inability to identify a safe context when encountering trauma-associated cues later in life. Although it is gaining attention that contextual amnesia is a critical etiologic factor for PTSD, there is no treatment currently available that can reverse contextual amnesia, and whether such treatment can prevent the development of PTSD is unknown. Here, we report that (I) a single dose of transcranial photobiomodulation (PBM) applied immediately after tone fear conditioning can reverse contextual amnesia. PBM treatment preserved an appropriately high level of contextual fear memory in rats revisiting the "dangerous" context, while control rats displayed memory impairment. (II) A single dose of PBM applied after memory recall can reduce contextual fear during both contextual and cued memory testing. (III) In a model of complex PTSD with repeated trauma, rats given early PBM interventions efficiently discriminated safety from danger during cued memory testing and, importantly, these rats did not develop PTSD-like symptoms and comorbidities. (IV) Finally, we report that fear extinction was facilitated when PBM was applied in the early intervention window of memory consolidation. Our results demonstrate that PBM treatment applied immediately after a traumatic event or its memory recall can protect contextual fear memory and prevent the development of PTSD-like psychopathological fear in rats.

Retinal neurodegeneration, an early characteristic of several blinding diseases, triggers glial activation, resulting in inflammation, secondary damage and visual impairment. Treatments that aim only at neuroprotection have failed clinically. Here, we examine the impact of modulating thioredoxin interacting protein (TXNIP) to the inflammatory secondary damage and visual impairment in a model of ischemia/reperfusion (IR). Wild type (WT) and TXNIP knockout (TKO) mice underwent IR injury by increasing intraocular pressure for 40 min, followed by reperfusion. An additional group of WT mice received intravitreal TXNIP-antisense oligomers (ASO, 100 µg/2 µL) 2 days post IR injury. Activation of Müller glial cells, apoptosis and expression of inflammasome markers and visual function were assessed. IR injury triggered early TXNIP mRNA expression that persisted for 14 days and was localized within activated Müller cells in WT-IR, compared to sham controls. Exposure of Müller cells to hypoxia-reoxygenation injury triggered endoplasmic reticulum (ER) stress markers and inflammasome activation in WT cells, but not from TKO cells. Secondary damage was evident by the significant increase in the number of occluded acellular capillaries and visual impairment in IR-WT mice but not in IR-TKO. Intervention with TXNIP-ASO prevented ischemia-induced glial activation and neuro-vascular degeneration, and improved visual function compared to untreated WT. Targeting TXNIP expression may offer an effective approach in the prevention of secondary damage associated with retinal neurodegenerative diseases.