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.

Alzheimer's disease (AD) is the most common form of dementia in the elderly, causing neuronal degeneration and cognitive deficits that significantly impair independence and quality of life for those affected and their families. Though AD is a major neurodegenerative disease with vast avenues of investigation, there is no effective treatment to cure AD or slow disease progression. The present work evaluated the therapeutic effect of long-term photobiomodulation (PBM) treatment with continuous-wave low-level laser on AD and its underlying mechanism. Methods: PBM was implemented for 2 min, 3 times per week for 16 months in 2-month-old transgenic AD rats. A battery of behavioral tests was performed to measure the effect of PBM treatment on cognitive dysfunction in AD rats. The effects of PBM therapy on typical AD pathologies, including amyloid plaques, intracellular neurofibrillary tangles, neuronal loss, neuronal injury, neuronal apoptosis, and neurodegeneration, were then assessed. The underlying mechanisms were measured using immunofluorescence staining, western blotting analysis, mass spectrometry, primary cortical and hippocampal cell cultures, and related assay kits. Results: PBM treatment significantly improved the typical AD pathologies of memory loss, amyloid plaques, tau hyperphosphorylation, neuronal degeneration, spine damage, and synaptic loss. PBM treatment had several mechanistic effects which may explain these beneficial effects, including 1) regulation of glial cell polarization and inhibition of neuroinflammation, 2) preservation of mitochondrial dynamics by regulating fission and fusion proteins, and 3) suppression of oxidative damage to DNA, proteins, and lipids. Furthermore, PBM enhanced recruitment of microglia surrounding amyloid plaques by improving the expression of microglial IL-3Rα and astrocytic IL-3, which implies a potential role of PBM in improving Aβ clearance. Finally, our results implicate neuronal hemoglobin in mediating the neuroprotective effect of PBM, as Hbα knockdown abolished the neuroprotective effect of PBM treatment. Conclusion: Collectively, our data supports the potential use of PBM treatment to prevent or slow the progression of AD and provides new insights into the molecular mechanisms of PBM therapy.

Rationale: Previous work has indicated that continuous theta-burst stimulation (cTBS), a modality of transcranial magnetic stimulation (TMS), may provide neuroprotection and improve neurological function after stroke by preserving the blood-brain barrier, altering glial polarization phenotypes, and supporting peri-infarct angiogenesis. The present study was performed to examine whether cTBS, a noninvasive neurostimulation technique, promotes neurogenesis in a photothrombotic (PT) stroke rat model and contributes to functional recovery. Methods: Beginning 3 h or 1 week after the induction of PT stroke, once-daily 5-min cTBS treatments were applied to the infarcted hemisphere for 6 days. Samples were collected 6 days, 22 days, and 35 days after PT stroke. Fluorescent labeling, Western blotting, and behavioral tests were performed accordingly. Results: We found that cTBS therapy significantly expanded the pool of neural progenitor cells (NPCs) and newly generated immature neurons in the cortical peri-infarct region after PT stroke. Likewise, the amount of DCX-positive immature neurons in the peri-infarct area was markedly elevated by cTBS. Application of cTBS strikingly diminished the PT-induced loss of NPCs and newly-formed neurons. In addition, the amount of newly generated mature neurons in the peri-infarct zone was significantly promoted by cTBS. Intriguingly, cTBS reduced reactive gliogenesis significantly while promoting oligodendrogenesis and preserving myelination. Mechanistic studies uncovered that cTBS upregulated brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2). Finally, cTBS-treated animals displayed improved motor functions. To be noted, temozolomide (TMZ), a drug that has been previously used to suppress neurogenesis, could reverse the beneficial effects of cTBS. Conclusions: Our findings provide new insight into the mechanism by which cTBS promotes functional recovery from stroke. We demonstrated that cTBS effectively enhances and sustains neurogenesis after PT stroke. Both early and delayed cTBS treatment could improve the survival of newly generated neurons and functional recovery, and inhibition of neurogenesis could reverse these therapeutic benefits. Mechanistically, cTBS was effective in upregulating the release of neurotrophic factors, protecting NPC and immature neurons, as well as suppressing excessive gliogenesis.

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.

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.

Rationale: The integrity and function of the blood-brain barrier (BBB) is compromised after stroke. The current study was performed to examine potential beneficial effects and underlying mechanisms of repetitive transcranial magnetic stimulation (rTMS) on angiogenesis and vascular protection, function, and repair following stroke, which are largely unknown. Methods: Using a rat photothrombotic (PT) stroke model, continuous theta-burst rTMS was administered once daily to the infarcted hemisphere for 5 min, beginning 3 h after PT stroke. This treatment was applied for 6 days. BBB integrity, blood flow, vascular associated proteins, angiogenesis, integrity of neuronal morphology and structure, and behavioral outcome were measured and analyzed at 6 and/or 22 days after PT stroke. Results: We report that rTMS significantly mitigated BBB permeabilization and preserved important BBB components ZO-1, claudin-5, occludin, and caveolin-1 from PT-induced degradation. Damage to vascular structure, morphology, and perfusion was ameliorated by rTMS, resulting in improved local tissue oxygenation. This was accompanied with robust protection of critical vascular components and upregulation of regulatory factors. A complex cytokine response was induced by PT, particularly at the late phase. Application of rTMS modulated this response, ameliorating levels of cytokines related to peripheral immune cell infiltration. Further investigation revealed that rTMS promoted and sustained post-ischemic angiogenesis long-term and reduced apoptosis of newborn and existing vascular endothelial cells. Application of rTMS also inhibited PT-induced excessive astrocyte-vasculature interactions and stimulated an A1 to A2 shift in vessel-associated astrocytes. Mechanistic studies revealed that rTMS dramatically increased levels of PDGFRβ associated with A2 astrocytes and their adjacent vasculature. As well, A2 astrocytes displayed marked amplification of the angiogenesis-related factors VEGF and TGFβ. PT induced a rise in vessel-associated expression of HIF-1α that was starkly intensified by rTMS treatment. Finally, rTMS preserved neuronal morphology, synaptic structure integrity and behavioral outcome. Conclusions: These results indicate that rTMS can exert powerful protective and restorative effects on the peri-infarct microvasculature after PT stroke by, in part, promoting HIF-1α signaling and shifting vessel-associated astrocytic polarization to the A2 phenotype. This study provides further support for the potent protective effects of rTMS in the context of ischemic stroke, and these findings implicate vascular repair and protection as an important underlying phenomenon.

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.

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.

Maladaptive fear memory processing after a traumatic event is a major contributor to the development of the comorbidities related to posttraumatic stress disorder (PTSD). An intervention to normalize this process could be a first-line treatment to prevent PTSD development. However, little progress has been made in identifying interventions that can prevent trauma survivors from developing PTSD. A treatment that could help trauma survivors cope with traumatic memories and decrease the prevalence of PTSD is thus in high demand. This study was designed to investigate the potential beneficial effects of early photobiomodulation (PBM) interventions to prevent PTSD-like comorbidities in animals. PTSD-like comorbidities in rats were induced by an underwater trauma (UWT) procedure, followed by multiple swimming sessions on later days for memory recall. Immediately after UWT and swimming, rats were restrained with or without PBM treatment (808 nm, 25 mW/cm2, 3 J/day). PTSD-like commodities, such as anxiety-like behavior, depression-like behavior, and cognitive dysfunction, were reproduced in UWT-rats. These comorbidities, however, could be prevented by early PBM interventions. By measuring the expression of immediate early genes (IEGs) as neuronal activity markers, we found that PBM treatment differentially regulated Arc and c-fos expression in the hippocampus and amygdala, two PTSD-related brain regions. Additionally, PBM boosted ATP production and regulated protein expression in the hippocampus following stress. Our results demonstrate that PBM can modulate brain activity in response to traumatic and stressful events and that early PBM intervention can prevent the occurrence of PTSD-like comorbidities in rats.

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.