A multiple Arc (mArc) tagging system to uncover the organizational principles of multiple memories.

Engrams or memory traces are the neuronal ensembles that collectively store individual experiences. Genetic strategies based on immediate early genes (IEGs), such as Arc/Arg3.1 , allow us to tag the ensembles active during memory encoding and compare them to those active during retrieval. However, these strategies only allow for the tagging of one neural ensemble. Here, we developed a multiple Arc (mArc) system that allows for the tagging of two Arc + ensembles. We validated this system by investigating how context, time, and valence influence neuronal ensemble reactivation in the dentate gyrus (DG). We show that similar contextual and valenced experiences are encoded in overlapping DG ensembles. We also find that ensembles are modulated by time, where experiences closer in time are encoded in more similar ensembles. These results highlight the dynamic nature of DG ensembles and show that the mArc system provides a powerful approach for investigating multiple memories in the brain. HIGHLIGHTS: The mArc system allows for the tagging of two Arc + ensembles in the same mouse DG ensembles labeled by the mArc system receive increased excitatory inputContext, valence, and time influence DG ensemble reactivationDG neural ensembles are reactivated less with increasing time.

Progressive Excitability Changes in the Medial Entorhinal Cortex in the 3xTg Mouse Model of Alzheimer's Disease Pathology.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by memory loss and progressive cognitive impairments. In mouse models of AD pathology, studies have found neuronal and synaptic deficits in hippocampus, but less is known about changes in medial entorhinal cortex (MEC), which is the primary spatial input to the hippocampus and an early site of AD pathology. Here, we measured neuronal intrinsic excitability and synaptic activity in MEC layer II (MECII) stellate cells, MECII pyramidal cells, and MEC layer III (MECIII) excitatory neurons at 3 and 10 months of age in the 3xTg mouse model of AD pathology, using male and female mice. At 3 months of age, before the onset of memory impairments, we found early hyperexcitability in intrinsic properties of MECII stellate and pyramidal cells, but this was balanced by a relative reduction in synaptic excitation (E) compared with inhibition (I; E/I ratio), suggesting intact homeostatic mechanisms regulating MECII activity. Conversely, MECIII neurons had reduced intrinsic excitability at this early time point with no change in synaptic E/I ratio. By 10 months of age, after the onset of memory deficits, neuronal excitability of MECII pyramidal cells and MECIII excitatory neurons was largely normalized in 3xTg mice. However, MECII stellate cells remained hyperexcitable, and this was further exacerbated by an increased synaptic E/I ratio. This observed combination of increased intrinsic and synaptic hyperexcitability suggests a breakdown in homeostatic mechanisms specifically in MECII stellate cells at this postsymptomatic time point, which may contribute to the emergence of memory deficits in AD.SIGNIFICANCE STATEMENT AD causes cognitive deficits, but the specific neural circuits that are damaged to drive changes in memory remain unknown. Using a mouse model of AD pathology that expresses both amyloid and tau transgenes, we found that neurons in the MEC have altered excitability. Before the onset of memory impairments, neurons in layer 2 of MEC had increased intrinsic excitability, but this was balanced by reduced inputs onto the cell. However, after the onset of memory impairments, stellate cells in MEC became further hyperexcitable, with increased excitability exacerbated by increased synaptic inputs. Thus, it appears that MEC stellate cells are uniquely disrupted during the progression of memory deficits and may contribute to cognitive deficits in AD.

Chronotate: An open-source tool for manual timestamping and quantification of animal behavior.

A core necessity to behavioral neuroscience research is the ability to accurately measure performance on behavioral assays, such as the novel object location and novel object recognition tasks. These tasks are widely used in neuroscience research and measure a rodent's instinct for investigating novel features as a proxy to test their memory of a previous experience. Automated tools for scoring behavioral videos can be cost prohibitive and often have difficulty distinguishing between active investigation of an object and simply being in close proximity to an object. As such, many experimenters continue to rely on hand scoring interactions using stopwatches, which makes it difficult to review scoring after-the-fact and results in the loss of temporal information. Here, we introduce Chronotate, a free, open-source tool to aid in manually scoring novel object behavior videos. The software consists of an interactive video player with keyboard integration for marking timestamps of behavioral events during video playback, making it simple to quickly score and review bouts of rodent-object interaction. In addition, Chronotate outputs detailed interaction bout data, allowing for nuanced behavioral performance analyses. Using this detailed temporal information, we demonstrate that novel object location performance peaks within the first 3 s of interaction time and preference for the novel location becomes reduced across the test session. Thus, Chronotate can be used to determine the temporal structure of interactions on this task and can provide new insight into the memory processes that drive this behavior. Chronotate is available for download at: https://github.com/ShumanLab/Chronotate.

Progressive excitability changes in the medial entorhinal cortex in the 3xTg mouse model of Alzheimer's disease pathology.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterized by memory loss and progressive cognitive impairments. In mouse models of AD pathology, studies have found neuronal and synaptic deficits in the hippocampus, but less is known about what happens in the medial entorhinal cortex (MEC), which is the primary spatial input to the hippocampus and an early site of AD pathology. Here, we measured the neuronal intrinsic excitability and synaptic activity in MEC layer II (MECII) stellate cells, MECII pyramidal cells, and MEC layer III (MECIII) excitatory neurons at early (3 months) and late (10 months) time points in the 3xTg mouse model of AD pathology. At 3 months of age, prior to the onset of memory impairments, we found early hyperexcitability in MECII stellate and pyramidal cells' intrinsic properties, but this was balanced by a relative reduction in synaptic excitation (E) compared to inhibition (I), suggesting intact homeostatic mechanisms regulating activity in MECII. Conversely, MECIII neurons had reduced intrinsic excitability at this early time point with no change in the synaptic E/I ratio. By 10 months of age, after the onset of memory deficits, neuronal excitability of MECII pyramidal cells and MECIII excitatory neurons was largely normalized in 3xTg mice. However, MECII stellate cells remained hyperexcitable and this was further exacerbated by an increased synaptic E/I ratio. This observed combination of increased intrinsically and synaptically generated excitability suggests a breakdown in homeostatic mechanisms specifically in MECII stellate cells at this post-symptomatic time point. Together, these data suggest that the breakdown in homeostatic excitability mechanisms in MECII stellate cells may contribute to the emergence of memory deficits in AD.

Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters.

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

Lycopene-rich diets modulate HDL functionality and associated inflammatory markers without affecting lipoprotein size and distribution in moderately overweight, disease-free, middle-aged adults: A randomized controlled trial.

Background: The consumption of lycopene-rich foods may lower cardiovascular disease (CVD) risk. Lycopene circulates in the blood bound to lipoproteins, including high-density lipoproteins (HDLs). Preliminary data from our group showed that increased consumption of tomato-based food or lycopene supplement in middle-aged subjects led to functional changes to HDL's sub-fractions, HDL2 and HDL3. These changes were also associated with a decrease in serum amyloid A (SAA), potentially enhancing their anti-atherogenic properties. Objective: We carried out a comprehensive randomized controlled intervention trial with healthy middle-aged volunteers to assess whether the consumption of tomato-based foods or lycopene supplements affects HDL functionality and associated inflammatory markers, and lipoprotein subfractions size and distribution. Design: Volunteers (225, aged 40-65 years) were randomly assigned to one of three dietary intervention groups and asked to consume a control diet (low in tomato-based foods, <10 mg lycopene/week), a lycopene-rich diet (224-350 mg lycopene/week), or the control diet with a lycopene supplement (70 mg lycopene/week). HDL2 and HDL3 were isolated by ultracentrifugation. Compliance was monitored by assessing lycopene concentration in serum. Systemic and HDL-associated inflammation was assessed by measuring SAA concentrations. HDL functionality was determined by monitoring paraoxonase-1 (PON-1), cholesteryl ester transfer protein (CETP), and lecithin cholesterol acyltransferase (LCAT) activities. The lipoprotein subfractions profile was assessed by NMR. Results: Lycopene in serum and HDL significantly increased following consumption of both the high tomato diet and lycopene supplement (p ≤ 0.001 for both). Lycopene, either as a tomato-rich food or a supplement, enhanced both serum- and HDL3-PON-1 activities (p ≤ 0.001 and p = 0.036, respectively), while significantly reducing HDL3-SAA-related inflammation (p = 0.001). Lycopene supplement also significantly increased HDL3-LCAT activity (p = 0.05), and reduced the activity of both HDL2- and HDL3-CETP (p = 0.005 and p = 0.002, respectively). These changes were not associated with changes in the subclasses distribution for all lipoprotein fractions or the size of lipoprotein subclasses. Conclusion: Our results showed that dietary lycopene can significantly enhance HDL functionality, without associated changes in particle size and distribution, by modulating the activity of HDL-associated enzymes. Concomitantly, dietary lycopene significantly decreased serum- and HDL3-associated SAA, confirming that SAA may represent a sensitive inflammatory biomarker to dietary change. Clinical Trial Register: (https://www.isrctn.com), ISRCTN34203810.

Propranolol Decreases Fear Expression by Modulating Fear Memory Traces.

BACKGROUND: Posttraumatic stress disorder can develop after a traumatic event and results in heightened, inappropriate fear and anxiety. Although approximately 8% of the U.S. population is affected by posttraumatic stress disorder, only two drugs have been approved by the Food and Drug Administration to treat it, both with limited efficacy. Propranolol, a nonselective ß-adrenergic antagonist, has shown efficacy in decreasing exaggerated fear, and there has been renewed interest in using it to treat fear disorders. METHODS: Here, we sought to determine the mechanisms by which propranolol attenuates fear by utilizing an activity-dependent tagging system, ArcCreERT2 x eYFP mice. 129S6/SvEv mice were administered a 4-shock contextual fear conditioning paradigm followed by immediate or delayed context reexposures. Saline or propranolol was administered either before or after the first context reexposure. To quantify hippocampal, prefrontal, and amygdalar memory traces, ArcCreERT2 x eYFP mice were administered a delayed context reexposure with either a saline or propranolol injection before context reexposure. RESULTS: Propranolol decreased fear expression only when administered before a delayed context reexposure. Fear memory traces were affected in the dorsal dentate gyrus and basolateral amygdala after propranolol administration in the ArcCreERT2 x eYFP mice. Propranolol acutely altered functional connectivity between the hippocampal, cortical, and amygdalar regions. CONCLUSIONS: These data indicate that propranolol may decrease fear expression by altering network-correlated activity and by weakening the reactivation of the initial traumatic memory trace. This work contributes to the understanding of noradrenergic drugs as therapeutic aids for patients with posttraumatic stress disorder.

Evaluation of RPE-Select: A Web-Based Respiratory Protective Equipment Selector Tool.

This article describes the evaluation of an open-access web-based respiratory protective equipment selector tool (RPE-Select, accessible at http://www.healthyworkinglives.com/rpe-selector). This tool is based on the principles of the COSHH-Essentials (C-E) control banding (CB) tool, which was developed for the exposure risk management of hazardous chemicals in the workplace by small and medium sized enterprises (SMEs) and general practice H&S professionals. RPE-Select can be used for identifying adequate and suitable RPE for dusts, fibres, mist (solvent, water, and oil based), sprays, volatile solids, fumes, gases, vapours, and actual or potential oxygen deficiency. It can be applied for substances and products with safety data sheets as well as for a large number of commonly encountered process-generated substances (PGS), such as poultry house dusts or welding fume. Potential international usability has been built-in by using the Hazard Statements developed for the Globally Harmonised System (GHS) and providing recommended RPE in picture form as well as with a written specification. Illustration helps to compensate for the variabilities in assigned protection factors across the world. RPE-Select uses easily understandable descriptions/explanations and an interactive stepwise flow for providing input/answers at each step. The output of the selection process is a report summarising the user input data and a selection of RPE, including types of filters where applicable, from which the user can select the appropriate one for each wearer. In addition, each report includes 'Dos' and 'Don'ts' for the recommended RPE. RPE-Select outcomes, based on up to 20 hypothetical use scenarios, were evaluated in comparison with other available RPE selection processes and tools, and by 32 independent users with a broad range of familiarities with industrial use scenarios in general and respiratory protection in particular. For scenarios involving substances having safety data sheets, 87% of RPE-Select outcomes resulted in a 'safe' RPE selection, while 98% 'safe' outcomes were achieved for scenarios involving process-generated substances. Reasons for the outliers were examined. User comments and opinions on the mechanics and usability of RPE-Select are also presented.