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A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Microglial cells (resident macrophages) feature rapid activation in CNS disease and can acquire multiple phenotypes exerting neuroprotection or neurotoxicity. The functional impact of surveying ("resting") microglia on neural excitability and neurotransmission in physiology is widely unknown, however. We addressed this issue in male rat hippocampal slice cultures (in situ) by pharmacological microglial ablation within days and by characterizing neuronal gamma-band oscillations (30-70 Hz) that are highly sensitive to neuromodulators and disturbances in ion and energy regulation. Gamma oscillations support action potential timing and synaptic plasticity, associate with higher brain functions like perception and memory, and require precise communication between excitatory pyramidal cells and inhibitory (GABAergic) interneurons. The slice cultures featured well-preserved hippocampal cytoarchitecture and parvalbumin-positive interneuron networks, microglia with ramified morphology, and low basal levels of IL-6, TNF-α, and nitric oxide (NO). Stimulation of slice cultures with the pro-inflammatory cytokine IFN-γ or bacterial LPS serving as positive controls for microglial reactivity induced MHC-II expression and increased cytokine and NO release. Chronic exposure of slice cultures to liposome-encapsulated clodronate reduced the microglial cell population by about 96%, whereas neuronal structures, astrocyte GFAP expression, and basal levels of cytokines and NO were unchanged. Notably, the properties of gamma oscillations reflecting frequency, number and synchronization of synapse activity were regular after microglial depletion. Also, electrical stimulus-induced transients of the extracellular potassium concentration ([K+ ]o ) reflecting cellular K+ efflux, clearance and buffering were unchanged. This suggests that nonreactive microglia are dispensable for neuronal homeostasis and neuromodulation underlying network signaling and rhythm generation in cortical tissue.

Type II IFN (IFN-γ) is a proinflammatory T lymphocyte cytokine that serves in priming of microglia—resident CNS macrophages—during the complex microglial activation process under pathological conditions. Priming generally permits an exaggerated microglial response to a secondary inflammatory stimulus. The impact of primed microglia on physiological neuronal function in intact cortical tissue (in situ) is widely unknown, however. We explored the effects of chronic IFN-γ exposure on microglia in hippocampal slice cultures, i.e., postnatal parenchyma lacking leukocyte infiltration (adaptive immunity). We focused on fast neuronal network waves in the gamma-band (30–70 Hz). Such gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and are exquisitely sensitive to metabolic and oxidative stress. IFN-γ induced substantial morphological changes and cell population expansion in microglia as well as moderate up-regulation of activation markers, MHC-II, CD86, IL-6, and inducible nitric oxide synthase (iNOS), but not TNF-α. Cytoarchitecture and morphology of pyramidal neurons and parvalbumin-positive inhibitory interneurons were well-preserved. Notably, gamma oscillations showed a specific decline in frequency of up to 8 Hz, which was not mimicked by IFN-α or IL-17 exposure. The rhythm disturbance was caused by moderate microglial nitric oxide (NO) release demonstrated by pharmacological microglia depletion and iNOS inhibition. In conclusion, IFN-γ priming induces substantial proliferation and moderate activation of microglia that is capable of slowing neural information processing. This mechanism might contribute to cognitive impairment in chronic brain disease featuring elevated IFN-γ levels, blood–brain barrier leakage, and/or T cell infiltration, well before neurodegeneration occurs.

Background: Carbapenem-resistant gram-negative bacteria are an urgent threat to healthcare safety around the world. In Vietnam, Although surveillance and control of multidrug-resistant organisms is a national priority, information on the burden of these resistant pathogens is still scarce. At University Medical Center Ho Chi Minh City, Vietnam, we aimed to better understand carbapenem-resistance through 2 phases: (1) assess proportion of carbapenem-resistant gram-negative organisms that are carbapanemase-producing (CP-CRO) and (2) assess transmission burden of carbapenemase-producing carbapenem-resistant Enterobacterieacea (CP-CRE) in the general intensive care unit (ICU). Methods: In the first phase, all gram-negative clinical isolates collected between November 2018 and April 2019 were tested for carbapenem-resistance using the disc-diffusion method and were defined as meropenem resistant using the Clinical and Laboratory Standards Institute 2018 break point (M100-Performance Standards for Antimicrobial Susceptibility Testing, 28th Edition). Carbapenem-resistant bacteria were tested for phenotypic carbapenemase-production using the Becton Dickinson Phoenix CPO Detect assay. In the second phase, we instituted CP-CRE rectal screening using CHROMagar mSuperCARBA media for all ICU patients from July through September 2019. Patients were screened on admission, and negative patients were rescreened every 2 days until discharge, death, or CRE-positive screening or culture. Admission prevalence and incidence of CP-CRE transmission was calculated among CP-CRE infected or colonized patients. Results: From November 2018 through April 2019, 599 gram-negative clinical isolates from 543 patient samples were identified. Of these, 108 were carbapenem-resistant; 107 (99%) of carbapenem-resistant isolates were carbapenemase-producing by phenotypic method. Most CP-CRO were Acinetobacter baumannii (45 of 107, 42%) or Klebsiella pneumoniae (39 of 107, 36%). During ICU CP-CRE colonization screening, the July positivity rate on admission was 40% (32 of 81), the August positivity rate on admission was 30% (21 of 71), and the September positivity rate on admission was 40% (30 of 75). Of those with negative admission screen, the proportion of new CP-CRE colonization in July was 45% (22 of 49), the proportion of new CP-CRE colonization in August was 64% (32 of 50), and the proportion of new CP-CRE colonization in September was 44% (20 of 45). Across all 3 months of screening, the proportions of CP-CRE that were Klebsiella, Citrobacter, or Enterobacter were 68% (118 of 174) and the proportion of CP-CRE that were Eschericia coli was 37% (56 of 174). The average number of days to turn from negative to positive screening result was 4.1. Conclusions: Our analysis demonstrates that nearly all carbapenem-resistant organisms at our hospital are carbapenemase producing. In the ICU, we identified a high burden of CP-CRE, attributable to high presence on admission and new acquisition in the ICU. An intervention package based on CDC-recommended enhanced infection control measures is being implemented to decrease CP-CRE transmission in the ICU.Funding: NoneDisclosures: None

Gamma oscillations (30–100 Hz) reflect a fast brain rhythm that provides a fundamental mechanism of complex neuronal information processing in the hippocampus and in the neocortex in vivo. Gamma oscillations have been implicated in higher brain functions, such as sensory perception, motor activity, and memory formation. Experimental studies on synaptic transmission and bioenergetics underlying gamma oscillations have primarily used acute slices of the hippocampus. This study tests whether organotypic hippocampal slice cultures of the rat provide an alternative model for cortical gamma oscillations in vitro. Our findings are that 1) slice cultures feature well-preserved laminated architecture and neuronal morphology; 2) slice cultures of different maturation stages (7–28 days in vitro) reliably express gamma oscillations at about 40 Hz as induced by cholinergic (acetylcholine) or glutamatergic (kainate) receptor agonists; 3) the peak frequency of gamma oscillations depends on the temperature, with an increase of ∼3.5 Hz per degree Celsius for the range of 28–36°C; 4) most slice cultures show persistent gamma oscillations for ∼1 hr during electrophysiological local field potential recordings, and later alterations may occur; and 5) in slice cultures, glucose at a concentration of 5 mM in the recording solution is sufficient to power gamma oscillations, and additional energy substrate supply with monocarboxylate metabolite lactate (2 mM) exclusively increases the peak frequency by ∼4 Hz. This study shows that organotypic hippocampal slice cultures provide a reliable model to study agonist-induced gamma oscillations at glucose levels near the physiological range. © 2015 Wiley Periodicals, Inc.

Fast neuronal network oscillations in the gamma-frequency band (30-100 Hz) provide a fundamental mechanism of complex neuronal information processing in the hippocampus and neocortex of mammals. Gamma oscillations have been implicated in higher brain functions such as sensory perception, motor activity and memory formation. The oscillations emerge from precise synapse interactions between excitatory principal neurons such as pyramidal cells and inhibitory GABAergic interneurons, and they are associated with high energy expenditure. However, both energy substrates and metabolic pathways that are capable to power cortical gamma oscillations have been less defined. Here, we investigated the energy sources fueling persistent gamma oscillations in the CA3 subfield of organotypic hippocampal slice cultures of the rat. This preparation permits superior oxygen supply as well as fast application of glucose, glycolytic metabolites or drugs such as glycogen phosphorylase inhibitor during extracellular recordings of the local field potential. Our findings are: (i) gamma oscillations persist in the presence of glucose (10 mmol/L) for greater than 60 minutes in slice cultures while (ii) lowering glucose levels (2.5 mmol/L) significantly reduces the amplitude of the oscillation. (iii) Gamma oscillations are absent at low concentration of lactate (2 mmol/L). (iv) Gamma oscillations persist at high concentration (20 mmol/L) of either lactate or pyruvate, albeit showing significant reductions in the amplitude. (v) The breakdown of glycogen significantly delays the decay of gamma oscillations during glucose deprivation. However, when glucose is present, the turnover of glycogen is not essential to sustain gamma oscillations. Our study shows that fast neuronal network oscillations can be fueled by different energy-rich substrates, with glucose being most effective.

Gamma oscillations (30-100 Hz) reflect a fast brain rhythm that provides a fundamental mechanism of complex neuronal information processing in the hippocampus and in the neocortex in vivo. Gamma oscillations have been implicated in higher brain functions, such as sensory perception, motor activity, and memory formation. Experimental studies on synaptic transmission and bioenergetics underlying gamma oscillations have primarily used acute slices of the hippocampus. This study tests whether organotypic hippocampal slice cultures of the rat provide an alternative model for cortical gamma oscillations in vitro. Our findings are that 1) slice cultures feature well-preserved laminated architecture and neuronal morphology; 2) slice cultures of different maturation stages (7-28 days in vitro) reliably express gamma oscillations at about 40 Hz as induced by cholinergic (acetylcholine) or glutamatergic (kainate) receptor agonists; 3) the peak frequency of gamma oscillations depends on the temperature, with an increase of ∼ 3.5 Hz per degree Celsius for the range of 28-36 °C; 4) most slice cultures show persistent gamma oscillations for ∼ 1 hr during electrophysiological local field potential recordings, and later alterations may occur; and 5) in slice cultures, glucose at a concentration of 5 mM in the recording solution is sufficient to power gamma oscillations, and additional energy substrate supply with monocarboxylate metabolite lactate (2 mM) exclusively increases the peak frequency by ∼ 4 Hz. This study shows that organotypic hippocampal slice cultures provide a reliable model to study agonist-induced gamma oscillations at glucose levels near the physiological range.