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Cholinergic stimulation of the hippocampal formation results in excitation and/or seizure. We report here, using whole-cell patch-clamp techniques in the hippocampal slice (34 -35°C), a cholinergic-dependent slow afterdepolarization (sADP) and long-lasting plateau potential (PP). In the presence of 20 M carbachol, action potential firing evoked by weak intracellular current injection elicited an sADP that lasted several seconds. Increased spike firing evoked by stronger depolarizing stimuli resulted in long-duration PPs maintained close to Ϫ20 mV. Removal of either Na ϩ or Ca 2ϩ from the external media, intracellular Ca 2ϩ ([Ca 2ϩ ] i ) chelation with 10 mM bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetra-acetic acid, or the addition of 100 M Cd 2ϩ to the perfusate abolished both the sADP and PP. The sADP was depressed and the PP was abolished by either 10 M nimodipine or 1 M -conotoxin, whereas 1.2 M tetrodotoxin was ineffective. The involvement of a Na ϩ /Ca 2ϩ exchanger was minimal because both the sADP and PP persisted after equimolar substitution of 50 mM Li ϩ for Na ϩ in the external media or reduction of the bath temperature to 25°C. Finally, in the absence of carbachol the sADP and PP could not be evoked when K ϩ channels were suppressed, suggesting that depression of K ϩ conductances alone was not sufficient to unmask the conductance. Based on these data, we propose that a Ca 2ϩ -activated nonselective cation conductance was directly enhanced by muscarinic stimulation. The sADP, therefore, represents activation of this conductance by residual [Ca 2ϩ ] i , whereas the PP represents a novel regenerative event involving the interplay between high-voltage-activated Ca 2ϩ channels and the Ca 2ϩ -activated nonselective cation conductance. This latter mechanism may contribute significantly to ictal depolarizations observed during cholinergic-induced seizures.

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The properties of GABA receptor-mediated responses were examined in noncultured astrocytes, acutely isolated from the mature rat hippocampus. Whole-cell patch clamping revealed a GABA-activated Cl- conductance that was mimicked by the GABAA receptor agonist muscimol and depressed by the GABAA antagonists bicuculline and picrotoxin. The GABAA-activated currents were potentiated by the barbiturate pentobarbital and the benzodiazepine diazepam. The benzodiazepine inverse agonist DMCM either enhanced or depressed the astrocytic GABAA-mediated responses, suggesting receptor heterogeneity with respect to pharmacologic profiles. In addition, GABA evoked an increase in [Ca2+]n measured by indo-1 fluorometry, which was depressed in the presence of verapamil or picrotoxin. A GABAA-induced depolarization, therefore, causes Ca2+ influx through voltage-gated Ca2+ channels. The expression and subcellular localization of GABAA receptors and its subunits were examined using immunohistochemical and fluorescent benzodiazepine binding techniques. Polyclonal antisera raised against the GABAA/benzodiazepine receptor, which recognizes multiple subunit isoforms, labeled receptors on the astrocytic cell body and most large processes. In contrast, antisera generated against either alpha 1 or beta 1 subunit peptides revealed immunoreactivity predominantly on a subset of processes. To determine the subcellular distribution of membrane-bound receptors, a fluorescent benzodiazepine derivative was superfused over live astrocytes and visualized with laser-scanning confocal microscopy. Specific fluorescence was distributed in discrete clusters on the cell soma and a subset of distal processes. Collectively, these data support the view that astrocytes, like neurons, express GABAA receptors and target subunit isoforms to distinct cellular localizations. Astrocytic GABAA receptors may be involved in both [Cl-]o and [pH]o homeostasis, and a GABA-evoked increase in [Ca2+]i could serve as a signal between GABAergic neurons and astrocytes.

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on behalf of the Lawson COVID-19 Study TeamObjectives: Coronavirus disease 2019 is caused by severe acute respiratory syndrome-coronavirus-2 infection to which there is no community immunity. Patients admitted to ICUs have high mortality, with only supportive therapies available. Our aim was to profile plasma inflammatory analytes to help understand the host response to coronavirus disease 2019. Design: Daily blood inflammation profiling with immunoassays. Setting: Tertiary care ICU and academic laboratory. Subjects: All patients admitted to the ICU suspected of being infected with severe acute respiratory syndrome-coronavirus-2, using standardized hospital screening methodologies, had daily blood samples collected until either testing was confirmed negative on ICU day 3 (coronavirus disease 2019 negative), or until ICU day 7 if the patient was positive (coronavirus disease 2019 positive). Interventions: None.Measurements and Main Results: Age-and sex-matched healthy controls and ICU patients that were either coronavirus disease 2019 positive or coronavirus disease 2019 negative were enrolled. Cohorts were well-balanced with the exception that coronavirus disease 2019 positive patients were more likely than coronavirus disease 2019 negative patients to suffer bilateral pneumonia. Mortality rate for coronavirus disease 2019 positive ICU patients was 40%. We measured 57 inflammatory analytes and then analyzed with both conventional statistics and machine learning. Twenty inflammatory analytes were different between coronavirus disease 2019 positive patients and healthy controls (p < 0.01). Compared with coronavirus disease 2019 negative patients, coronavirus disease 2019 positive patients had 17 elevated inflammatory analytes on one or more of their ICU days 1-3 (p < 0.01), with feature classification identifying the top six analytes between cohorts as tumor necrosis factor, granzyme B, heat shock protein 70, interleukin-18, interferon-gammainducible protein 10, and elastase 2. While tumor necrosis factor, granzyme B, heat shock protein 70, and interleukin-18 were elevated for all seven ICU days, interferon-gamma-inducible protein 10 transiently elevated on ICU days 2 and 3 and elastase 2 increased over ICU days 2-7. Inflammation profiling predicted coronavirus disease 2019 status with 98% accuracy, whereas elevated heat shock protein 70 was strongly associated with mortality. Conclusions: While many inflammatory analytes were elevated in coronavirus disease 2019 positive ICU patients, relative to healthy controls, the top six analytes distinguishing coronavirus disease 2019 positive ICU patients from coronavirus disease 2019 negative ICU patients were tumor necrosis factor, granzyme B, heat shock protein 70, interleukin-18, interferon-gamma-inducible protein 10, and elastase 2.

The authors analyzed blood metabolites in nine children with epilepsy prior to starting the ketogenic diet (KD) and 3 to 4 weeks after KD therapy. Elevated beta-hydroxybutyrate and cortisol levels were observed in all children on the KD. Free fatty acids increased 2.2-fold on the KD, with significant elevations in most polyunsaturated fatty acids (PUFA; arachidonate increased 1.6- to 2.9-fold and docosahexaenoate increased 1.5- to 4.0-fold). The rise in total serum arachidonate correlated with improved seizure control. Elevated PUFA may represent a key anticonvulsant mechanism of the KD.

Objectives: Coronavirus disease 2019 continues to spread rapidly with high mortality. We performed metabolomics profiling of critically ill coronavirus disease 2019 patients to understand better the underlying pathologic processes and pathways, and to identify potential diagnostic/prognostic biomarkers. Design: Blood was collected at predetermined ICU days to measure the plasma concentrations of 162 metabolites using both direct injection-liquid chromatography-tandem mass spectrometry and proton nuclear magnetic resonance. Setting: Tertiary-care ICU and academic laboratory. Subjects: Patients admitted to the ICU suspected of being infected with severe acute respiratory syndrome coronavirus 2, using standardized hospital screening methodologies, had blood samples collected until either testing was confirmed negative on ICU day 3 (coronavirus disease 2019 negative) or until ICU day 10 if the patient tested positive (coronavirus disease 2019 positive). Interventions: None. Measurements and Main Results: Age- and sex-matched healthy controls and ICU patients that were either coronavirus disease 2019 positive or coronavirus disease 2019 negative were enrolled. Cohorts were well balanced with the exception that coronavirus disease 2019 positive patients suffered bilateral pneumonia more frequently than coronavirus disease 2019 negative patients. Mortality rate for coronavirus disease 2019 positive ICU patients was 40%. Feature selection identified the top-performing metabolites for identifying coronavirus disease 2019 positive patients from healthy control subjects and was dominated by increased kynurenine and decreased arginine, sarcosine, and lysophosphatidylcholines. Arginine/kynurenine ratio alone provided 100% classification accuracy between coronavirus disease 2019 positive patients and healthy control subjects ( p = 0.0002). When comparing the metabolomes between coronavirus disease 2019 positive and coronavirus disease 2019 negative patients, kynurenine was the dominant metabolite and the arginine/kynurenine ratio provided 98% classification accuracy ( p = 0.005). Feature selection identified creatinine as the top metabolite for predicting coronavirus disease 2019-associated mortality on both ICU days 1 and 3, and both creatinine and creatinine/arginine ratio accurately predicted coronavirus disease 2019-associated death with 100% accuracy ( p = 0.01). Conclusions: Metabolomics profiling with feature classification easily distinguished both healthy control subjects and coronavirus disease 2019 negative patients from coronavirus disease 2019 positive patients. Arginine/kynurenine ratio accurately identified coronavirus disease 2019 status, whereas creatinine/arginine ratio accurately predicted coronavirus disease 2019-associated death. Administrat...