Radiographers and COVID-19 pneumonia: Diagnostic performance using CO-RADS.

INTRODUCTION: A more structured role of radiographers is advisable to speed up the management of patients with suspected COVID-19. The purpose of our study was to evaluate the diagnostic performance of radiographers in the detection of COVID-19 pneumonia on chest CT using CO-RADS descriptors. METHODS: CT images of patients who underwent RT-PCR and chest CT due to COVID-19 suspicion between March and July 2020 were analysed retrospectively. Six readers, including two radiologists, two highly experienced radiographers and two less experienced radiographers, independently scored each CT using the CO-RADS lexicon. ROC curves were used to investigate diagnostic accuracy, and Fleiss'κ statistics to evaluate inter-rater agreement. RESULTS: 714 patients (419 men; 295 women; mean age: 64 years ±19SD) were evaluated. CO-RADS> 3 was identified as optimal diagnostic threshold. Highly experienced radiographers achieved an average sensitivity of 58.7% (95%CI: 52.5-64.7), an average specificity of 81.8% (95%CI: 77.9-85.2), and a mean AUC of 0.72 (95%CI: 0.68-0.75). Among less experienced radiographers, an average sensitivity of 56.3% (95%CI: 50.1-62.2) and an average specificity of 81.5% (95%CI: 77.6-84.9) were observed, with a mean AUC of 0.71 (95%CI: 0.68-0.74). Consultant radiologists achieved an average sensitivity of 60.0% (95%CI: 53.7-65.8), an average specificity of 81.7% (95%CI: 77.8-85.1), and a mean AUC of 0.73 (95%CI: 0.70-0.77). CONCLUSION: Radiographers can adequately recognise the classic appearances of COVID-19 on CT, as described by the CO-RADS assessment scheme, in a way comparable to expert radiologists. IMPLICATIONS FOR PRACTICE: Radiographers, as the first healthcare professionals to evaluate CT images in patients with suspected SARS-CoV-2 infection, could diagnose COVID-19 pneumonia by means of a categorical reporting scheme at CT in a reliable way, hence playing a primary role in the early management of these patients.

Activity-dependent decrease in NMDA receptor responses during development of the visual cortex.

Plasticity of the developing visual system has been regarded as the best model for changes of neuronal connections under the influence of the environment. N-methyl-D-aspartate (NMDA) receptors are crucial for experience-dependent synaptic modifications that occur in the developing visual cortex. NMDA-mediated excitatory postsynaptic currents (EPSCs) in layer IV neurons of the visual cortex lasted longer in young rats than in adult rats, and the duration of the EPSCs became progressively shorter, in parallel with the developmental reduction in synaptic plasticity. This decrease in NMDA receptor-mediated EPSC duration is delayed when the animals are reared in the dark, a condition that prolongs developmental plasticity, and is prevented by treatment with tetrodotoxin, a procedure that inhibits neural activity. Application of L-glutamate to outside-out patches excised from layer IV neurons of young, but not of adult, rats activated prolonged bursts of NMDA channel openings. A modification of the NMDA receptor gating properties may therefore account for the age-dependent decline of visual cortical plasticity.

Social Justice and the Promotion of the Common Good in Medical Missions to Low-Resourced Countries.

The article highlights the importance of critically examining medical missions to low-resourced countries in light of a bioethical focus on social justice and the promotion of the common good.

Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons.

In the hilar region of the rat hippocampus, large spontaneous excitatory postsynaptic currents (sEPSCs) mediated by non-NMDA glutamate receptors are present in both excitatory spiny mossy cells and inhibitory aspiny hilar interneurons, making these neurons ideal candidates for a comparative study using the tight seal whole-cell recording technique. Although sEPSCs have similar amplitude distributions, the rise and decay times are significantly slower in spiny versus aspiny neurons. Similar kinetic differences are observed in synaptic currents evoked by mossy fiber stimulation. These results demonstrate a physiological difference between the excitatory drive to excitatory and inhibitory neurons in the hilus that certainly contributes to differences in synaptic strength and that may be applicable to other brain regions. Furthermore, since the development or modification of individual spines or groups of spines may affect synaptic strength, these results may be pivotal in establishing a role for spines in modulating synaptic activity.

Functional diversity of GABA-activated Cl- currents in Purkinje versus granule neurons in rat cerebellar slices.

In rat cerebellar slices, we compared whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) with Cl- currents resulting from pulses of GABA (1 mM, < 2 ms) to outside-out patches from Purkinje and granule neurons. sIPSCs in Purkinje cells decayed with a single fast exponential, as previously reported, whereas in granule cells sIPSC decay was best described by the sum of a fast and a slow exponential curve, with a variable contribution of the slow component to the peak current. GABA pulses to nucleated patches from granule cells elicited Cl- currents with decays similar to sIPSC decays, whereas in patches from Purkinje neurons GABA pulses produced Cl- currents decaying largely with a fast component, but often followed by a slower exponential. GABA concentration steps produced rapidly desensitizing currents in patches from both cerebellar neurons. In distinct cerebellar neurons, specific functional properties of GABAA receptors may relate to the presence of distinct receptor subtypes.

Neurosteroids act on recombinant human GABAA receptors.

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Mapping homeostatic synaptic plasticity using cable properties of dendrites.

When chronically silenced, cortical and hippocampal neurons homeostatically upregulate excitatory synaptic function. However, the subcellular position of such changes on the dendritic tree is not clear. We exploited the cable-filtering properties of dendrites to derive a parameter, the dendritic filtering index (DFI), to map the spatial distribution of synaptic currents. Our analysis indicates that young rat cortical neurons globally scale AMPA receptor-mediated currents, while mature hippocampal neurons do not, revealing distinct homeostatic strategies between brain regions and developmental stages. The DFI presents a useful tool for mapping the dendritic origin of synaptic currents and the location of synaptic plasticity changes.

Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons.

In rat cerebellar granule neurons, mRNA and protein levels of the NR2A and NR2C subunits of the NMDA receptor increase during the second postnatal week. At this time, mRNA and protein levels of the NR2B subunit begin to fall. To investigate targeting of NMDA receptor subunits, we performed whole-cell recordings from rat cerebellar granule neurons at different times during development and investigated the pharmacological and biophysical properties of mossy fiber-evoked NMDA EPSCs. Isolated NMDA EPSCs from newly formed synapses in the first postnatal week exhibited partial block by the NR2B subunit-specific antagonist (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101,606). By the end of the second postnatal week, NMDA EPSCs were virtually unaffected by the NR2B antagonist. In parallel, NMDA EPSC decay times decreased over a similar developmental time course. We compared properties of synaptic NMDA receptors with extrasynaptic receptors that are present on the cell body with rapid application of glutamate to excised nucleated patches. Deactivation of patch responses accelerated with development and closely resembled evoked NMDA EPSCs in rats of the same age. However, patch responses were highly sensitive to CP 101,606 through the second postnatal week, and sensitivity was seen in some neurons up to the fourth postnatal week. Spermine potentiated peak NMDA patch responses from postnatal days 10-14 rats but had little effect on evoked NMDA EPSCs. Our data suggest selective targeting of a distinct NMDA receptor subtype to synaptic receptor populations in cerebellar granule neurons. Later in development, similar changes occur in the extrasynaptic receptor population.

Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate.

To obtain insights into the spatiotemporal characteristics and mechanism of Ca(2+)-dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in astrocytes trigger synchronous and repetitive [Ca(2+)](i) elevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A(1). We conclude that [Ca(2+)](i) oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H(+) ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.

GABA(A) receptor alpha1 subunit deletion prevents developmental changes of inhibitory synaptic currents in cerebellar neurons.

Developmental changes in miniature IPSC (mIPSC) kinetics have been demonstrated previously in cerebellar neurons in rodents. We report that these kinetic changes in mice are determined primarily by developmental changes in GABA(A) receptor subunit expression. mIPSCs were studied by whole-cell recordings in cerebellar slices, prepared from postnatal day 11 (P11) and P35 mice. Similar to reports in granule neurons, wild-type cerebellar stellate neuron mIPSCs at P11 had slow decay kinetics, whereas P35 mIPSCs decayed five times faster. When mIPSCs in cerebellar stellate neurons were compared between wild-type (+/+) and GABA(A) receptor alpha1 subunit-deficient (-/-) littermates at P35, we observed dramatically slower mIPSC decay rates in -/- animals. We took advantage of the greater potency of imidazopyridines for GABA current potentiation with alpha1 subunit-containing receptors to characterize the relative contribution of alpha1 subunits in native receptors on inhibitory synapses of cerebellar granule neurons. Zolpidem-induced prolongation of mIPSC decay was variable among distinct cells, but it increased during development in wild-type mice. Similarly, Zolpidem prolongation of mIPSC decay rate was significantly greater in adult +/+ mice than in knock-outs. We propose that an increased alpha1 subunit assembly in postsynaptic receptors of cerebellar inhibitory synapses is responsible for the fast inhibitory synaptic currents that are normally observed during postnatal development.

Interleukin-10 prevents glutamate-mediated cerebellar granule cell death by blocking caspase-3-like activity.

Interleukin-10 (IL-10) has been shown to reduce neuronal degeneration after CNS injury. However, the molecular mechanisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apoptosis upon exposure to toxic concentrations of glutamate (100-500 microm) or NMDA (300 microm). Pretreatment of cerebellar granule cells with IL-10 (1-50 ng/ml) elicited a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Most importantly, IL-10 reduced the number of apoptotic cells when added to the cultures together or 1 hr after glutamate. Using patch-clamping and fluorescence Ca(2+) imaging techniques, we examined whether IL-10 prevents glutamate toxicity by blocking the function of NMDA channel. IL-10 failed to affect NMDA channel properties and to reduce NMDA-mediated rise in intracellular Ca(2+). Thus, this cytokine appears to prevent glutamate toxicity by a mechanism unrelated to a blockade of NMDA receptor function. Various proteases, such as caspase-3, and transcription factors, such as nuclear factor kappaB (NF-kappaB), have been proposed to participate in glutamate-mediated apoptosis. Thus, we examined whether IL-10 modulates the activity of these apoptotic markers. IL-10 blocked both the glutamate-mediated induction of caspase-3 as well as NF-kappaB DNA binding activity, suggesting that the neuroprotective properties of IL-10 may rely on its ability to block the activity of proapoptotic proteins.

New perspectives in the functional role of GABA(A) channel heterogeneity.

Gamma-aminobutyric acid A (GABA(A)) channels responsible for inhibitory synaptic transmission possess a consistent heterogeneity of structure in terms of distinct constitutive subunits. During the past 10 years, considerable progress has been made in understanding the magnitude of this large diversity. Structural requirements for clinically important drugs such as benzodiazepines and barbiturates have been elucidated, and the anatomical distribution in distinct neuronal populations and the developmental profiles of individual subunits have been elucidated with various techniques. However, the relevance of subunit heterogeneity to synaptic transmission is still largely lacking. Recently, substantial progress has been achieved in understanding the crucial role of desensitization as a molecular determinant in defining the duration and frequency responses of inhibitory synaptic transmission. This development, together with a combination of different experimental approaches, including patch-clamp recordings and ultrafast agonist applications in brain slices and mammalian cells expressing recombinant GABA(A) receptor, has begun to shed light on a possible role for subunit composition of synaptic receptors in shaping the physiological characteristics of synaptic transmission. Nowhere else in the central nervous system is the anatomical and developmental profile of GABA receptor heterogeneity as well understood as it is in the cerebellum. This review summarizes advances in the understanding of functional correlates to subunit heterogeneity in the cerebellum relevant for inhibitory synaptic function.

Pharmacologic significance of the structural heterogeneity of the GABAA receptor-chloride ion channel complex.

Gamma-Aminobutyric acidA (GABAA) receptors are heterooligomeric proteins with an apparent high degree of variability in the specific assembly of their component subunits. Although the precise nucleotide and deduced amino acid sequences of many of the various GABAA receptor subunits are known, the exact quaternary structures of the native receptors are unknown. Recombinant expression of receptors with different combinations of subunits produces a variety of structurally different receptors with varying Cl- channel function and sensitivities to modulation by drugs such as benzodiazepines. Differences in the regional distribution of GABAA receptor subtypes in brain, coupled with the observed differences in the relative affinities of various anxiolytic and hypnotic drugs among these receptor subtypes, suggests a new strategy for drug development that is the targeting of drugs to specific subpopulations of GABAA receptors. This is a review of the recent striking progress in understanding the heterogeneity of the GABAA receptors and its possible significance.

NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons.

The peptide transmitter N-acetylaspartylglutamate (NAAG) selectively activates the group II metabotropic glutamate receptors. Several reports also suggest that this peptide acts as a partial agonist at N-methyl-D-aspartate (NMDA) receptors but its putative antagonist effects have not been directly tested. To do this, we used whole cell recordings from cerebellar granule cells (CGC) in culture that allow the highest possible resolution of NMDA channel activation. When CGC were activated with equimolar concentrations of NMDA and NAAG, the peptide failed to alter the peak current elicited by NMDA. Very high concentrations of NAAG (100-200 microM) did not significantly reduce the current elicited by 10 microM NMDA or 0.1 microM glutamate, while 400 microM NAAG produced only a very small (less than 15%) reduction in these whole cell currents. Similarly, NAAG (400 microM) failed to significantly alter the average decay time constant or the peak amplitude of NMDA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs). We conclude that high concentrations of the peptide do not exert physiologically relevant antagonist actions on synaptic NMDA receptor activation following vesicular release of glutamate. As an agonist, purified NAAG was found to be at least 10,000-fold less potent than glutamate in increasing "background" current via NMDA receptors on CGC. Inasmuch as it is difficult to confirm that NAAG preparations are completely free from contamination with glutamate at the 0.01% level, the peptide itself appears unlikely to have a direct agonist activity at the NMDA receptor subtypes found in CGC. Recent reports indicate that enhancing the activity of endogenous NAAG may be an important therapeutic approach to excitotoxicity and chronic pain perception. These effects are likely mediated by group II mGluRs, not NMDA receptors.

Kynurenic acid inhibits the activation of kainic and N-methyl-D-aspartic acid-sensitive ionotropic receptors by a different mechanism.

The action of kynurenic acid on currents elicited by the activation of amino acid receptors was investigated in primary cultures of cortical neurons prepared from neonatal rats. Kynurenic acid was tested on currents elicited by both N-methyl-D-aspartic acid (NMDA) and kainate, using patch-clamp recording techniques in "outside-out" and "whole-cell" configurations. The inhibition by kynurenic acid was compared with that elicited by amino-phosphono-valeric acid (APV). Whole-cell currents, elicited by increasing doses of NMDA, were antagonized competitively by APV and non-competitively by kynurenic acid (ID50 70 microM); in contrast, kynurenic acid inhibited competitively the whole-cell currents elicited by kainic acid (ID50 500 microM). The non-competitive inhibition by kynurenic acid of the whole cell currents elicited by NMDA was antagonized competitively by glycine, a specific positive allosteric modulator of NMDA receptors; on the other hand glycine failed to change the inhibition by APV of the NMDA-elicited responses. Thus, kynurenic acid inhibits NMDA receptors allosterically (non-competitively) and kainic acid receptors isosterically (competitively).

A pyridazinyl derivative of gamma-aminobutyric acid (GABA), SR 95531, is a potent antagonist of Cl- channel opening regulated by GABAA receptors.

The antagonistic effects of a pyridazinyl derivative of GABA (SR 95531) were investigated on GABA-induced Cl- currents in neonatal rat cortical neurons in primary culture. Three different methods were used: direct application of GABA onto the cell, induction of inhibitory postsynaptic currents (IPSCs) and membrane patch-clamp recordings. Using the first technique, SR 95531 appeared to be more potent than bicuculline methiodide while both drugs seemed equally potent in reducing the IPSC amplitudes and the opening rate of Cl- channels regulated by GABAA receptors in patch-clamp membranes.

Distinct effect of pregnenolone sulfate on NMDA receptor subtypes.

Using rapid agonist applications to transfected HEK-293 cells, we investigated pregnenolone sulfate (PS) effects on deactivation and desensitization of recombinant NMDA receptors subtypes. PS prolonged the deactivation of responses produced by brief applications of L-glutamate with all subunit combinations tested. The action of PS was larger on NR1a/NR2A than on NR1a/NR2B channels. PS slowed the rate of macroscopic desensitization of the responses with all subunit combinations tested. In contrast, PS had little effect on current rise time and had much reduced action on responses with L-cysteate, a low affinity agonist. Our results suggest that PS decreases agonist unbinding. However, this action is counteracted by decreased desensitization. Since desensitization produces slow deactivating components, particularly with NR1a/NR2B receptors, this underlies the decreased PS effect with these subtypes. Indeed PS action was mainly observed on the fast component of deactivation. Furthermore, prolongation of NR1a/NR2A responses was similar to that of responses from NR1b/NR2B receptor, a subtype characterized by reduced desensitization. PS prolongation of evoked NMDA receptor mediated synaptic currents from cortical neuronal primary culture(s) was not significantly different from that of responses with NR1a/NR2B receptors indicating that native receptors in these neurons comprised at least some heteromeric combinations of these two subunits.

Pharmacological modulation of GABAergic transmission in cultured cerebellar neurons.

GABA activates specific ion channels in post-natal cerebellar neurons in primary culture generating Cl- currents that can be recorded with the whole-cell patch-clamp technique. Evoked and spontaneous GABA-mediated synaptic activity can be recorded from cells kept in culture for a few days. Benzodiazepine and beta-carboline derivatives which bind with high affinity to the domains for allosteric regulation of GABA receptors facilitated and inhibited directly applied GABA responses and synaptically evoked Cl- currents recorded under voltage-clamp.

Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid is highly enriched in neuronal membranes, and several studies suggest that DHA is critical for neuronal development. We have investigated the effects of exogenously applied DHA on voltage-gated K+ channels using patch-clamp techniques. DHA produced a concentration-dependent inhibition of the sustained outward current in isolated neocortical neurons. This blocking action was examined in more detail with two cloned neuronal K+ channels (Kv1.2 and Kv3.1a) expressed in mammalian fibroblasts. DHA produced a potent inhibition of depolarization-activated K+ currents from cells expressing these channels (Kd values, 1.8 +/- 0.1 muM and 690 +/- 60 nM, for Kv1.2 and Kv3.1a, respectively, at +40 mV). The DHA block of both channel types was rapidly reversed (approximately 2 sec) by bovine serum albumin, which binds the fatty acid. Micromolar concentrations of extracellular Zn2+ non-competitively antagonized DHA inhibition of Kv1.2 channels, whereas there was little effect on DHA block of Kv3.1a channels. Experiments with membrane patches from Kv1.2 transfected cells demonstrated that the DHA block occurred from the outside, suggesting that the fatty acid interacts directly with an external domain of the ion channel. DHA may serve as a local messenger molecule that selectively modulates the activity of certain voltage-gated K+ channels in a Zn2(+)-dependent fashion.

Anandamide, an endogenous cannabinoid, inhibits Shaker-related voltage-gated K+ channels.

Anandamide has been identified in porcine brain as an endogenous cannabinoid receptor ligand and is believed to be a counterpart to the psychoactive component of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC). Here we report that anandamide directly inhibits (IC50, 2.7 muM) Shaker-related Kv1.2 K+ channels that are found ubiquitously in the mammalian brain. Delta 9-THC also inhibited Kv1.2 channels with comparable potency (IC50, 2.4 muM), as did several N-acyl-ethanolamides with cannabinoid receptor binding activity. Potassium current inhibition occurred through a pertussis toxin-insensitive mechanism and was not prevented by the cannabinoid receptor antagonist SR141716A. Utilizing excised patches of Kv1.2 channel-rich membrane as a rapid and sensitive bioassay, we found that phospholipase D stimulated the release of an endogenous anandamide-like K+ channel blocker from rat brain slices. Structure-activity studies were consistent with the possibility that the released blocker was either anandamide or another N-acyl-ethanolamide.