From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells

Genetic diversity and the emergence of ethnic groups in Central Asia

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Phenotypic plasticity and water flux rates of Citrus root orders under salinity

Knowledge about the root system structure and the uptake efficiency of root orders is critical to understand the adaptive plasticity of plants towards salt stress. Thus, this study describes the phenological and physiological plasticity of Citrus volkameriana rootstocks under severe NaCl stress on the level of root orders. Phenotypic root traits known to influence uptake processes, for example frequency of root orders, specific root area, cortical thickness, and xylem traits, did not change homogeneously throughout the root system, but changes after 6 months under 90 mM NaCl stress were root order specific. Chloride accumulation significantly increased with decreasing root order, and the Cl− concentration in lower root orders exceeded those in leaves. Water flux densities of first-order roots decreased to <20% under salinity and did not recover after stress release. The water flux densities of higher root orders changed marginally under salinity and increased 2- to 6-fold in second and third root orders after short-term stress release. Changes in root order frequency, morphology, and anatomy indicate rapid and major modification of C. volkameriana root systems under salt stress. Reduced water uptake under salinity was related to changes of water flux densities among root orders and to reduced root surface areas. The importance of root orders for water uptake changed under salinity from root tips towards higher root orders. The root order-specific changes reflect differences in vulnerability (indicated by the salt accumulation) and ontogenetic status, and point to functional differences among root orders under high salinity

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

Selective Pharmacological Modulation of Pyramidal Neurons and Interneurons in the CA1 Region of the Rat Hippocampus

The hippocampus is a complex network that consists of tightly regulated interaction between excitation and inhibition. In Alzheimer’s disease (AD) and other neurodegenerative disorders in which cognitive functions such as learning and memory are severely impaired, the inhibition-excitation balance in the hippocampal neuronal circuitry is impaired. Increasing evidence suggest that, due to the role of N-methyl-D-aspartate receptor (NMDAR) in learning and memory, dysfunctions in NMDAR functions may play a pivotal role in the pathogenesis of numerous neurodegenerative disorders. The uncompetitive NMDAR antagonist memantine, clinically used for the treatment of AD, may restore the inhibition-excitation balance in the hippocampus. The mechanism through which memantine exerts its action is, however, not clear. To better understand the effect of memantine on the hippocampal neuronal circuit, we studied the pharmacology of memantine on the NMDAR currents of pyramidal cells (PCs) and interneurons (Ints) in the CA1 region of the hippocampus. Using whole-cell patch-clamp recordings on acute rat hippocampal slices, we found that memantine antagonism of NMDAR currents was more effective in PCs than Ints. In particular, using specific NMDAR subunit antagonists, we found that the effect of memantine varies depending on the molecular make up of the NMDAR that the neurons express. Our results open new avenues for studies on the mechanism of action of memantine

Spatial-temporal changes in synaptic potentiation after long-term exposure to amyloid-beta in hippocampal slice cultures integrated with multi-electrode arrays.: Brain Res.

In press: YesNRC publication: Ye

Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling: Am J Physiol Cell Physiol

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca2+-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A1 receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca2+ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca2+ during preconditioning, a Ca2+/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning modelsNRC publication: Ye

Choline-mediated depression of hippocampal synaptic transmission

Choline is a micronutrient essential for the structural integrity of cellular membranes, and its presence at synapses follows either depolarization-induced pre-synaptic release or degradation of acetylcholine. Previous studies using whole-cell recording have shown that choline can modulate inhibitory input to hippocampal pyramidal neurons by acting upon nicotinic acetylcholine receptors (nAChRs) found on interneurons. However, little is known about how choline affects neuronal activity at the population level; therefore, we used extracellular recordings to assess its influence upon synaptic transmission in acutely prepared hippocampal slices. Choline caused a reversible depression of evoked field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependant manner (10, 500, and 1000 \ub5M). When applied after the induction of long-term potentiation, choline-mediated depression (CMD) was still observed, and potentiation returned on wash-out. Complete blockade of CMD could not be achieved with antagonists for the ?7 nAChR, to which choline is a full agonist, but was possible with a general nAChR antagonist. The ability of choline to increase paired-pulse facilitation, and the inability of applied gamma-aminobutyric acid (GABA) to mediate further depression of fEPSPs, suggests that the principal mechanism of choline's action was on the facilitation of neurotransmitter release. Our study provides evidence that choline can depress population-level activity, quite likely by facilitating the release of GABA from interneurons, and may thereby influence hippocampal function.Peer reviewed: YesNRC publication: Ye