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

Cellular distribution of the nicotinic acetylcholine receptor \u3b17 subunit in rat hippocampus

The hippocampus is a region of the mammalian brain that has been extensively studied due to its role in many forms of memory. To better understand hippocampal function, significant attention has focused upon the cellular distribution of ligand-gated ion channels. Despite strong cholinergic innervation from the basal forebrain and a dense expression of nicotinic acetylchoine receptors (nAChRs), the cellular distribution of subunits forming these receptors has received little attention. We used organotypic hippocampal slice cultures (OHSCs) to study native \u3b17 subunits, which, unlike other nAChR subunits, form a homomeric receptor. Cell-surface biotinylation, cross-linking of surface proteins, and sub-cellular fractionation all revealed a very limited presence of the subunit at the plasma membrane. In contrast, subunits of other receptors displayed significant surface expression. Notably, subunits in adult hippocampal tissue were distributed in a fashion similar to that observed in OHSCs. To monitor \u3b17 subunits contained in functional nAChRs, a colourimetric assay using \u3b1-bungarotoxin (a specific \u3b17 nAChR antagonist) was developed, and revealed a majority of binding at the cell surface. To change \u3b17 subunit distribution, OHSCs were treated with compounds known to affect other ionotropic receptors\u2014 insulin, genistein, and elevated external K\u207a; however, neither subunit surface expression nor antagonist binding was affected. Our data reveal that hippocampal neurons possess a large internal population of \u3b17 subunits under basal conditions, which persists during stimuli affecting tyrosine phosphorylation or neuronal activity. The nature of the internal pool of \u3b17 subunits remains to be determined, but should have important implications for hippocampal activity.Peer reviewed: YesNRC 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

Preconditioning induces tolerance by suppressing glutamate release in neuron culture ischemia models

This study determined how preconditioned neurons responded to oxygen-glucose deprivation (OGD) to result in neuroprotection instead of neurotoxicity. Neurons preconditioned using chronically elevated synaptic activity displayed suppressed elevations in extracellular glutamate ([glutamateex]) and intracellular Ca\ub2+ (Ca\ub2+ in) during OGD. The glutamate uptake inhibitor TBOA induced neurotoxicity, but at a longer OGD duration for preconditioned cultures, suggestive of delayed up-regulation of transporter activity relative to non-preconditioned cultures. This delay was attributed to a critically attenuated release of glutamate, based on tolerance observed against insults mimicking key neurotoxic signaling during OGD (OGD-mimetics). Specifically, in the presence of TBOA, preconditioned neurons displayed potent protection to the OGD-mimetics: ouabain (a Na+/K+ ATPase inhibitor), high 55 mM KCl extracellular buffer (plasma membrane depolarization), veratridine (a Na+ ionophore), and paraquat (intracellular superoxide producer), which correlated with suppressed [glutamateex] elevations in the former two insults. Tolerance by preconditioning was reversed by manipulations that increased [glutamateex], such as by exposure to TBOA or GABAA receptor agonists during OGD, or by exposure to exogenous NMDA or glutamate. Pre-synaptic suppression of neuronal glutamate release by preconditioning, possibly via suppressed exocytic release, represents a key convergence point in neuroprotection during exposure to OGD and OGD-mimetics. \ua9 2012 National Research Council Canada. Journal of Neurochemistry \ua9 2012 International Society for Neurochemistry.Peer reviewed: YesNRC publication: Ye

Hippocampal slice cultures integrated with multi-electrode arrays allow for the study of spatial-temporal changes in synaptic potentiation after chronic exposure to amyloid-beta

NRC publication: Ye

Mechanisms of 1S,3R- trans -ACPD-induced neuroprotection in rat hippocampal slices exposed to in vitro ischemia

NRC publication: Ye

Polymer neurochip: development of a microfluidic chip for neuronal network patch-clamp analysis

Peer reviewed: YesNRC publication: Ye