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Drug-induced seizure liabilities produce significant compound attrition during drug discovery. Currently available in vitro cytotoxicity assays cannot predict all toxicity mechanisms due to the failure of these assays to predict sublethal target-specific electrophysiological liabilities. Identification of seizurogenic and other electrophysiological effects at early stages of the drug development process is important to ensure that safe candidate compounds can be developed while chemical design is taking place, long before these liabilities are discovered in costly preclinical in vivo studies. The development of a high throughput and reliable in vitro assay to screen compounds for seizure liabilities would de-risk compounds significantly earlier in the drug discovery process and with greater dependability. Here we describe a method for screening compounds that utilizes rat cortical neurons plated onto multiwell microelectrode array plates to identify compounds that cause neurophysiological disruptions. Changes in 12 electrophysiological parameters (spike train descriptors) were measured after application of known seizurogenic compounds and the response pattern was mapped relative to negative controls, vehicle control and neurotoxic controls. Twenty chemicals with a variety of therapeutic indications and targets, including GABAA antagonists, glycine receptor antagonists, ion channel blockers, muscarinic agonist, δ-opioid receptor agonist, dopaminergic D2/adrenergic receptor blocker and nonsteroidal anti-inflammatory drugs, were tested to assess this system. Sixteen of the seventeen seizurogenic/neurotoxic compounds tested positive for seizure liability or neurotoxicity, moreover, different endpoint response patterns for firing rate, burst characteristics and synchrony that distinguished the chemicals into groups relating to target and seizurogenic response emerged from the data. The negative and vehicle control compounds had no effect on neural activity. In conclusion, the multiwell microelectrode array platform using cryopreserved rat cortical neurons is a highly effective high throughput method for reliably screening seizure liabilities within an early de-risking drug development paradigm.

Neuronal activity in the central nervous system evokes localized changes in blood flow, a response termed neurovascular coupling or functional hyperaemia. Modern functional imaging methods, such as functional magnetic resonance imaging (fMRI), measure signals related to functional hyperaemia in order to determine localization of brain function and to diagnose disease. The cellular mechanisms that underlie functional hyperaemia, however, are not well understood. Glial cells have been hypothesized to be intermediaries between neurons and blood vessels in the control of neurovascular coupling, owing to their ability to release vasoactive factors in response to neuronal activity. Using an in vitro preparation of the isolated, intact rodent retina, we have investigated two likely mechanisms of glial control of the vasculature: glial K(+) siphoning and glial induction of vasoactive arachidonic acid metabolites. Potassium siphoning is a process by which a K(+) current flowing through glial cells transfers K(+) released from active neurons to blood vessels. Since slight increases in extracellular K(+) can cause vasodilatation, this mechanism was hypothesized to contribute to neurovascular coupling. Our data, however, suggest that glial K(+) siphoning does not contribute significantly to neurovascular coupling in the retina. Instead, we suggest that glial cells mediate neurovascular coupling by inducing the production of two types of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), which dilate and constrict vessels, respectively. We show that both light flashes and direct glial stimulation produce vasodilatation or vasoconstriction mediated by EETs and 20-HETE, respectively. Further, we show that the type of vasomotor response observed (dilatation or constriction) depends on retinal levels of nitric oxide. Our data also demonstrate that glial cells are necessary intermediaries for signalling from neurons to blood vessels, since functional hyperaemia does not occur when neuron-to-glia communication is interrupted. These results indicate that glial cells play an important role in mediating functional hyperaemia and suggest that the regulation of blood flow may involve both vasodilating and vasoconstricting components.

Neuronal activity evokes localized changes in blood flow, a response termed neurovascular coupling. One widely recognized hypothesis of neurovascular coupling holds that glial cell depolarization evoked by neuronal activity leads to the release of K+ onto blood vessels (K+ siphoning) and to vessel relaxation. We now present two direct tests of this glial cell-K+ siphoning hypothesis of neurovascular coupling. Potassium efflux was evoked from glial cells in the rat retina by applying depolarizing current pulses to individual cells. Glial depolarizations as large as 100 mV produced no change in the diameter of adjacent arterioles. We also monitored light-evoked vascular responses in Kir4.1 knock-out mice, where functional Kir K+ channels are absent from retinal glial cells. The magnitude of light-evoked vasodilations was identical in Kir4.1 knock-out and wild-type animals. Contrary to the hypothesis, the results demonstrate that glial K+ siphoning in the retina does not contribute significantly to neurovascular coupling.

This article reviews calcium signaling in three specialized types of glial cells: Müller cells of the retina, Bergmann glial cells of the cerebellum, and radial glial cells of the developing cortex. Müller cells generate spontaneous and neuronal activity-evoked increases in Ca(2+). Neuron to Müller cell signaling is mediated by neuronal release of ATP and activation of glial P2Y receptors. Müller cells, in turn, modulate neuronal excitability and mediate vasomotor responses. Bergmann glial cells also generate spontaneous and activity-evoked Ca(2+) increases. Neuron to Bergmann glia signaling is mediated by neuronal release of nitric oxide, noradrenaline, and glutamate. In Bergmann glia, Ca(2+) increases control the structural and functional interactions between these cells and Purkinje cell synapses. In the ventricular zone of the developing cortex, radial glial cells generate spontaneous Ca(2+) increases that propagate as Ca(2+) waves through clusters of neighboring glial cells. These Ca(2+) increases control cell proliferation and neurogenesis.

Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.