Tonic extracellular glutamate and ischaemia: glutamate antiporter system xc - regulates anoxic depolarization in hippocampus.

In stroke, the sudden deprivation of oxygen to neurons triggers a profuse release of glutamate that induces anoxic depolarization (AD) and leads to rapid cell death. Importantly, the latency of the glutamate-driven AD event largely dictates subsequent tissue damage. Although the contribution of synaptic glutamate during ischaemia is well-studied, the role of tonic (ambient) glutamate has received far less scrutiny. The majority of tonic, non-synaptic glutamate in the brain is governed by the cystine/glutamate antiporter, system xc - . Employing hippocampal slice electrophysiology, we showed that transgenic mice lacking a functional system xc - display longer latencies to AD and altered depolarizing waves compared to wild-type mice after total oxygen deprivation. Experiments which pharmacologically inhibited system xc - , as well as those manipulating tonic glutamate levels and those antagonizing glutamate receptors, revealed that the antiporter's putative effect on ambient glutamate precipitates the ischaemic cascade. As such, the current study yields novel insight into the pathogenesis of acute stroke and may direct future therapeutic interventions. KEY POINTS: Ischaemic stroke remains the leading cause of adult disability in the world, but efforts to reduce stroke severity have been plagued by failed translational attempts to mitigate glutamate excitotoxicity. Elucidating the ischaemic cascade, which within minutes leads to irreversible tissue damage induced by anoxic depolarization, must be a principal focus. Data presented here show that tonic, extrasynaptic glutamate supplied by system xc - synergizes with ischaemia-induced synaptic glutamate release to propagate AD and exacerbate depolarizing waves. Exploiting the role of system xc - and its obligate release of ambient glutamate could, therefore, be a novel therapeutic direction to attenuate the deleterious effects of acute stroke.

Electrolyte transport pathways induced in the midgut epithelium of Drosophila melanogaster larvae by commensal gut microbiota and pathogens.

KEY POINTS: The digestive tract of larval and adult Drosophila is an excellent analogue of the mammalian gut. Enterocytes of the posterior midgut are separated by septa, with no paracellular path, and therefore perform both immune and transport functions. Using microperfusion electrophysiology, we show that larvae emerging from the embryo into sterile medium have symmetrical apical and basal membrane conductances while larvae emerging into non-sterile medium have apical membranes fivefold more conductive than basal membranes. The channels inserted into the apical membranes could originate in microbiata or host and mediate recognition of microbes. Entomopathogenic cyclic peptide toxins deplete intracellular ions reversibly, forming transient ion channels that do not conduct water, unlike an ionophore like nystatin that depletes ions irreversibly. We show the feasibility of studying the interaction of a single microbial species, or tractable combinatorials of microbial species, with only enterocytes in the primary epithelial barrier. ABSTRACT: Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post-embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non-sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non-specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non-sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter-enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter.

Synaptic and Network Contributions to Anoxic Depolarization in Mouse Hippocampal Slices.

Ischemic stroke remains the third leading cause of death and leading cause of adult disability worldwide. A key event in the pathophysiology of stroke is the anoxic depolarization (AD) of neurons in the ischemic core. Previous studies have established that both the latency to AD and the time spent in AD prior to re-oxygenation are predictors of neuronal death. The present studies used hippocampal slices from male and female mice to investigate the electrophysiological events that affect latency to AD after oxygen deprivation. The results confirm that the epoch between AD and re-oxygenation largely determines the magnitude of synaptic recovery after anoxic challenge. Using a selective antagonist of adenosine A1 receptors, we also confirmed that adenosine released during anoxia (ANOX) suppresses synaptic glutamate release; however, this action has no effect on AD latency or the potential for post-anoxic recovery of synaptic transmission. In contrast, antagonism of AMPA- and NMDA-type glutamate receptors significantly prolongs the latency to AD and alters the speed and synchrony of associated depolarizing waves. Experiments using slices with fields Cornu ammonis 3 (CA3) and Cornu ammonis 1 (CA1) disconnected showed that AD latency is longer in CA1 than in CA3; however, the early AD in CA3 is propagated to CA1 in intact slices. Finally, AD latency in CA1 was found to be longer in slices from female mice than in those from age-matched male mice. The results have implications for stroke prevention and for understanding brain adaptations in hypoxia-tolerant animals.

Cellular Obstruction Clearance in Proximal Ventricular Catheters Using Low-Voltage Joule Heating.

OBJECTIVE: Proximal obstruction due to cellular material is a major cause of shunt failure in hydrocephalus management. The standard approach to treat such cases involves surgical intervention which unfortunately is accompanied by inherent surgical risks and a likelihood of future malfunction. We report a prototype design of a proximal ventricular catheter capable of noninvasively clearing cellular obstruction. Methods: In-vitro cell-culture methods show that low-intensity ac signals successfully destroy a cellular layer in a localized manner by means of Joule heating induced hyperthermia. A detailed electrochemical model for determining the temperature distribution and ionic current density for an implanted ventricular catheter supports our experimental observations. RESULTS: In-vitro experiments with cells cultured in a plate as well as cells seeded in mock ventricular catheters demonstrated that localized heating between 43 °C and 48 °C caused cell death. This temperature range is consistent with hyperthermia. The electrochemical model verified that Joule heating due to ionic motion is the primary contributor to heat generation. CONCLUSION: Hyperthermia induced by Joule heating can clear cellular material in a localized manner. This approach is feasible to design a noninvasive self-clearing ventricular catheter system. SIGNIFICANCE: A shunt system capable of clearing cellular obstruction could significantly reduce the need for future surgical interventions, lower the cost of disease management, and improve the quality of life for patients suffering from hydrocephalus.