Research ArticleSynaptic and Network Contributions to Anoxic Depolarization in Mouse Hippocampal Slices
Introduction
Ischemic stroke is the third leading cause of death worldwide, and a primary contributor to death and disability in both developing and developed countries (Burke et al., 2012). On average, someone in the United States suffers an ischemic stroke every 40 s and dies from one approximately every 4 min (Benjamin et al., 2018). The loss of blood supply to brain tissue (ischemia) and consequent depletion of ATP induces a rapid cascade of reactions that leads to neuronal death beginning within minutes and continuing for hours and days thereafter. Much of what is known about the neuronal responses to stroke has been learned from studies of brain slices or isolated neurons in primary culture in vitro after exposure to total (anoxia) or partial (hypoxia) oxygen deprivation. The initial events proceed as follows (Lipton, 1999): Oxygen or combined oxygen/glucose deprivation leads to ATP depletion, that may produce a very transient initial membrane hyperpolarization due to opening of ATP-dependent K+ channels. ATP breakdown leads to a build-up of adenosine that is released into the extracellular compartment, binding to presynaptic A1 receptors which partially antagonize synaptic glutamate release. The rundown of membrane pumps in the absence of ATP causes the neurons to slowly depolarize, leading to synaptic release of glutamate and, with further depolarization, the release of glutamate from reverse operation of glutamate transporters (Rossi et al., 2000), which synergize with K+ efflux to massively depolarize the cells.
These ischemia-driven reactions ultimately result in the loss of membrane potentials, or anoxic depolarization (AD), in affected neurons. Importantly, it has become widely-accepted that the AD event is the spark that ignites the neurotoxic cascade leading to cell death (Ayata, 2018, Hartings et al., 2017, Leao, 1947). Both the latency to AD and the time remaining in AD prior to re-oxygenation are important for the extent of cell damage in ischemia: shorter AD latencies lead to greater cell death while shorter delays between AD and re-oxygenation lead to greater survival (Jarvis et al., 2001, Kaminogo et al., 1998, Kostandy, 2012).
The fact that certain brain regions are selectively vulnerable to ischemic damage implies that there may be factors inherent either in the neurons themselves or in the networks in which they reside that influence the extent or rapidity with which they respond to metabolic insults. Cornu ammonis 1 (CA1) neurons in the hippocampus are particularly sensitive to ischemia, as they are the first to die after global ischemia in the brain (Schmidt-Kastner and Freund, 1991). For isolated neurons, AD may be directly related to the effects of metabolic inhibition on membrane ion pumps. However, the situation is more complicated in vivo and in slices since neurons in intact networks are affected by glutamate and potassium released from neighboring neurons and may be affected by synaptic connections with more distant neurons.
In addition, studies of animals which live in chronically hypoxic environments or experience hypoxia intermittently show that isolated brain slices from these animals take longer to undergo AD than slices from appropriate comparison species (Geiseler et al., 2016, Larson et al., 2014, Larson and Park, 2009). The contributions of individual steps in the anoxic cascade to the overall AD latency in these hypoxia-tolerant animals or, indeed, in typical laboratory rodents are not well understood. The present work was undertaken to examine which of the steps can be modified to extend AD latency in order to identify targets for investigation in hypoxia-tolerant animals that may represent putative therapeutic approaches for stroke.
Experiments were designed to investigate local synaptic and network factors that influence the latency to AD in the CA1 field of mouse hippocampal slices in vitro. Slices were maintained in an interface-type chamber design in which oxygen deprivation alone is used to model ischemia and produces rapid AD similar to that which occurs in stroke (Croning and Haddad, 1998). The results indicate that (1) while adenosine release and action on A1 adenosine receptors antagonize synaptic glutamate release during anoxic challenge, it has no significant effect on the latency to AD; (2) antagonism of NMDA- and AMPA-type glutamate receptors prolongs the latency to AD; (3) neurons in field Cornu ammonis 3 (CA3) show AD earlier than those in CA1 when the two regions are disconnected; however the early AD in CA3 is propagated from the CA3 network to CA1 in intact slices; and (4) slices from female animals show delayed AD relative to those from males.
Section snippets
Animals
Most experiments were performed using 2–4 month old male CD-1 mice acquired from Charles River Laboratories (Wilmington, MA). Two experiments used C57Bl/6 (B6) mice obtained from Jackson Laboratories (Bar Harbor, ME). One experiment used B6 mice (2–4 months old) to examine the electrophysiological recovery of slices re-oxygenated at varying times after AD. A second experiment used B6 mice (8–9 months old) to examining sex differences in anoxia sensitivity. All efforts were made to maximize use
Anoxia induces rapid loss of synaptic and action potentials and a depolarizing wave that originates in the dendrites and propagates to the soma
Fig. 1 shows the positioning of electrodes as well as recordings of synaptic potentials in the dendritic layer (stratum radiatum) and antidromic potentials in the cell body layer (s. pyramidale) before, during, and after a brief episode of oxygen deprivation (nominal anoxia). Synaptic (Fig. 1B) and antidromic (Fig. 1C) responses were recorded alternately at 10 s intervals throughout the experiment. After a baseline period of at least 10 min in normoxic conditions (95% O2/5% CO2), the slice was
Discussion
The present study used the mouse hippocampal slice to investigate the steps in the ischemic cascade that contribute to the timing of AD. Clinically, the AD event is rapidly followed by the “early” cell death within the ischemic core; this differs from the “late” cell death taking place during reperfusion in the penumbra. This is an important distinction, because there are currently no treatments available to affect the ischemic core. Current interventions, whether preventative or
Acknowledgements
We thank Heba Akbari, Maribell Heredia, Sylvia Pawlowski, Uzma Saleha, Hanaa Siddiqi, and Yulianna Yaroshuk for assistance with some of the experiments.
Funding Source
This work was supported by the National Science Foundation (IOS grants #0744979 and #1655494), The National Institutes of Health (HD084209), and the Campus Research Board of the University of Illinois at Chicago.
Declarations of Interest
None.
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