Cortical spreading depression-induced preconditioning in mouse neocortex is lamina specific.

Cortical spreading depression (CSD) is able to confer neuroprotection when delivered at least 1 day in advance of an ischemic event. However, its ability to confer neuroprotection in a more immediate time frame has not previously been investigated. Here we have used mouse neocortical brain slices to study the effects of repeated episodes of CSD in layer V and layer II/III pyramidal neurons. In layer V, CSD evoked at 15-min intervals caused successively smaller membrane depolarizations and increases in intracellular calcium compared with the response to the first CSD. With an inter-CSD interval of 30 min this preconditioning effect was much less marked, indicating that preconditioning lasts between 15 and 30 min. A single episode of CSD also provided a degree of protection in oxygen-glucose deprivation (OGD) by significantly lengthening the time a cell could withstand OGD before anoxic depolarization occurred. In layer II/III pyramidal neurons no preconditioning by CSD on subsequent episodes of CSD was observed, demonstrating that the response of pyramidal neurons to repeated CSD is lamina specific. The A1 receptor antagonist 8-cyclopentyl theophylline (8-CPT) reduced the layer V preconditioning in a concentration-related manner. Inhibition of extracellular formation of adenosine by blocking ecto-5'-nucleotidase with α,ß-methyleneadenosine 5'-diphosphate prevented preconditioning in most but not all cells. Block of equilibrative nucleoside transporters 1 and 2 with dipyramidole alone or in combination with 6-[(4-nitrobenzyl)thio]-9-ß-d-ribofuranosylpurine also prevented preconditioning in some but not all cells. These data provide evidence that rapid preconditioning of one CSD by another is primarily mediated by adenosine.

Changes in membrane potential and the intracellular calcium concentration during CSD and OGD in layer V and layer II/III mouse cortical neurons.

Cortical spreading depression (CSD) is an episode of electrical silence following intense neuronal activity that propagates across the cortex at ∼3-6 mm/min and is associated with transient neuronal depolarization. CSD is benign in normally perfused brain tissue, but there is evidence suggesting that repetitive CSD contributes to infarct growth following focal ischemia. Studies to date have assumed that the cellular responses to CSD are uniform across neuronal types because there are no data to the contrary. In this study, we investigated the effect of CSD on membrane potential and the intracellular calcium concentration ([Ca(2+)](i)) of mouse layer V and layer II/III pyramidal neurons in brain slices. To place the data in context, we made similar measurements during anoxic depolarization induced by oxygen and glucose deprivation (OGD). The [Ca(2+)](i) was quantified using the low-affinity ratiometric indicator Fura-4F. During both CSD- and OGD-induced depolarization, the membrane potential approached 0 mV in all neurons. In layer V pyramids OGD resulted in an increase in [Ca(2+)](i) to a maximum of 3.69 ± 0.73 (SD) µM (n = 12), significantly greater than the increase to 1.81 ± 0.70 µM in CSD (n = 34; P < 0.0001). Membrane potential and [Ca(2+)](i) returned to nearly basal levels following CSD but not OGD. Layer II/III neurons responded to CSD with a greater peak increase in [Ca(2+)](i) than layer V neurons (2.88 ± 0.6 µM; n = 9; P < 0.01). We conclude there is a laminar difference in the response of pyramidal neurons to CSD; possible explanations are discussed.