Multiphoton
in vivo imaging of neurons has become increasingly useful to study dynamic events in the ischemic brain such as spine morphology and Ca
2+ homeostasis (
Misgeld and Kerschensteiner, 2006). Here, for the first time, we demonstrate the
in vivo imaging of the neuronal ER and its dynamic structural changes in pyramidal cortical neurons in mice using two-photon laser scanning microscopy. The gross morphology of the dendrites in the living mouse was similar to that
in vitro. Hence, under basal conditions, the neuronal ER lumen was continuous, displaying high degree of FRAP, as in cultured neurons and hippocampal slice cultures (
Kucharz et al, 2009;
Kucharz et al, 2011 (in press)).
The ER in primary cultures and organotypic slices undergoes rapid and reversible fragmentation upon NMDA receptor stimulation, while inhibition of NMDA receptors or removal of extracellular Ca
2+ abolishes ER fragmentation (
Kucharz et al, 2009;
Kucharz et al, 2011 (in press)). Cardiac arrest results in an immediate loss of cerebral blood flow, followed by depletion of tissue glucose, ATP and PCr, and subsequently depolarization of neuronal plasma membranes (
Hansen, 1985). The shortage of energy supply leads to inhibition of Ca
2+ extrusion from neuronal cytosol and increase in intracellular Ca
2+ levels (
Silver and Erecinska, 1992). Noteworthy, the depolarization of cell membranes and intracellular Ca
2+ increase is rapid and occurs 1 to 2
minutes after cessation of blood flow to the brain (
Hansen, 1985;
Xie et al, 1995). In our study, using potassium-induced CA we validated the occurrence of ER fragmentation
in vivo in ischemic conditions. During the first 1 to 2 minutes after CA, the ER morphology appeared intact. Subsequently, ER fragmented rapidly (<15
seconds), which corresponded to the time at which Ca
2+ enters the brain cells (
Silver and Erecinska, 1992). This result strongly suggests that ER fragmentation during CA is regulated by the same mechanisms as
in vitro, that is, Ca
2+ entry to the cell (
Kucharz et al, 2009;
Kucharz et al, 2011 (in pres;
Subramanian and Meyer, 1997). The rapid reduction in ER continuity in the brain was validated by loss of FRAP, and corresponded to the results from
in vitro studies (
Kucharz et al, 2009;
Kucharz et al, 2011 (in pres). The ER fragmentation was not restricted to selected neurons, but was a global process, observed in all cortical neurons. Although we speculate that the influx of Ca
2+ into neurons during ischemic conditions is responsible for the rapid fragmentation of ER, the injected potassium levels in the blood could potentially enter the brain causing ER fragmentation. This is unlikely, since the potassium must then cross the blood–brain barrier and enter the brain during the few cardiac strokes expected after KCl injection. If this occurred, an instantaneous fragmentation would be expected. Instead, ER fragmentation occurs >1 minute after the injection and coincides with the well-described effect of anoxic depolarization implicated in previous studies on ER structural rearrangements in the brain (
Banno and Kohno, 1996).
The CA experiments are terminal; however, in the transient middle cerebral artery occlusion experimental model of stroke, the infarct core with irreversibly injured tissue is surrounded by penumbra area with cells that have the potential to recover and exhibit trains of transient membrane depolarizations. A transient ER fragmentation in these conditions may contribute to neuroprotection by limiting the diffusion of Ca
2+ within ER, as proposed previously (
Kucharz et al, 2009;
Kucharz et al, 2011 (in press)). Whether the reversible ER fission occurs in penumbra and if it contributes to cell survival are of subject of future research.
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