Two-photon imaging of glial ion and water homeostasis in health and disease

Abstract

The vital role of glial cells in the nervous system has only been revealed relatively recently as these cells are electrically silent. Technical advances that allow direct visualization of glial activity have therefore been paramount to the recent glial renaissance. One such technique, two-photon laser scanning microscopy (2PLSM), forms the basis of our thesis. 2PLSM offers the necessary spatial and temporal resolution to study glial cells in intact living brain. Exploiting the strengths of 2PLSM, we chose to examine key roles of glial water and ion homeostasis in health and disease. Our first study (Paper I) delineated a novel aquaporin-4 (AQP4) mediated glial signaling pathway that is activated in response to hypo-osmotic brain edema, and which may contribute to the high mortality of this condition. In the second study (Paper II) we showed that AQP4 plays a crucial role in oxygen microdistribution during cortical spreading depression (CSD), a wave of tissue depolarization closely linked to migraine. In the next study (Paper III), we found that general anesthetics directly suppress astrocyte calcium signals, and this effect may explain some of the sedative effects of these drugs. In Paper IV we explored the paravascular circulation of cerebrospinal fluid (CSF) through brain, and found that this compartment served both as a highway for selective lipid transport and signal mediation between astrocytes. In Paper V we used 2PLSM to show a pathological activation of microglia in the advanced stages of hepatic encephalopathy (HE), which may be linked to the development of fatal brain edema in severe cases of this condition. Finally, in Paper VI we use a congenital disorder of ammonia handling to show that the immediate neurotoxicity of ammonia is mediated by a direct shortcircuiting of astrocyte potassium buffering. This secondarily causes an impairment of inhibitory neurotransmission, and is not related to astrocyte swelling as was previously hypothesized. Blocking the downstream accumulation of chloride in neurons using Na+-K+-2Cl- cotransporter subtype-1 (NKCC1) inhibitor bumetanide, we were able to reverse ammonia neurotoxicity and thus develop a clinically relevant therapy. Taken together, using in vivo 2PLSM imaging we were able to elucidate important roles of glial cells in normal brain function and debilitating neurological disorders.