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.
List of papers. Papers I, III and V are removed from the thesis due to publisher restrictions.
Paper I: Thrane AS, Rappold PM, Fujita T, Torres A, Bekar LK, Takano T, Peng W, Rangroo Thrane V, Enger R, Haj-Yasein NN, Skare Ø, Holen T, Klungland A, Ottersen OP, Nedergaard M and Nagelhus EA. Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema. Proc Natl Acad Sci. 2011;108 (2):846-51. (A.S.T., P.M.R. and T.F. are joint first authors, commented upon in Nat Rev Neurosci, February 2011). doi:10.1073/pnas.1015217108
Paper II: Thrane AS, Takano T, Rangroo Thrane V, Wang F, Peng W, Ottersen OP, Nedergaard M and Nagelhus EA. In vivo NADH fluorescence imaging indicates effect of aquaporin-4 deletion on oxygen microdistribution in cortical spreading depression. (A.S.T., T.T. and V.R.T are joint first authors) Resubmitted after review to J Cereb Blood Flow Metab. Published as: J Cereb Blood Flow Metab. 2013 Jul;33(7):996-9. doi:10.1038/jcbfm.2013.63
Paper III: Thrane AS, Rangroo Thrane V, Zeppenfeld D, Lou N, Xu Q, Nagelhus EA and Nedergaard M. General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex. Proc Natl Acad Sci. 2012; 109(46):18974-9. (A.S.T. and V.R.T are joint first authors) doi:10.1073/pnas.1209448109
Paper IV: Rangroo Thrane V, Thrane AS, Plog B, Thiyagarajan M, Gundersen GA, Iliff J, Deane R, Nagelhus EA, and Nedergaard M. Paravascular microcirculation facilitates rapid lipid transport and astrocyte signaling in the brain. Submitted to Nat Neurosci. (V.R.T and A.S.T. are joint first authors) Published as: Sci Rep. 2013;3:2582. doi:10.1038/srep02582 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.
Paper V: Rangroo Thrane V, Thrane AS, Chang J, Alleluia V, Nagelhus EA and Nedergaard M. Real-time analysis of microglial activation and motility in hepatic and hyperammonemic encephalopathy. Neuroscience. 2012; 220:247-55. (V.R.T and A.S.T. are joint first authors) doi:10.1016/j.neuroscience.2012.06.022
Paper VI: Rangroo Thrane V, Thrane AS, Wang F, Cotrina ML, Smith NA, Chen M, Kang N, Fujita T, Nagelhus EA and Nedergaard M. Ammonia compromises astrocyte potassium buffering and impairs neuronal inhibition without causing swelling in vivo. Pending resubmission after review to Nat Med. (V.R.T and A.S.T. are joint first authors) Published as: Nat Med. 2013 Dec;19(12):1643-8. doi:10.1038/nm.3400