Chloride Cotransporters as a Molecular Mechanism underlying Spreading Depolarization-Induced Dendritic Beading.
Spreading depolarizations (SDs) are waves of sustained neuronal and glial depolarization that propagate massive disruptions of ion gradients through the brain. SD is associated with migraine aura and recently recognized as a novel mechanism of injury in stroke and brain trauma patients. SD leads to neuronal swelling as assessed in real time with two-photon laser scanning microscopy (2PLSM). Pyramidal neurons do not express aquaporins and thus display low inherent water permeability, yet SD rapidly induces focal swelling (beading) along the dendritic shaft by unidentified molecular mechanisms. To address this issue, we induced SD in murine hippocampal slices by focal KCl microinjection and visualized the ensuing beading of dendrites expressing EGFP by 2PLSM. We confirmed that dendritic beading failed to arise during large (100 mOsm) hyposmotic challenges, underscoring that neuronal swelling does not occur as a simple osmotic event. SD-induced dendritic beading was not prevented by pharmacological interference with the cytoskeleton, supporting the notion that dendritic beading may result entirely from excessive water influx. Dendritic beading was strictly dependent on the presence of Cl(-), and, accordingly, combined blockade of Cl(-)-coupled transporters led to a significant reduction in dendritic beading without interfering with SD. Furthermore, our in vivo data showed a strong inhibition of dendritic beading during pharmacological blockage of these cotransporters. We propose that SD-induced dendritic beading takes place as a consequence of the altered driving forces and thus activity for these cotransporters, which by transport of water during their translocation mechanism may generate dendritic beading independently of osmotic forces.


CONCLUSIONS
Spreading depolarization occurs during pathological conditions such as stroke, brain injury, and migraine and is characterized as a wave of massive ion translocation between intracellular and extracellular space in association with recurrent transient focal swelling (beading) of dendrites. Numerous ion channels have been demonstrated to be involved in generation and propagation of spreading depolarization, but the molecular machinery responsible for the dendritic beading has remained elusive. Using real-time in vitro and in vivo two-photon laser scanning microscopy, we have identified the transport mechanisms involved in the detrimental focal swelling of dendrites. These findings have clear clinical significance because they may point to a new class of pharmacological targets for prevention of neuronal swelling that consequently will serve as neuroprotective agents.
Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark, and.
Annette B
AB
Steffensen
Brain and Behavior Discovery Institute and.
Jeremy
J
Sword
Brain and Behavior Discovery Institute and.
Deborah
D
Croom
Brain and Behavior Discovery Institute and Department of Neurosurgery, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912 skirov13@gmail.com macaulay@sund.ku.dk.
Sergei A
SA
Kirov
Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark, and skirov13@gmail.com macaulay@sund.ku.dk.
Nanna
N
MacAulay
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D056547:CA1 Region, Hippocampal / Q000166:cytology* / Q000502:physiology; D000073883:CX3C Chemokine Receptor 1; D002704:Chlorates / Q000494:pharmacology; D013181:Cortical Spreading Depression / Q000187:drug effects / Q000502:physiology*; D003712:Dendrites / Q000378:metabolism / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D005260:Female; D005665:Furosemide / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010042:Ouabain / Q000494:pharmacology; D017966:Pyramidal Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D027981:Symporters / Q000235:genetics* / Q000378:metabolism*; D053778:Thiazolidines / Q000494:pharmacology
D019086:Bridged Bicyclo Compounds, Heterocyclic; D000073883:CX3C Chemokine Receptor 1; C095235:CX3CR1 protein, human; D002704:Chlorates; D019707:Receptors, Chemokine; D049994:Sodium Potassium Chloride Symporter Inhibitors; D027981:Symporters; D053778:Thiazolidines; D010042:Ouabain; D005665:Furosemide; D000255:Adenosine Triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C037067:latrunculin A; C022842:chloric acid
Cl-cotransporters; cotransporters; dendritic beading; neuronal swelling; spreading depression; two-photon microscopy