title: Determination of transmembrane water fluxes in neurons elicited by glutamate ionotropic receptors and by the cotransporters KCC2 and NKCC1: a digital holographic microscopy study.
abstract: Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.
authors.affiliation: Brain and Mind Institute, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. pascal.jourdain@epfl.ch
authors.forename: Pascal
authors.initials: P
authors.lastname: Jourdain
authors.forename: Nicolas
authors.initials: N
authors.lastname: Pavillon
authors.forename: Corinne
authors.initials: C
authors.lastname: Moratal
authors.forename: Daniel
authors.initials: D
authors.lastname: Boss
authors.forename: Benjamin
authors.initials: B
authors.lastname: Rappaz
authors.forename: Christian
authors.initials: C
authors.lastname: Depeursinge
authors.forename: Pierre
authors.initials: P
authors.lastname: Marquet
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Magistretti
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004058:Diffusion / Q000187:drug effects; D005260:Female; D005665:Furosemide / Q000494:pharmacology; D006696:Holography / Q000379:methods; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011810:Quinoxalines / Q000494:pharmacology; D058468:Receptors, Ionotropic Glutamate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012815:Signal Processing, Computer-Assisted; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000378:metabolism*; D014867:Water / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D005665:Furosemide; D011810:Quinoxalines; D058468:Receptors, Ionotropic Glutamate; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D014867:Water; D000096922:K Cl- Cotransporters; C578878:Slc12a2 protein, mouse; C056723:FG 9041