Ketamine Inhibits ATPEvoked Exocytotic Release of

Authors: Matjaž Stenovec Eva Lasič Mićo Božić Saša Trkov Bobnar Randy F Stout Vladimir Grubišić Vladimir Parpura Robert Zorec

Publish Date: 2015/12/12

Volume: 53, Issue: 10, Pages: 6882-6896

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Abstract

In the brain astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules such as brainderived neurotrophic factor BDNF Recent studies uncovered a role of ketamine an anesthetic and antidepressant in the regulation of BDNF expression and in the disruption of astrocytic Ca2+ signaling but it is unclear whether it affects astroglial BDNF release We investigated whether ketamine affects ATPevoked Ca2+ signaling and exocytotic release of BDNF at the singlevesicle level in cultured rat astrocytes Cells were transfected with a plasmid encoding preproBDNF tagged with the pHsensitive fluorescent protein superecliptic pHluorin BDNFpHse to load vesicles and measure the release of BDNFpHse when the exocytotic fusion pore opens and alkalinizes the luminal pH In addition cellattached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane Intracellular Ca2+ activity was monitored with Fluo4 and confocal microscopy which was also used to immunocytochemically characterize BDNFpHseladen vesicles As revealed by doublefluorescent micrographs BDNFpHse localized to vesicles positive for the soluble Nethylmaleimidesensitive fusion protein attachment protein receptor SNARE proteins vesicleassociated membrane protein 2 VAMP2 VAMP3 and synaptotagmin IV Ketamine treatment decreased the number of ATPevoked BDNFpHse fusion/secretion events P  005 the frequency of ATPevoked transient P  0001 and fullfusion exocytotic P  005 events along with a reduction in the ATPevoked increase in intracellular Ca2+ activity in astrocytes by ~70  P  0001 The results show that ketamine treatment suppresses ATPtriggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca2+ excitabilityThis work was supported by the Slovenian Research Agency grant nos P3 310 J3 3236 J3 4051 J34146 J3 6790 VP’s work is supported by the National Institutes of Health HD078678 We thank Dr Masami Kojima and Dr James E Rothman for kindly providing the preproBDNFEGFP and pCMVSpHse plasmids respectively All DNA plasmids were sequenced at The Heflin Center Genomics Core at UABExocytotic release of brainderived neurotrophic factor BDNF BDNFpHse a fusion protein of BDNF and superecliptic phluorin pHse allowed us to monitor its release ATP stimulation induces fusion of vesicles at the plasma membrane PM When a vesicle fuses with the PM the acidic vesicular lumen connects to the alkaline extracellular space and pHse fluorescence intensity increases a left This is followed by a decrease in the pHse fluorescence intensity due to release of BDNFpHse a middle BDNFpHse is released by full fusion events a middle and Fig 5 as revealed by NH4Cl application at the end of experiments a right and Fig 2 Acidic environment inside nonfusing vesicle quenches pHse fluorescence b left The fluorescence intensity increases when the vesicle lumen is artificially alkalinized by NH4Cl b right Endogenously expressed proBDNF was omitted for clarity Not drawn to scale GIF 12 kb

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