Journal Article

Ketamine Inhibits ATP-Evoked Exocytotic Release of Brain-Derived Neurotrophic Factor from Vesicles in Cultured Rat Astrocytes

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

2016

Overview

In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived 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 Ca 2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca 2+ signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca 2+ activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events ( P  < 0.05), the frequency of ATP-evoked transient ( P  < 0.001) and full-fusion exocytotic ( P  < 0.05) events, along with a reduction in the ATP-evoked increase in intracellular Ca 2+ activity in astrocytes by ~70 % ( P  < 0.001). The results show that ketamine treatment suppresses ATP-triggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca 2+ excitability.

• Publisher: Springer US,Springer Nature B.V
• Subject: Adenosine Triphosphate - pharmacology / Animals / Astrocytes - drug effects / Astrocytes - metabolism / Biomarkers - metabolism / Biomedical and Life Sciences / Biomedicine / Brain / Brain-Derived Neurotrophic Factor - metabolism / Calcium - metabolism / Calcium Signaling - drug effects / Cell Biology / Cells, Cultured / Cellular biology / Cytoplasmic Vesicles - metabolism / Exocytosis - drug effects / Hydrogen-Ion Concentration / Ketamine - pharmacology / Membrane Fusion - drug effects / Membranes / Molecular biology / Neurobiology / Neurology / Neurosciences / Plasma / Purines - metabolism / Rats, Wistar / SNARE Proteins - metabolism / Subcellular Fractions - metabolism