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 Ca2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ excitability.
COBISS.SI-ID: 32520921
Ketamine is an anesthetic that exhibits analgesic, psychotomimetic, and rapid antidepressant effects that are of particular neuropharmacological interest. Recent studies revealed astrocytic Ca2+ signaling and regulated exocytosis as ketamine-targeted processes. Thus high-resolution cell-attached membrane capacitance measurements were performed to examine the influence of ketamine on individual vesicle interactions with the plasma membrane in cultured rat astrocytes. Ketamine evoked long-lasting bursts of repetitive opening and closing of the fusion pore that were both time- and concentration-dependent. Moreover, acute application and subanesthetic doses of ketamine elicited a significant increase in the occurrence of bursts that were characterized by a decreased fusion pore conductance, indicating that the fusion pore was stabilized in a narrow configuration. The time- and concentration-dependent increase in burst occurrence was correlated with a decrease in full fission events. This study has demonstrated a novel effect of ketamine manifested as stabilization of a fusion pore incapable of transiting to full vesicle fission, suggestive of an inhibitory effect on vesicle retrieval. This until now unrecognized effect of ketamine on the vesicle fusion pore might play a role in astroglial release and (re)uptake of molecules, modulating synaptic activity. This study demonstrates a novel effect of ketamine on the fusion pore. High-resolution cell-attached membrane capacitance measurements revealed that ketamine evokes long-lasting flickering of a narrow fusion pore that is incapable of transiting to full fission. Astrocytic vesicle fusion/retrieval modified by subanesthetic ketamine doses most likely affects gliotransmission and indicates a non-neuronal mechanism of ketamine action that may contribute to its behavioral effects.
COBISS.SI-ID: 32795609
Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.
COBISS.SI-ID: 32590041