1. Fluoxetine increases the correlation of kinetic parameters
Analysis of kinetically distinguished synaptic subtypes in hippocampal neurons.
Andreas W. Henkel
Department of Physiology, Faculty of Medicine, Jabriya, Kuwait University, Kuwait
Abstract:
Neural transmission and information processing requires not only quantitative modulation of transmitter release but also temporal coordination of the information flow. It is known that action potentials trigger fast synaptic exocytosis by influx of calcium ions. Here we describe that the onset of synaptic exocytosis in specialized individual synapses can be delayed by several seconds after the start of electrical stimulation.
Hippocampal neurons from newborn rats were cultured in vitro until they obtained functionally mature synapses. Synaptic vesicles were fluorescently labeled using 600 pulses at 30 Hz to label the whole recycling pool. After thorough wash-out synapses were destained in two consecutive stimulation cycles, where the first served as internal control. Neurons were either treated with 1 µM fluoxetine or control solution during the second cycle and kinetic parameters from both cycles were compared.
Mean exocytosis of FM followed roughly a single exponential decay curve during the stimulation period. Detailed analysis, however, revealed that the general synaptic population was composed of at least 4 kinetically distinguished subtypes (1. single-exponential exocytosis, 2. delayed exocytosis, 3. two-component exocytosis and linear exocytosis). FM-exocytosis kinetics were significantly altered by short term application of fluoxetine during the second stimulation cycle, which decreased the number of delayed synapses by more than 50 % and increased the speed of synaptic exocytosis significantly by rising the percentage of fast two-component synapses. Synapses in control experiments showed the opposite effects when they were stimulated in a second stimulation cycle. Exocytosis of synapses, expressing synaptopHlourin, was never delayed after start of stimulation.
Considering earlier findings on synaptic release of FM1-43, our results suggest that the mode of exocytosis is switched between full vesicle fusion and “kiss-and-run” within the same synapse. The delay of FM release could be explained by “kiss-and-run” type of exocytosis since the dye couldn’t leave a vesicle through a narrow fusion pore. Both, H+ ions and neurotransmitter molecules, however, can leave the vesicle lumen through a pore, and therefore allow synaptopHlourin to monitor exocytosis.
Fluoxetine increases the correlation of kinetic parameters
Results:
Mean exocytosis of FM1-43 followed roughly a single exponential decay curve during the stimulation period. Detailed analysis revealed that the global synaptic population was composed of at least four kinetically distinguished exocytosis subtypes: 1. single-exponential type, 2. delayed type, 3. two-component type and 4. linear type. The distribution of these subtypes was different on somata and dendrites. FM1-43 exocytosis kinetics were significantly altered by application of fluoxetine during the second stimulation cycle, which decreased the number of delayed synapses by more than 50 % compared to controls and increased the rate of synaptic exocytosis significantly by rising the percentage of fast two-component synapses. Synapses in control experiments showed the opposite effects when they were stimulated in a second stimulation cycle. However, exocytosis in synapses, expressing synaptopHlourin as a pH-sensitive reporter fluorophore, was never delayed after start of electrical stimulation.
Fluoxetine changes the prevalence of synaptic subtypes.
A. The percentage of delayed synapses in the whole population increases during a 2nd series of stimulation, indicating fatigued synapses. Fluoxetine application before the 2nd stimulation cycle prevents this effect. B. The fast releasing two-component synapse subpopulation decreases during a 2nd series of stimulation, while application of fluoxetine causes an increase.
Fluoxetine stabilizes kinetic exocytosis parameters between 2 successive series of stimulation.
The plots represent the mean data from 8 individual experiments. The upper 3 panels show the correlation between the first and the second stimulation in control cells. The exocytotic delay (ExoDelay) and the time of maximal release velocity (t-Vmax) are increased and not correlated. The maximal release velocity (Vmax) is largely unchanged and correlated. Application of fluoxetine causes no increase in ExoDelay,
t-Vmax time. All parameters remain correlated between both stimulation series.
seconds
Mean destaining curve. The red section of the curve represents the mean of 998 individual synaptic distaining curves during stimulation. It closely follows a single exponential function.
Distribution of synapses.
Bright fluorescent spots represent active hippocampal synapses, loaded with FM1-43.
Methods:
Hippocampal neurons from newborn rats were cultured in vitro until they obtained functionally mature synapses. Synaptic vesicles were fluorescently labeled, using 600 pulses at 30 Hz to label the whole recycling pool. After thorough wash-out, synapses were destained in two consecutive stimulation cycles, where the first served as internal control. Neuronal cells were either treated with 1 µM fluoxetine or control solution during the second cycle and kinetic parameters from both cycles were compared.
A. High-resolution distribution of kinetically distinguished synapses.
Small areas on neuronal somata and dendrites harbor different kinetic synaptic subtypes in close vicinity. Graphs show color-coded de-staining-curves (dF/Fmax; circles) and rate-curves (dF’/Fmax’; lines) of individual synapses.
B. Large - scale spatial distribution of kinetically distinguished synapses.
Delayed and slow releasing synapses are preferentially located on neuronal somata, while fast releasing synapses are located on neuronal processes (dendrites).
SynaptopHlourin transfected synapses show a concomitant onset of exocytosis.
A. Synapses transfected with synaptopHlourin. B. Fluorescence increase (exocytosis) starts exactly with first electric stimulus simultaneously on all synapses, but magnitude and rates show variations. C. Synapses labeled with FM1-43. D. Fluorescence decrease (exocytosis) starts exactly with first electric stimulus only in a subpopulation of synapses (upper set of curves). Delayed synapses exhibit a significant prolongation of destaining onset. E. Kinetically distinguished subpopulations of synapses are characterized by different onset of exocytosis in FM1-43 stained synapses, while all synaptopHlourin transfected synapses (subpopulation definitions based on fluorescence-rate increase) show immediate onset of exocytosis directly at start of electrical stimulation.
Discussion:
Individual synapses exhibit very different exocytosis kinetics that can be modulated by fluoxetine. It is not clear so far, which mechanism controls this phenomena. We hypothesize, that at least the delayed FM1-43 release is due to “kiss-and-run” exocytosis, because the dye cannot leave a vesicle through a narrow fusion pore. Delayed dilation of the pore could explain the late onset of dye release. Both, H+ ions and neurotransmitter molecules can leave the vesicle lumen through a pore, and therefore allow synaptopHlourin to monitor exocytosis.
Monitoring exocytosis.
FM1-43 and other styryl dyes have been used to monitor exocytosis cells of cultured hippocampal neurons. The amphipathic structure of the dyes proposes the following mechanism of action: From the extracellular fluid dye molecules reversibly partition into the outer leaflet of all surface membranes, but they cannot penetrate to the cytoplasm. If dye-labeled membrane is internalized the dye becomes trapped in endocytosed vesicles. Subsequent washing of the pre-paration in dye-free medium removes dye only from surface membranes. Subsequent stimulation in dye-free medium, triggers exocytosis and causing the synapse to destain.
Sequential stain exocytosis assay for hippocampal neurons.
Hippocampal neurons were stained with FM1–43 in the absence of fluoxetine, washed and pictures were taken during the period, indicated by dots. Fluoxetine was added (red line) after the first staining staining/destaining series and remained in the bath during the further rest of the experiment. The staining/destaining procedure was repeated in a second series in presence of the drug and pictures were taken. These two resulting picture series were subsequently compared and analyzed.
Acknowledgment:
Supported by Grant MY 01/11 from Kuwait University. We thank Oliver Welzel for provision of synaptopHlourin-transfected neurons and Anju Devassy for technical assistance. Experiments were done in the Research Unit for Genomics, Proteomics and Cellomics Sciences of HSC Kuwait University
Contact: Andreas W. Henkel, Ph.D.,
Destaining curves of four kinetic synaptic subpopulations. A classical synapse follows a single exponential decay curve while a two-component synapse destains almost entirely during the first half of the stimulation period. A linear destaining synapse exhibits a constant rate of destaining over the whole stimulation period while a delayed synapse starts to destain with a delay of a few seconds after the onset of stimulation.