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The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity

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An Erratum to this article was published on 13 March 2017

Abstract

Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer™5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.

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Acknowledgments

We thank Andreas W. Henkel for fruitful discussions and Katrin Ebert and Elke Meyer for excellent technical assistance. We would also like to thank Peter Gmeiner and Alina Tabor for assistance with the spectrophotometer.

Funding

This work was supported by the Interdisciplinary Center of Clinical Research (IZKF) in Erlangen (Project E10) (to Teja W. Groemer and Volker Eulenburg).

Conflict of Interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6, 91054, Erlangen, Germany

    Jasmin Jung, Kristina Loy, Eva-Maria Schilling, Mareike Röther, Jan M. Brauner, Johannes Kornhuber, Oliver Welzel & Teja W. Groemer

  2. Institute of Physiology and Pathophysiology, Friedrich-Alexander-University of Erlangen-Nuremberg, Universitaetsstr. 17, 91054, Erlangen, Germany

    Tobias Huth & Christian Alzheimer

  3. Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6, 91054, Erlangen, Germany

    Ursula Schlötzer-Schrehardt

Authors
  1. Jasmin Jung
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  2. Kristina Loy
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  3. Eva-Maria Schilling
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  4. Mareike Röther
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  5. Jan M. Brauner
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  6. Tobias Huth
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  7. Ursula Schlötzer-Schrehardt
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  8. Christian Alzheimer
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  9. Johannes Kornhuber
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  10. Oliver Welzel
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  11. Teja W. Groemer
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Corresponding author

Correspondence to Teja W. Groemer.

Additional information

Jasmin Jung and Kristina Loy, as well as Oliver Welzel and Teja W. Groemer contributed equally to this work.

An erratum to this article is available at http://dx.doi.org/10.1007/s12035-017-0476-8.

Electronic supplementary material

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Fig. S1

Fluoxetine does not influence protein levels in hippocampal neurons. Exemplary Western Blot. Top, first lane, molecular weight marker. Lysates of fluoxetine-treated cells were compared with lysates of untreated cells. Antibodies against dynamin, synaptotagmin1, synaptobrevin2 and CDK5 were used to compare the protein levels. GAPDH served as a loading control. Bottom, Quantification of intensities of blotted bands (Wilcoxon rank sum test: n.s.; N=3) (JPEG 248 kb)

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Fig. S2

Fluoxetine increases the number of release-competent synaptic vesicles in repeatedly stimulated neurons. A Exemplary images of spH-transfected neurons treated with 1 μM fluoxetine or DMSO under the v-ATPase inhibitor folimycin, respectively, before (pre) and after the first, second and third stimulation round (1 st stim to 3 rd stim). Scale bar, 10 μm. Cells were repeatedly stimulated with 600 pulses at 30 Hz with decreasing inter-stimulation intervals after 30, 300, 500, 600, 650 and 675 seconds, respectively. As no further fluorescence increase was observed after the 3rd stimulation, corresponding data was omitted. B Fluorescence profiles for control and fluoxetine-treated conditions for the 1st to the 3rd stimulation. The 1st stimulations did not differ, whereas in the 2nd and 3rd stimulation round fluoxetine mobilized more synaptic vesicles (two-sample Kolmogorov–Smirnov test, 1st stimulation: p = 0.33, 2nd stimulation: p<0.001, 3rd stimulation: p<0.001). C Stimulationinduced fluorescence changes of neurons treated with 1 μM fluoxetine or vehicle (DMSO). Fluorescence changes were normalized to their first amplitude, respectively. In cells treated with fluoxetine more synaptic vesicles were mobilized in the 2nd and 3rd stimulation round (two-sample t test, 1st stimulation: p = 0.50, 2nd stimulation: p = 0.02, 3rd stimulation: p = 0.06; N =4) (JPEG 436 kb)

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Fig. S3

Fluoxetine does not influence synaptic vesicle endocytosis. A Illustration of αGFPCypHerTM5E labeling of spH-transfected neurons and its fluorescence properties. Hippocampal neurons expressing synapto-pHluorin, where some proteins are located on the cell surface. CypHerTM5E-labeled antibodies against the protein GFP (pHluorin) added to extracellular solution. Fluorescence of CypHerTM5E is reduced in neutral pH 7.4. Upon an electrical stimulus vesicles fuse with the plasma membrane and αGFP-CypHerTM5E binds to the new available synapto-pHluorin proteins. Furthermore the intravesicular protons are lost. After compensatory endocytosis synaptic vesicles are reacidified and the fluorescence of CypHerTM5E increases. Thus, αSyt1-CypHerTM5E enables the measurement of exocytosis and endocytosis/reacidification of synaptic vesicles. B Representative images of hippocampal neurons expressing spH labeled with αGFP-CypHer™5E for detection of acutely recycling synaptic vesicles. Images are shown before, during and after stimulation with 600 pulses at 30 Hz. Scale bar, 10 μm. C Average fluorescence profiles of simultaneously detected spH and αSyt1-CypHerTM5E upon a stimulation with 600 pulses at 30 Hz with and without 1 μM fluoxetine. D Quantification of fluorescence changes due to electrical stimulus of 600 pulses at 30 Hz (two-sample t test, spH: p = 0.58, CypHer™5E: p = 0.91; N = 4). E Fluoxetine has no effect on endocytosis kinetics in primary hippocampal neurons. Average fluorescence profiles of αSyt1-CypHerTM5E upon a stimulation with 600 pulses at 30 Hz before and after shortterm incubation with 1 μM fluoxetine or vehicle. Fluorescence profiles were normalized for 4 better comparison of endocytosis kinetics. Inset, typical image of neurons, where recycling synaptic vesicles were labeled with αSyt1-CypHerTM5E (JPEG 327 kb)

High Resolution Image (TIFF 24.5 mb)

Fig. S4

Fluoxetine increases vesicle exocytosis by recruitment of additional vesicles and leads to enhanced vesicle turnover. A Exemplary images of two stimulations under 80 nM folimycin. Folimycin prevents reacidification of synaptic vesicles and thus spH quenching, and is therefore a measure of net exocytosis. Scale bar, 10 μm. B Fluorescence read outs after first and second stimulation with 600 pulses at a frequency of 10 Hz. The first control stimulation evokes a similar fluorescence increase in both settings whereas the increase at the second stimulus is larger after short-term incubation with 1 μM Fluoxetine compared to the DMSO control. C Quantification of fluorescence increase normalized to the corresponding control stimulus (two-sample t test; 1st stimulation: p = 0.99, 2nd stimulation: p = 0.05; N = 4 each). D Incubation of hippocampal neurons transfected with spH with and without fluoxetine reveals higher vesicle turnover in fluoxetine-treated cells compared to DMSO control. Neurons were incubated with αGFP-CypHer™5E and drug for 10 min at 37 °C under 40 mM KCl. CypHer™5E signals were normalized to corresponding spH signals to handle different expression level due to transfection. Scale bar, 10 μm. (E) Quantification of fluorescence signals revealed more recycling vesicles in fluoxetine-treated neurons (two-sample t test, p = 0.05; N = 4 each). F Fluoxetine increases FM 1-43 loading. After short-term incubation with 1 μM fluoxetine or DMSO for 20 min at 37 °C neurons were stained with 2.5 μM FM 1-43 using 1,200 pulses at 40 Hz. Fluorescence was determined at dye-loaded synapses and normalized to the control (G; two-sample t test, p = 0.01; N = 5 each) (JPEG 264 kb)

High Resolution Image (TIFF 20.0 mb)

Fig. S5

Fluoxetine does not influence emission spectra and intensity of FM 1-43, CypHer™5E or Alexa488. A Emission spectra of FM 1-43 with (blue) and without (black) 1 μM fluoxetine. All solutions were mixed with 20 % CHAPS. B The spectrum of the pHdependent dye CypHer™5E coupled to an anti-synaptotagmin1 antibody remains unaltered under 1 μM fluoxetine (blue). All solutions were prepared at intravesicular pH of 5.3 at which CypHer™5E is in its fluorescent state. C Emission profile of Alexa488 coupled to an antisynaptotagmin1 antibody with (blue) and without (black) 1 μM fluoxetine (N = 3 each) (JPEG 133 kb)

High Resolution Image (TIFF 6.71 mb)

Fig. S6

Short-term application of 1 μM fluoxetine did not influence the ultrastructure of hippocampal synapses. A Fluoxetine had no impact on the synapse number. Exemplary detail images of hippocampal synapses, where recycling synaptic vesicles were labeled using αSyt1- Alexa488. Top, Images taken by conventional epifluorescence microscopy. Bottom, Corresponding images taken by dSTORM super resolution microscopy. More details of synaptic morphology are clearly visible compared to the conventional technique. Scale bar, 1 μm. B Quantification of the number of neighboring synapses in a 50 μm2 circular area (twosample t test: p = 0.49; N = 7 each). C Fluoxetine did not alter vesicle numbers. Transmission electron microscopy affirmed the results obtained with fluorescence microscopy. Representative ultrastructural images of hippocampal synapses. Short-term application of 1 μM fluoxetine did not affect the ultrastructural morphology of hippocampal synapses showing pre- and postsynaptic densities, synaptic clefts, and accumulations of synaptic vesicles. Scale bar, 0.5 μm. D Quantification of synaptic vesicles (two-sample t test, p = 0.5656, N = 20 each) (JPEG 539 kb)

High Resolution Image (TIFF 14.2 mb)

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Jung, J., Loy, K., Schilling, EM. et al. The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity. Mol Neurobiol 49, 916–930 (2014). https://doi.org/10.1007/s12035-013-8569-5

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