A new means to measure neurotransmission optically using fluorescent dopamine analogs.
The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct “reserve” synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.
Hui Zhang and Niko G. Gubernator contributed equally to this work.
In this video, we demonstrate a method to visualize neurotransmission optically using fluorescent dopamine analogs. FFN511 is the first generation of FFNs we have developed. Although it was designed by targeting the neuronal vesicular monoamine transporter (VMAT2) that carries monoamine neurotransmitters from the cytoplasm into synaptic vesicles, and specifically labels dopamine terminals in the striatum and presumed catecholamine and/or serotonin terminals in the cortex (as shown in the Science paper), an appropriate loading period is critical for the specificity since FFN511 is relatively hydrophobic. We found that incubation for longer than 40 minutes will result in extensive nonspecific staining in striatal slices. The specificity can be straightforwardly determined by destaining using a high concentration of KCl, which should cause the release of FFN within functional synaptic vesicles. Exposure of the slice to FFN511 for less than 15 minutes will result in weak fluorescent signal due to insufficient loading of the dye in the terminals. In this protocol, we use 100μM ADVASEP-7 to remove the dye bound to extracellular tissue. This step is not necessary, but if it is omitted, the washout time in ACSF needs to be prolonged. In summary, depending on the preparation you chose, you will need to modify the concentration and loading period to determine optimal labeling.
The authors have nothing to disclose.
D. Sames thanks The G. Harold & Leila Y. Mathers Charitable Foundation and Columbia University’s Initiatives in Science and Engineering.
D. Sames and D. Sulzer thank the McKnight Foundation for The McKnight Technological Innovations in Neuroscience Award
D. Sulzer thanks NIDA, NIMH, and the Picower and Parkinson’s Disease Foundations.
H. Zhang thanks NARSAD.
R.H. Edwards thanks the Michael J. Fox Foundation, the National Parkinson
Foundation, NIDA and NIMH.
We thank Robert Burke for 6-OHDA injections and advice, Mark Sonders for useful discussion, Merek Siu for imaging analysis programming, and Jan Schmoranzer for technical support with the TIRF microscopy setup.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one) | Dalibor Sames’s laboratory at Columbia University | |||
ADVASEP-7 | CyDex, Overland Park, KS | AR-OA7-005 | ||
RC-27L Recording chamber | Warner Instrument | 64-0375 | ||
PELCO PrepEze 6-Well Holder | Ted Pella, Inc. | 36157-1 | For slice incubation | |
Master-8 pulse stimulator and Iso- Flex stimulus isolator | AMPI, Jerusalem, Israel |