The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order 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.

We introduce pH responsive fluorescent false neurotransmitters (pH responsive FFNs) as novel probes that act as vesicular monoamine transporter (VMAT) substrates and ratiometric fluorescent pH sensors. The development of these agents was achieved by systematic molecular design that integrated several structural elements, including the aminoethyl group (VMAT recognition), halogenated hydroxycoumarin core (ratiometric optical pH sensing in the desired pH range), and N-or C-alkylation (modulation of lipophilicity). Of fourteen compounds that were synthesized, the probe Mini202 was selected based on the highest uptake in VMAT2-transfected HEK cells and desirable optical properties. Using Mini202, we measured the pH of catecholamine secretory vesicles in PC-12 cells (pH ~ 5.9) via two-photon fluorescence microscopy. Incubation with methamphetamine led to an increase in vesicular pH (pH ~ 6.4), consistent with a proposed mechanism of action of this psychostimulant, and eventually to redistribution of vesicular content (including Mini202) from vesicles to cytoplasm. Mini202 is sufficiently bright, photostable and suitable for two-photon microscopy. This probe will enable fundamental neuroscience and neuroendocrine research as well as drug screening efforts.As part of a broad program aimed at optical imaging of metabolic and signaling enzymes in cells and tissues,1 , 2 we became interested in visualization of neurotransmission. Recently we have introduced fluorescent false neurotransmitters (FFNs), probes that act as optical tracers that provide the first means to image neurotransmitter release from individual presynaptic terminals in the brain. Monoamine neurotransmitters are accumulated in synaptic vesicles by vesicular monoamine transporter 2 (VMAT2), which translocates the monoamine (e.g., dopamine) from cytosol to the lumen of synaptic vesicles.5 Similarly, in chromaffin cells of adrenal medulla, epinephrine and norepinephrine are accumulated in secretory vesicles by a closely related protein, VMAT1 ( Figure 1). The vesicular lumen is acidic (pH ~ 5-6) due to the action of vacuolar-H + ATPase, which imports H + at the expense of ATP hydrolysis. The pH gradient between the cytoplasm and the vesicular lumen in turn provides the driving force for the accumulation of transmitters in the vesicles. Thus the pH gradient is a key parameter regulating synaptic plasticity as it controls the vesicular transmitter content and amount of transmitter released during vesicle fusion. The pH gradient between the cytosol and the vesicular lumen is ATP dependent and thus closely coupled to the metabolic state of the presynaptic terminals. Despite the importance of this parameter and great efforts focused on development of fluorescent pH indicators,6 there are currently no small molecule probes available for selective measurement of pH in synaptic or secretory vesicles. The pHsensitive synaptopHluorin protein can be used to estimate pH in synaptic vesicles of cultured neurons.7 Alternatively, construction of avidin-chimera prot...

This study describes the design of sensitive, selective, and fluorogenic reporter substrates for monoamine oxidase (MAO) enzymes. This was achieved by an iterative effort, guided by PET and TICT photophysical concepts, which led to the development of irreversible redox switches based on a facile oxidation-cyclization reporting mechanism. Specifically, enzymatic oxidation of the ethylamino group in probe 9 proceeded via a putative aldehyde intermediate, which subsequently underwent spontaneous and intramolecular condensation with the aniline amino group furnishing an indole product in an irreversible fashion. This overall change resulted in a significant change in the emission intensity. When expressed in terms of brightness, the origins of this emission switch may be rationalized by the changes in quantum yield and absorbance strength. The fluorescence readout directly correlated with the kinetics of the oxidative step (i.e., reporting mechanism was fast, the intermediate aldehyde was not detected). Probe 9 is a good substrate for MAO B (Km = 510 +/- 40 muM, kcat = 21 min-1) with the kinetic parameters comparable to physiological substrates. This probe not only allows for direct and continuous measurement of MAO activity in mitochondria and tissue homogenates, but more importantly sets the stage for future studies in intact cells and organs.

Ongoing efforts in our laboratories focus on design of optical reporters known as fluorescent false neurotransmitters (FFNs) that enable the visualization of uptake into, packaging within, and release from individual monoaminergic neurons and presynaptic sites in the brain. Here, we introduce the molecular probe FFN246 as an expansion of the FFN platform to the serotonergic system. Combining the acridone fluorophore with the ethylamine recognition element of serotonin, we identified FFN54 and FFN246 as substrates for both the serotonin transporter and the vesicular monoamine transporter 2 (VMAT2). A systematic structure-activity study revealed the basic structural chemotype of aminoalkyl acridones required for serotonin transporter (SERT) activity and enabled lowering the background labeling of these probes while maintaining SERT activity, which proved essential for obtaining sufficient signal in the brain tissue (FFN246). We demonstrate the utility of FFN246 for direct examination of SERT activity and SERT inhibitors in 96-well cell culture assays, as well as specific labeling of serotonergic neurons of the dorsal raphe nucleus in the living tissue of acute mouse brain slices. While we found only minor FFN246 accumulation in serotonergic axons in murine brain tissue, FFN246 effectively traces serotonin uptake and packaging in the soma of serotonergic neurons with improved photophysical properties and loading parameters compared to known serotonin-based fluorescent tracers.

The synthesis of @-tide beta-strand peptidomimetics has been improved such that oligomers now can be obtained from solution- and solid-phase synthesis protocols approaching the efficiency and flexibility of peptide chemistry. These methods enable the synthesis of @-tide oligomers with a variety of amino acids and with lengths up to 13 units. [reaction: see text]

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. Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=1562 ProtocolThis method was used in the research reported in Gubernator et al., Science 324 (5933). 1441-1444 (2009 3. Extract the whole brain from the mouse, and mount it on the vibratome tray placed on the vibratome using Krazy®Glue. Brain extraction and mounting must be done within 2 minutes. During this time, make sure to keep the ACSF ice cold and oxygenated to keep the tissue healthy. 4. Cut coronal striatal brain slices at 250μm thickness between bregma +1.54 to +0.62 1 . Three whole slices will be obtained which can then be cut into 6 half slices with a needle. 5. Incubate the brain slices in oxygenated ACSF at room temperature for at least 1 hour before probe loading. Slices display good FFN511 loading and remain active for imaging up to 4 hours after slice preparation.1. Prepare FFN511 loading solution (10μM FFN511 in ACSF); also prepare 100μM ADVASEP-7 in ACSF. All solutions should be prepared freshly and oxygenated at least 15 minutes prior to loading into the slices. 2. Individually incubate slice with FFN511 loading solution for 30 minutes at room temperature. 3. Then remove the dye bound to extracellular tissue by incubating the loaded slice in oxygenated 100μM ADVASEP-7 ACSF for 30 minutes at room temperature 2 . Slices are now ready for imaging.

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