Fluorescent False Neurotransmitters Visualize Dopamine Release from Individual Presynaptic Terminals

Abstract

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.

Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors

“…The structures are shown in Figure 2. The polymers are abbreviated as follows: N1−N12 are ssDNA sequences, N13 is the ssRNA sequence (GU) 15 , and PL1−PL12 are phospholipids with different head and tail groups. P1−P5 are different amphiphilic polymers with a hydrophobic group that binds to the carbon surface and a hydrophilic group that renders the conjugate water-soluble.…”

“…In general, aromatic (and redox-active) neurotransmitters such as dopamine showed stronger responses than non-redox-active neurotransmitters such as glycine. The SWCNT−polymer conjugate with the strongest fluorescence increase, N1, corresponds to (GT) 15 DNA-wrapped SWCNTs. We used this specific DNA sequence as an exemplary model to study the fluorescence enhancement of nucleic acid−SWCNT conjugates to dopamine and to investigate the mechanism of this molecular recognition.…”

“…Glass substrates were functionalized with (3-aminopropyl)triethoxysilane (APTES) in ethanol (1% APTES, 1% water) and then mounted under the flow chamber. Next, 1 mg/L (GT) 15 -or (GU) 15 -wrapped SWCNT solution in PBS was incubated in the channels to adsorb SWCNTs to the surface. For the reversibility and single-SWCNT experiments, dopamine (100 μM) and PBS were periodically washed over the surface (flow rate 50 μL/min).…”

Successful Prediction of Substrate-binding Pocket in SLC17 Transporter Sialin

“…Virtual HTS studies of broader databases not restricted to sialic acids should provide a variety of molecular tools, such as pharmacological chaperones (16) able to correct the trafficking defect of sialin R39C in Salla disease (11,15), cell-permeant inhibitors allowing the testing of the proposed aspartatergic role of sialin (17) in a physiological context or, if this role is confirmed, false fluorescent neurotransmitters mimicking the vesicular release of aspartate. The latter compounds are fluorescent substrates used as optical probes to monitor transmitter secretion in cell cultures and brain slices and currently available only for mono- aminergic neurotransmission (62). As the transport activity of recombinant sialin is easier to test than those of VGLUT and VNUT proteins, the validated sialin models may also guide similar modeling and virtual HTS studies of these vesicular transporters to identify cell-permeant inhibitors and false fluorescent neurotransmitter targeted to glutamatergic and purinergic synapses.…”

Influence of Phasic and Tonic Dopamine Release on Receptor Activation

“…where I DA is the tissue content of dopamine (Bannon et al, 1981), R is the average fraction of terminal contents released per action potential (Gubernator et al, 2009), is total density of dopamine release sites (Doucet et al, 1986), and N 0 is the number of dopamine molecules released in successful vesicle fusion (Pothos et al, 1998) (for numerical values, see Table 1). Definition of firing patterns.…”