Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain

Abstract

We recently introduced fluorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater fluorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the measurement of individual synaptic terminal activity by following fluorescence loss upon stimulation. Thus, FFN102 enables not only the identification of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allowing for the study of both the microanatomy and functional plasticity of the normal and diseased nervous system. dopamine reporter | secretion kinetics | molecular design | multiphoton imaging D opamine neurotransmission plays a key role in habit learning, motivation, reward, and motor function (1), and altered dopamine neurotransmission is associated with disorders such as Parkinson's disease, schizophrenia, and drug addiction (2-4). As a "social" neurotransmitter that overflows relatively long distances beyond its presynaptic terminals, dopamine's extrasynaptic concentration is principally determined by the combination of exocytotic neurotransmitter release and reuptake by the plasma membrane dopamine transporter (DAT) (5). Psychostimulants, such as cocaine and amphetamine (AMPH), increase extracellular dopamine via interactions with DAT.Extracellular dopamine concentration, particularly in the striatum where it is present at high levels, has been characterized by microdialysis (6, 7) and rapid electrochemical detection using carbon fiber cyclic voltammetry (8, 9) and amperometry (10). The excellent temporal resolution of the electrochemical methods is well suited for measuring changes in extrasynaptic dopamine concentration associated with neuronal activity. However, these approaches usually measure the release and reuptake of dopamine from large sets of striatal dopamine release sites and lack the spatial resolution required to study synaptic transmission at the level of individual presynaptic terminals.Optical methods provide vastly improved spatial resolution so that processes by which specific synapses are modulated can be studied. The first group of fluorescent reporters for the study of presynaptic function were the endocytic FM dyes (11), which act as tracers of exocytosis and endocytosis. Thes...

Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles

“…6 C and D). Interestingly, the 10% decrease in mean fluorescence is comparable to the decrease found using similar stimulation in the striatum with the false fluorescent neurotransmitter FFN102 before separating responders from nonresponders (11). Again, removing Ca 2+ was inhibitory.…”

“…Furthermore, pCA experiments suggest that VMAT-containing organelles in the soma are sparse, and therefore obscured by other acidic organelles or are not acidified. It is also possible that 20 min of pCA treatment was too short to detect a somatic CYAM response, because the false fluorescent neurotransmitter FFN102 is released from the soma of DA neurons only after 40 min of incubation with 1 μM amphetamine (11). Nevertheless, the region-specific differences in CYAM responses suggest that the soma, in contrast to terminals, is not optimized for vesicular release of DA and CYAM.…”

APP+, a Fluorescent Analogue of the Neurotoxin MPP+, Is a Marker of Catecholamine Neurons in Brain Tissue, but Not a Fluorescent False Neurotransmitter

“…This contrasts with the recently developed probe FFN102, which was designed to produce increased fluorescence when released to the extracellular space from acidic synaptic vesicles. 11 These results indicate that only a small portion of the APP+ fluorescence signal originates from the releasable synaptic vesicular pool, while the majority of the APP+ signal is derived from an intracellular mixture of labeled nonexocytotic compartments and cellular structures including mitochondria. Our findings are similar to those obtained with the antihypotensive agent amezinium (4-amino-6-methoxy-1-phenyl-pyridazinium salt) reported by others.…”

“…We have previously shown that FFN102-loaded presynaptic terminals in dorsal striatum were completely destained through the action of 40 mM KCl. 11 With APP+, however, we found that KCl only partially destained terminal fields compared to ACSF-treated control ( Figure 7). Analysis was accomplished by measuring mean fluorescence intensity of a field of background-subtracted puncta, before and during KCl treatment, and comparing the results to those obtained with a control imaged without KCl treatment.…”

“…As discussed above, FFN102 is to date the most selective FFN probe for dopamine neurons, featuring strong DAT dependence and ∼90% colocalization with TH-GFP signal in dorsal striatum, and thus may serve as a reliable reference signal. 11 We found that 74.1 ± 6.9% (mean ± SD, n = 3) of APP+ puncta were FFN102 positive ( Figure 5D−F). These results indicate that APP+ labels fine axonal processes of dopamine neurons in the dorsal striatum with selectivity > 70% as defined by colocalization to two reference signals.…”