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...

SUMMARY Degeneration of dopamine (DA) neurons in Parkinson’s disease (PD) causes hypokinesia, but DA replacement therapy can elicit exaggerated voluntary and involuntary behaviors that have been attributed to enhanced DA receptor sensitivity in striatal projection neurons. Here we reveal that in hemiparkinsonian mice, striatal D1 receptor-expressing medium spiny neurons (MSNs) directly projecting to the substantia nigra reticulata (SNr) lose tonic presynaptic inhibition by GABAB receptors. The absence of presynaptic GABAB response potentiates evoked GABA release from MSN efferents to the SNr and drives motor sensitization. This alternative mechanism of sensitization suggests a synaptic target for PD pharmacotherapy.

Summary Modulation of corticostriatal synaptic activity by dopamine is required for normal sensorimotor behaviors. After loss of nigrostriatal dopamine axons in Parkinson's disease, l-DOPA and dopamine D2-like receptor agonists are used as replacement therapy, although these drugs also trigger sensitized sensorimotor responses including dyskinesias and impulse control disorders. In mice, we lesioned dopamine projections to left dorsal striatum and assayed unilateral sensorimotor deficits with the corridor test as well as presynaptic corticostriatal activity with the synaptic vesicle probe, FM1-43. Sham-lesioned mice acquired food equivalently on both sides, while D2 receptor activation filtered the less active corticostriatal terminals, a response that required coincident co-activation of mGlu-R5 metabotropic glutamate and CB1 endocannabinoid receptors. Lesioned mice did not acquire food from their right, but overused that side following treatment with l-DOPA. Synaptic filtering on the lesioned side was abolished by either l-DOPA or a D2 receptor agonist, but when combined with a CB1 receptor antagonist, l-DOPA or D2 agonists normalized both synaptic filtering and behavior. Thus, high-pass filtering of corticostriatal synapses by the coordinated activation of D2, mGlu-R5, and CB1 receptors is required for normal sensorimotor response to environmental cues.

Optical imaging is a valuable tool for investigating alterations in membrane turnover and vesicle trafficking. Established techniques can easily be adapted to study the mechanisms of synaptic dysfunction in models of neuropsychiatric disorders and neurodegenerative diseases, such as drug addiction, Parkinsonism, and Huntington's disease. Fluorescent endocytic tracers, including FM1-43, have been used to optically monitor synaptic vesicle fusion and measure synaptic function in various preparations, including chromaffin cells, dissociated cell cultures, and brain slices. In this chapter, we describe a technique that provides a direct measure of pathway-specific exocytosis from glutamatergic corticostriatal terminals. Neurotransmitter release and reuptake from recycling synaptic terminals is tightly regulated and alterations in vesicular turnover or in the availability of neuromodulators that act presynaptically can be features of neurodegenerative conditions (1). Optical tracers that label individual axon terminals in an activity-dependent manner have become useful tools in neurobiology and are responsible for improving our understanding about membrane trafficking and synaptic activity. When combined with standard electrophysiological techniques, optical recordings enable synapse modeling by showing how neuromodulators select subsets of presynaptic terminals, leading to changes in postsynaptic activation.Perhaps the first optical tracer to monitor neurotransmitter release used horseradish peroxidase, which in the presence of appropriate substrates can produce a colored or electron-dense

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