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

Karpowicz, Richard J.; Dunn, Matthew R.; Sulzer, David; Sameš, Dalibor

doi:10.1021/cn400038u

Cited by 37 publications

(56 citation statements)

References 53 publications

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“…First, CYAM and IDT307 were imaged. IDT307 labeling was not punctate like CYAM, because this DAT substrate fills DA neuron processes but likely does not get packaged into vesicles (8). Importantly, punctate CYAM fluorescence was present in IDT307-labeled structures (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…4A). To verify this result independently, CYAM-treated slices were imaged before and after incubation with IDT307 (also called APP + ), a fluorescent substrate of plasma membrane monoamine transporters (7)(8)(9). Because the DA transporter (DAT) is the only somatic monoamine transporter in the SNc, somatic IDT307 labeling identifies DA neurons.…”

Section: Resultsmentioning

confidence: 99%

“…IDT307 has a wide 2P excitation spectrum ranging from ∼700-950 nm, with a peak at 870 nm (8). Its emission spectrum also overlaps with the emission spectrum of CYAM, at 450-600 nm (8).…”

Section: Methodsmentioning

confidence: 97%

“…In experiments for which it was important to localize CYAM puncta to monoaminergic neurons, the fluorescent monoamine transporter substrate, IDT307 (also known as APP + ) was used (7)(8)(9). IDT307 has a wide 2P excitation spectrum ranging from ∼700-950 nm, with a peak at 870 nm (8).…”

Section: Methodsmentioning

confidence: 99%

“…IDT307 has a wide 2P excitation spectrum ranging from ∼700-950 nm, with a peak at 870 nm (8). Its emission spectrum also overlaps with the emission spectrum of CYAM, at 450-600 nm (8). To eliminate the problem of overlap, 2P images of CYAM fluorescence were taken before a 10-min application of 0.5-1 μM IDT307 (a generous gift from Randy Blakely, Vanderbilt University, Nashville, TN).…”

Section: Methodsmentioning

confidence: 99%

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

Tucker

Block

Levitan

2015

Proc. Natl. Acad. Sci. U.S.A.

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Based on lysotracker red imaging in cultured hippocampal neurons, antipsychotic drugs (APDs) were proposed to accumulate in synaptic vesicles by acidic trapping and to be released in response to action potentials. Because many APDs are dopamine (DA) D2 receptor (D2R) antagonists, such a mechanism would be particularly interesting if it operated in midbrain DA neurons. Here, the APD cyamemazine (CYAM) is visualized directly by two-photon microscopy in substantia nigra and striatum brain slices. CYAM accumulated slowly into puncta based on vacuolar H + -ATPase activity and dispersed rapidly upon dissipating organelle pH gradients. Thus, CYAM is subject to acidic trapping and released upon deprotonation. In the striatum, Ca 2+ -dependent reduction of the CYAM punctate signal was induced by depolarization or action potentials. Striatal CYAM overlapped with the dopamine transporter (DAT). Furthermore, parachloroamphetamine (pCA), acting via vesicular monoamine transporter (VMAT), and a charged VMAT, substrate 1-methyl-4-phenylpyridinium (MPP + ), reduced striatal CYAM. In vivo CYAM administration and in vitro experiments confirmed that clinically relevant CYAM concentrations result in vesicular accumulation and pCA-dependent release. These results show that some CYAM is in DA neuron VMAT vesicles and suggests a new drug interaction in which amphetamine induces CYAM deprotonation and release as a consequence of the H + countertransport by VMAT that accompanies vesicular uptake, but not by inducing exchange or acting as a weak base. Therefore, in the striatum, APDs are released with DA in response to action potentials and an amphetamine. This synaptic corelease is expected to enhance APD antagonism of D2Rs where and when dopaminergic transmission occurs.M ost antipsychotic drugs (APDs) are competitive dopamine (DA) D2 receptor (D2R) antagonists (1, 2). The standard view is that APDs, by being weak bases that are in equilibrium with an uncharged form, cross the blood-brain barrier to access extracellular receptor binding sites from the circulation. However, because APDs are weak bases, they have been proposed to accumulate also in acidic intracellular organelles by acidic trapping (i.e., the less abundant uncharged basic hydrophobic form enters organelles passively and then is protonated to become membrane-impermeant). This hypothesis was first explored by examining a D2 antagonist with a covalently added fluorophore and APD displacement of acridine orange (3). More recently, APDs were found to be released from brain tissue by depolarization, which is expected for any positively charged drug. However, follow-up experiments in hippocampal neuron cultures showed that APDs displace the acidophilic dye lysotracker red, implying there is overlap in intracellular distribution between APDs and lysotracker red. Furthermore, lysotracker red was found to accumulate in hippocampal neuron synaptic vesicles by acidic trapping without displacing native neurotransmitter and to be released in response to electrical stimulation. Thus, b...

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

“…IDT307 has a wide 2P excitation spectrum ranging from ∼700-950 nm, with a peak at 870 nm (8). Its emission spectrum also overlaps with the emission spectrum of CYAM, at 450-600 nm (8).…”

Section: Methodsmentioning

confidence: 97%