The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

Abstract

Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the “weak base hypothesis” that amphetamine reduces synaptic vesicle transmitter and quantal size.

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…H + -ATPase activity establishes the high intravesicular H + concentration upon which VMAT2 transport depends, and reducing H + -ATPase function or increasing vesicular pH impairs VMAT2 transport activity (for review, see Sulzer et al, 2005;Fleckenstein et al, 2007). This can affect VMAT2 function: AMPH and its analogs may function as weak bases to disrupt the synaptic vesicle pH and contribute to VMAT2 dysfunction (Sulzer et al, 1993;Omiatek et al, 2013; for review, see Fleckenstein et al, 2007) (Fig. 3B).…”

“…After synthesis, VMAT2 transports DA from the cytoplasmic space into synaptic vesicles within presynaptic terminals. VMAT2 activity largely dictates quantal size, affecting the scale of subsequent neurotransmitter release (Pothos et al, 2000;Omiatek et al, 2013).…”

Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes

“…[2] With this method, we then successfully quantified the vesicular contents in individual vesicles isolated from pheochromocytoma (PC12) cells and mouse brain tissue. [3] Recently, Compton and coworkers have also investigated the electrochemistry of liposomes, in this case encapsulated with ascorbic acid as they impact electrodes. [4] We have now characterized the contents of mammalian vesicles isolated from adrenal chromaffin cells by single adsorption and rupture events at electrodes without separation.…”

“…Thus, as we and others have published recently, only part of the quantal contents is released during exocytosis, which is consistent with our results from the cell-free model. [3b, 5, 11] The peak parameters for these data are summarized in Table S1.…”

Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision–Adsorption Events at an Electrode

“…2,3,6,7 Vesicle electrochemical cytometry provides a robust approach for obtaining this information and opens the door to a wide variety of experiments for which quantitative knowledge of the contents of vesicles is important.…”