Vesicular expression and release of ATP from dopaminergic neurons of the mouse retina and midbrain

Abstract

Vesicular nucleotide transporter (VNUT) is required for active accumulation of adenosine tri-phosphate (ATP) into vesicles for purinergic neurotransmission, however, the cell types that express VNUT in the central nervous system remain unknown. This study characterized VNUT expression within the mammalian retina and brain and assessed a possible functional role in purinergic signaling. Two native isoforms of VNUT were detected in mouse retina and brain based on RNA transcript and protein analysis. Using immunohistochemistry, VNUT was found to co-localize with tyrosine hydroxylase (TH) positive, dopaminergic (DA) neurons of the substantia nigra and ventral tegmental area, however, VNUT expression in extranigral non-DA neurons was also observed. In the retina, VNUT labeling was found to co-localize solely with TH-positive DA-cells. In the outer retina, VNUT-positive interplexiform cell processes were in close contact with horizontal cells and cone photoreceptor terminals, which are known to express P2 purinergic-receptors. In order to assess function, dissociated retinal neurons were loaded with fluorescent ATP markers (Quinacrine or Mant-ATP) and the DA marker FFN102, co-labeled with a VNUT antibody and imaged in real time. Fluorescent ATP markers and FFN102 puncta were found to co-localize in VNUT positive neurons and upon stimulation with high potassium, ATP marker fluorescence at the cell membrane was reduced. This response was blocked in the presence of cadmium. These data suggest DA neurons co-release ATP via calcium dependent exocytosis and in the retina this may modulate the visual response by activating purine receptors on closely associated neurons.

Imaging Adenosine Triphosphate (ATP)

“…The analogues mant-ATP and deac-ATP have been extensively used to study kinase and ATPase activities in solution (Fili and Toseland, 2014). For example, mant-ATP has been used to monitor vesicular ATP release from dopaminergic neurons (Ho et al , 2015), and deac-ATP has been used to study the kinetics of myosin Va movement on actin using total internal reflection fluorescence (TIRF) microscopy (Sakamoto et al , 2008). Analogues of ATP modified with Cy3 or BODIPY fluorescent dyes are also available, which shift excitation bands into the visible range to reduce phototoxicity.…”

“…A-Band fluorescence: excitation of the protonated chromophore form of the fluorescent protein. B-Band fluorescence: excitation of the deprotonated chromophore form of the fluorescent protein; CFP, cyan fluorescent protein; Cy5, cyanine dye; YFP, yellow fluorescent protein. a (Akopova et al , 2012); b (Ho et al , 2015); c (Zheng et al , 2009); d (Manfredi et al , 2002); e (Rangaraju et al , 2014); f (Imamura et al , 2009); g (Yaginuma et al , 2014); h (Tantama et al , 2013) …”

Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling

“…VNUT co-localizes with VGLUT1 or VGAT in some populations of synaptic vesicles, as determined by quantitative immunoelectron microscopy [60]. VNUT is also expressed in tyrosine hydroxylase-positive dopaminergic neurons of the substantia nigra and ventral tegmental area, and in subpopulations of rat dorsal root ganglion neurons [61,62]. VNUT is also present in the nerve terminals of enteric musculomotor neurons and co-localizes with vesicular acetylcholine transporter (VAchT) [63].…”

Lysosomal solute carrier transporters gain momentum in research

“…Deficiency of lysosomal proteins in Niemann–Pick type C1 disease or Niemann–Pick type C2 diminish cholesterol efflux from the lysosomal compartment, leading to abnormal lysosomal cholesterol accumulation. Correspondingly, a recent finding identified SLC17A9 in lysosome‐mediated adenosine triphosphate (ATP) release . Because ATP is an important neurotransmitter, lysosomal exocytosis maintains regulated ATP release from neurons and/or astrocytes.…”