ABSTRACT
Neurotransmission at dopaminergic synapses has been studied with techniques that provide high temporal resolution, but cannot resolve individual synapses. To elucidate the spatial dynamics and heterogeneity of individual dopamine boutons, we developed fluorescent false neurotransmitter 200 (FFN200), a vesicular monoamine transporter 2 (VMAT2) substrate that selectively traces monoamine exocytosis in both neuronal cell culture and brain tissue. By monitoring electrically evoked Ca(2+) transients with GCaMP3 and FFN200 release simultaneously, we found that only a small fraction of dopamine boutons that exhibited Ca(2+) influx engaged in exocytosis, a result confirmed with activity-dependent loading of the endocytic probe FM1-43. Thus, only a low fraction of striatal dopamine axonal sites with uptake-competent VMAT2 vesicles are capable of transmitter release. This is consistent with the presence of functionally 'silent' dopamine vesicle clusters and represents, to the best of our knowledge, the first report suggestive of presynaptically silent neuromodulatory synapses.
Subject(s)
Corpus Striatum/metabolism , Dopamine/metabolism , Exocytosis/physiology , Fluorescent Dyes/metabolism , Presynaptic Terminals/metabolism , Synaptic Vesicles/metabolism , Animals , Cells, Cultured , Corpus Striatum/chemistry , Dopamine/analysis , Female , Fluorescent Dyes/analysis , HEK293 Cells , Humans , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neurotransmitter Agents/analysis , Neurotransmitter Agents/metabolism , Organ Culture Techniques , Presynaptic Terminals/chemistry , Synaptic Vesicles/chemistryABSTRACT
Chemical synapses are not only fundamental functional units of the brain but also anatomical and functional biomarkers of numerous brain disorders. Therefore, new experimental readouts of synaptic function are needed--with the spatial resolution of single synapses and the scale to image large ensembles of synapses in specific circuits--for the study of both acute and chronic effects of pharmacological agents on synaptic plasticity in living mammals. In this article we discuss the design and use of fluorescent false neurotransmitters (FFNs) as an important step in the development of versatile synaptic imaging platforms. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.
Subject(s)
Neurotransmitter Agents/metabolism , Optical Imaging , Synapses/metabolism , Synaptic Transmission/physiology , Animals , Fluorescent Dyes , HumansABSTRACT
To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation inâ vivo and inâ vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.
Subject(s)
Fluorescent Dyes/metabolism , Neurons/metabolism , Animals , Boron Compounds/chemistry , Boron Compounds/metabolism , Cells, Cultured , Fluorescent Dyes/chemistry , Microscopy, Confocal , Microscopy, Video , Neurons/cytology , Rats , Staining and LabelingABSTRACT
CD38 is a type II transmembrane glycoprotein with multiple functions. It acts as an ecto-enzyme as well as a receptor. The enzymatic activity catalyzes the formation of two potent Ca(2+) releasing agents: cyclic adenosine diphosphate ribose (cADPR) from nicotinamide adenine dinucleotide (NAD) and nicotinic acid adenine dinucleotide phosphate (NAADP) from NAD phosphate (NADP). The receptor function of CD38 leads to the phosphorylation of intracellular signaling proteins and the up-regulation of cytokine production in immune cells. These two functions of CD38 underlie its involvement in various biological processes, such as hormone secretion, immune cell differentiation, and immune responses. Clinically, CD38 is used as a negative prognosis marker for chronic lymphatic leukemia (CLL). However, a clear molecular understanding of CD38's role in physiology and pathology is still lacking. To facilitate the study of CD38 at cellular and molecular levels, here we report a mechanism-based method for fluorescently labeling CD38 on live cells. This labeling method does not interfere with the receptor function of CD38 and the downstream signaling. The labeling method is thus a useful tool to study the receptor function of CD38 in live cells. In addition, since the mechanism-based labeling also inhibits the enzymatic activity of CD38, it should be useful for dissecting the receptor function of CD38 without interference from its enzyme function in complicated biological processes.
