Surface D2 Autoreceptor Expression on Substantia Nigra Dopamine Neuron Dendrites

G-Protein coupled D2 receptors expressed on dopamine neurons, i.e. D2-autoreceptors (D2Rs), negatively regulate transmitter release and cell firing in axonal terminals and somatodendritic compartments. However, as the membrane distribution of D2Rs at somatodendritic signaling sites has not been fully characterized, functional mechanistic details remain uncertain. This study utilized a knockin mouse to examine surface D2Rs linked at the N-terminus to a superecliptic pHlourin green fluorescent protein epitope (SEP) in dopamine neurons of the substantia nigra. By incubating live slices with a highly specific anti-SEP antibody, the selective labeling of plasma membrane associated receptors was achieved. The SEP-D2Rs appeared as puncta like structures apposed to the surface of dendrites and soma of dopamine neurons. The percentage of D2R expression apposed to TH positive structures varied linearly with the density of TH fibers observed. TH-associated SEP-D2Rs displayed a cell surface density of 0.180 puncta per µm2, which corresponds to an average frequency of 1 punctum every 1.326 µm using nearest neighbor analysis. The distinct punctate appearance of the anti-SEP staining indicates there is a population of D2Rs organized in discrete clusters along the plasma membrane, an arrangement that can spatially localize signaling and influence GPCR efficacy. The results also indicate that D2Rs are spatially distributed on the plasma membrane at a frequency higher than previously reported.

Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

Comparing dopamine release, uptake, and D2 autoreceptor function across the ventromedial to dorsolateral striatum in adolescent and adult rats

Adolescents have increased vulnerability for the development of a range of psychiatric disorders, including substance abuse disorders (SUD) and mood disorders. Adolescents also have increased rates of sensation seeking and risk taking. The mesolimbic dopamine system plays a role in all these behaviors and disorders and reorganization of the dopamine system during adolescence may be important in mediating these developmental changes in behavior and vulnerability. Here, we used ex vivo fast scan cyclic voltammetry to examine developmental differences in dopamine release and its local circuitry regulation across the striatum. We found that adolescents have significantly decreased dopamine release in the nucleus accumbens core across a range of stimulation frequencies that model tonic and phasic firing of mesolimbic dopamine neurons. We show this is not mediated by differences in rate of dopamine uptake, but may be driven by hypersensitive dopamine autoreceptors, indicated by increased inhibition in dopamine release following agonism of D2/D3 receptors, in the adolescent nucleus accumbens core. Additionally, we observed increases in dopamine uptake in the dorsomedial striatum. No other significant differences between release, uptake, or D2 autoreceptor function was observed between adolescent and adult rats in all brain areas tested (nucleus accumbens shell, nucleus accumbens core, dorsomedial striatum, and dorsolateral striatum). These developmental differences in dopamine release may be important in mediating some of the unique behavioral repertoire seen in adolescents, such as increases in sensation seeking, and its associated vulnerabilities.

Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000–1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 µm. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.