Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization.

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.

Ethnoracial inequities in access to gender-affirming mental health care and psychological distress among transgender adults.

PURPOSE: Transgender people face known barriers to accessing mental health care generally, and gender-affirming care in particular. However, little research has been done to evaluate the impact of systemic racism on access to gender-affirming mental health care (GAMHC) among transgender people of color (TPOC). METHODS: We conducted a cross-sectional, secondary analysis of data on 20,967 respondents to the 2015 United States Transgender Survey who reported a desire for GAMHC services related to their gender transition. We estimated inequities across ethnoracial groups in access to GAMHC, and measured the association between severe psychological distress and access to GAMHC among TPOC. RESULTS: We found decreased access to GAMHC across all TPOC groups. Inequities in access to GAMHC were most severe among assigned male at birth respondents in the Black/African-American group (aOR 0.51, 95% CI 0.37-0.71), Latino/a/e/Hispanic group (aOR 0.52, 95% CI 0.42-0.65), and Native American group (aOR 0.59, 95% CI 0.38-0.94). Among all respondents, severe psychological distress was highest among Native American respondents (47.4%), Latino/a/e/Hispanic (47.1%) respondents, and other/multiracial respondents (46.7%) and lowest among whites (39.9%). Further, among all TPOC, access to GAMHC was associated with decreased odds of severe psychological distress (aOR 0.74, 95% CI 0.62-0.87). CONCLUSION: These results illustrate the need for research that explicitly addresses the intersectional experiences of transgender communities, and the structural drivers of inequities in access to gender-affirming care.

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter.

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.