@article {69839, title = {Whole-brain, all-optical interrogation of neuronal dynamics underlying gut interoception in zebrafish}, journal = {bioRxiv}, year = {2025}, month = {2025 Mar 30}, abstract = {

Internal signals from the body and external signals from the environment are processed by brain-wide circuits to guide behavior. However, the complete brain-wide circuit activity underlying interoception{\textemdash}the perception of bodily signals{\textemdash}and its interactions with sensorimotor circuits remain unclear due to technical barriers to accessing whole-brain activity at the cellular level during organ physiology perturbations. We developed an all-optical system for whole-brain neuronal imaging in behaving larval zebrafish during optical uncaging of gut-targeted nutrients and visuo-motor stimulation. Widespread neural activity throughout the brain encoded nutrient delivery, unfolding on multiple timescales across many specific peripheral and central regions. Evoked activity depended on delivery location and occurred with amino acids and D-glucose, but not L-glucose. Many gut-sensitive neurons also responded to swimming and visual stimuli, with brainstem areas primarily integrating gut and motor signals and midbrain regions integrating gut and visual signals. This platform links body-brain communication studies to brain-wide neural computation in awake, behaving vertebrates.

}, doi = {10.1101/2025.03.26.645305}, url = {https://www.biorxiv.org/content/early/2025/03/30/2025.03.26.645305}, author = {Chen, Weiyu and James, Ben and Ruetten, Virginia M. S. and Banala, Sambashiva and Wei, Ziqiang and Fleishman, Greg and Rubinov, Mikail and Fishman, Mark C. and Engert, Florian and Lavis, Luke D. and Fitzgerald, James E. and Ahrens, Misha B.} } @article {69543, title = {Elucidating and Optimizing the Photochemical Mechanism of Coumarin-Caged Tertiary Amines.}, journal = {J Am Chem Soc}, year = {2024}, month = {2024 Jul 18}, abstract = {

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φ \< 1\%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φ \> 20\%-a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

}, issn = {1520-5126}, doi = {10.1021/jacs.4c03092}, author = {Banala, Sambashiva and Jin, Xiao-Tao and Dilan, Tanya L and Sheu, Shu-Hsien and Clapham, David E and Drenan, Ryan M and Lavis, Luke D} } @article {67946, title = {2,7-Diaminobenzopyrylium Dyes Are Live-Cell Mitochondrial Stains2,7-Diaminobenzopyrylium Dyes Are Live-Cell Mitochondrial Stains}, journal = {ACS Bio \& Med Chem Au}, volume = {2}, year = {2022}, month = {2022 Jun 15}, pages = {307-12}, abstract = {

Small-molecule fluorescent stains enable the imaging of cellular structures without the need for genetic manipulation. Here, we introduce 2,7-diaminobenzopyrylium (DAB) dyes as live-cell mitochondrial stains excited with violet light. This amalgam of the coumarin and rhodamine fluorophore structures yields dyes with high photostability and tunable spectral properties.

}, issn = {2694-2437}, doi = {10.1021/acsbiomedchemau.1c00068}, url = {https://pubs.acs.org/doi/10.1021/acsbiomedchemau.1c00068}, author = {Banala, Sambashiva and Tkachuk, Ariana N. and Patel, Ronak and Kumar, Pratik and Brown, Timothy A. and Lavis, Luke D.} } @article {65368, title = {3D ATAC-PALM: super-resolution imaging of the accessible genome.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Apr 01}, pages = {430-6}, abstract = {

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA{\textendash}fluorescence in situ hybridization (FISH), RNA{\textendash}FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

}, issn = {1548-7091}, doi = {10.1038/s41592-020-0775-2}, url = {http://www.nature.com/articles/s41592-020-0775-2}, author = {Xie, Liangqi and Dong, Peng and Chen, Xingqi and Hsieh, Tsung-Han S. and Banala, Sambashiva and De Marzio, Margherita and English, Brian P. and Qi, Yifeng and Jung, Seol Kyoung and Kieffer-Kwon, Kyong-Rim and Legant, Wesley R. and Hansen, Anders S. and Schulmann, Anton and Casellas, Rafael and Zhang, Bin and Betzig, Eric and Lavis, Luke D. and Chang, Howard Y. and Tjian, Robert and Liu, Zhe} } @article {64780, title = {Probing nicotinic acetylcholine receptor function in mouse brain slices via laser flash photolysis of photoactivatable nicotine.}, journal = {Journal of Visualized Experiments : JoVE}, year = {2019}, month = {2019 Jan 25}, abstract = {

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

}, issn = {1940-087X}, doi = {10.3791/58873}, author = {Arvin, Matthew C and Wokosin, David L and Banala, Sambashiva and Lavis, Luke D and Drenan, Ryan M} } @article {49293, title = {Imaging dynamic and selective low-complexity domain interactions that control gene transcription.}, journal = {Science (New York, N.Y.)}, volume = {361}, year = {2018}, month = {2018 Jun 21}, pages = {eaar2555}, abstract = {

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs), but how they drive transactivation remains unclear. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for novel drugs targeting gene regulatory interactions implicated in disease.

}, issn = {1095-9203}, doi = {10.1126/science.aar2555}, author = {Chong, Shasha and Dugast-Darzacq, Claire and Liu, Zhe and Dong, Peng and Dailey, Gina M and Cattoglio, Claudia and Heckert, Alec and Banala, Sambashiva and Lavis, Luke and Darzacq, Xavier and Tjian, Robert} } @article {49244, title = {Nicotinic cholinergic receptors in VTA glutamate neurons modulate excitatory transmission.}, journal = {Cell Reports}, volume = {23}, year = {2018}, month = {2018 May 22}, pages = {2236-2244}, abstract = {

Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2 neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2 neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2018.04.062}, author = {Yan, Yijin and Peng, Can and Arvin, Matthew C and Jin, Xiao-Tao and Kim, Veronica J and Ramsey, Matthew D and Wang, Yong and Banala, Sambashiva and Wokosin, David L and McIntosh, J Michael and Lavis, Luke D and Drenan, Ryan M} } @article {49121, title = {Photoactivatable drugs for nicotinic optopharmacology.}, journal = {Nature Methods}, volume = {15}, year = {2018}, month = {2018 Mar 26}, pages = {347-50}, abstract = {

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

}, issn = {1548-7105}, doi = {10.1038/nmeth.4637}, author = {Banala, Sambashiva and Arvin, Matthew C and Bannon, Nicholas M and Jin, Xiao-Tao and Macklin, John J and Wang, Yong and Peng, Can and Zhao, Guiqing and Marshall, John J and Gee, Kyle R and Wokosin, David L and Kim, Veronica J and McIntosh, J Michael and Contractor, Anis and Lester, Henry A and Kozorovitskiy, Yevgenia and Drenan, Ryan M and Lavis, Luke D} }