Ultrastructural localization of DREADDs in monkeys

• Type of Material: Article
• Authors: Adriana Galvan, Emory University; Jessica Raper, Emory University; Xing Hu, Emory University; Jean-Francois Pare, Emory University; Jordi Bonaventura, National Institute on Drug Abuse; Christopher T. Richie, National Institute on Drug Abuse; Michael Michaelides, National Institute on Drug Abuse; Sascha A. L. Mueller, University of Wisconsin; Patrick H. Roseboom, University of Wisconsin; Jonathan A. Oler, University of Wisconsin; Ned H. Kalin, University of Wisconsin; Randy Hall, Emory University; Yoland Smith, Emory University
• Language: English
• Date: 2019-09-01
• Publisher: Wiley
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2019 John Wiley & Sons, Inc. All rights reserved.
• Final Published Version (URL): http://dx.doi.org/10.1111/ejn.14429
• Title of Journal or Parent Work: European Journal of Neuroscience
• Volume: 50
• Issue: 5
• Start Page: 2801
• End Page: 2813
• Grant/Funding Information: This work was funded by National Institutes of Health Office of Research Infrastructure Programs OD P51-OD011132 to the YNPRC, P51-OD011106 to the WNPRC, grant R01-MH046729 to NHK and National Institute on Drug Abuse Intramural Research Program support DA00069 to MM.
• Keywords: Axis; HA-tag; Expression; Glutamate; Science & Technology; hM3Dq; Protein-coupled receptors; Life Sciences & Biomedicine; hM4Di; Nonhuman primates; Neurosciences & Neurology; immunogold; mCherry; Neurosciences
• Research Categories: Biology, Molecular; Biology, Neuroscience; Biology, Cell; Health Sciences, Medicine and Surgery

Abstract

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.