A single-vector intersectional AAV strategy for interrogating cellular diversity and brain function

• Published in: Nature neuroscience 2024-07, Vol.27 (7), p.1400-1410
• Main Authors: Hughes, Alex C., Pittman, Brittany G., Xu, Beisi, Gammons, Jesse W., Webb, Charis M., Nolen, Hunter G., Chapman, Phillip, Bikoff, Jay B., Schwarz, Lindsay A.
• Format: Article
• Language: English
• Subjects: 13/44; 38/91; 631/1647/338; 631/378/3920; 64/110; Animal Genetics and Genomics; Animals; Behavioral Sciences; Biological Techniques; Biomedical and Life Sciences; Biomedicine; Brain; Brain - cytology; Brain - metabolism; Brain - physiology; Brain research; Dependovirus - genetics; Design; Design parameters; Enkephalins - genetics; Enkephalins - metabolism; Genetic Vectors; Locus coeruleus; Locus Coeruleus - metabolism; Male; Mice; Mice, Inbred C57BL; Narcotics; Neurobiology; Neurons; Neurons - metabolism; Neurons - physiology; Neurosciences; Norepinephrine; Norepinephrine - metabolism; Parameter modification; Payloads; Plasmids; Prodynorphin; Protein Precursors - genetics; Protein Precursors - metabolism; Protein synthesis; Proteins; Rats; Recombinase; Rodents; technical-report; Toolkits
• ISSN: 1097-6256; 1546-1726; 1546-1726
• DOI: 10.1038/s41593-024-01659-7

As discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple features. Here we developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), an adeno-associated virus-based, single-construct, intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver molecular cargo to specific neuronal subtypes. ConVERGD offers benefits over existing intersectional expression platforms, such as expanded intersectional targeting with up to five recombinase-based features, accommodation of larger and more complex payloads and a vector that is easy to modify for rapid toolkit expansion. In the present report we employed ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus that co-express the endogenous opioid gene prodynorphin ( Pdyn ). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn -expressing NE + locus coeruleus neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents. Intersectional adeno-associated viruses are important for neuroscience research but can be limited by complex and bulky design parameters. Hughes et al. present a unique and space-saving approach that simplifies toolkit development and provides expanded functionality.