A Novel Single Vector Intersectional AAV Strategy for Interrogating Cellular Diversity and Brain Function

Abstract

As the discovery of cellular diversity in the brain accelerates, so does the need for functional tools that target cells based on multiple features, such as gene expression and projection target. By selectively driving recombinase expression in a feature-specific manner, one can utilize intersectional strategies to conditionally promote payload expression only where multiple features overlap. We developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), a single-construct intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches. ConVERGD offers benefits over existing platforms, such as expanded intersectionality, the ability to accommodate larger and more complex payloads, and a vector design that is easily modified to better facilitate rapid toolkit expansion. To demonstrate its utility for interrogating neural circuitry, we employed ConVERGD to target an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus (LC) identified via single-cell transcriptomic profiling to co-express the stress-related endogenous opioid gene prodynorphin (Pdyn). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE+ LC neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

Neuropeptide Y neurons of the locus coeruleus inhibit noradrenergic system activity to reduce anxiety

“…Although species differences might contribute to the observed discrepancy, it is also plausible that methodological limitations previously overestimated NPY-NE colocalization in the LC proper. Using more advanced techniques, recent single-cell and single-nucleus RNA sequencing data corroborated lack of NPY-expressing noradrenergic neurons, both in the mouse 31,60 and in the human 61 LC. Notably, similar to our observations, NPY transcripts were detected outside the LC nuclear core 31 , further validating the existence of a previously undetected discrete pool of NPY-expressing neurons populating the mouse pericoerulean space.…”

Differential control of intestine function by genetically defined enteric neurons

“…In select cases, a combination of two marker genes allowed for faithful isolation of the desired neural population ( Figure 1D ). To use ‘intersectional genetic’ strategies, we generated a series of adaptable dual recombinase dependent AAV reporters by taking advantage of a recently generated small self-cleaving type III hammerhead ribozyme variant (T3H48) with traditional recombinase sites 58,59 . In brief, when the T3H48 sequence is in the 3’ UTR of a transgene, it is rapidly destabilized and degraded, thereby preventing protein translation.…”