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

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.

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

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.

Diet composition, not calorie intake, rapidly alters intrinsic excitability of hypothalamic AgRP/NPY neurons in mice.

Obesity is a chronic condition resulting from a long-term pattern of poor diet and lifestyle. Long-term consumption of high-fat diet (HFD) leads to persistent activation and leptin resistance in AgRP neurons in the arcuate nucleus of the hypothalamus (ARH). Here, for the first time, we demonstrate acute effects of HFD on AgRP neuronal excitability and highlight a critical role for diet composition. In parallel with our earlier finding in obese, long-term HFD mice, we found that even brief HFD feeding results in persistent activation of ARH AgRP neurons. However, unlike long-term HFD-fed mice, AgRP neurons from short-term HFD-fed mice were still leptin-sensitive, indicating that the development of leptin-insensitivity is not a prerequisite for the increased firing rate of AgRP neurons. To distinguish between diet composition, caloric intake, and body weight, we compared acute and long-term effects of HFD and CD in pair-fed mice on AgRP neuronal spiking. HFD consumption in pair-fed mice resulted in a significant increase in AgRP neuronal spiking despite controls for weight gain and caloric intake. Taken together, our results suggest that diet composition may be more important than either calorie intake or body weight for electrically remodeling arcuate AgRP/NPY neurons.