Precise opto- and chemo-genetic inactivation of cortico-cortical projection pathways in a non-human primate model

A fundamental challenge in neuroscience is to understand the functional architecture of brain circuits. Selective targeting of neural circuits has been possible in transgenic mouse Cre-lines, but in non-human primates (NHPs) comparable methods have been limited. While viral approaches in NHPs using AAV’s to deliver opsins reliably produce expression in cortex, targeting specific cell classes is challenging and when applied in awake animals can produce effects that are difficult to interpret or weak. Our preliminary data highlight that there is a unique opportunity in NHP models to achieve greater specificity in opto-genetic manipulations by targeting projection pathways. Specifically, highly efficient retrograde viral vectors have been recently developed to target specific populations of neurons based on their projection patterns. One way to label neurons is to use a viral vector that can travel retrogradely from axon terminals in the target area back to cell bodies in the source area. However, retrograde vectors, such as AAV2-retro, will also directly infect cell bodies in the target area resulting in the labeling of reciprocal projection pathway. This represents a major challenge as nearly all connection pathways in the primate cortex are reciprocally connected. To solve this problem, a two-vector Cre-intersectional labeling method can be adopted, which we have previously established in rodent studies and in our preliminary data have applied in a non-human primate model, the marmoset monkey. The goal of the current proposal is to test and validate this intersectional viral method for inactivating a specific pathway (V1 to MT) in the visual cortex of awake marmosets. Specifically, in Aim 1, we will develop a Cre-intersectional strategy that targets a projection pathway and apply it to the pathway from visual area V1 to MT. We will optogenetically inactivate this pathway, confirm the suppression of projection neurons in V1, and evaluate its impact on downstream visual processing in area MT. In Aim 2, we will target the same projection pathway but instead use a chemogenetic approach with hM4Di, which will suppress projection neuron activity and synaptic transmission through a non- invasive systemic administration of the DCZ ligand. Successful implementation of these methods will provide versatile tools for investigating the functional role of projection pathways between primate cortical or sub-cortical areas, while also enabling non-invasive modulation through DCZ administration. This approach could ultimately contribute towards studies in other NHP models and human gene therapies. Project Number: 1R21NS143084-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: JUDE MITCHELL (+1 co-PI) | Institution: UNIVERSITY OF ROCHESTER, ROCHESTER, NY | Award Amount: $264,250 | Activity Code: R21 | Study Section: Neuroscience of Basic Visual Processes Study Section[NBVP] View on NIH RePORTER: https://reporter.nih.gov/project-details/11302441