Projection-Specific Intersectional Optogenetics for Precise Excitation and Inhibition in the Marmoset Brain
The primate cerebral cortex relies on long-range connections to integrate information between functionally specialized areas. Investigating these processes requires tools that can selectively modulate specific projection pathways. While cell-class-specific optogenetics can modulate local circuits, these approaches often lack pathway specificity. Projection-specific optogenetics offers greater precision, especially in primates, where cortical areas are spatially and functionally well-separated. To address challenges in translating this approach from rodents to primates, we developed a mouse-to-marmoset pipeline. We first validated that optogenetic targeting of inhibitory neurons (AAV9-Dlx-ChR2) effectively silenced local cortical areas in marmosets. We then tested selective excitation and inhibition of defined projection pathways. By intersecting retrogradely delivered Cre-recombinase (AAVretro-Cre) with locally injected Cre-dependent opsins (AAV8-FLEx-ChR2 or Jaws), we achieved efficient, direction-specific labeling of both callosal and longitudinal projections. This intersectional strategy enabled precise excitatory and inhibitory control of cortical activity using distinct light wavelengths, advancing projection-specific optogenetics for investigating primate brain circuit function.

Graphical Abstract

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org.highwire.dtl.DTLVardef@c6fd34org.highwire.dtl.DTLVardef@1997b0org.highwire.dtl.DTLVardef@35bb75org.highwire.dtl.DTLVardef@1a607aa_HPS_FORMAT_FIGEXP M_FIG A: Local suppression of cortical activity that is non-selective across cell classes can be accomplished by excitation of inhibitory interneurons expressing mDlx-ChR2 yielding broad inhibition. B: By contrast, targeting a specific cortico-cortical projection pathway for suppression can be accomplished through an intersectional approach. For a successful intersection, a retrograde AAV carrying hSyn-Cre is injected at the projection terminal and a locally expressing AAV carrying CAG-FLEx-opsin is injected at the soma. These viruses meet at the nucleus and yield opsin expression restricted to projection neurons. C: Inhibition or excitation is possible in targeted projection neurons due to co-expression of the blue light triggered excitatory opsin ChR2 or the red shifted inhibitory opsin Jaws.

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