Oculomotor Roles of the Claustrum and its Projection to Frontal Cortex in Primates

Abstract

The claustrum is the most densely connected brain structure by volume, yet little is known about its functions centuries after it was first described in the literature. Its thin shape and deep location make it challenging to target with classical neurophysiological techniques. In rodents, the study of the claustrum has benefited from recent genetic technologies, like optogenetics. These approaches have implicated the claustrum in a wide range of functions including premotor planning, attention, addiction, place encoding, sleep, salience processing, and pain. Nonetheless, a unified role for the claustrum across brain systems remains to be uncovered. In the animal model most homologous to humans – the non-human primate (NHP) - only five physiological studies of the claustrum are published. The cell- and circuit-specific genetic approaches that have worked well for studies of the rodent claustrum have yet to be translated to the macaque. The overall goal of this dissertation project was to refine and apply viral-mediated genetic techniques in macaques to study the role of the primate claustrum in sensation, cognition, and movement. We selected the well-characterized macaque oculomotor system as a testbed for these experiments. Specifically, we focused on the projection from the claustrum to the frontal eye field (FEF), a key node in the oculomotor system involved in vision, eye movements, and visual cognition. Our central question was: what role – if any – does the claustro-FEF projection play in eye movements?In our quest to identify a virus suitable for our optogenetics experiments in macaques, we anatomically characterized the labeling produced by the retrograde virus AAV2-retro after injecting it in various brain areas, including FEF, in six macaques (Chapter 2). We found that rAAV2-retro was a promising candidate for viral-mediated genetic techniques in macaques. When rAAV2-retro with a CAG promoter was injected in FEF, it produced robust, reliable, and safe retrograde claustrum labeling that was comparable in density and location to that achieved with conventional retrograde tracers. We replicated this finding in two monkeys. Thus, we concluded that rAAV2-retro was suitable for our optogenetic studies of the claustro-FEF projection. Next, we injected a rAAV2-retro-CAG viral construct encoding the inhibitory opsin Jaws into the FEF of a monkey trained on memory-guided and visually guided saccades (MGS and VGS). We optically inhibited putative claustro-FEF neurons while the monkey performed the saccade tasks (Chapter 3). We found that inhibition of putative FEF-projecting claustrum neurons significantly worsened the monkey’s MGS task performance, especially when inhibition was applied at target onset or during the delay period of contralateral target trials. Inhibition during the VGS task had comparatively minimal behavioral effects. Lastly, we used Neuropixels probes to record from claustrum neurons, including a subset of putative claustro-FEF neurons identified via phototagging (Chapter 4). We found that neurons in the general population, as well as putative claustro-FEF neurons, carried visual, delay, saccadic, and reward signals. The population of putative phototagged neurons was enriched in task-related activity compared to the population of non-phototagged claustrum neurons, and delay activity was especially enriched in putative claustro-FEF neurons. Overall, the approaches we developed in this dissertation project permitted the first circuit-specific physiological studies of the claustrum in non-human primates. The results provide the first quantitative description of saccade-related activity in the primate claustrum, which differs in intriguing ways from activity in other oculomotor areas, suggesting unique contributions of the claustrum to visual-saccadic behavior. The outcome of this work, including its technological innovations, elucidates the functions of the claustrum beyond what has been achievable by more conventional approaches, laying the foundation for more sophisticated studies of the claustrum in monkeys.