Abstract
Optogenetics offers unprecedented possibilities to investigate cortical networks. Yet, the number of successful optogenetic applications in non-human primates is still low, and the consequences of opsin expression in the primate brain are not well documented. We assessed histologically if we can target cerebrocortical networks with three common optogenetic constructs (AAV2/5-CaMKIIα-eNpHR3.0-mCherry, -ChR2-eYFP, -C1V1-mCherry). The frontal eye field or the dorsal premotor area of rhesus macaques were virally injected, and the resulting transduction spread, expression specificity, and opsin trafficking into axons projecting to parietal and visual areas were examined. After variable periods (2–24 months), expression was robust for all constructs at the injection sites. The CaMKIIα promoter driven-expression was predominant, but not exclusive, in excitatory neurons. In the case of eNpHR3.0-mCherry and ChR2-eYFP, opsins were present in axonal projections to target areas, in which sparse, retrogradely transduced neurons could also be found. Finally, the intracellular distribution of opsins differed: ChR2-eYFP had almost exclusive membrane localization, while eNpHR3.0-mCherry and C1V1-mCherry showed additional intracellular accumulations, which might affect neuronal survival in the long-term. Results indicate that all three constructs can be used for local neuronal modulation, but axonal stimulation and long-term use require additional considerations of construct selection and verification.
Highlights
Optogenetics offers unprecedented possibilities to investigate cortical networks
All opsin genes were fused with a fluorescent marker (ChR2 with enhanced yellow fluorescent protein—eYFP and eNpHR3.0 and C1V1 with monomeric red fluorescent protein—mCherry), expression was driven by the CaMKIIα promoter, and all constructs were packaged into AAV2/5 vectors
We examined the posterior parietal cortex (PPC; including medial intraparietal area: MIP, and lateral intraparietal area: LIP) and extrastriate visual areas, which are at a substantial distance, and are known to receive input from the injected frontal regions (PMd → PPC31–33, frontal eye field (FEF) → LIP34, and FEF → MT/MST34,35)
Summary
Optogenetics offers unprecedented possibilities to investigate cortical networks. Yet, the number of successful optogenetic applications in non-human primates is still low, and the consequences of opsin expression in the primate brain are not well documented. The prospect of selectively stimulating the axons of projection neurons of area A at their termination zone in a non-injected area B offers a unique way to establish causal interactions that underlie cognitive processes in behaving NHPs13. These two approaches probe different aspects of the neural circuitry and address distinct questions, as shown in r odents[14,15] and NHPs ( see[16] vs.). While such verifications are routinely done in rodents, in NHPs such information is often unobtainable in time, due to the long-term use of experimental animals
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