Author:

Keywords:

MONKEY, VIRAL VECTORS, PROMOTER SPECIFICITY

Abstract:

Adeno-associated viral (AAV) vectors are widely used for the delivery of genes coding for light-sensitive proteins for in vivo optogenetics. Although different AAV vectors have been profoundly studied in rodents (Clearly et al. 2008, Taymans et al. 2007, Yizhar et al. 2011), information on their transduction properties in nonhuman primates remains limited (Dodiya et al. 2010, Markakis et al. 2010) certainly for cortical structures (Diester et al. 2011). In this study, we aimed to compare transduction selectivity and sensitivity of various promoters in an AAV2/7 serotype in the cortex of macaque monkeys. Specifically, we injected equal volumes of different AAV2/7 vectors encoding eGFP driven by either a short (0.4kb) or a long (1.3 kb) version of the CaMKII or a universal CMV promoter in primary visual cortex of two rhesus monkeys. Immunohistochemistry revealed distinct eGFP-expression in V1 neurons for the two AAV2/7-CaMKII-viral vectors. More specifically, expression under the control of the CaMKII 0.4 promoter showed eGFP+-cells in layers 2 to 6, with an especially strong labeling in all sub-compartments of layer 4. A profoundly different spread and laminar transduction pattern emerged after injection with the long CaMKII-promoter. First of all, the total transduced volume was ~ 10 fold larger with the long CaMKII promoter. Also, eGFP+-cells were only visible in layers 2, 4a/b, 5 and 6, but prominently absent in layer 4C. Furthermore, we tested the cell specificity of the CaMKII-promoter transduced cells with stainings for parvalbumin (inhibitory cells) and phosphate-activated glutaminase (excitatory cells). The short CaMKII-promoter showed expression exclusively in excitatory neurons, while the longer CaMKII 1.3 promoter also showed expression in few inhibitory neurons. Furthermore, injections of both CaMKII-AAV2/7-vectors resulted in retinotopic-specific retrograde projections (fibers and axon terminals) within the LGN and the inferior pulvinar. Retrogradely labeled cells in the LGN were only visible after injection of the long CaMKII-promoter in V1. These results suggest that both AAV2/7-CaMKII vectors are efficient in transducing V1-neurons, but that compared to the short CaMKII promoter, the 1.3 kb-CaMKII promoter results in more widespread transduction in different layers and also drives expression in a small fraction of inhibitory cells. Additionally, the CMV-promoter in AAV2/7-viral vectors was surprisingly weak in primate V1. In general, these results suggest that quite different functional and behavioral effects may be expected in optogenetic monkey studies even when using the same AAV serotype with similar promoter sequences.