UC San Diego

Neuronal tracing methods are essential tools to understand the fundamental architecture of the neural circuits and their connection to the overall functional behavior of the brain. Various viral vectors have been developed to achieve cell-type specific and directional specific labeling of the neuronal connections. The incorporation of the viral tracers with optogenetics techniques can further provide precise control over the viral propagation. Herein, a novel approach to guide the infectious pattern of the Rabies Virus (RV) retrograde tracer with light is described. The key concept is to use Baculovirus (BV) as a helper virus to deliver all the functional components prior to the rabies infection. These functional components include the optogenetic tool parts, a fluorescent CRE reporter to mark RV infected cells, and other genes necessary for RV spread. GoldenGate DNA assembly method was used to efficiently construct three large BV expression cassettes in bacteria plasmids that can be transposed into the baculovirus genome in a single TN7 transposon recombination step. The actual imaging of BV and RV infected cells and stimulation of optogenetics tools for the spread of RV can be achieved with CMOS sensor and OLED display panels. By integrating these commercially available electronics with proper microcontrollers, a synchronized communication protocol for optogenetic stimulation, image capturing, and file transferring was established in a lensless imaging setup. The OLED and CMOS sensor’s large field of view and low cost allows this tracing method to scale many orders of magnitude greater than using microscopy-based methods.