As previously mentioned, performing virus injections in rats is suitable for validation of virus efficacy and can be used in lieu of primate histology. Our protocol is detailed next. The viral construct to be tested was injected into two cortical areas (usually motor, somatosensory, or posterior parietal cortical areas) from the same hemisphere in two rats. As a control, a viral construct already tested and of known efficiency was injected into the same cortical areas on the other hemisphere. The injections were performed as follows: rats were mounted on a stereotaxic frame (Model 1730, Kopf Instruments) under isoflurane (2%) anesthesia, and under aseptic conditions, a small skin incision () was made to expose the skull above the target areas for injections. Small burr holes () were made at each site to expose the brain with the dura intact. Through each burr holes, the virus was injected at two depths (0.5 and 1.5 mm) with the same micropump-syringe system described previously mounted to a stereotaxic micromanipulator. The injection amount and speed were similar to that used in primates, i.e., of virus per depth at a speed of and 5 min waiting time after injections. After injections were completed, the burr holes were covered with bone wax and the skin was sutured. After allowing at least 3 weeks for opsin expression, the rats were anesthetized with either isoflurane () or ketamine/xylazine () and the sites injected with the investigated virus were exposed with two craniotomies (). The location of viral expression was first determined by investigating the yellow fluorescent protein (YFP) fluorescence (YFP is coexpressed with the opsin) at the brain surface. For this purpose, we delivered 473-nm laser light over the craniotomy and observed the YFP fluorescence with a stereomicroscope carrying a YFP filter. Usually, YFP fluorescence signal was visible at the surface throughout the craniotomy. After verifying the expression and finding the site of peak expression by observing the fluorescence, we validated neuromodulation by inserting an optrode into the region of peak fluorescence intensity and delivering occasional light pulses (, 561 nm) of 1 to 2 s duration and observing single and multiunit spiking activity. Once the functional expression was validated, the rats were perfused with 2% paraformaldehyde to prepare thick histological slices. The slices were investigated for the extent of YFP expression under a fluorescence microscope. This time, the YFP fluorescence intensities and pattern of expressions due to injections of the tested and control viruses were compared. If they appeared to be similar, we concluded that the new viral construct was suitable for primate use.