How VTA afferent projections are modified by cocaine or appetitive learning is best answered by optogenetic approaches. By injecting CaMKII-driven-ChR2 AAV virus to VTA afferent regions (mPFC, PLH and PPTg), it becomes possible now to isolate specific glutamatergic afferent pathway to quantitate synaptic properties. Preliminary recording results showed that, in terms of short-term plasticity, mPFC-to-VTA synapses showed pair-pulse facilitation and both PLH-to-VTA and PPTg-to-VTA synapses showed pair-pulse depression. In terms of AMPA/NMDA ratio, mPFC-to-VTA synapses showed smallest ratio. Moreover, cocaine selectively potentiated PPTg-to-VTA but not PLH-to-VTA glutamatergic synapses, suggesting existence of input specificity in the VTA. Ongoing experiments are trying to provide electrophysiological or anatomical evidence to explain observed input specificity. To further examine the functional roles of VTA afferents, I used a modified Pavlovian cue-reward association learning, which contained two stages of learning: single tone-reward association learning and two-tone discrimination learning. Instead of using channelrhodopsin (ChR2), Halorhodopsin (Halo3.0), which hyperpolarized neurons while activated by 532 nm light, was selectively expressed in VTA afferent regions (mPFC, PLH or PPTg). The experimental manipulation is to decrease specific afferent input to VTA during acquisition. Preliminary results showed that inhibition of PPTg-to-VTA or mPFC-to-VTA afferents dampen single tone-reward association learning. Inhibition of PLH-to-VTA afferent did not affect the performance in the same learning task. However, inhibition of PLH-to-VTA afferent disrupted two-tone discrimination learning. Further experiments will be needed to understand how information is processed during acquisition by various VTA afferents.