The use of zebrafish will also open new avenues for addressing these issues. Our tracing data of anatomical connections from the activated

area indicate that this area sends efferents to the Vd, the presumptive zebrafish striatum that expresses precursor genes for Substance P and Enkephalin, two markers of projection neurons in the mammalian striatum (Figures S4K and S4L). Moreover, our tracing data showed that the activated area receives afferents from the midbrain multimodal sensory relay nucleus, the preglomerular nucleus (PG) (Figure 4E, see Supplemental Information and Figures S4M–S4T). Thus, the visual stimulus (i.e., cue) and the somatosensory STI571 supplier PD-1/PD-L1 inhibitor 2 stimulus (i.e., electric shock) information from sensory organs probably enter the activated area of the telencephalon via the PG during learning. Based on its connectivity

and developmental origin, fish PG has been proposed to be part of the thalamus (Mueller and Wullimann, 2009). These results suggest that neurons in the activated area may be a part of the neural circuit homologous to the mammalian corticobasal ganglia circuit. Recently, it was anatomically shown that lamprey, the oldest phylogenetic group of vertebrates, possesses a well-conserved basal ganglia circuit (Stephenson-Jones et al., 2011). Zebrafish can be a good system to further test whether the canonical and functional circuit homologous to the corticobasal ganglia

circuit in mammals is conserved anatomically and functionally in evolution. All surgical and experimental procedures were reviewed and approved by the Animal Care and Use Committees of the RIKEN Brain Science Institute. See also full experimental procedures in the Supplemental Experimental Procedures for Ca2+ imaging, electrophysiological recordings, and other histological studies. Either transgenic HuC:IP or wild-type adult zebrafish were trained in a shuttle tank divided into two compartments of equal size by a hurdle ( Figure 1A). MycoClean Mycoplasma Removal Kit The fish had to cross a hurdle to avoid a mild electric shock delivered as a punishment upon presentation of a red LED lamp given as a cue (avoidance). When a fish achieved the learning criterion by making eight avoidance responses in ten trials, or when a maximum of 60 trials was reached, the training session was terminated. Fish that achieved the learning criterion within three consecutive sessions were considered learners. As control groups, we trained either HuC:IP or wild-type fish in three conditions: cue-alone group, shock-alone group, and cue-shock unpaired group. Cue-alone group fish were given only cue, and shock-alone group fish were given only shock for 35 trials for the first session and ten trials for each of the second and third sessions.