The neostriatum (caudate-putamen, CPu), input nuclei of the basal ganglia, plays a key role in the motor control and cognitive function. Medium-sized spiny neurons (MSNs) constitute more than 90% of the neostriatal neurons and can be divided in two different subtypes, direct and indirect pathway neurons. The direct pathway neurons express dopamine D1 receptor, and project to the substantia nigra reticulata (SNr) and entopeduncular nucleus (EP). In contrast, indirect pathway neurons are characterized by the expression of D2 receptor and send axons only to the external globus pallidus (GPe). However, it was recently demonstrated that not only indirect but also direct pathway neurons innervate the GPe (Fujiyama et al., 2012). In the present study, we investigated whether or not direct and indirect pathway neurons located in vicinity send axon fibers to the same region(s) of the GPe in a topological manner.

To visualize the direct and indirect pathway axons separately, we injected adeno-asociated virus (AAV2/1) into the CPu of D1-Cre transgenic mice. The virus expresses GFP or tdTomato in the presence or absence of Cre, respectively, by using a Cre-dependent genetic switch (FLEx switch). Thus, D1-positive direct pathway MSNs were labeled with GFP, whereas indirect pathway MSNs and some interneurons were visualized by tdTomato. Since MSNs have numerous spines on the dendrites, we can easily distinguish indirect pathway MSNs from interneurons.

After we observed the location of the direct (GFP) and indirect (tdTomato) pathway neurons in the CPu, we analyzed their axonal arboraizations in the GPe. Both types of neurons often made two axonal bushes at the edge of the GPe, and sent varicose axons to mostly ovelaping regions in the GPe. In addition, when both types of neurons were located medialy or lateraly in the CPu, those arborization was observed medialy or lateraly in the GPe, respectively.