While it is presumed that the dense collection of axons located GDC-0068 supplier in layer V is primarily targeting projection neurons located within this layer, the target of the lost presynaptic terminals cannot be known for certain, and the reduction in synaptophysin-GFP puncta may not be specific to any particular postsynaptic partner. In order to evaluate the functional consequences of reduced Boc expression

and its effect on connectivity to postsynaptic partners located in either layer II/III or layer V, we used an optogenetic approach. We coelectroporated Channelrhodopsin-2 (ChR2) ( Nagel et al., 2003) into layer II/III cortical neurons, thus allowing us to photoactivate the cells and identify their postsynaptic target cells ( Petreanu et al., 2007) ( Figures 8A and 8C). We examined the strength of layer II/III to layer V connectivity by flashing the cells with blue light, causing the ChR2-electroporated neurons to fire action potentials. We

found a significant reduction in the excitatory postsynaptic current (EPSC) amplitude in layer V neurons in which functional Boc had been disrupted when compared to controls ( Figures 8E and 8F). We also tested for a similar deficit in Shh conditional knockouts and found an 8-fold reduction in EPSC amplitude, suggesting that loss of either ligand in the target cell or receptor from the input neuron yields similar deficits in layer V connectivity selleck products ( Figure 8F). In contrast, when we compared the strength of layer II/III to layer II/III connections, we did not find a significant difference in EPSC amplitude between conditions where Boc function is perturbed and control conditions ( Figure 8E). Taken together, these findings suggest that the reduction in presynaptic terminal formation observed science when Boc function is disrupted is specific to layer II/III to layer V cortical circuits, while the strength of connectivity of layer II/III to layer II/III cells remains unchanged. Using a combination of genetics,

anatomy, and electrophysiology, we have shown that Shh and its receptor Boc are required for the formation of specific cortical microcircuits. A common theme in developmental biology is the multitude of cellular functions a signaling molecule will regulate depending upon the cellular context in which it is expressed. Shh is most well known and best characterized for its functions in regulating unspecified populations of stem and progenitor cells to regulate the fate of their progeny. However, as development proceeds, so too does the context of expression, where Shh function shifts from specifying the cell types within the spinal cord to guiding axons to their targets.