, 2004). Perhaps Shh function is critical not only for the development of ventral structures in these patients, but it may also have roles in human cortical circuit formation as well. It will be interesting to determine if changes in behavior or learning and memory result as a consequence of the loss of Shh or Boc in the cerebral cortex of mutant mice. Precision Selleck Birinapant and specificity of synaptic connectivity is essential for normal brain function. In the cerebral cortex axons must traverse both short and long distances in order to make connections with their proper synaptic partners. Identifying the molecular mediators of synaptic specificity will

be key to understanding mechanisms regulating the construction of cortical circuits. Here we have shown that

the secreted protein, Sonic Hedgehog, functioning through its receptor Boc, has a role in conferring synaptic specificity to neurons that are part of a stereotypical cortical microcircuit. Freshly dissected P21 and 8-week-old mouse brains were incubated in the dark in Golgi solution A+B (FD Rapid Golgi Stain Kit, PK401, FD NeuroTechnologies) for 2–3 weeks. After incubation, all brains were washed thoroughly with Solution C for 2 days at room temperature, and then mouse brains were blocked and embedded in OCT embedding medium (Tissue-Tek). Coronal sections (150 μm) through the medial somatosensory cortex were cut with a Leica CM3050 cryostat and mounted on 3% gelatin-coated

slides. Staining procedures were followed as described (FD NeuroTechnologies), and slides were dehydrated in ethanol and mounted with Permount (Fisher LY294002 in vivo Scientific) for microscopy. Pyramidal neurons from layers II, III, and V of the primary motor and medial somatosensory cortex were used for analysis. Animals were maintained according to protocols approved by the Institutional Animal Care and Use Committee at UCSF. Plasmids were introduced into the developing cortex in vivo by intraventricular injection and electroporation. Intraventricular injections were carried out in E14 timed-pregnant mice, where the morning of the plug is designated embryonic day 1 (E1). Electroporations were performed using an Electro Square Porator ECM830 4-Aminobutyrate aminotransferase (Genetronics) (5 pulses, 50V, 100 ms, 1 s interval). DNA was prepared in endotoxin-free conditions and 1 μl was injected per brain at the following ratios 3:1:1.5:0.5 (3 Boc/control-shRNA: 1 CAG-H2BGFP-2A-MyrTdTomato: 1 AAV-CAG-DIO-Synaptophysin-GFP; 0.5 CAG-Cre), 2:1:1 (2Boc/control-shRNA: 1 CAG-H2BGFP-2A-MyrTdTomato: 1 CAG-ChR2). Boc hairpin RNA vectors were purchased from Open Biosystems (SM2446B12, SM2438G6). Spine density (spines per μm) along Golgi-stained neurons in P21 and adult brains were viewed in coronal sections of pyramidal neurons in layer II/III and V on the basal dendrite between 100–200 μm from the soma.