2 to 4.8 mm angled at 45° along the long axis to ensure targeting to the MC layer (Figure 1A). In this study, as reported previously by Kay and Laurent (1999) and Rinberg et al. (2006), no spikes were detected while the electrodes traversed the granule cell layer. Once the electrode reached the ventral MC, layer spikes with amplitudes ranging from 100 to 2000 μV were detected with spontaneous firing frequency characteristic of MCs (Figures S1A and S5, MCL). As shown in Figure S5, recording from the granule cell layer yielded significantly smaller voltage deviations. Recordings from electrodes

displaying only such small voltage deviations were infrequent and PI3K inhibitor were not analyzed to avoid contamination by granule-cell generated multiunit activity. Because granule cell signals were too small to be detected when thresholding based upon recordings in the MC layer, these cells almost certainly do not contribute to the multiunit activity detected in the MC layer. Once the MC layer was reached,

the arrays were fixed in place with titanium skull screws and nail acrylic with one of the titanium screws serving as the ground. Although the electrodes do not record spikes from the granule cells, we term the recorded units “suspected MCs” because our measurements may include some internal tufted cells. All animal procedures were performed under a protocol approved by the institutional animal care and use committee of the University of Colorado Anschutz Medical Campus. Surgical procedures for cannula implantation were based upon the work of Wesson et al. (2008). Briefly, animals were anesthetized as described above, and lidocaine click here was injected into the epidermis above the frontal nasal bone as a local anesthetic. An incision was made down the midline and the skull was cleaned with 3% H202. Next, a hole was drilled 1 mm anterior to the frontal/nasal fissure and 1 mm lateral from the

midline. A hollow cannula was then lowered into the hole and fixed in place with nail acrylic. Mice were anesthetized with nembutal (100 mg/kg) and perfused with 4% paraformaldehyde. Fixed heads were placed in PBS containing 5% Prohance Sodium butyrate (Bracco Diagnostics Inc, Princeton, NJ) and1% distilled H2O for 2 weeks prior to imaging. Imaging experiments were conducted on a Bruker Biospec 7-T horizontal-bore system (Bruker Inc, Billerica, MA) controlled with Paravision 4.0 software. The brain specimens were placed inside a sealed container filled with Fomblin liquid (Solvay Slexis, West Deptford, NJ) to minimize artifacts arising from air-tissue interface. A standard 3D Fast Spin Echo sequence was used to acquire the 256 images for each head (repetition time, 500 ms; echo time, 8.6 ms; echo train length, 4; number of averages, 4; scan time, 11 hr 22 min). The imaging resolution was 78 μm isotropic. Volumes were constructed using ImageJ 1.42q software and final images were contrast enhanced using Photoshop 6.0.