We refer to synapses as the sum of all contacts between an axon and the target neuron; contact or contact site as morphologically identifiable apposition between the pre-and postsynaptic membrane. Release site is a physiologically identifiable site of quantal release. A contact can have one or more release sites. Images were acquired with a cooled CCD camera

(Till Imago-QE) in TillVision software. For fluorescence, http://www.selleckchem.com/products/PLX-4032.html the image was binned at 2 × 2; with a 40× objective, each pixel represented one-third micrometer. An LED with peak wavelength at 470 nm and total power of 210 mW (Thor Labs) provided illumination; each image was acquired over a 15 ms period at an overall rate of 15–20 Hz. Fluorescence intensity analysis was performed in TillVision and Microsoft Excel. A region of interest around a hotspot was selected, and two identically sized flanking background regions were averaged and subtracted from the fluorescence signal before carrying out ΔF/F analysis. To determine hotspot dimensions (Figure 2),the ΔF/F image along the longitudinal axis of the dendrite underwent a single pass of 3-pixel (1 μm) boxcar smoothing and traces were aligned at their peaks. We verified that

the high-affinity Ca indicator OGB was not saturated by synaptic input (Figure S4). Hotspot intensity is reported as the average of the first five image buy MAPK Inhibitor Library frames (300–340 ms) following stimulation. Successes of Ca hotspots on a sweep by sweep basis were defined as occurring when the ΔF/F of at least two of the five image frames following synaptic stimulation exceeded 2× the SD of the baseline period. During paired-pulse and 10-pulse analysis, successes of Ca hotspots were defined as occurring when the running integral of the five image frames following the second (or 10th) synaptic stimulation exceeded the mean + 2× SD of the integral of interleaved

single- (or 9-) stimulus trials, MYO10 after all trials were scaled to the same baseline. Slices containing biocytin-filled neurons were processed with diaminobenzidine (DAB) according to standard methods (see Supplemental Experimental Procedures). Neurons were imaged on a Zeiss Imager A1 microscope with a 63× objective (oil, NA 1.4). Cells were traced in Neurolucida 8 (MBF Bioscience). The live fluorescence image and the reconstructed outline were then compared to identify the precise location of the hotspot, and a marker was placed at the corresponding dendritic location. Analysis of morphology was then carried out in Neurolucida Explorer 4. Most reconstructed neurons displayed axons and dendrites that arborized exclusively or predominantly in L4 (axons: 40/49 neurons; dendrites: 39/50). The remaining neurons extended processes to L2/3 and/or L5, and rarely beyond. All reconstructed neurons (50/50) were aspiny. QX-314 in the recording solution precluded characterization based on firing properties.