(A) Dendrites of frog (Rana pipiens) olfactory bulb mitral cells filled with Calcium Green-1 dextran conjugate by local application of dye to the secondary dendrites. The dye was first dried onto a micropipet and then delivered to the tissue as described in the Basic Protocol. Dendrites were imaged 2 to 3 hr after application of the dye to an intact olfactory bulb maintained in vitro. Panel 1 is a wide-field CCD image focusing on the glomerular layer and showing many dendritic tufts in different glomeruli. The left edge of the image is 700 µm from the dye application site. Panel 2 is a CCD image of one glomerulus with more than one apical dendrite projecting to it. Panel 3 is a maximum intensity projection of 20 optical sections obtained at 1-µm intervals using a two-photon laser scanning microscope. Scale bar corresponds to 50 µm for panel 1, 20 µm for panel 2, and 40 µm for 3. (B) Physiological responses recorded from an olfactory bulb stained as described for A. The upper pair of overlapped traces are fluorescence changes measured in dendrites of a single glomerulus, while the lower, vertically offset traces are the corresponding local field potential responses. Thick traces are responses evoked by a brief puff of odorant applied to the nose in an isolated nose brain preparation maintained in vitro (Delaney and Hall, 1996). Note the odor-evoked oscillations in the local field potential and the large long-lasting elevation of [Ca²⁺] in the dendrites. Thin traces are responses evoked by a single electrical stimulus of the olfactory nerve for the same tuft and local field potential recording site. Odor and nerve stimulation responses have been aligned along the time axis so that the onset of the local field potentials are coincident; local field potential traces are offset vertically for comparison purposes. Scale bars (at 30% F/F) are 400 µV and 0.5 sec. (C) Neurons and presynaptic terminals stained by pressure injection of Oregon Green 488 BAPTA-1 dextran conjugate into frog accessory olfactory bulb (AOB). Panel 1 is a CCD image of fluorescence from the AOB (lower left), the diagonally projecting accessory olfactory tract containing the axons of mitral cells, and the mitral cell presynaptic terminals in the amygdala (upper right). A stimulating electrode is positioned on the AO tract. Action potential evoked fluorescence transients recorded from the terminals in the amygdala are shown in Figure 2.5.2. Panel 2 shows mitral cell somata 150 µm below the surface of a live AOB imaged using two-photon laser scanning microscopy presented as a maximum intensity projection of fifteen optical sections obtained at 1-µm intervals from live tissue. Panel 3 is a maximum intensity projection of three confocal images at 3-µm intervals. Scale bars are 500 µm for 1, 25 µm for 2, and 20 µm for 3.