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Fig. 8. Disconnection of the olfactory bulb from piriform cortex by transection of the olfactory peduncle (bulbotomy) causes apoptosis of relay pyramidal neurons located superficially in layer II after early signaling that involves interneurons in outer layer I. (A) This c-fos/CV-stained preparation taken from an animal 20 h after lesion shows the differentiation between neurons undergoing apoptosis (arrowhead) and activated neurons in layer I (arrows). The c-fos immunoreactive nucleus to the right of the apoptotic neuron belongs to a perivascular cell that becomes activated after the initiation of apoptosis (1) (capillary is indicated with an asterisk). (B) Preparations dually stained for c-fos and GFAP show that activated cells in layer I , as represented in A, include both neurons and glia. Large c-fos(+) nuclei (black arrows) belong to GFAP(-), presumably neuronal, profiles, whereas smaller c-fos(+) nuclei belong to GFAP(+) astrocytes (indicated with red arrows in main frame and also in the Inset). Olfactory tract is at the bottom of the main frame, whereas top corresponds to the beginning of layer II. (Scale bars, 20 m m.)

1. Capurso, S. A., Calhoun, M. E., Sukhov, R. R., Mouton, P. R., Price, D. L. & Koliatsos, V. E. (1997) J. Neurosci. 17, 7372-7384.

Fig. 9. Identification of bulbotomy-induced interneurons in piriform cortex as reelin(+) neurons based on colocalization of CR-50 with c-fos. Dual immunofluorescence for reelin and c-fos reveals activation of two sampled layer I neurons (one on the left and the other on the right column) 14 h after bulbotomy. (Scale bar, 20 m m.)

Fig. 10. A significant portion of layer I neurons activated by bulbotomy colocalize neuronal nitric oxide synthase (nNOS) with glutamic acid decarboxylase (GAD). (A-C) Illustrations of the outer layer I of piriform cortex from a dually stained section prepared 14 h after bulbotomy. nNOS-immunoreactive neurons are represented with numbers 1-8 in A, of which only nos. 1, 2, 7, and 8 colocalize GAD (see green perikaryal fluorescence in B and yellow profiles from image merging in C). (Inset) A higher magnification of outlined region in C to show details of single and double-labeled neurons and also punctate-terminal versus diffuse-perikaryal GAD staining. (Scale bar, 20 m m.) ot, olfactory tract.

Fig. 11. The combination of anterograde tracing of bulb axons/terminals with biotin dextran amine (red) and reelin immunocytochemistry (green) shows that reelin (+) interneurons in layer Ia receive direct contacts from mitral cells. (Left) The epifluorescent/double label rendition (EF) of two confocal images (CF) presented at Right, focusing on two mitral cell contacts on the right lower aspect of the depicted interneuron. The terminal-rich area at the bottom is the olfactory tract. The red-green apposition is further confirmed by X and Y virtual sectioning. Inset contains the confocal image of another example of a reelin(+) neuron contacted by mitral cell boutons (arrowheads in bottom). (Scale bar, 20 m m.)