Figure 3.

Long-term labeling of cFos-LacZ⁺ neurons activated by fear conditioning. A, Experimental design for cFos-LacZ labeling. Four aCSF injections were performed once a day for four consecutive days to habituate the cFos response to the injection procedure before FDG administration. Control mice were injected with FDG alone without prior training or subsequent contextual exposures. Mice of the training group were injected with FDG immediately after training and left undisturbed for 5 consecutive days. The extinction group consisted of mice exposed to training, FDG injection, and five extinction trials. B, FDG signals (green) were weak in the control group (top). Note strong FDG signals in the groups injected with FDG immediately after training with (middle panel) or without extinction (bottom panel). Background was subtracted using the same threshold for each section to eliminate interference of autofluorescence. C, Control section obtained from a mouse with FDG injected into the brain ventricles showing the effect of a diffusion gradient from the ventricles into the hippocampal tissue on the size of FDG⁺ signals. The size of the signals was smaller than was observed in vitro or after surface application (Nirenberg and Cepko, 1993), probably because of dilution after diffusing within the brain tissue. The size of FDG signals decreased from 10 μm (neurons close to the chorioid plexus) to 0.5–3 μm (100 μm laterally within the hippocampal tissue). D, Image of cresyl violet staining showing an intact CA1 area immediately dorsal to the injection site. E, Micrographs showing lack of cFos-LacZ⁺ labeling under control conditions when primary (left) or secondary (middle) antibody (Ab) was omitted or FDG was not injected (right). F, Micrographs showing nuclear colocalization of FDG and cFos signals. G, Using DAPI as a nuclear counterstain, we determined that most nuclei contained one FDG punctum. Red arrows, cFos⁺ cells; blue arrows, DAPI⁺ nuclei.