(A) Schematic representation of the bidirectional TetO construct used to express two different ORs (rI7 and M72) from a single locus. In situ hybridization demonstrated that approx. 90% of OSNs expressed rI7 (B) and only approx. 5% of OSNs expressed M72 (C); more detail is presented in supplemental data (Fig. S1). The projection pattern of OSNs expressing tgORs in OiS-animals was monitored using the co-expressed marker gene (D–F). The MOB exhibited a normal distribution and size range of glomeruli. Whole-mount fluorescence of the dorsal OB revealed rI7 and GFP-expressing neurons innervate nearly all glomeruli (D). Although the M72 transgene is expressed in ∼5% of mature OSNs in OiS-mice, staining for LacZ revealed very few glomerular targets for M72-expressing neurons (E, arrowed). The image shown (lateral view) is typical of LacZ staining in this line with most stained fibers restricted to the ventro-caudal region of the bulb. (This is quite different from LacZ staining in other tgOR-LacZ-lines where targeted glomeruli are generally very clearly labeled even when an equal number of MOE neurons express LacZ thus this does not reflect restriction of LacZ to the cell bodies of OSNs.) (F) GFP-fluorescence (green) in a coronal section through the MOB of an OiS-mouse counterstained with DAPI (gray) demonstrates that all glomeruli contained rI7 and GFP-positive fibers. Scale bars: B & C, 100 µm; D & E, 1 mm; F, 200 µm. Medial (M), Anterior (A), and Ventral (V) directions in whole-mount images are indicated.

In situ hybridization was used to monitor expression of c-fos as a measure of neuronal activity in the MOE. Control (A) and OiS- (B) mice showed essentially no c-fos expression after exposure to mineral oil. Exposure of mice to filter paper carrying a 20 µl drop of 10% octanal in mineral oil (MO) induced moderate c-fos expression in a subset (25–40%) of OSNs of control animals (C) and strong expression in the vast majority (≥90%) of OiS-line OSNs (D–E). No systematic differences in the intensity or density of c-fos staining were observed between individual exhibiting no response (D) or strong (E) seizures. Scale bar, 50 µm.

(A) Schematic diagram of the main olfactory bulb (MOB) showing the location of periglomerular (PG), mitral and tufted (M/T), and granular (GC) cells. (B–F) Representative images from in situ hybridization of sections through the MOB using a c-fos probe: mineral oil exposure of control (B) and OiS- (D) mice revealed background c-fos expression that was concentrated in the granule cell layer. Only slight increases in c-fos staining were observed when control mice were (C) challenged with octanal. In contrast, octanal exposure resulted in dramatic differences in MOB neuronal activity of OiS-mice that reflected whether the mouse exhibited symptoms of seizure (E, F). Animals that did not exhibit any symptoms displayed a c-fos expression pattern (E) that resembled c-fos expression in control mice (B, C) or in OiS animals exposed to mineral oil (D). In contrast, OiS-mice that showed strong seizures exhibited a prominent increase in c-fos expression in the M/T but no significant change in activity of PG cells (see G, H for quantitation). In addition, octanal induced seizures were characterized by prominent labeling of the entire granule cell layer. Scale bars: B–F, 500 µm; data are mean ± s.e.m, n = 3 mice; **denotes p<0.01.

In situ hybridization for c-fos was used to monitor the spread of neuronal activity in the piriform cortex and other regions of the brain (see also Fig. 5). Representative coronal sections at approx. Bregma +1.0 of control (A) and OiS-mouse brains (B, C) are shown; boxed area (piriform cortex) in each panel is shown magnified to the right. After exposure to 10% octanal, control mice showed c-fos expression in a sparse and randomly distributed population of cells in the piriform cortex and other regions of the brain (A). A similar pattern of neural activity was observed in OiS-mice if they exhibited no seizure-like symptoms (B). Mice exhibiting strong symptoms of seizures in response to octanal displayed a robust increase in c-fos expression in the piriform cortex with labeling of many neurons in all layers (C). Seizures also triggered massive neural activity in most other regions of the forebrain. (D) Quantitation of c-fos positive cells in the piriform cortex. Scale bars: A–C, 1 mm; 500 µm for magnified boxed area, data are mean ± s.e.m, n = 3 mice; **denotes p<0.01.

In situ hybridization for c-fos was used to monitor the spread of neuronal activity in the brain; shown are representative coronal sections at approx. Bregma −1.3 to −2.0 of control (A) and OiS-mouse brains (B–D). After exposure of mice to 10% octanal, control animals showed a sparse and randomly distributed population of cells expressing c-fos (A). Similar neural activity was observed in OiS-mice exposed to mineral oil (B) or to 10% octanal if mice exhibited no seizure-like symptoms (C). Mice that experienced strong seizures in response to octanal (D) displayed a robust increase in c-fos expression in piriform cortex (Pir) and other M/T cell target areas like the olfactory amygdala (Am). In addition, we always observed increased neuronal activity in many areas of the brain including pronounced activity in the hippocampus (Hip) including the dentate gyrus (D). Scale bar, 1 mm.

Controlled delivery of octanal using an olfactometer results in neural activity patterns that closely resemble those observed when mice are passively exposed to odor. In situ hybridization for c-fos was used to monitor the spread of neuronal activity after exposure of mice to defined concentrations and gradients of octanal. Control mice exposed to 5% octanal for 1 minutes in the olfactometer (A, D, G) exhibit c-fos expression patterns in the MOE (A), MOB (D) and brain (G), including piriform cortex (boxed, G) that closely resemble those observed when control animals were passively exposed to odorant (Figs. 2–4). OiS mice exposed to a gradient of 0–5% octanal over a period of 4 minutes in the olfactometer show activation of the MOE (B) but little change in c-fos expression in the MOB (E) or brain (H). In contrast, OiS mice that were exposed to 5% octanal for 1 minute and exhibited strong seizures not only showed pronounced activation of the MOE (C) but also of M/T and GC cells in the MOB (F) as well as much of the forebrain including piriform cortex (I); see Fig. S3 for quantitation. Scale bars: A–C, 50 µm; D–F, 200 µm; G–I, 1 mm.