BDNF promoter-mediated beta-galactosidase expression in the olfactory epithelium and bulb

Abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the generation and differentiation of new olfactory sensory neurons (OSNs) and in the regulation of branching of OSN axons in their target glomeruli. However, previous reports of BDNF mRNA and protein expression in olfactory epithelium and olfactory bulb (OB) have been inconsistent, raising questions on the proposed roles for BDNF. Here, we report on beta-galactosidase (beta-gal) expression in adult gene-targeted mice where the BDNF promoter drives expression of the Escherichia coli lacZ gene (BDNF(lacZneo) mice). We find that beta-gal is expressed in a small subset of OSNs with axons that reach the olfactory nerve layers throughout the OB. In the OB, we find expression of beta-gal in gamma-aminobutyric acidergic but not dopaminergic periglomerular cells and external tufted cells and in interneurons located in the mitral cell layer. Our results are inconsistent with the regulation of generation and differentiation of new OSNs elicited by the release of BDNF from horizontal basal cells. The results are consistent with a role for BDNF in competitive branching of OSN axons within the glomeruli of the OB.

Figure 1

Cell populations of the OE and OB. Left panel: Diagrammatic representation of the OE. BG, Bowman's gland; CN I, cranial nerve I, the olfactory nerve; OEC, olfactory ensheathing cell; Sus, sustentacular cell. Right panel: Cartoon of the OB. GC, granule cell; MC, mitral cell. This figure was modified from a previously published image (Huard et al. 1998) reproduced with permission of Wiley-Liss Inc., a subsidiary of John Wiley and Sons, Inc.

Figure 2

β-Gal immunoreactivity in the OE. (A–C) Double-label immunohistochemistry with β-gal antibody (green) and PGP 9.5, a marker for mature OSNs (red). (A) β-gal alone. (B) PGP 9.5 alone. (C) Merged image of (A) and (B). (D–F) Images of β-gal-positive cells where immunoreactivity was found throughout the cell. BL: basal lamina. Scale bars: 20 μm. Arrowheads denote cilia, and arrows point to axonal processes.

Figure 3

Diagram showing the distribution of β-gal-positive cells in the adult mouse OE. A red asterisk (*) denotes the location of a β-gal-positive cell. (A) Anterior OE. (B) Approximately 800 μm caudal to (A). (C) Posterior OE, approximately 800 μm caudal to (B).

Figure 4

β-gal-positive fibers in the olfactory nerve. (A) β-gal-positive fibers (green) traverse the olfactory nerve layer following the route of PGP 9.5–positive fibers (red). (B) Image of the OB nerve layer and the glomerular layer. CGRP in red; β-gal in green. Notice the lack of overlap of the CGRP and β-gal immunoreactivity. Asterisks mark β-gal-positive PG cells surrounding the glomeruli. Scale bar 20 μm.

Figure 5

Double-label immunohistochemistry (IHC) for PGP 9.5 (red) and β-gal (green) in the OB of BDNF^lacZneo heterozygous mice. (A, D) β-gal IHC (green). (B, E) PGP 9.5 IHC (red). (C, F) Combined β-gal/PGP 9.5 images. (A) to (C) are lower magnification than (D) to (F). Arrows indicate PGP 9.5+/β-gal+ juxtaglomerular cells. Arrowheads indicate PGP 9.5+/β-gal+ cells in the EPL. Square in (C) indicates region shown in (D) to (F). Scale bar: 20 μm.

Figure 6

Double-label immunohistochemistry (IHC) for β-gal in green and either tyrosine hydroxylase (TH, A through F) or GAD67 (G through L) in red in the OB of BDNF^lacZneo heterozygous mice. TH and GAD67 are markers for 2 different populations of PG cells. (A, D) β-gal IHC (green). (B, E) Corresponding TH IHC (red). (C, F) Combined β-gal/TH images. Square in (C) indicates region shown in (D) to (F). (G, J) β-gal IHC (green). (H, K) Corresponding GAD67 IHC (red). (I, L) Combined β-gal/GAD67 images. Arrows indicate β-gal-negative/GAD67-positive cells. Square in (C) indicates region shown in (D) to (F). Scale bar: 20 μm.

Figure 7

Immunohistochemistry (IHC) for GFAP and β-gal in the OB of BDNF^lacZneo heterozygous mice. (A, D) β-gal IHC (green). (B, E) GFAP IHC (red). (C, F) Combined β-gal/GFAP images. Square in (C) indicates region shown in (D) to (F). Scale bar: 20 μm.

Figure 8

Whole mount showing β-gal-reactive cells in blue. Notice the variability of β-gal-positive juxtaglomerular cell density throughout the OB surface. (A) Dorsal view of BDNF^lacZneo mouse OBs and rostral cortex processed with X-gal reaction. (B) Ventral view. (C) Ventromedial view. Arrow indicates putative necklace glomeruli. (D) β-gal immunohistochemistry of putative necklace glomerulus. Scale bar: 20 μm.