Activity-dependent changes in cholinergic innervation of the mouse olfactory bulb

Abstract

The interplay between olfactory activity and cholinergic modulation remains to be fully understood. This report examines the pattern of cholinergic innervation throughout the murine main olfactory bulb across different developmental stages and in naris-occluded animals. To visualize the pattern of cholinergic innervation, we used a transgenic mouse model, which expresses a fusion of the microtubule-associated protein, tau, with green fluorescence protein (GFP) under the control of the choline acetyltransferase (ChAT) promoter. This tau-GFP fusion product allows for a remarkably vivid and clear visualization of cholinergic innervation in the main olfactory bulb (MOB). Interestingly, we find an uneven distribution of GFP label in the adult glomerular layer (GL), where anterior, medial, and lateral glomerular regions of the bulb receive relatively heavier cholinergic innervation than other regions. In contrast to previous reports, we find a marked change in the pattern of cholinergic innervation to the GL following unilateral naris occlusion between the ipsilateral and contralateral bulbs in adult animals.

Figure 1. ChAT-GFP colabels with anti-ChAT antibody and distinctly labels cholinergic fibers with a greater signal to noise ratio so that finer details of the cholinergic fibers can be resolved.

A. Single color channel (A; GFP & B; ChAT) and merged (C) confocal images of a glomerulus (dashed line) in the main olfactory bulb.

Figure 2. Micrographs of main olfactory tissue from Chat-GFP mice show diffuse labeling throughout the bulb with some regions of high labeling in the GL.

A–C . High-resolution composite micrographs (shown for visualization only) of MOB tissue. Plane orientation: Sg - sagittal, Cr - coronal, Hz - horizontal; a = anterior, p = posterior, d = dorsal, v = ventral, l = lateral, m = medial. Layer labels: nl - nerve; gl - glomerular; epl - external plexiform; gr - granule cell; ml - mitral cell; ipl - internal plexiform. Scale bar indicates 500 µm. A. Parasagittal section. Arrow points to region of relatively heavy GFP labeling in the anterior region of the bulb. Arrow head indicates region where relatively little labeling is found. B. Coronal section. oc - orbital cortex. aob - accessory olfactory bulb. aon - anterior olfactory nucleus. C. Micrograph of a coronal cross-section of the MOB and AOB. Arrow points to heavily staining glomeruli shown in inset. C inset . High-resolution micrograph of glomeruli with a relatively high amount of GFP staining. D . High-resolution micrograph of the AOB showing that the relatively light GFP staining in the anterior portion of the structure did not co-label with G_oα labeled in red. E–F . Heavily-stained GFP glomeruli do not co-label with PDE 2A (E) or PDE 4A (F). Red = PDE#A, Green = GFP. Scale bar = 100 µm.

Figure 3. The pattern and intensity of GFP staining in the GL and GCL of the MOB across different developmental stages in ChAT-GFP mice.

A–D. Representative cross-sections of MOB cut horizontally from four different age groups: Post-natal day (PD) 02, 06, 12, and adult. Scale bar indicates 100 µm. E–H Average intensity maps of GFP-staining across the glomerular surface of each MOB. Abscissa: rostro-caudal position realigned to the standard bulb where 0 µm corresponds to the anterior end of the bulb. Ordinate: cylindrical coordinates realigned so that 0° corresponds to dorsal, 90° to lateral, 180° to ventral, 270° to medial surfaces of the bulb. The color bar indicates the level of corrected green intensity in the GL of the MOB with warm colors indicating higher levels of green intensity. I. Intensity map from H overlaid on a standardized, three-dimensional representation of the inner GL of the MOB showing the heterogenous staining pattern on the lateral surface of the bulb. Axes range: d – dorsal, v- ventral; r – rostral, c – caudal; m – medial, l – lateral. J–L. Changes in GFP intensity in the glomerular (GL) and granule cell (GC) layers of the bulb across age groups. J. The level of GFP intensity in the GL layer significantly increases up to post-natal day (PD) 12 and then decreases in adult animals (Anova p-value: 3×10⁻³⁶). A multiple comparison test using Tukey's HSD criterion shows: ** PD12 mean GL intensity is significantly greater than all other age groups. * PD06 and Adult mean GL intensities are significantly greater than PD02, but significantly lower than PD12. They are not significantly different from each other. K. The level of GFP intensity in the GC layer significantly levels out at PD6 and then decreases in adult animals (Anova p-value: 3×10⁻³⁶). L. GC intensity subtracted from GL intensity highlighting the change in relative intensity between GC and GL during development.

Figure 4. Standardized and normalized GFP intensity maps show significant differences in the distribution of GFP intensity between PD12 and adult age MOBs.

A–B. GFP intensity maps realigned to standardized rostro-caudal and dorso-ventral positions. C. A map of p-values generated from a bin-wise Mann-Whitney U test comparing bins with identical cylindrical coordinates across the PD 12 and adult GFP intensity maps. Each p-value is calculated from a U-test comparing bins from intensity maps across both occluded and non-occluded bulbs that have identical cylindrical coordinates. p-values that fall below the false discovery rate (0.014, hatch on the color bar) are circumscribed in black and indicate regions on the map that exhibit a statistically significant change in the level of GFP intensity D–F. Probability map from A–C overlaid on a 3D reconstruction of the olfactory bulb (standard bulb). Wire mesh in F indicates the bins compared in C.

Figure 5. Naris occlusion abolishes differential GFP staining pattern in adult animals.

A. Significantly lower TH Intensity in occluded bulbs as compared to non-occluded bulbs confirmed that the occluded bulbs had reduced olfactory activity in both PD12 animals and adult animals (t-test, p-value = 1×10⁻² and 3.6×10⁻⁴, respectively). B. PD12 animals: mean GFP intensity did not vary significantly between occluded and non-occluded bulbs in either the glomerular or granule cell layers of PD12 bulbs. C. Adult animals: mean GFP intensity fell significantly in both the glomerular and granule cell layers in the occluded bulbs of adult animals (t-test, p-values: 2.2×10⁻³ and 2.4×10⁻⁸, respectively). D–E. Average intensity map in the occluded bulb (E) showed a markedly reduced differential patterning of GFP intensity throughout the GL as compared to the non-occluded bulb. F. p-value map generated from a binwise Mann-Whitney U test where p-value is indicated by color. Statistically significant p-values that fall below the false discovery rate (0.02, as indicated by the black line on the color bar) are circumscribed in black. These circumscribed bins indicate regions of the glomerular layer that differ significantly in GFP intensity between the occluded and non-occluded bulbs. G. The ratio of statistically significant bins per column of positive bins in the U-test map increases from the caudal end of the bulb towards the rostral end as discussed in the text. Magenta- colored bars (indicated by * line) in the bar plot differ significantly from the mean of the green bars (paired-ratio t-test, corrected for multiple comparisons using the false discovery rate, p-value = 0.0071).