Adult neurogenesis and specific replacement of interneuron subtypes in the mouse main olfactory bulb

Abstract

Background: New neurons are generated in the adult brain from stem cells found in the subventricular zone (SVZ). These cells proliferate in the SVZ, generating neuroblasts which then migrate to the main olfactory bulb (MOB), ending their migration in the glomerular layer (GLL) and the granule cell layer (GCL) of the MOB. Neuronal populations in these layers undergo turnover throughout life, but whether all neuronal subtypes found in these areas are replaced and when neurons begin to express subtype-specific markers is not known.

Results: Here we use BrdU injections and immunohistochemistry against (calretinin, calbindin, N-copein, tyrosine hydroxylase and GABA) and show that adult-generated neurons express markers of all major subtypes of neurons in the GLL and GCL. Moreover, the fractions of new neurons that express subtype-specific markers at 40 and 75 days post BrdU injection are very similar to the fractions of all neurons expressing these markers. We also show that many neurons in the glomerular layer do not express NeuN, but are readily and specifically labeled by the fluorescent nissl stain Neurotrace.

Conclusion: The expression of neuronal subtype-specific markers by new neurons in the GLL and GCL changes rapidly during the period from 14-40 days after BrdU injection before reaching adult levels. This period may represent a critical window for cell fate specification similar to that observed for neuronal survival.

Figure 1

BrdU-positive cells and also cells staining for the biochemical subtype markers CB, CR, GABA, TH, and NC are differentially distributed throughout the olfactory bulb. Low magnification images (10×) of sagittal sections through the olfactory bulb show positive immunoreactivity for (A) BrdU, (B) CB, (C) CR, (D) GABA, (E) TH, and (F) NC predominately within the GLL and GCL. Scale bars, 100 μm. CB, calbindin; CR, calretinin; TH, tyrosine-hydroxylase; NC, N-copine; GLL, glomerular layer; GCL, granule cell layer.

Figure 2

Adult born neurons express CR, CB, and TH. 40× images with arrowheads indicating double positive cells for BrdU (red) and the subtypes CR, CB, and TH (green). Scale bars, 37 μm. (A and B) BrdU co-labels with CR in the GLL (A) and the GCL (B). BrdU co-labels with CB (C) and TH (D) in the GLL. CB, calbindin; CR, calretinin; TH, tyrosine-hydroxylase; GLL, glomerular layer; GCL, granule cell layer.

Figure 3

Adult born neurons express GABA and NC. 40× images of sagittal sections with arrowheads indicating double positive cells for BrdU (red) and the subtypes GABA and NC (green). Scale bars, 37 μm. (A and B) BrdU co-labels with GABA in the GLL (A) and the GCL (B). (C and D) BrdU co-labels with NC in the GCL (C) and TH (D) in the GLL. NC, N-copine; GLL, glomerular layer; GCL, granule cell layer.

Figure 4

The neuronal specific marker NeuN does not label all GLL neurons. 10× images of sagittal sections shown to compare the immunofluorescent staining of NeuN (A) to that of the fluorescent Nissl stain NT (B) within the olfactory bulb. NT stains a much larger number of cells in the GLL. Scale bars, 148 μm. NeuN, neuronal nuclei; GLL, glomerular layer; NT, NeuroTrace; GCL, granule cell layer; MCL, mitral cell layer; EPL, external plexiform layer.

Figure 5

Adult born neurons express the neuron specific marker NT in the SVZ and proximal RMS 12 hours after the final BrdU injection. 40× images of sagittal sections through the olfactory bulb of SVZ showing BrdU (red) and NT (green). Co-localization is seen within the SVZ (A) and the proximal RMS (B). Scale bars, 37 μm. NT, NeuroTrace; SVZ, sub-ventricular zone; RMS, rostral migratory stream, BrdU, bromodeoxyuridine.

Figure 6

The fractions of biochemical markers expressed by adult born neurons are similar to those expressed by the overall population of olfactory bulb neurons labeled with NT. The percentage of BrdU or NT positive cells that also express NC, CR, GABA, TH, and CB in the GCL (A) and GLL (B). Data for all markers are similar except for GABA, which is expressed in significantly more new neurons (BrdU+) than in the overall population (NT+). Also shown, data from confocal imaging of new GCL neurons double labeled for Calretinin, GABA and N-copein. BrdU, bromodeoxyuridine; NT, NeuroTrace; NC, N-copine; CR, calretinin; TH, tyrosine-hydroxylase; CB, calbindin; GCL, granule cell layer; GLL, glomerular layer.

Figure 7

The biochemical subtype expression of adult born neurons is dynamic from 14–75 days after the final BrdU injection. The percentage of BrdU cells that co-expressed NC, CR, GABA, TH, and CB in the GCL (A) and GLL (B) at 7, 14, 20, 40 and 75 days post final BrdU injection. BrdU, bromodeoxyuridine; NC, N-copine; CR, calretinin; TH, tyrosine-hydroxylase; CB, calbindin; GCL, granule cell layer; GLL, glomerular layer.