Immunohistochemical identification of discrete subsets of rat olfactory neurons and the glomeruli that they innervate

Abstract

Glomeruli at the posterior margin of the main olfactory bulb differ in several respects from those located in the remainder of the bulb; e.g., the olfactory sensory neurons (OSNs) that project here exhibit a distinct biochemical phenotype and signal transduction pathway, the microcircuitry of the glomeruli is substantially altered, and the glomeruli are activated by unconventional odorants. In the present work, we report that the monoclonal antibodies 2C6 and MAb213 label distinct subsets of OSNs in the olfactory epithelium (OE), including their axons to their terminations in the main olfactory bulb (MOB). Neurons immunopositive with 2C6 are concentrated in the cul-de-sacs of ectoturbinates 1 and 2 and of endoturbinate IV. Unlike the vast majority of OSNs, 2C6(+) neurons express olfactory marker protein (OMP) at a low level, but their failure to stain with anti-GAP-43 labeling indicates that the OMP "weak" neurons are nonetheless mature. Glomeruli positive for 2C6 are bilaterally symmetrical and occupy reproducible positions along the posterior margin of the MOB. Three of these are very large, and we refer to them as the lateral, posterior ventral, and anterior ventral 2C6(+) necklace glomeruli. MAb213(+) neurons are concentrated in the posteriormost tips of the cul-de-sacs and recesses at the reflection of the OE at the cribriform plate. Like 2C6(+) neurons, MAb213(+) OSNs are weakly labeled with anti-OMP but are fully mature. MAb213(+) glomeruli are also bilaterally symmetrical; they occupy reproducible positions along the posterior margin of the MOB. The three largest glomeruli occupy lateral, posterior ventral, and posterior positions; the first two are found close to the aforementioned 2C6(+) glomeruli. MAb213 also intensely labels one of the glomeruli of the modified glomerular complex, a string of small glomeruli ventrally, and another string dorsal to the accessory olfactory bulb. Acetylcholinesterase (AChE) histochemical staining of adjacent sections showed that many, but not all, MAb213(+) glomeruli colocalize with dense or moderate AChE staining. Thus, it is likely that the "necklace olfactory glomeruli" (Shinoda et al., 1990, 1993) and the phosphodiesterase (PDE2)(+) glomeruli (Juilfs et al., 1997) are a subset(s) of the MAb213(+) glomeruli. On the other hand, 2C6(+) glomeruli are not associated with AChE staining. These data indicate that the 2C6(+) glomeruli comprise a novel subset in the posterior MOB. In addition to the 2C6(+) and MAb213(+) necklace glomeruli, there is another distinct set of glomeruli at the posterior margin of the bulb that are OMP(-), 2C6(-), and MAb213(-). In summary, the current work indicates that glomeruli at the posterior margin of the bulb, which are necklace glomeruli in terms of location and appearance, are actually heterogeneous and may subserve specialized functions within the olfactory system.

Expression of Pax6 and Sox2 in adult olfactory epithelium

“…Some of the Pax6 + neurons are not stained by MAb213. We suspect that the Pax6 + /MAb213 − neurons are another subgroup of necklace neurons despite lacking the MAb213 epitope, because MAb213 − necklace neurons have been demonstrated previously (Ring et al, 1997).…”

“…That these intensely positive Pax6‐expressing cells are limited to specific regions of the OE is not particularly consistent with that interpretation. Rather, the positioning of such intensely Pax6 + cells across the epithelium is highly reminiscent of the distribution of a population of neurons that projects to the necklace glomeruli at the posterior margins of the glomerular sheet; a large percentage of these atypical or necklace neurons also stains heavily with the mouse monoclonal antibody designated MAb213 (Ring et al, 1997). Accordingly, we performed double labeling with anti‐Pax6 and MAb213 to assess the possibility that these densely Pax6 + cells are necklace neurons (Fig.…”

“…Monoclonal antibody 213‐4.4 (MAb 213) was raised against formaldehyde‐fixed full‐length TGF‐α (Oncogene Science, Uniondale, NY), We have previously shown that MAb 213 labels a subset of olfactory neurons that projects to the necklace glomeruli of the main olfactory bulb under the conditions of fixation used here (Ring et al, 1997). Comparison with other antibodies against TGF‐α indicates that the neuronal labeling is not against the growth factor but against some fixation‐induced, but useful, alternative antigen, which has not been identified (Ring et al, 1997).…”

Neural regeneration and the peripheral olfactory system

“…5). That interpretation is confirmed by demonstrations of mistargeting of neuronal subsets defined by OR expression (i.e., assaying P2 fibers in mice after MeBr lesion) (Iwema and Schwob, 2001) or by expression of other molecular markers that apparently correlate with OR expression, for example MAb 2A4, which labels neurons projecting to a single pair of glomeruli in the normal rat (Carr et al, 1994, 1998), and MAbs 213 and 2C6, which label distinct sets of neurons projecting to largely nonoverlapping groups of necklace glomeruli (Ring et al, 1995, 1997). In these examples, it appears that at least some axons approach the correct glomerular subarray, but the tight convergence onto one or a few grouped glomeruli is seriously distorted (Ring et al, 1995; Carr et al, 1998).…”

“…The example of the necklace glomeruli is particularly informative, as those glomeruli can be identified from animal to animal solely on the basis of position and structure in conventional histological sections (Ring et al, 1997). In cases for which the lesion is severe, the staining with MAb 2C6 offers a clear demonstration that fibers that would normally target the identifiable glomeruli in the posterior bulb end up in more anterior regions, i.e., the fibers approach the usual target area, defined broadly as the set of glomeruli adjacent or near to the normal necklace, but fall short and do not converge onto a bona fide necklace glomerulus (Ring et al, 1995).…”