IMMUNOLOCALIZATION OF RETINOIC ACID RECEPTORS IN THE MAMMALIAN OLFACTORY SYSTEM AND THE EFFECTS OF OLFACTORY DENERVATION ON RECEPTOR DISTRIBUTION K. K. YEE,* AND N. E. RAWSON Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104-3308, USA Abstract—All-trans retinoic acid (ATRA), a metabolite of vi- tamin A, binds to retinoic acid receptors (RARs) to mediate gene transcription in target cells. We previously found that an ATRA supplement enhanced olfactory recovery rate in adult mice after olfactory bulb deafferentation. In this study, we examined the cellular localization of RAR, RAR, and RAR and the effects of surgery and ATRA treatment using immu- nocytochemistry. Mice received a left olfactory nerve tran- section with the right side serving as internal control. One day after surgery, the mice were given either ATRA mixed with sesame oil or just sesame oil. In the unoperated olfac- tory bulb, only RAR immunoreactivity (ir) was observed. In the unoperated right olfactory epithelium, RAR-ir was found in flask-shaped cells located in the supporting cell layer, in cell clusters above the basal cell layer, in cells in the lamina propria, in some respiratory cells and in the olfactory bulb. The flask-shaped cells did not immunostain for either neu- rons or sustentacular cells. RAR-ir was localized only in the respiratory cells while no RAR-ir was observed in the olfac- tory epithelium. The density of RAR-ir cells was higher in the operated left olfactory epithelium and highest after ATRA treatment. This study demonstrates the presence of RARs in the olfactory system, provides additional support that the ATRA-signaling pathway may be involved in the recovery of the olfactory epithelium after injury, and suggests a role for an unstudied cell type in that process. © 2005 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: deafferentation, regeneration, vitamin A, mi- crovillar, mouse. All-trans retinoic acid (ATRA; RA, retinoic acid) has been implicated in morphogenesis during fetal development of the olfactory pathway (LaMantia et al., 1993). Several studies also suggest that retinoids are involved in neuro- genesis in the adult olfactory system. For instance, in vitamin A-deficient adult rats, proliferation of olfactory basal cells increased while mRNA expression levels of olfactory maker protein, a specific neural marker for ma- ture olfactory receptor neurons (ORNs), decreased to 40% of control levels (Asson-Batres et al., 2003). Also, the addition of ATRA to immortalized olfactory clonal cell lines induced differentiation (Illing et al., 2002) and enhanced axonal outgrowth in explant culture of E11.5 embryonic olfactory epithelia on laminin (Whitesides et al., 1998), suggesting ATRA is important in the growth and mainte- nance of mature ORNs. Our laboratory has shown that an oral supplement of ATRA enhanced the rate of olfactory recovery after lesion of the olfactory nerves, suggesting ATRA is also involved in regeneration of the olfactory system (Yee and Rawson, 2000). In the body, ATRA is synthesized from vitamin A and acts as a physiological ligand for three isotypes of nuclear RA receptors (RAR, RAR, and RAR) that belong to the steroid/thyroid/retinoid receptor family (reviewed in Mangelsdorf et al., 1994). These receptors form ho- modimers or heterodimers with retinoid X receptors (RXRs) and bind to specific DNA sequences termed RA response elements to regulate gene transcription. RA sig- naling components have been identified in the normal adult olfactory system. Proteins and mRNA of RARs and RXRs have been detected in the olfactory epithelium (OE) and olfactory bulb (OB) of postnatal and adult mouse (Krezel et al., 1999; Zhang, 1999; Zetterstrom et al., 1999). How- ever, no study has identified the cellular location of RAR expression in the olfactory system after injury or deter- mined the effects of ATRA treatment on RAR expression. In this study, we used a surgery-induced injury model where the olfactory nerves were cut, which led to the degeneration of mature ORNs and produced anosmia or loss of smell, defined as an inability to locate a piece of buried food within 3 min. With time, new ORNs repopu- lated the OE, their olfactory nerves innervated the OBs, and olfactory function was restored (Yee and Costanzo, 1995). Immunocytochemical experiments were conducted to localize RAR, RAR, and RAR expression in the injured olfactory system of young adult mice over different periods of recovery. The results showed that RAR- immunoreactivity (ir) was present in the OE and OB. RAR-ir was observed only in the respiratory cells, whereas no RAR-ir was observed anywhere in the OE or OB. Specifically, RAR-ir was observed in respiratory cells, flask-shaped cells and small clusters in the OE, and cells in the lamina propria. The density of RAR-ir cells increased after surgery and was significantly higher after ATRA treatment. While the functions of the RAR-ir cells in the OE are currently unknown, we propose that these cells *Corresponding author. Tel: +1-215-898-5672; fax: +1-215-898-2084. E-mail address: karenyee@monell.org (K. K. Yee). Abbreviations: ATRA, all-trans retinoic acid; DAB, diaminobenzidine; EDTA, ethylenediaminetetraacetic acid; ir, immunoreactivity; LNX, left olfactory nerve transection; NCAM, neural cell adhesion molecule; OB, olfactory bulb; OE, olfactory epithelium; OMP, olfactory marker pro- tein; ORN, olfactory receptor neuron; PBS, phosphate buffer solution; RA, retinoic acid; RAR, retinoic acid receptor; RT-PCR, reverse transcriptase–polymerase chain reaction; RXR, retinoic acid orphan (X) receptor; SUS, sustentacular. Neuroscience 131 (2005) 733–743 0306-4522/05$30.00+0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.11.011 733