CILIARY NEUROTROPHIC FACTOR-IMMUNOREACTIVITY IN OLFACTORY SENSORY NEURONS T. LANGENHAN, a M. SENDTNER, b B. HOLTMANN, b P. CARROLL c AND E. ASAN a * a Institute of Anatomy and Cell Biology, University of Wuerzburg, Koel- likerstr. 6, 97070 Wuerzburg, Germany b Institute for Clinical Neurobiology, University of Wuerzburg, Josef- Schneider-Str. 11, 97080 Wuerzburg, Germany c INSERM U 583, INM, Institut des Neurosciences de Montpellier, Hopital St. Eloi, 80 rue Augustin Fliche, BP 74103,34091 Montpellier Cedex 5, France Abstract—Ciliary neurotrophic factor (CNTF) has been impli- cated in processes of neuroprotection, axonal regeneration and synaptogenesis in the lesioned CNS. In the olfactory system, which is characterized by particularly robust neuroplasticity throughout life, the concentration of CNTF is high even under physiological conditions. In the present study, the cellular lo- calization of CNTF-immunoreactivity was studied in the rat and mouse olfactory epithelium. In both species, individual olfac- tory sensory neurons (ONs) displayed intense CNTF-immuno- reactivity. The number of CNTF-ir ONs varied interindividually in rats and was lower in mice than in rats. In olfactory epithelia of mice expressing -galactosidase under control of the CNTF promoter, cells of the ON layer were immunoreactive for the reporter protein. CNTF-ir ONs were olfactory marker protein- positive and growth associated protein 43-negative. CNTF-ir ONs lacked apoptotic markers, and the number of specifically labeled ONs was apparently unchanged after light chemical lesioning of the epithelium, indicating that CNTF-immunoreac- tivity was not associated with ON death. Electron microscopy of CNTF-ir ON axons in innervated olfactory bulb glomeruli docu- mented that they formed typical ON axonal synapses with target neurons. Three dimensional reconstructions of bulb pairs showed a striking similarity of the positions of glomeruli inner- vated by CNTF-ir ON axons in left and right bulbs of individual animals and interindividually. The number of innervated glo- meruli differed interindividually in rats and was lower in mice than in rats. The results show that in rodents CNTF-immunore- activity occurs in a subset of mature, functionally competent ONs. The localization of target glomeruli suggests that CNTF- immunoreactivity may be associated with the expression and/or activation of specific olfactory receptor proteins. © 2005 Pub- lished by Elsevier Ltd on behalf of IBRO. Key words: olfactory epithelium, neuronal cell death, olfac- tory information processing, electron microscopy. Olfactory sensory neurons (ONs) are the only neurons situated in a surface epithelium, and resemble other epi- thelial cells in that they form typical intercellular junctions with cells surrounding them, creating an epithelial barrier (Miragall et al., 1988; Asan and Meier-Stiegen, 1998). Despite these unusual properties, ONs are true neurons. The single axons of individual ONs run directly into the telencephalon, and terminate in one particular glomerulus of the ipsilateral olfactory bulb (OB). Each ON expresses only one of a large family (1000 members in mice) of odorant receptor proteins (OR). Individual ORs possess high affinity for specific molecular features of odorants, and thus endow the subpopulation of ONs which synthesize them with characteristic odorant response sensitivities. All ONs expressing the same OR project to a limited number of bilaterally symmetrically localized OB glomeruli (Mom- baerts et al., 1996), where they form conventional syn- apses with bulbar projection and interneurons. The topo- graphical arrangement of glomeruli activated by specific odorants represents an olfactory sensory map which ap- pears to be fundamental for olfactory information process- ing (Schwob, 2002; for reviews see Nagao et al., 2002). Since they are directly exposed to the environment, ONs are vulnerable, and massive ON death can be caused by toxic or infectious agents in the air (e.g. Cowan and Roskams, 2002). An acute or gradual loss of the sense of smell, which would be life-threatening at least in macros- matic animals, is prevented by continuous ON neurogen- esis from neuronal precursors situated in the olfactory epithelium (OE; e.g. Mackay-Sim and Chuah, 2000; Schwob, 2002; Nibu, 2002). Newly formed neurons have to regrow their axons into the predestined glomeruli, and to form synapses with target neurons in the OB (Schwob, 2002). Thus, neurogenesis, targeted axogenesis and syn- aptogenesis are permanently occurring in the peripheral olfactory system. Additionally, olfactory learning processes afford continuous plasticity of existing synapses in the OB (Matsuoka et al., 1997). In rodents and primates, more- over, interneurons of the OB are continuously generated in the forebrain subventricular zone, migrate along the “ros- tral migratory stream” into the OB, and are integrated into preexisting neuronal circuits (e.g. Gheusi et al., 2000). Neurotrophic factors are known to be essential for neuroplasticity in the nervous system both during embry- onic development and in the adult and have been shown to be expressed in the peripheral olfactory system at high levels throughout life (Schwob et al., 1992; Mackay-Sim *Corresponding author. Tel: +49-931-312715; fax: +49-931-312712. Abbreviations: a-CNTFm, monoclonal mouse-anti-rat ciliary neurotro- phic factor; BL, basal cell layer; CASP, activated caspase 3; cha- CNTFp, polyclonal chicken-anti-ciliary neurotrophic factor; CNTF, cil- iary neurotrophic factor; DAB, 3,3=-diaminobenzidine; DAPI, 4=,6-dia- midin-2=-phenylindol-dihydrochlorid; GA, glutaraldehyde; ga-CNTFp, polyclonal goat-anti-ciliary neurotrophic factor; GAP43, growth-asso- ciated protein of 43 kD; GOD, glucose oxidase; -ir, -immunoreactive; NDS, normal donkey serum; NGS, normal goat serum; NiDAB, nickel- intensified 3,3=-diaminobenzidine; OB, olfactory bulb; OE, olfactory epithelium; OL, olfactory sensory neuron layer; OMP, olfactory marker protein; ON, olfactory sensory neuron; OR, odorant receptor protein; PBS, phosphate-buffered saline; PFA, paraformaldehyde; ra-CNTFp, polyclonal rabbit-anti-rat ciliary neurotrophic factor; RT, room temper- ature. Neuroscience 134 (2005) 1179 –1194 0306-4522/05$30.00+0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.05.017 1179