MOLECULAR NEUROSCIENCE NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 12 No 18 21 December 2001 4081 Neurotrophin receptor expression and responsiveness by postnatal cerebral oligodendroglia G. D. Starkey, S. Petratos, K. A. Shipham, H. Butzkueven, T. Bucci, K. Lowry, 1 S.-S. Cheema 1 and T. J. Kilpatrick CA Development and Neurobiology Group, The Walter and Eliza Hall Institute of Medical Research, Post Of®ce, The Royal Melbourne Hospital Parkville 3050 Victoria; 1 Department of Anatomy Monash University Clayton 3800 Victoria, Australia CA Corresponding Author Received 6 September 2001; accepted 24 October 2001 The low af®nity neurotrophin receptor (p75 NTR ) is implicated in promoting oligodendrocytic death after nerve growth factor (NGF) stimulation but NGF and neurotrophin-3 (NT-3) can also potentiate oligodendrocytic survival. We show regional variability in p75 NTR expression within the central nervous system of the postnatal rat; expression is readily detectable by immunohistochemistry upon a subset of CNPase-positive oligodendroglia in optic nerve but not within the cerebrum. Nevertheless, oligodendroglia isolated from the cerebrum and cultured for 16 hours express p75 NTR as well as the trkC but not the TrkA gene. Viability was not, however, in¯uenced by exposure to either NGF or NT-3. Cells overexpressing p75 NTR remained unresponsive to NGF but exhibited potentiated survival with NT-3, correlating with the differential expression pro®le of their high af®nity receptors. NeuroReport 12:4081± 4086 & 2001 Lippincott Williams & Wilkins. Key words: Development; Oligodendrocyte; p75 NTR ; Rodent INTRODUCTION Oligodendrocytes are responsible for the myelination of the CNS. During development their numbers are regulated by differential survival of the newly differentiated oligo- dendroglia, due to either limited availability of growth factors [1] or activation of cell surface receptors that can induce cell death. The death of cultured oligodendrocytes can be induced by nerve growth factor (NGF), via activa- tion of the low af®nity neurotrophin receptor (p75 NTR ) [2], although the relevance of this ®nding to the in vivo situation is unclear as the experiments were performed on oligodendrocytes that had been subjected to long-term culture. The p75 NTR is reported to be expressed only at low levels by progenitors but at higher levels by cultured mature oligodendrocytes, as assessed by Western blot and RTPCR [3]. However, cultured oligodendroglia can also express both TrkA and TrkC, the high af®nity receptors for NGF and neurotrophin-3 (NT-3), respectively [3±5], and some investigators have shown that neurotrophins can promote the survival of cultured oligodendrocytes, pre- sumably via activation of these high af®nity receptors. In particular, Cohen et al. [3] have found that NGF poten- tiated the survival of oligodendrocytes that expressed both p75 NTR and TrkA and Barres et al. [6] reported that oligodendrocytes isolated from the postnatal optic nerve exhibit unregulated survival in response to NT-3. In this study we focus on identifying the neurotrophin receptor expression and neurotrophin responsiveness of postnatal oligodendroglia either within or freshly isolated from the brain, rather than amongst cells subjected to prior long-term culture. We directed the study to postnatal day 3, a developmental stage at which oligodendroglia are susceptible to apoptotic death [1]. We found evidence for regulated expression of p75 NTR and differential expression of both TrkA and C, but that viability of the cells was not in¯uenced by exposure to NGF or NT-3 except with over- expression of p75 NTR where the cells exhibited potentiated survival in response to NT-3. MATERIALS AND METHODS Immunohistochemistry: Wistar rat pups at postnatal day (P) 3 were perfusion ®xed with 4% paraformaldehyde and the heads were paraf®n embedded. Coronal sections were cut at 10 ìm, mounted, dewaxed and processed for immu- nohistochemistry. The tissue was double labeled with anti- p75 NTR and cell type speci®c antibodies. The sections were incubated with proteinase K (20 ìg/ml at 378C for 30 min) and washed in PBS. They were post-®xed in 4% parafor- maldehyde, incubated for 2 hr at room temperature (RT) with blocking buffer (10% fetal calf serum (FCS) and 0.3% Triton-X100) and for 2 h at RT with polyclonal anti-human p75 NTR antibody (Promega, USA; 1:200). The sections were