Exp Brain Res (2010) 205:215–221 DOI 10.1007/s00221-010-2355-7 123 RESEARCH ARTICLE DiVerential regulation of axon outgrowth and reinnervation by neurotrophin-3 and neurotrophin-4 in the hippocampal formation Daniel Hechler · Francesco Boato · Robert Nitsch · Sven Hendrix Received: 11 August 2009 / Accepted: 30 June 2010 / Published online: 17 July 2010  Springer-Verlag 2010 Abstract In this study, we investigated the hypothesis whether neurotrophins have a diVerential inXuence on neu- rite growth from the entorhinal cortex depending on the presence or absence of hippocampal target tissue. We investigated organotypic brain slices derived from the entorhinal-hippocampal system to analyze the eVects of endogenous and recombinant neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on neurite outgrowth and reinnerva- tion. In the reinnervation assay, entorhinal cortex explants of transgenic mice expressing enhanced green Xuorescent protein (EGFP) were co-cultured with wild-type hippo- campi under the inXuence of recombinant NT-3 and NT-4 (500 ng/ml). Both recombinant NT-3 and NT-4 signiW- cantly increased the growth of EGFP+ nerve Wbers into the target tissue. Consistently, reinnervation of the hippocampi of NT-4 ¡/¡ and NT-3 +/¡ NT-4 ¡/¡ mice was substantially reduced. In contrast, the outgrowth assay did not exhibit reduction in axon outgrowth of NT-4 ¡/¡ or NT-3 +/¡ NT-4 ¡/¡ cortex explants, while the application of recombinant NT-3 (500 ng/ml) induced a signiWcant increase in the neurite extension of cortex explants. Recombinant NT-4 had no eVect. In summary, only recombinant NT-3 stimulates axon outgrowth from cortex explants, while both endogenous and recombinant NT-3 and NT-4 synergistically promote reinnervation of the denervated hippocampus. These results suggest that endogenous and exogenous NT-3 and NT-4 diVerentially inXuence neurite growth depending on the presence or absence of target tissue. Keywords NT-3 · NT-4 · Hippocampus · Dentate gyrus · K252a · EGFP Introduction After nerve lesion, neurotrophin-3 (NT-3) and neurotro- phin-4 (NT-4) and their receptors exhibit altered expres- sion patterns and functional impact on axon regeneration (Schnell et al. 1994; Prang et al. 2001; Tobias et al. 2003; Alto et al. 2009). Several studies have shown that NT-3 and NT-4 promote axon outgrowth from neuronal cells in single cell cultures: neurite outgrowth from neuroblastoma cells was strongly enhanced by NT-3 and NT-4 (Fryer et al. 1997), while NT-3 was a potent inducer of terminal arborization in single cell cultures of embryonic mouse dorsal root ganglia (DRG) cells and rat trigeminal gan- glion neurons (Ulupinar et al. 2000). NT-4 increased neu- rite outgrowth from DRG neurons of newborn rats as well as from retinal ganglion cells of rats (Cohen et al. 1994; Jerregard et al. 2000). In addition to promote axon outgrowth from isolated neurons, there is increasing evidence that neurotrophins are key factors in reinnervating denervated target tissues. For example, following spinal cord injury, transplantation of The authors D. Hechler and F. Boato contributed equally to this study. D. Hechler · S. Hendrix Institute for Cell Biology and Neurobiology, Center for Anatomy, Charité, Universitätsmedizin, Berlin, Germany F. Boato · S. Hendrix (&) Department of Functional Morphology & BIOMED Institute, Hasselt University, Campus Diepenbeek, Agoralaan Gebouw C, 3590 Diepenbeek, Belgium e-mail: sven.hendrix@uhasselt.be F. Boato · R. Nitsch Institute for Microscopic Anatomy and Neurobiology, University Medicine Mainz, Johannes Gutenberg University Mainz, Mainz, Germany