EARLY REMODELING IN AN INDUCIBLE ANIMAL MODEL OF RETINAL DEGENERATION S. NAGAR, 1 V. KRISHNAMOORTHY, 1† P. CHERUKURI, 1‡ V. JAIN AND N. K. DHINGRA* National Brain Research Centre, Nainwal Road, NH-8, Manesar (Gur- gaon) Haryana 122050, India Abstract—Photoreceptor degeneration is followed by signif- icant morphological changes in the second-order retinal neu- rons in humans and in several genetic animal models. How- ever, it is not clear whether similar changes occur when photoreceptor degeneration is induced nongenetically, rais- ing the question whether these changes are a general effect of deafferentation independent of the cause of degeneration. We addressed this by inducing selective photoreceptor de- generation with N-methyl-N-nitrosourea (MNU) and studying its effects on inner retinal neurons in a mouse for up to 3 months, using immunocytochemistry and iontophoretic la- beling. To develop objective measures of photoreceptor de- generation and of retinal remodeling, we measured several retinal proteins using immunoblot analysis, and quantified gross visual ability of the animal in a visual cliff test. The MNU-induced progressive degeneration of rods and cones was associated with declining levels of postsynaptic density 95 protein in the retina, and with deteriorating visual perfor- mance of the animal. Müller glial cells showed enhanced reactivity for glial fibrillary acidic protein as demonstrated by immunocytochemistry, which also reflected in increased lev- els of the protein as demonstrated by immunoblotting. Hori- zontal cells and rod bipolar cells progressively lost their dendritic processes, which correlated with a slight decline in the levels of calbindin and protein kinase C alpha respec- tively. Horizontal cell axons, immunoreactive for nonphos- phorylated neurofilaments, showed sprouting into the inner nuclear layer. Ganglion cells and their synaptic inputs, probed by immunolocalizing -III-tubulin, neurofilaments, bassoon and synaptophysin, appeared to be unaffected. These results demonstrate that MNU-induced photoreceptor degeneration leads to retinal remodeling similar to that ob- served in genetic models, suggesting that the remodeling does not depend on the etiopathology that underlies photo- receptor degeneration. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: N-methyl-N-nitrosourea, MNU, retinal remodeling, photoreceptor degeneration, mouse. Retinal degenerative diseases, such as age-related mac- ular degeneration (AMD) and retinitis pigmentosa (RP) are characterized by photoreceptor degeneration, and are among the leading causes of blindness (Margalit and Sadda, 2003; Hartong et al., 2006). In humans and in several genetic animal models the loss of photoreceptors is followed by significant morphological changes in the inner retinal neurons, including dendritic retraction and neuritic sprouting in bipolar and horizontal cells (Milam et al., 1998; Fariss et al., 2000; Strettoi and Pignatelli, 2000; Park et al., 2001; Strettoi et al., 2002, 2003; Jones et al., 2003; Marc et al., 2003; Pignatelli et al., 2004; Cuenca et al., 2005; Jones and Marc, 2005; Gargini et al., 2007; Sullivan et al., 2007; Barhoum et al., 2008). It has been suggested that these secondary changes are similar to those observed after deafferentation in other parts of the brain, and are a consequence of photoreceptor loss irrespective of the underlying cause (Jones et al., 2003; Marc et al., 2003). This hypothesis is supported by the finding that the photoreceptor degeneration leads to similar morphological changes in various genetic models whether the degeneration is early and fast, or delayed and slow (Strettoi et al., 2002, 2003; Jones et al., 2003; Pig- natelli et al., 2004; Gargini et al., 2007; Barhoum et al., 2008). However, in these animal models a genetic muta- tion is present since conception, which could potentially affect retinal cells even before the onset of photoreceptor degeneration, raising the question of whether the second- ary changes are simply the result of photoreceptor loss or are developmental defects, or both. For example, in a transgenic pig with rhodopsin gene mutation the cone- driven inner retinal function is reported to be defective even when the cones themselves are normal (Banin et al., 1999). Furthermore, morphological remodeling in bipolar and horizontal cells has not been unequivocally demon- strated in various non-genetic (inducible) animal models (Peichl and Bolz, 1984; Fisher and Lewis, 2003; Linberg et al., 2006; Marc et al., 2008; Liang et al., 2008). In the present study, we asked whether bipolar and horizontal cells undergo morphological remodeling when selective photoreceptor degeneration is induced nonge- netically. Several inducible models are available that can be produced by physical insults, such as exposure to strong light and intravitreal insertion of iron particles, or 1 These authors have contributed equally. *Corresponding author. Tel: 91-124-2338922-26221; fax: 91- 124-2338910. † Present address of V. Krishnamoorthy: Max Planck Institute of Neu- robiology, Am Klopferspitz 18, 82152 Martinsried-Munich, Germany. ‡ Present address of P. Cherukuri: Developmental Neurobiology Lab- oratory, European Neuroscience Institute Göttingen, Grisebachstr. 5, 370777 Göttingen, Germany. E-mail address: naren@nbrc.ac.in (N. K. Dhingra). Abbreviations: AMD, age-related macular degeneration; BSA, bovine serum albumin; DAPI, 4=,6-diamidino-2-phenylindole; DiI, 1,1=-diocta- decyl-3,3,3=,3=-tetramethylindocarbocyanine perchlorate; EDTA, eth- ylene diamine tetraacetic acid; GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; INL, inner nuclear layer; IPL, inner plexiform layer; MNU, N-methyl-N-nitrosourea; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PBS, phosphate- buffered saline; PKC, protein kinase C alpha; PNA, peanut aggluti- nin; PSD-95, postsynaptic density 95; RP, retinitis pigmentosa; SDS, sodium dodecyl sulfate. Neuroscience 160 (2009) 517–529 0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.02.056 517