ANATOMICAL CHANGES IN THE PRIMARY VISUAL CORTEX OF THE CONGENITALLY BLIND CRX/ MOUSE Y. GOLDSHMIT, a S. GALLEY, b D. FOO, a E. SERNAGOR b AND J. A. BOURNE a * a Australian Regenerative Medicine Institute, Monash University, VIC, 3800 Australia b Institute of Neuroscience, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK Abstract—Mutations in the human cone-rod homeobox (Crx) gene are associated with retinal dystrophies such as Leber Congenital Amaurosis (LCA), characterized by complete or near complete absence of vision from birth. The photoreceptors of Crx/ mice lack outer segments, and therefore cannot cap- ture light signals through rods and cones, thus resulting in a lack of normal retinal ganglion cell activity from birth. Using specific antibodies to subsets of neurons and markers of activ- ity, we examined the impact of this absence of sensory input on the development of the primary visual cortex (V1) in early post- natal Crx/ mice, before wiring of the visual system is com- plete, and in adulthood. We revealed that Crx/ mice did not exhibit gross anatomical differences in V1; however, they ex- hibited significantly fewer calcium-binding protein (parvalbu- min and calbindin-D28k) expressing interneurons, as well as reduced nonphosphorylated neurofilament expression in V1. These results reveal that the Crx mutation and lack of light stimulation through the photoreceptor pathway regulate the development and phenotype of different neuronal populations in V1 but not its general morphology. We conclude, therefore, that photoreceptor-mediated visual input during development is crucial for the normal postnatal development and maturation of subsets of cortical neurons. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: retinal dystrophy, photoreceptor, neurofilament, calcium-binding proteins, visual development, mutant. The cone-rod homeobox transcription factor (Crx) plays a pivotal role in the morphological differentiation of both cone and rod photoreceptors, and is the earliest expressed photo- receptor marker in the retina (Chen et al., 1997; Furukawa et al., 1997). Mutation of the human Crx gene results in either congenital blindness or photoreceptor degeneration (Freund et al., 1997; Swain et al., 1997; Freund et al., 1998; Sohocki et al., 1998). The most severe form of inherited retinal blind- ness is Leber congenital amaurosis (LCA), which exhibits complete or near complete absence of vision from birth. Research into the molecular basis of LCA in the last 12 years has revealed the underlying disease genes, demonstrating that the Crx gene is involved in 70% of the cases (den Hollander et al., 2008). Crx-/- mice exhibit a phenotype that closely reflects LCA. Indeed, electroretinograms reveal a complete lack of cone/rod response in the Crx-/- retina (Foxman et al., 1985). The photoreceptors of these mice lack outer seg- ments, resulting in the complete absence of vision from birth (Furukawa et al., 1997). Degeneration of the outer retinal layers commences postnataly at 1 month of age and continues for 3– 4 months, resulting in the complete abla- tion of the outer nuclear and plexiform layers. Despite marked degeneration of the outer retinal layers, the inner retina remains largely unaltered. In the developing vertebrate retina, ganglion cells fire spontaneous bursts of action potentials long before the eye becomes exposed to sensory experience after birth. These early bursts are synchronized between neighbouring retinal ganglion cells (RGCs), yielding unique spatiotemporal patterns: “waves” of activity sweep across large retinal areas every few minutes. Both at retinal and extraretinal levels, these embryonic retinal waves are believed to guide the wiring of the visual system using Hebbian mechanisms of synaptic strengthening (Sernagor and Mehta, 2001). However in the Crx-/- mouse, RGC waves disappear earlier than normal, becoming replaced by large slow oscillations and strong bursting, persisting beyond the onset of de- generation (Adams et al., 2008), but still transmit through to the visual centers of the brain (Pignatelli et al., 2004; Morrow et al., 2005). Visual experience has been clearly demonstrated to play an important role in the patterning of the visual centers of the brain (e.g. Wiesel and Hubel, 1963; Hubel and Wiesel, 1970). Ongoing development of visual sys- tem areas, especially with respect to the maturation of the neuronal circuitry, is highly dependent on organized retinal activity (Chapman, 2000). At the level of the lateral geniculate nucleus (LGN), for example, patterned retinal activity is required for the formation of eye-spe- cific regions, and this input remains important for the maintenance of areal segregation (Chapman, 2000; Zhou et al., 2003; Demas et al., 2006). However, little is known about whether early retinal activity shapes the development and maturation of cellular subtypes in the visual cortex, although it has been demonstrated that such activity mediates binocular competition important for shaping receptive fields in V1 (Huberman, 2006). Furthermore, studies in the mouse during the precritical period have demonstrated that V1 is susceptible to ex- *Corresponding author. Tel: +61-3-9902-9622; fax: +61-3-9902-9862. E-mail address: James.Bourne@armi.monash.edu.au (J. A. Bourne). Abbreviations: Crx, cone-rod homeobox gene; LCA, Leber congenital amaurosis; LGN, lateral geniculate nucleus; NeuN, neuronal nuclei specific protein; NNF, nonphosphorylated neurofilament; P, postnatal day; PB, phosphate buffer; PBS, phosphate buffer saline; PFA, para- formaldehyde; RGC, retinal ganglion cell; S1, primary somatosensory cortex; V1, primary visual cortex; wt, wild-type. Neuroscience 166 (2010) 886 – 898 0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.12.039 886