BEHAVIORAL NEUROSCIENCE Seasonal rewiring of the songbird brain: an in vivo MRI study Geert De Groof, 1 Marleen Verhoye, 1,2 Vincent Van Meir, 1 Jacques Balthazart 3 and Annemie Van der Linden 1 1 Bio-Imaging Lab, University of Antwerp, CGB, Groenenborgerlaan 171, B-2020 Antwerp, Belgium 2 Vision Lab, University of Antwerp, CDE, Universiteitsplein, Wilrijk (Antwerp), Belgium 3 Center for Cellular and Molecular Neurobiology, University of Lie `ge, Lie ` ge, Belgium Keywords: diffusion tensor imaging, optic chiasma, seasonal plasticity, song control system, starling (sturnus vulgaris) Abstract The song control system (SCS) of songbirds displays a remarkable plasticity in species where song output changes seasonally. The mechanisms underlying this plasticity are barely understood and research has primarily been focused on the song nuclei themselves, largely neglecting their interconnections and connections with other brain regions. We investigated seasonal changes in the entire brain, including the song nuclei and their connections, of nine male starlings (Sturnus vulgaris). At two times of the year, during the breeding (April) and nonbreeding (July) seasons, we measured in the same subjects cellular attributes of brain regions using in vivo high-resolution diffusion tensor imaging (DTI) at 7 T. An increased fractional anisotropy in the HVC–RA pathway that correlates with an increase in axonal density (and myelination) was found during the breeding season, confirming multiple previous histological reports. Other parts of the SCS, namely the occipitomesencephalic axonal pathway, which contains fiber tracts important for song production, showed increased fractional anisotropy due to myelination during the breeding season and the connection between HVC and Area X showed an increase in axonal connectivity. Beyond the SCS we discerned fractional anisotropy changes that correlate with myelination changes in the optic chiasm and axonal organization changes in an interhemispheric connection, the posterior commissure. These results demonstrate an unexpectedly broad plasticity in the connectivity of the avian brain that might be involved in preparing subjects for the competitive and demanding behavioral tasks that are associated with successful reproduction. Introduction One of the most important developments that has taken place in neuroscience in the past 25 years is the realization that the brain is not the fixed structure it was thought to be but rather displays extensive dynamic changes. These changes constitute what is commonly called neuroplasticity. Understanding their specific nature and control mechanisms represents a critical step towards a full understanding of brain functioning. Some of the most dramatic brain structural modifications are the seasonal changes affecting a connected set of brain nuclei controlling singing behavior, the song control system (SCS) in oscine songbirds. In most temperate zone species, reproduction is a seasonal phenom- enon. In a specific group of birds belonging to the order Passeriformes behaviors associated with reproduction, such as singing, are performed at higher rates during the breeding season (Phillmore et al., 2006). In parallel, a seasonal variation in the volume of song control nuclei has been observed (e.g., Nottebohm, 1981; Kirn et al., 1989; Brenowitz et al., 1991, 1998; Bernard & Ball, 1995, 1997; Smith et al., 1995; Smith, 1996; Caro et al., 2005). Due to the magnitude of these changes, sometimes as large as a 99% increase (Nottebohm, 1981), seasonal variation in the brain of songbirds has emerged as one of the best model systems for the study of naturally occurring brain plasticity (Tramontin & Brenowitz, 2000; Ball et al., 2004; Brenowitz, 2004). Most of the gray matter structures known to be plastic are part of the SCS (Nottebohm, 1981; Hill & DeVoogd, 1991; Tramontin & Brenowitz, 1999, 2000; Tindemans et al., 2003; Van Meir et al., 2004, 2006; Thompson & Brenowitz, 2005). Recently, improvements in in vivo magnetic resonance imaging (MRI) techniques have made it possible to assess neuroplasticity of the SCS (Tindemans et al., 2003; Van der Linden et al., 2004; Van Meir et al., 2004, 2006). Diffusion tensor imaging (DTI) has also been implemented for the analysis of the songbird brain (De Groof et al., 2006) in the form of a high resolution variant of the method used to assess changes in the brain during human brain development (Huppi & Dubois, 2006; Cascio et al., 2007; Lebel et al., 2008) or neurodegeneration (Ulug et al., 1999; Rose et al., 2000; Taber et al., 2002). DTI is an MRI method that uses water diffusion as a highly sensitive marker of the microarchitecture of cellular membranes. The noninvasive in vivo nature of the technique allows for longitudinal studies in individuals following changes with time. In the present study, we repeatedly imaged by DTI the brain of nine individual male starlings during the breeding and the nonbreeding Correspondence: Dr G. De Groof, as above. E-mail: geert.degroof@ua.ac.be Received 17 July 2008, revised 21 October 2008, accepted 22 October 2008 European Journal of Neuroscience, Vol. 28, pp. 2475–2485, 2008 doi:10.1111/j.1460-9568.2008.06545.x ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience