Neuroinform (2012) 10:341–350 Magnetic Resonance Imaging All participants underwent high resolution MRI T1- weighted, T2-weighted and FLAIR imaging at 3 T (Philips Intera T30, T/R head coil). All MRI modalities were used to exclude the possibility of brain lesions in patients and con- trols. For volumetric analysis, we acquired T1-weighted structural MRIs using a high-resolution 3D turbo-field- echo sequence (matrix 256 ×256× 160 over a field of view of 25.6 × 25.6 × 16 cm3 reconstructed after zero filling to 512 × 512 × 320 cubic voxels with an edge length of 0.5 mm). Prior to morphometric analyses, all MR images were intensity inhomogeneity corrected and resampled to isotropic voxels of 1× 1 ×1 mm (256× 256 ×160 slices) us- ing in-house software (Eval 3.0). To confirm that there was no systematic difference in head size between patient and control groups, we automatically obtained relative brain size, VSCALE (global tissue volumes normalized for head size) and CSF volume estimates from the 3D T1-weighted images of all subjects using the SIENAX protocol (Smith, et al. 2002) integrated into FSL software (http:// www.fmrib.ox.ac.uk/fsl/siena/index.html). There were no inter-group differences in relative brain size (p 0 0.40), VSCALE (p 0 0.96) or CSF volume (p 0 0.95). A. Stereology The Cavalieri method of design-based stereology in con- junction with point counting (Gundersen and Jensen 1987; Gundersen, et al. 1999; Mayhew 1992; Roberts, et al. 2000) was used as an unbiased estimator of the volume of the left and right thalamus in all subjects. By using the Cavalieri method, volume is directly estimated from equidistant and parallel MR images of the brain with a uniform random starting position. A second level of sampling is required to estimate the section area from each image by applying point counting within the ROI. The mathematical justification and implementation of the methodology is simple and it can be applied to structures of arbitrary shape (Garcia-Finana, et al. 2009). This technique has been frequently applied to reli- ably estimate brain volume and surface area on MR images (Acer, et al. 2010; Bas, et al. 2009; Cowell, et al. 2007; Eriksen, et al. 2010; Hallahan, et al. 2011; Howard, et al. 2003; Jelsing, et al. 2005; Keller, et al. 2009; Keller, et al. 2007; Keller, et al. 2002a; Keller, et al. 2009b; Keller, et al. 2002b; Lux, et al. 2008; Mackay, et al. 1998; Mackay, et al. 2000; Ronan, et al. 2006; Salmenpera, et al. 2005; Sheline, et al. 1996), and more widely applied to study other aspects of anatomy with and without the use of MRI. Stereology has been shown to be at least as precise as tracing and thresh- olding volumetry techniques and substantially more time efficient, with validation relative to post-mortem measure- ments (Garcia-Finana, et al. 2003; Garcia-Finana, et al. 2009; Keller and Roberts 2009; Keshavan, et al. 1995). Windows- 343 compatible Easymeasure software (Keller, et al. 2007; Puddephat 1999) was used for point counting on MR images. Given that the borders of different thalamic nuclei are almost indistinguishable on conventional MRI, the thalamus was sampled as an entire complex, including the anterior thalamic nucleus, mediodorsal thalamic nucleus, lateral dorsal thalamic nucleus, ventral lateral thalamic nucleus, ventral postero-lateral/medial thalamic nuclei, and pulvinar (Duvernoy 1999). The lateral and medial geniculate nuclei were not included in measurements, similar to previous studies (Natsume, et al. 2003; Qiu, et al. 2009). On axial sections, point counting began on the dorsal most section, where the area of the lateral dorsal thalamic nucleus was demarcated laterally by the corona radiata and medially by the lateral ventricles. As measurements progressed ventrally, the posterior limb of the internal capsule bordered the tha- lamus laterally. On ventral sections, care was taken to de- lineate the thalamic nuclei from the neighbouring pars medialis of the globus pallidus, and at the most ventral sections, to segregate the pulvinar and area of the ventral posterolateral nucleus from the emerging hypothalamus an- teriorly, superior colliculus posteromedially, and the hippo- campus posteriorly / posterolaterally. Figure 1 shows point counting within the thalamic ROI relative to the automated FreeSurfer approach described below. More information on the MRI-based anatomy of the thalamus with respect to post-mortem sections can be found at http://www.psychia- try.uiowa.edu/mhcrc/pdf/papers/thalamus.pdf. The sampling density (i.e. size of points, distance be- tween sections) were optimized to achieve a coefficient of error (CE) of less that 5 % (Roberts, et al. 2000), an ap- proach that has been adopted in our stereological analysis of the hippocampus (Keller, et al. 2002a; Keller, et al. 2002b), putamen (Keller, et al. 2011a), prefrontal region (Keller, et al. 2009a), planum temporale (Keller, et al. 2007; Keller, et al. 2011b), Broca’s area (Keller, et al. 2007; Keller, et al. 2009b) and insula (Keller, et al. 2011b). The CE is essen- tially a statistical estimate of how accurate the stereological volume estimation is for each structure. Separation between test points on the square grid used for point counting was 0.312 cm (i.e., 4 pixels) and slice interval was 1 mm (sin- gular axial MR sections). Thalamic transect area was obtained by multiplying the total number of points recorded by the area corresponding to each test point. An estimate of thalamic volume was obtained as the sum of the estimated areas of the structure transects on consecutive systematic sections multiplied by the distance between sections. Between approximately 400–550 points were recorded on approximately 20 systematic random sections. B. FreeSurfer FreeSurfer software (http://surfer.nmr.mgh.harvard.edu/) was used to obtain thalamic volumes for all subjects using