PET Acquisition and Analysis

[¹¹C]-WAY100635 radiochemistry and input function measurement was performed as previously described (Parsey, et al., 2010). Briefly, WAY100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride) was reacted with radioactive [¹¹C]-CO₂ to produce the final radiotracer. Mean injected dose was 6.49 +/− 0.4 mCi. During the scan, arterial blood was sampled every 5 seconds automatically for the first two minutes, and manually at longer intervals after (6, 12, 20, 40, and 60 minutes). These samples were centrifuged for 10 minutes and radioactivity in the separated plasma was counted using a gamma counter (Wallac 1480 Wizard 3M Automatic Gamma Counter). To measure metabolite levels, samples collected at 2, 6, 12, 20, 40, and 60 minutes were processed by protein precipitation with acetonitrile followed by purification via high pressure liquid chromatography (HPLC). The fraction of unmetabolized [¹¹C]-WAY100635 was calculated in these samples, and these fractions were fit to the Hill function, with the value at time zero constrained to 100%. The input function was the product of the interpolated unmetabolized fraction and total counts. The calculated input function values were fit to a sum of three exponentials from the time of the peak to the final data point, whereas the early rising part of the curve was fit to a straight line. These fitted values were then used as input to the kinetic analysis. Plasma free fraction (f_P) was obtained by centrifuging 200 μL aliquots of plasma mixed with tracer; f_P was calculated as the ratio of ultrafiltrate to total plasma activity concentrations.

PET images were acquired on an ECAT EXACT HR+ (Siemens/CTI, Knoxville, TN) as described in Parsey et al., 2010. Emission data were collected for 110 minutes at 20 frames of increasing duration. Image processing was performed through MATLAB (The Mathworks, Natick, Massachusetts), with extension to Functional Magnetic Resonance Imaging of the Brain’s (FMRIB’s) Linear Image Registration Tool (FLIRT v.5.2 (Jenkinson and Smith, 2001)) for coregistration, Brain Extraction Tool v1.2 (Smith, 2002) for skull-stripping, Statistical Parametric Mapping (SPM5) segmentation routines (Ashburner and Friston, 2005) to segment gray matter, white matter, and CSF, and the Advanced Normalization Toolbox (ANTs (Avants, et al., 2008; Avants, et al., 2011)) for normalization. Frame-by-frame rigid body registration was performed to a reference frame in order to correct for subject motion.

A 5-HT_1A defined raphe nucleus (RN) ROI was delineated as described previously (Delorenzo, et al., 2013) (Figure 1). Briefly, an average 5-HT_1A voxel-binding map was created using a previous [¹¹C]-WAY100635 study of 52 healthy controls (Parsey, et al., 2010). PET images were warped into a high resolution template space (Holmes, et al., 1998) using ANTs (Avants, et al., 2008; Avants, et al., 2011) and averaged. A thresholding technique was used to extract the RN, as binding is higher in this region than in surrounding areas. The RN was then applied to individual participant’s MRI by first warping their MRI to the template space, then using those parameters to inverse-warp the RN ROI into the individual participant’s space.

A: Template brain. B: Average BP_F voxel image for [¹¹C]-WAY100635 PET scans. C: Merged PET and MRI image. D: Raphe nucleus region of interest overlaid on template MRI.

PET time activity curves obtained from the RN ROI were fit with a two-compartment model with rate constants of radiotracer between blood and tissue compartments constrained to that of the cerebellar white matter. We used these time activity curves and arterial input functions to generate a distribution volume, V_T. BP_F and BP_ND were then calculated. BP_F is defined as specific regional binding (regional volume of distribution or V_T minus nonspecific volume of distribution or V_ND) normalized to the free fraction of radiotracer in arterial blood or f_p; thus BP_F = (V_T-V_ND)/f_p. BP_ND is defined as specific binding normalized to binding in a reference region posited to have no specific binding; thus, BP_ND = (V_T-V_ND)/V_ND. To derive V_ND, we used the white matter of the cerebellum as a reference region, as this region was found to be optimal for [¹¹C]-WAY100635 imaging (Hirvonen, et al., 2007; Parsey, et al., 2005). Standard errors (SE) for both measures were calculated by bootstrapping both PET and plasma data (Parsey, et al., 2000).