Test-retest Reproducibility of Quantitative Binding Measures of [C]Ro15-4513, a PET Ligand for GABA Receptors Containing Alpha5 Subunits

Introduction: Alteration of γ-aminobutyric acid "A" (GABA) receptor-mediated neurotransmission has been associated with various neurological and psychiatric disorders. [C]Ro15-4513 is a PET ligand with high affinity for α5-subunit-containing GABA receptors, which are highly expressed in limbic regions of the human brain (Sur et al., 1998). We quantified the test-retest reproducibility of measures of [C]Ro15-4513 binding derived from six different quantification methods (12 variants).

Methods: Five healthy males (median age 40 years, range 38-49 years) had a 90-min PET scan on two occasions (median interval 12 days, range 11-30 days), after injection of a median dose of 441 MegaBequerels of [C]Ro15-4513. Metabolite-corrected arterial plasma input functions (parent plasma input functions, ppIFs) were generated for all scans. We quantified regional binding using six methods (12 variants), some of which were region-based (applied to the average time-activity curve within a region) and others were voxel-based: 1) Models requiring arterial ppIFs - regional reversible compartmental models with one and two tissue compartments (2kbv and 4kbv); 2) Regional and voxelwise Logan's graphical analyses (Logan et al., 1990), which required arterial ppIFs; 3) Model-free regional and voxelwise (exponential) spectral analyses (SA; (Cunningham and Jones, 1993)), which also required arterial ppIFs; 4) methods not requiring arterial ppIFs - voxelwise standardised uptake values (Kenney et al., 1941), and regional and voxelwise simplified reference tissue models (SRTM/SRTM2) using brainstem or alternatively cerebellum as pseudo-reference regions (Lammertsma and Hume, 1996; Gunn et al., 1997). To compare the variants, we sampled the mean values of the outcome parameters within six bilateral, non-reference grey matter regions-of-interest. Reliability was quantified in terms of median absolute percentage test-retest differences (MA-TDs; preferentially low) and between-subject coefficient of variation (BS-CV, preferentially high), both compounded by the intraclass correlation coefficient (ICC). These measures were compared between variants, with particular interest in the hippocampus.

Results: Two of the six methods (5/12 variants) yielded reproducible data (i.e. MA-TD <10%): regional SRTMs and voxelwise SRTM2s, both using either the brainstem or the cerebellum; and voxelwise SA. However, the SRTMs using the brainstem yielded a lower median BS-CV (7% for regional, 7% voxelwise) than the other variants (8-11%), resulting in lower ICCs. The median ICCs across six regions were 0.89 (interquartile range 0.75-0.90) for voxelwise SA, 0.71 (0.64-0.84) for regional SRTM-cerebellum and 0.83 (0.70-0.86) for voxelwise SRTM-cerebellum. The ICCs for the hippocampus were 0.89 for voxelwise SA, 0.95 for regional SRTM-cerebellum and 0.93 for voxelwise SRTM-cerebellum.

Conclusion: Quantification of [C]Ro15-4513 binding shows very good to excellent reproducibility with SRTM and with voxelwise SA which, however, requires an arterial ppIF. Quantification in the α5 subunit-rich hippocampus is particularly reliable. The very low expression of the α5 in the cerebellum (Fritschy and Mohler, 1995; Veronese et al., 2016) and the substantial α1 subunit density in this region may hamper the application of reference tissue methods.