Fig 1.
The experimental setup used for data acquisition and analysis of rat rsfMRI.
(A) Experimental setup for fMRI data acquisition on a 470-MHz MRI using a 1H transmit/receive head surface coil. (B) Processed Blood-Oxygen-level Dependent (BOLD) signals from regions of the reward system. After skull stripping, atlas registration, motion and drift correction, and intensity normalization, images were band-pass filtered between 0.01–0.1Hz. (C) Anatomical location of selected seed regions within the reward system that were used for cross-correlation analysis (to generate voxel-wise maps of Pearson’s r coefficient that was later z-transformed prior to group comparisons). Shown are both a standard anatomical atlas and high-resolution MRI-based atlas maps. Green arrows highlight the seed region. Overlays on the rat brain atlas show connectivity with corresponding seed regions based on Fisher’s z-transformed r coefficient values (r ≥ 0.35) [29].
Fig 2.
KB220Z increases functional connectivity in the rat brain reward system seed regions.
Composite resting state functional connectivity maps for placebo and KB220Z for seed ROI placed on (A) the accumbens, only left seed is shown (n = 7 placebo and n = 8 KB220Z). (B) The anterior thalamic nucleus and (C) the dorsal hippocampus. Green arrows and circles indicate highly correlated voxels within the seed region itself. Connectivity maps are set at a lower statistical threshold of t = 4, p < 0.005 (cluster size corrected).
Fig 3.
Representative cross-correlation maps show five subjects: Placebo compared to KB220Z treated rats.
The maps correspond to resting state connectivity for the NAc (highlighted in green in the atlas map above the figure; only left seed is shown). Note the distributed but significant connectivity between various brain regions and the NAc in the placebo subjects. KB220Z increased connectivity, especially between left-right accumbens, dorsal striatum, and limbic cortical areas such as the anterior cingulate, prelimbic and infralimbic regions. Correlation maps for representative subjects presented at a threshold between 0.3 ≤ z ≥ 1.2.
Fig 4.
Composite three-dimensional functional connectivity maps comparing KB220Z and placebo.
The top row shows the segmented 3D ROI used as seed for the placebo and KB220Z maps seen below them. High clustering of voxels occurs within the seed regions for both placebo and KB220Z groups. Greater connectivity based on the number of voxels showing high correlation coefficient values is observed in the KB220Z maps. Difference maps (KB220Z minus placebo) are shown in the bottom row. Maps are set at a lower statistical threshold of p < 0.005 (voxel cluster size corrected).
Fig 5.
Group statistical maps comparing KB220Z and placebo treatments.
Data are shown for three different seed regions: (A) accumbens (ACb), (B) anterior thalamic nucleus (ATN) and (C) dorsal hippocampus (dHPC), only left representations are shown here. Voxel-wise statistical t-tests (KB220Z vs. Placebo, p < 0.05, two tailed, heteroscedastic variances; cluster-size corrected using 3dClustSim on AFNI were used to compare the two groups. Other ROI indicated by green arrows are the dorsal striatum (dSTR), anterior cingulate (ACg), accumbens (ACb), somatosensory cortex (SSC); and the mediodorsal thalamus (ATN/MD).
Fig 6.
Heat maps of cross-correlation coefficient values (Fisher’s z-transformed) for seed regions and 65 rat brain atlas ROI.
(A) Data summary for placebo treatment. (B) Data summary for KB220Z treatment. (C) Difference map of correlation coefficient values greater in KB220Z than placebo. The scale bar color indicates degree of connectivity (cool-blue < warm-yellow < hot-orange/red). Note the overall similar pattern of connectivity within and outside brain reward regions between A and B. The degree of connectivity is strengthened with KB220Z treatment compared to placebo (B vs. A) and is observed in the difference heat map in (C). ROI that correspond to each row in the color-coded maps are shown on the far left. The seed region corresponding to each column are left accumbens (L ACb), anterior cingulate cortex (ACg), dorsal hippocampus (dHPC), infralimbic area (IL), prelimbic area (PrL), anterior thalamus (AT) and somatosensory cortex (SSC).
Fig 7.
Resting state functional connectivity is higher with KB220Z than with placebo.
Plots show differences in functional connectivity between various ROI and three seed regions. Data are presented as mean correlation (z transformed) ± standard error. * Significantly different than placebo t221 > 2.1 p < 0.05 (p values FDR corrected at q = 0.05).
Fig 8.
KB220Z effects resting state functional connectivity within the limbic cortex.
Mean connectivity maps are at a threshold z value of 0.35 for visualization of resting state networks (scale bar indicates connectivity strength based on z value).
Fig 9.
KB220Z increased the volume of connectivity.
Connectivity volume was assessed by applying a correlation threshold value of z = 0.3 to all subjects and quantifying the volume above this threshold. Voxels were then converted to mm3 based on 3D voxel resolution. Shown are connectivity volume data for various regions including (A), nucleus accumbens, (B) mediodorsal thalamus, (C) infralimbic cortex, (D) dorsal hippocampus, (E) anterior cingulate cortex, and (F) somatosensory cortex. *Significantly different from placebo t ≥ 3.4 p ≤ 0.05 with multiple comparison corrections using the Holm-Sidak method. Sixty-five ROI were analyzed for each seed in A-F and areas of the brain with increases in functional connectivity with KB220Z treatment are shown.