Whole-brain activation signatures of weight-lowering drugs

Abstract

Objective: The development of effective anti-obesity therapeutics relies heavily on the ability to target specific brain homeostatic and hedonic mechanisms controlling body weight. To obtain further insight into neurocircuits recruited by anti-obesity drug treatment, the present study aimed to determine whole-brain activation signatures of six different weight-lowering drug classes.

Methods: Chow-fed C57BL/6J mice (n = 8 per group) received acute treatment with lorcaserin (7 mg/kg; i.p.), rimonabant (10 mg/kg; i.p.), bromocriptine (10 mg/kg; i.p.), sibutramine (10 mg/kg; p.o.), semaglutide (0.04 mg/kg; s.c.) or setmelanotide (4 mg/kg; s.c.). Brains were sampled two hours post-dosing and whole-brain neuronal activation patterns were analysed at single-cell resolution using c-Fos immunohistochemistry and automated quantitative three-dimensional (3D) imaging.

Results: The whole-brain analysis comprised 308 atlas-defined mouse brain areas. To enable fast and efficient data mining, a web-based 3D imaging data viewer was developed. All weight-lowering drugs demonstrated brain-wide responses with notable similarities in c-Fos expression signatures. Overlapping c-Fos responses were detected in discrete homeostatic and non-homeostatic feeding centres located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures as well as the dopaminergic system.

Conclusions: Whole-brain c-Fos expression signatures of various weight-lowering drug classes point to a discrete set of brain regions and neurocircuits which could represent key neuroanatomical targets for future anti-obesity therapeutics.

Keywords: Anti-Obesity drugs; Imaging; Light sheet fluorescence microscopy; Obesity; c-Fos; iDISCO.

Figure 1

Web-based whole-brain imaging data viewer (Gubra 3D Experience/G3DE, https://g3de.gubra.dk/). (A) User interface of the online data browsing system showing the quantitative data for all treatment groups in selected appetite-regulating brain region (nucleus of the solitary tract, NTS). (B) Brain regions with compound-induced statistically significantly regulation of c-Fos+ cell numbers (p < 0.05 vs. corresponding vehicle control group). Example of data filtered to show only semaglutide-induced regulation of c-Fos expression (c-Fos + cell counts) in the central amygdalar nucleus (CEA) in each individual mouse (dot plot with indication of average number of c-Fos+ cells ± S.E.M.). (C) Representative group-average c-Fos expression heatmaps for each individual drug tested. Lower panel: Corresponding dorsal and coronal view of selected brain region. (D) Online movies showing whole-brain c-Fos responses to weight-lowering drugs (selected coronal plane at the level of the nucleus of the solitary tract, NTS). The 12 appetite-regulating regions are delineated in the coronal slice-by-slice fly-through movies. Upper panel: Heatmaps showing vehicle-subtracted average whole-brain c-Fos expression in response to weight-lowering compounds. Statistically significant changes (p < 0.05; Dunnett's test negative binomial generalized linear model, FDR < 0.05 for p-value adjustment) in c-Fos expression in response to treatment with weight-lowering compounds compared to corresponding vehicle controls are depicted in red (upregulation) and blue (downregulation). Lower panel: P-value maps from voxel-based statistical analysis visualising whole-brain c-Fos responses to individual weight-lowering drugs. Statistically significant changes between the treatment and vehicle group (p < 0.05) are indicated by graded purple colour.

Figure 2

3D mapping and quantification of whole-brain c-Fos responses to acute treatment with various weight-loss promoting compounds. Quantification and statistical analysis of c-Fos expression was performed in 308 brain regions. (A) Lorcaserin (7 mg/kg, i.p.), (B) rimonabant (10 mg/kg, i.p.), (C) bromocriptine (10 mg/kg, i.p.), (D) sibutramine (10 mg/kg, p.o.), (E) semaglutide (0.04 mg/kg, s.c.) and (F) setmelanotide (4 mg/kg, s.c.). All samples were registered into an LSFM-based mouse brain atlas. Heatmaps (dorsal view) depict vehicle-subtracted average whole-brain c-Fos expression (n = 7-8 mice per group) responses to the individual drug. Brain areas with statistically significant changes in c-Fos expression (p < 0.05; Dunnett's test negative binomial generalised linear model, FDR < 0.05 for p-value adjustment) are delineated in red (upregulation) or blue (downregulation) compared to corresponding vehicle controls. Coronal slice-by-slice fly-through of the heatmaps can be found in the G3DE imaging viewer. Bar plots show the differences in total numbers of c-Fos+ cells detected in compound and corresponding vehicle-dosed mice (∗p <0.05, ∗∗∗p < 0.001; Dunnett's test negative binomial generalised linear model). (G) Principal component analysis (PCA) of whole-brain c-Fos expression. The PCA plot illustrates the degree of separation between individual drug effects on global c-Fos expression patterns (large markers indicate group average). (H) PCA loading plot depicting the coefficients of the top 15 most influential brain regions driving the clustering of data points in PCA plot. Abbreviations: Vehicle IP1, 0.1% Tween-80 in saline (intraperitoneal); Vehicle IP2, 5% DMSO + 5% chremophor in saline (intraperitoneal); Vehicle PO, 0.5% hydroxypropyl methylcellulose (peroral); Vehicle SC, 0.1% bovine serum albumin in phosphate-buffered saline (PBS, subcutaneous); PARN, parvicellular reticular nucleus; MDRNd, dorsal medullary reticular nucleus; MY-mot, motor-related part of medulla; PB, parabrachial nucleus; NTS, nucleus of the solitary tract; IRN, intermediate reticular nucleus; IMD, intermediodorsal nucleus of the thalamus, LP, lateral posterior nucleus of the thalamus; DG, dentate gyrus; LGv, ventral part of the lateral geniculate complex; APN, anterior pretectal nucleus; PPT, posterior pretectal nucleus; HIP, hippocampal region; SCm, motor-related superior colliculus; CA3, field CA3 of the Ammon's horn.

Figure 3

Overlapping and specific c-Fos expression signatures of weight-lowering drugs in major appetite-regulating brain regions. (A) Anatomical map (dorsal view) depicting 12 selected brain regions involved in appetite regulation. (B) Summary of drug-induced c-Fos induction across the 12 individual brain regions (p < 0.05; Dunnett's test negative binomial generalised linear model, FDR < 0.05 for p-value adjustment). (C) Fold-change (log2 scale, mean ± S.E.M.) in c-Fos positive cell counts in the 12 selected brain regions (rostro-caudal order) compared to corresponding vehicle controls. Dunnett's test negative binomial generalised linear model with p-value adjustment for multiple comparisons using FDR < 0.05 was applied for statistical analysis (∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001). Abbreviations: ACB, nucleus accumbens; ARH, arcuate hypothalamic nucleus; AP, area postrema; CEA, central amygdalar nucleus; DMH, dorsomedial nucleus of the hypothalamus; DMX, dorsal motor nucleus of the vagus nerve; LHA, lateral hypothalamic area; NTS, nucleus of the solitary tract; PB, parabrachial nucleus; PVH, paraventricular hypothalamic nucleus; SNc, substantia nigra pars compacta; VTA, ventral tegmental area.

Figure 4

Whole-brain c-Fos expression signatures in response to weight-lowering compounds. Significant changes in c-Fos+ cell counts (p < 0.05) following administration of each individual weight-lowering compound. Dunnett's test negative binomial generalised linear model with p-value adjustment for multiple comparisons using FDR (cut-off of 0.05) was applied for statistical analysis. Regulated brain regions are categorised anatomically and ranked according to the number of drugs demonstrating a similar effect. With the exception of reduced c-Fos+ cell counts in the thalamic suprageniculate nucleus (SGN, significantly down-regulated by liraglutide only), all significantly regulated areas exhibited increased c-Fos+ cell counts following drug treatment as compared to corresponding vehicle controls. Major appetite-regulating regions are indicated in red (ACB, nucleus accumbens; ARH, arcuate hypothalamic nucleus; AP, area postrema; CEA, central amygdalar nucleus; DMH, dorsomedial nucleus of the hypothalamus; DMX, dorsal motor nucleus of the vagus nerve; LHA, lateral hypothalamic area; NTS, nucleus of the solitary tract; PB, parabrachial nucleus; PVH, paraventricular hypothalamic nucleus; VTA, ventral tegmental area). For other abbreviated brain regions, see the web-based imaging data viewer.

Figure 5

Subregional differentiation of c-Fos responses in the parabrachial nucleus in response to weight-lowering compounds. Voxel-wise statistical analysis of c-Fos expression was performed on pre-processed segmentation images of c-Fos+ cells using the pTFCE method and FWER approach for p-value adjustments. Resulting spatial p-value distributions (p < 0.05) are shown for the parabrachial nucleus in a representative coronal cross-section for different compound treatments (right column). Levels of statistical significance are indicated by graded purple colours. Vehicle-subtracted group means of c-Fos expression are depicted in the left column (red, upregulation; blue downregulation as compared to corresponding vehicle controls). The signal appearing in the neighbouring regions of parabrachial nucleus has been marked on both p-value distribution and c-Fos expression visualizations for clarity. Coronal slice-by-slice fly-through of whole brain p-value distribution resulting from voxel-based statistical analysis is exemplified in Figure 1D and can be seen in the web-based imaging data viewer.