MHFD: Microglial morphology in the PVH

(A) Image analysis pipeline. Maximum projection of an Iba1 stained microglial cell in the PVH (Ai). Confocal images through labeled cells were used to generate 3D reconstructions (Aii). Each 3D rendering was then used to create a skeletonized model by using the Filaments tool in Imaris. Polyhedrons were generated around each cell using the Convex Hull function of Imaris to estimate the total tissue “territory” occupied by the microglial cell (Aiii). (B-E) Maximum projection images of microglial cells (green) and labeled AgRP terminals (red) in the PVH of mice at P16 (B,C) or P30 (D,E) that were either exposed to MHFD (C, E) or NCD (B, D). (F-J) Graphical comparisons between groups to show that MHFD increased microglial ramification complexity (F), microglial cell territory (G), cell volume (H) and process length (I) and AgRP terminals (K). The density of microglia in the PVH decreased between P16 and P30, irrespective of diet (J). Bars represent the mean ± SEM and each point represents one animal. *P<.05, **P<.005. Abbreviations: MHFD, maternal high fat diet during lactation; NCD, Normal Chow Diet; PVH, paraventricular nucleus of the hypothalamus.

MHFD: Microglial morphology in the ARH

Maximum projection images of microglial cells (green) in the ARH of mice at P16 (A, B) or P30 (C,D) that were either exposed to MHFD (B, D) or NCD (A, C). Graphical comparisons between groups to show that microglial ramification complexity (E) remained the same, regardless of age or diet. Microglial cell territory (F), cell volume (G) and process length (H) increased between P16 and P30, but were not changed as a result of diet. The density of microglia in the ARH decreased between P16 and P30, irrespective of diet (I). There were no changes in numbers of AgRP neurons (J). Bars represent the mean ± SEM and each point represents one animal. *P<.05, **P<.005. Abbreviations: ARH, arcuate nucleus of the hypothalamus; MHFD, maternal high fat diet during lactation; NCD, Normal Chow Diet.

MHFD: Microglial morphology in the BST

Maximum projection images of microglial cells (green) and labeled AgRP terminals (red) in the BST of mice at P16 (A, B) or P30 (C,D) that were either exposed to MHFD (B, D) or NCD (A, C). Graphical comparisons between groups to show that microglial ramification complexity (E) remained the same, regardless of age or diet. Microglial cell territory (F), cell volume (G) and process length (H) did not significantly change between P16 and P30 and were not changed as a result of diet. The density of microglia in the BST decreased between P16 and P30, irrespective of diet (I). The density of AgRP terminals increased between P16 and P30, but there was no effect of maternal diet (J). Bars represent the mean ± SEM and each point represents one animal. *P<.05. Abbreviations: BST, bed nucleus of the stria terminalis; MHFD, maternal high fat diet during lactation; NCD, Normal Chow Diet.

Microglial depletion during lactation period

Images of microglial cells (green) to assess microglia number (A,B). Maximum projection images of labeled AgRP terminals (red) to assess AgRP terminal density in distinct compartments of the PVH at P55 long after the period of daily injections (C-F). Graphical comparison between groups to show that daily postnatal PLX5622 injections caused a 33% decrease in microglia in the PVH (G). Maximum projections of confocal images to illustrate the density of AgRP labeling in the PVH of NCD offspring (C,D) and MHFD offspring (E,F). (G-J) Graphical comparison to illustrate the effects of postnatal PLX5622 treatments on microglia in the PVH (G), body weight (I) and the density of AgRP terminals in the PVHmpd (H) and PVHpml (J). Bars represent the mean ± SEM and each point represents one animal. *P<.05, **P<.005, ***P<.0005. Abbreviations: AgRP, agouti-related peptide; CSF1R, Colony-Stimulating Factor 1 Receptor; MHFD, maternal high fat diet during lactation; MPD, medial parvocellular compartment of the PVH; PML, posterior magnocellular compartment of the PVH; PVH, paraventricular nucleus of the hypothalamus.

Microglial interaction with AgRP axon terminals in the PVH and ARH

(A-D) Maximum projection images of microglial cells (green) labeled AgRP terminals (red), and CD68 (lysosomal associated membrane protein and phagocytic capacity marker, pink). (E-H) Digital 3D reconstructions shown in (A-D) after application of a digital zoom to more clearly illustrate engulfment of labeled AgRP terminals by microglia (I-L). (M-P) Graphical comparisons between groups to illustrate the effects of age and MHFD exposure on CD68 expression and AgRP terminal engulfment. Bars represent the mean ± SEM and each point represents one animal. *P<.05, **P<.005. Abbreviations: AgRP, agouti-related peptide; ARH, arcuate nucleus or the hypothalamus; CD68, Cluster of Differentiation 68; MHFD, maternal high fat diet during lactation; PVH, paraventricular nucleus of the hypothalamus.