Microglia are Required for Developmental Specification of AgRP Innervation in the Hypothalamus of Offspring Exposed to Maternal High Fat Diet During Lactation

Reviewer #1 (Public review):

Summary:

In this manuscript, Mendoza-Romero et al. investigate the effects of maternal high-fat diet (MHFD) on microglia and AgRP synaptic terminals in the hypothalamus of postnatal mice during lactation. The study employs 3D microglial morphology reconstruction and genetically targeted axonal labeling, offering a detailed examination of microglial changes and their implications for AgRP terminal density and body weight regulation, focusing on the PVN and ARC nuclei. The authors also use pharmacological (e.g., PLX5622) elimination of microglia to test the sufficiency of microglia to shape PVN AgRP+ synapses.

Strengths:

This is a well-written paper with a thorough introduction and discussion.

The impact of microglia on hypothalamic synaptic pruning is poorly characterized, so the findings herein are especially interesting.

Weaknesses:

(1) A cartoon paradigm of the HFD treatment window would be a helpful addition to Figure 1. Relatedly, the authors might consider qualifying MHFD as 'lactational MHFD.' Readers might miss the fact that the exposure window starts at birth.

(2) More details on the modeling pipeline are needed either in Figure 1 or text. Of the ~50 microglia that were counted (based on Figure 1J), were all 50 quantified for the morphological assessments? Were equal numbers used for the control and MHFD groups? Were the 3D models adjusted manually for accuracy? How much background was detected by IMARIS that was discarded? Was the user blind to the treatment group while using the pipeline? Were the microglia clustered or equally spread across the PVN?

(3) Suggest toning back some of the language. For example: "...consistent with enhanced activity and surveillance of their immediate microenvironment" (Line 195) could be "...perhaps consistent with...". Likewise, "profound" (Lines 194, 377) might be an overstatement.

(4) Representative images for AgRP+ cells (quantified in Figure 2J) are missing. Why not a co-label of Iba1+/AgRP+ as per Figure 1, 3? Also, what was quantified in Figure 2J - soma? Total immunoreactivity?

(5) For the PLX experiment:
a) "...we depleted microglia during the lactation period" (Line 234). This statement suggests microglia decreased from the first injection at P4 and throughout lactation, which is inaccurate. PLX5622 effects take time, upwards of a week. Thus, if PLX5622 injections started at P4, it could be P11 before the decrease in microglia numbers is stable. Moreover, by the time microglia are entirely knocked down, the pups might be supplementing some chow for milk, making it unclear how much PLX5622 they were receiving from the dam, which could also impact the rate at which microglia repopulation commences in the fetal brain. Quantifying microglia across the P4-P21 treatment window would be helpful, especially at P16, since the PVN AgRP microglia phenotypes were demonstrated and roughly when pups might start eating some chow.

b) I am surprised that ~70% of the microglia are present at P21. Does this number reflect that microglia are returning as the pups no longer receive PLX5622 from milk from the dam? Does it reflect the poor elimination of microglia in the first place?

(6) Was microglia morphology examined for all microglia across the PVN? It is possible that a focus on PVNmpd microglia would reveal a stronger phenotype? In Figure 4H, J, AgRP+ terminals are counted in PVN subregions - PVNmpd and PVNpml, with PVNmpd showing a decrease of ~300 AgRP+ terminals in MHFD/Veh (rescued in MHFD/PLX5622). In Figure 1K, AgRP+ terminals across what appears to be the entire PVN decrease by ~300, suggesting that PVNmpd is driving this phenotype. If true, then do microglia within the PVNmpd display this morphology phenotype?

(7) What chow did the pups receive as they started to consume solid food? Is this only a MHFD challenge, or could the pups be consuming HFD chow that fell into the cage?

(8) Figure 5: Does internalized AgRP+ co-localize with CD68+ lysosomes? How was 'internalized' determined?

(9) Different sample sizes are used across experiments (e.g., Figure 4 NCD n=5, MHFD n=4). Does this impact statistical significance?

Reviewer #2 (Public review):

Summary:

Microglia sense stressors and other environmental factors during the postnatal period in rodents and can sculpt developing circuits by promoting or pruning synaptic connections, depending on the brain region and context. Here, the authors examine the contributions of microglia to the effects of maternal high-fat diet during lactation (MHFD) to reduce the formation of projections from AgRP neurons in the ARH to the PVH, a critical node in circuits regulating energy balance. Using detailed histomorphometric analyses of Iba-1+ cells in 3 hypothalamic nuclei (ARH, PVH, and BNST) at two-time points (P16 and P30), the authors show that microglial volume and complexity increase while cell numbers decrease across this period. Exposure to MHFD is associated with an increase in the complexity/volume of microglia at P16 in the PVH but not in the other brain regions or time points assessed. The authors cite this as evidence of "spatial-specific" effects. They also demonstrate that reducing the number of microglia using a pharmacological approach (injection of the CSFR inhibitor from P4-P21) in pups exposed to MHFD enhances AgRP outgrowth to the PVH and reduces body weight at weaning, effectively reversing the effects of MHFD. The central claim in the manuscript is that microglia in the PVH "sculpt the density of AgRP inputs to the PVH" in a spatially restricted manner.

Strengths:

(1) Detailed 3-D reconstructions of Iba-1 staining in microglia are used to perform unbiased and comprehensive analyses of microglial complexity and to quantify the spatial relationship between microglial processes and AgRP terminals.

(2) The rationale for exploring whether the effects of maternal HFD on the formation of AgRP projections to the PVH is mediated via changes in microglia is supported by the literature. For example, microglial development in the postnatal hippocampus and cortex is sensitive to maternal factors, such as inflammation, with lasting effects on circuit formation and function.

(3) Here the authors explored whether changes in microglia contribute to the effects of maternal HFD feeding during lactation on the formation of AgRP to PVH circuits that are important for the regulation of food intake and energy expenditure.

Weaknesses:

(1) Under chow-fed conditions, there is a decrease in the number of microglia in the PVH and ARH between P16 and P30, accompanied by an increase in complexity/volume. With the exception of PVH microglia at P16, this maturation process is not affected by MHFD. This "transient" increase in microglial complexity could also reflect premature maturation of the circuit.

(2) The key experiment in this paper, the ablation of microglia, was presumably designed to prevent microglial expansion/activation in the PVH of MHFD pups. However, it also likely accelerates and exaggerates the decrease in cell number during normal development regardless of maternal diet. Efforts to interpret these findings are further complicated because microglial and AgRP neuronal phenotypes were not assessed at earlier time points when the circuit is most sensitive to maternal influences.

(3) Microglial loss was induced broadly in the forebrain. Enhanced AgRP outgrowth to the PVH could be caused by actions elsewhere, such as direct effects on AgRP neurons in the ARH or secondary effects of changes in growth rates.

(4) Prior publications from the authors and other groups support the idea that the density of AgRP projections to the PVH is primarily driven by factors regulating outgrowth and not pruning. The failure to observe increased engulfment of AgRP fibers by PVH microglia is surprising. Therefore, not surprising. The possibility that synaptic connectivity is modulated by microglia was not explored.

Reviewer #3 (Public review):

Summary:

The authors interrogated the putative role of microglia in determining AgRP fiber maturation in offspring exposed to a maternal high-fat diet. They found that changes in specific parts of the hypothalamus (but not in others) occur in microglia and that the effect of microglia on AgRP fibers appears to be beyond synaptic pruning, a classical function of these brain-resident macrophages.

Strengths:

The work is very strong in neuroanatomy. The images are clear and nicely convey the anatomical differences. The microglia depletion study adds functional relevance to the paper; however, the pitfalls of the technology regarding functional relevance should be discussed.

Weaknesses:

There was no attempt to interrogate microglia in different parts of the hypothalamus functionally. Morphology alone does not reflect a potential for significant signaling alterations that may occur within and between these and other cell types.

The authors should discuss the limitations of their approach and findings and propose future directions to address them.