Conditional microglial depletion in rats leads to reversible anorexia and weight loss by disrupting gustatory circuitry
Introduction
Microglia are the brain’s primary immune cells, critical for the response to pathological conditions such as stroke and neurodegenerative disease (Cunningham, 2013, Gomez-Nicola et al., 2013). They also contribute to brain homeostasis in the healthy individual, having lifelong roles in synaptic pruning (Chu et al., 2010, Schafer et al., 2012), neuronal apoptosis (Paolicelli et al., 2014, Tremblay et al., 2010) and facilitating synaptic activity (Chu et al., 2010, Weinhard et al., 2018). Such activity can be modulated by non-pathogenic low-level inflammation, including with obesity and high fat diet (HFD) (Miller and Spencer, 2014). As such, both acute and longer-term HFD result in microglial proliferation and hypo-ramification in the hypothalamus, reflective of microglial activation (Thaler et al., 2012). Microglial activation can occur rapidly after HFD consumption and correlates with the degree of weight gain (Douglass et al., 2017, Thaler et al., 2012). Given this highly dynamic role in the response to HFD, it seems likely that microglia play an equally important part in regulating satiety signalling under normal conditions. However, data in this regard are still inconclusive.
Acutely, in the rat and mouse, hypothalamic microglial activation leads to changes in arcuate nucleus (ARC) neuronal firing in slice preparations consistent with a suppressive effect on food intake and inhibiting microglial activity prevents this (Reis et al., 2015). Hypothalamic microglial activation can also activate pro-opiomelanocortin (POMC) neurons in vivo in rats and mice, resulting in manifestations of sickness behavior, including anorexia (Jin et al., 2016, Tu et al., 2017). Microglial inhibition with intracerebroventricular (icv) minocycline can increase food intake (Reis et al., 2015) whereas microglial inhibition with a colony stimulating factor 1 receptor (CSFR1) antagonist in the diet does not affect it (Valdearcos et al., 2014). The few studies that have reported weight data in models of microglial ablation conclude that these cells may not fundamentally contribute to regulating feeding in healthy chow-fed animals; at least weight is not significantly affected at one week after microglial ablation. However, acute microglial ablation-induced weight changes have not been investigated (Djogo et al., 2016). The role of microglia in acutely regulating food intake and energy balance therefore remains unclear. Additionally, specific microglial-ablation studies have, until now, relied exclusively on the mouse as a model; models in additional species are needed to facilitate cross-species comparisons and inform on potential for extrapolation of mechanistic insights to humans.
To specifically test microglia’s role in regulating feeding behavior and satiety signalling, we used CRISPR/Cas9 genome editing to develop a Cx3cr1-Dtr transgenic Wistar rat with a diphtheria toxin receptor (Dtr) gene expressed in the promoter for the fractalkine receptor (Cx3cr1) expressed on microglia and monocytes (Fig. S1). This model allows acute ablation of microglia and monocytes upon application of diphtheria toxin (DT). Here we report that acute depletion of Cx3cr1-expressing cells leads to a pronounced and reversible weight loss that is largely accounted for by a reduction in food intake. This anorexia persists despite robust activation of several independent compensatory mechanisms that would otherwise drive food intake and / or energy conservation to preserve weight gain. We show it is likely that microglial ablation leads to this powerful anorectic effect by disrupting the gustatory circuitry connecting the hypothalamus with the paraventricular nucleus of the thalamus (PVT), resulting in a strong distaste for foods that are normally highly palatable. These findings demonstrate that, in the rat, microglia have an essential role in feeding and may therefore be a potential target for temporarily suppressing the drive to eat in cases such as obesity.
Section snippets
Animals
All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, with approval from the RMIT University Animal Ethics Committee (AEC 1512, 1612, 1621, 1708). Rat generation was performed at SAGE Labs, which operated under approved animal protocols overseen by SAGE’s Institutional Animal Care and Use Committee (IACUC) in accordance with the Guide for the Care and Use of Laboratory Animals. SAGE Labs is an Association for
Acute microglial ablation causes pronounced reversible anorexia and weight loss
To investigate the role of microglia in satiety and weight regulation, we gave DT to Cx3cr1-Dtr and wild-type male rats and examined feeding and metabolic parameters. The Dtr expression, in the absence of DT, did not affect any measured parameters. Thus, numbers of pups born per litter (Fig. S3A), neonatal and adult weights (Fig. S3B), brain (hypothalamic) Cx3cr1 expression (Fig. S3C), number and density of microglia (Fig. S3D, E), circulating and spleen monocytes (Fig. S3F-H), and exploratory
Discussion
Previous studies have reported conflicting findings as to the role of microglia in regulating satiety and food intake under normal conditions (Reis et al., 2015, Valdearcos et al., 2014) and there has been a near-exclusive reliance on the mouse in such studies. Our data here provide strong evidence that microglia are crucial in the regulation of food intake and weight in the rat. Thus, this is the first study to show a direct role for microglia in feeding regulation in normal animals in vivo.
Acknowledgements
This project was supported by funding from a National Health and Medical Research Centre Career Development Fellowship II (APP1128646) to SJS, an RMIT University PhD Scholarship and Publication Grant to SND and an RMIT University Vice Chancellor’s Postdoctoral Fellowship to LS.
Author contributions
SJS conceived of and designed the study. SND performed all experiments with the contribution of LS, AS, HW, IZ, and MR. SJS and SND performed statistical analyses and wrote the manuscript. All authors helped interpret the data, revised the manuscript for critical content, and approved the final version of the manuscript.
Declaration of interests.
The authors declare no competing interests.
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