Probes for CRE

Abstract

The central melanocortin system plays a crucial role in the control of energy balance. Although the decreased energy expenditure and increased adiposity of melanocortin-3 receptor (Mc3R)-null mice suggest the importance of Mc3R-regulated neurons in energy homeostasis, the roles for specific subsets of Mc3R neurons in energy balance have yet to be determined. Because the lateral hypothalamic area (LHA) contributes to the control of energy expenditure and feeding, we generated Mc3rcre mice to determine the roles of LHA Mc3R (Mc3RLHA) neurons in energy homeostasis. We found that Mc3RLHA neurons overlap extensively with LHA neuron markers that contribute to the control of energy balance (neurotensin, galanin, and leptin receptor) and project to brain areas involved in the control of feeding, locomotion, and energy expenditure, consistent with potential roles for Mc3RLHA neurons in these processes. Indeed, selective chemogenetic activation of Mc3RLHA neurons increased locomotor activity and augmented refeeding after a fast. Although the ablation of Mc3RLHA neurons did not alter food intake, mice lacking Mc3RLHA neurons displayed decreased energy expenditure and locomotor activity, along with increased body mass and adiposity. Thus, Mc3R neurons lie within LHA neurocircuitry that modulates locomotor activity and energy expenditure and contribute to energy balance control.

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.

Abstract

KEY POINTS:

Alpha-melanocyte stimulating hormone (α-MSH) is an anorexigenic peptide, and injection of the α-MSH analog MTII into the ventral tegmental area (VTA) decreases food and sucrose intake and food reward. Melanocortin-3 receptors (MC3R) are highly expressed in the VTA, suggesting that the effects of intra-VTA α-MSH may be mediated by α-MSH changing the activity of MC3R-expressing VTA neurons. α-MSH increased the firing rate of MC3R VTA neurons in acute brain slices from mice, but did not affect the firing rate of non-MC3R VTA neurons. The α-MSH induced increase in MC3R neuron firing rate is likely activity dependent, and was independent of fast synaptic transmission and intracellular Ca2+ levels. These results help us to better understand how α-MSH acts in the VTA to affect feeding and other dopamine dependent behaviors.

ABSTRACT:

The mesocorticolimbic dopamine system, the brain's reward system, regulates multiple behaviors including food intake and food reward. There is substantial evidence that the melanocortin system of the hypothalamus, an important neural circuit controlling feeding and body weight, interacts with the mesocorticolimbic dopamine system to affect feeding, food reward, and body weight. For example, melanocortin-3 receptors (MC3Rs) are expressed in the ventral tegmental area (VTA), and our lab previously showed that intra-VTA injection of the MC3R agonist, MTII, decreases home-cage food intake and operant responding for sucrose pellets. The cellular mechanisms underlying the effects of intra-VTA α-MSH on feeding and food reward are unknown, however. To determine how α-MSH acts in the VTA to affect feeding, we performed electrophysiological recordings in acute brain slices from mice expressing EYFP in MC3R neurons to test how α-MSH affects the activity of VTA MC3R neurons. α-MSH significantly increased the firing rate of VTA MC3R neurons without altering the activity of non-MC3R expressing VTA neurons. In addition, the α-MSH-induced increase in MC3R neuron activity was independent of fast synaptic transmission and intracellular Ca2+ levels. Finally, we show that the effect of α-MSH on MC3R neuron firing rate is likely activity dependent. Overall, these studies provide an important advancement in the understanding of how α-MSH acts in the VTA to affect feeding and food reward.