Cold induces brain region-selective neuronal activity-dependent lipid metabolism

Reviewer #1 (Public Review):

Summary:

This study focuses on metabolic changes in the paraventricular hypothalamic (PVH) region of the brain during acute periods of cold exposure. The authors point out that in comparison to the extensive literature on the effects of cold exposure in peripheral tissues, we know relatively little about its effects on the brain. They specifically focus on the hypothalamus, and identify the PVH as having changes in Atgl and Hsl gene expression changes during cold exposure. They then go on to show accumulation of lipid droplets, increased Fos expression, and increased lipid peroxidation during cold exposure. Further, they show that neuronal activation is required for the formation of lipid droplets and lipid peroxidation.

Strengths:

A strength of the study is trying to better understand how metabolism in the brain is a dynamic process, much like how it has been viewed in other organs. The authors also use a creative approach to measuring in vivo lipid peroxidation via delivery of a BD-C11 sensor through a cannula to the region in conjunction with fiber photometry to measure fluorescence changes deep in the brain.

Weaknesses:

Although the topic and findings are of interest, there are a few key weaknesses in the study that would improve the work if addressed. One weakness was many of the experiments were done in a manner that could not distinguish between the contributions of neurons and glial cells, limiting the extent of conclusions that could be made. While this is not easily doable for all experiments, it can be done for some. For example, the Fos experiments in Figure 3 would be more conclusive if done with the labeling of neuronal nuclei with NeuN, as glial cells can also express Fos. To similarly show more conclusively that neurons are being activated during cold exposure, the calcium imaging experiments in Figure S3 can be done with cold exposure. Additionally, many experiments are only done with the minimal three animals required for statistics and could be more robust with additional animals included. Another weakness is that the authors do not address whether manipulating lipid droplet accumulation or lipid peroxidation has any effect on PVH function (e.g. does it change neuronal activity in the region?).

Reviewer #2 (Public Review):

Summary:

Cold-induced lipid metabolism is well-established in adipose tissues. The authors set out to determine whether cold could alter brain lipid metabolism. By QPCR analysis of brain punches after acute cold, they found that mRNA expressions of several lipolysis-related genes were upregulated compared to RT controls. By combining fluorescent sensors and in vivo fiberphotometry, they observed cold-induced lipid peroxidation/lipolysis, which could be blocked by pharmacological inhibitors of neuronal activity (muscimol and kynurenic acid). The brain is not traditionally considered an organ with high lipid metabolism (vs carbohydrate); therefore, the observation and hypothesis proposed by the authors are unexpected and can be interesting. However, the experiments and data were rather preliminary and superficial and did not support the authors' conclusions. In addition, the main hypothesis, in relationship to the role of cold/temperature, remains incoherent and needs a major update.

Strengths:

A set of relatively novel and interesting observations.

Creative use of several in vivo sensors and techniques.

Weaknesses:

(1) The physiological relevance of lipolysis and thermogenesis genes in the PVH. The authors need to provide quantitative and substantial characterizations of lipid metabolism in the brain beyond a panel of qPCRs, especially considering these genes are likely expressed at very low levels. mRNA and protein level quantification of genes in Fig 1, in direct comparison to BAT/iWAT, should be provided. Besides bulk mRNA/protein, IHC/ISH-based characterization should be added to confirm to cellular expression of these genes.

(2) The fiberphotometry work they cited (Chen 2022, Andersen 2023, Sun 2018) used well-established, genetically encoded neuropeptide sensors (e.g., GRABs). The authors need to first quantitatively demonstrate that adapting BD-C11 and EnzCheck for in vivo brain FP could effectively and accurately report peroxidation and lipolysis. For example, the sensitivity, dynamic range, and off-time should all be calibrated with mass spectrometry measurements before any conclusions can be made based on plots in Figures 4, 5, and 6. This is particularly important because the main hypothesis heavily relies on this unvalidated technique.

(3) Generally, the histology data need significant improvement. It was not convincing, for example, in Figure 3, how the Fos+ neurons can be quantified based on the poor IF images where most red signals were not in the neurons.

(4) The hypothesis regarding the direct role of brain temperature in cold-induced lipid metabolism is puzzling. From the introduction and discussion, the authors seem to suggest that there are direct brain temperature changes in responses to cold, which could be quite striking. However, this was not supported by any data or experiments. The authors should consolidate their ideas and update a coherent hypothesis based on the actual data presented in the manuscript.