Periodic Reporting for period 3 - Acclimatize (Hypothalamic mechanisms of thermal homeostasis and adaptation) Reporting period: 2021-09-01 to 2023-02-28 Summary of the context and overall objectives of the project The overarching goal of my ERC project “Acclimatize”, is to dissect and understand molecular substrates, neuronal circuits and mechanisms of plasticity that allow humans ––and our favorite model organism the laboratory mouse–– to acclimatize to hot environmental temperatures. We focus on the thermoregulatory center in the hypothalamus, the so-called preoptic area (POA).The central orchestration of body temperature control is a largely unexplored part of neurobiology, at least from the perspective of specific and selective, neuronal populations and their underlying pathways and neuronal circuits. The overall objectives of the ERC proposal Acclimatize are to:i. identify and characterize molecules that participate in internal deep brain temperature detection ii. define and describe neuronal populations and their connectivity involved in temperature acclimationiii. assess their mechanism of acclimation-induced plasticity. Regulation of body temperature is essential ––illustrated by the fact that body temperature is monitored in all hospitalized patients. Temperature is an omnipresent, integral part of every aspect of our daily lives, down to the kinetics of every enzymatic reaction. In particular, body temperature shapes our circadian rhythm, it is affecting infectious processes, is a variable in energy metabolism and is part of hormonal regulation (as emphasized by the fact that body temperature is used as a predictor for fertility during estrous or menstrual cycles).Therefore, understanding the regulation of body temperature has multiple interaction points to various biomedically relevant areas, such as infectious disease, chronobiology, energy metabolism and others. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the initial phase of the project that started in September 2018 I focused on recruiting additional personnel, purchasing and setting up new equipment for the project, in particular the new electrophysiology set-up that we equipped with optogenetics capabilities to perform ex-vivo circuit mapping of thermoregulatory pathways as detailed below. Additionally, my team and I established technologies as well as trained personnel in the respective techniques. As part Aim 1, I defined several sub-goals, one of which was the detailed characterization of the function of Trpm2, an ion channel we had previously implicated in thermoregulation (Song et al, Science, 2016). Using a long line of complex electrophysiological experiments carried out as part of our ERC project established that the ion channel constitutes a synaptic temperature sensor. This study is currently under consideration at the journal Neuron, one of the major research outlets for Neuroscience research.Another sub-goal of aim 1 is to identify neuroendocrine factors (neuropeptides) that are released to modulate thermoregulation. A graduate student, Carolina Sousa, has taken on that project. She set up a novel microdialysis system in the lab to obtain samples of interstitial brain fluid and cerebrospinal fluid from the hypothalamic region of awake and freely moving mice. Indeed, we were able to identify neuropetides with dynamic release patterns during temperature stimulation. These neuropeptide candidates a currently in the process of being verified by ELISA assays and by a targeted mass spectrometry approach in collaboration with the Haefeli group at the University Clinic at Heidelberg, who are experts in quantitative targeted mass spectrometry.Aim 2 of the proposal Acclimatize was conceived to understand and elucidate the connectivity of thermoregulatory neurons and to establish the inputs and outputs of body temperature-regulating POA circuits. We started out by characterizing output specificity of a POA neuron population that recently had been identified to mediate hypothermia. Due to competition within this research field and before publication, we currently opt for not revealing the nature of this neuronal population. We therefore name these preoptic cells here Acclimation-Activated Neurons (AANs), for reasons that become apparent below. By means of histological fiber tracing we found that AANs connect to several downstream hypothalamic and extra-hypothalamic areas. Using optogenetic circuit mapping in vivo, we find that there is pathway selectivity and recruitment of peripheral thermal effector organs by specific POA pathways: some of which controlling brown adipose tissue thermogenesis, while others stimulating cutaneous vasodilation which in mice manifests as tail vasodilation. These results are remarkablefor two reasons: First, pathway selectivity for different peripheral thermal effector organs have been proposed but thus far largely remained elusive. Also, one of the identified downstream target regions, the paraventricular hypothalamic nucleus (PVH) ,has thus far not been recognized as a major thermoregulatory outlet. These data have not been published yet, in part due to Covid restrictions and delays. We aim to finalize a manuscript after further verification and characterization these pathways towards the end of this year/beginning of the coming year. Aim 3 The goal of aim 3 is to understand the adaptive ability to cope with hot environmental temperatures referred to as acclimation. While the peripheral cardiovascular and energy-metabolic changes are quite well described, the neuro-central pathways that orchestrate these peripheral changes have largely remained unexplored. We hypothesized (as detailed in my ERC proposal) that the preoptic thermoregulatory center that response to heat and acutely mediates heat-loss responses is also responsible to mediate long-term adaptive responses. We indeed found that the aforementioned AANs increase their neuronal activity upon long-term heat acclimation. To obtain these data took longer than originally anticipated because one of my co-workes funded by the ERC project, Dr. Jörg Pohle, had to leave the project. Dr. Wojciech Ambroziak eventually took over this project part and is still continuing to work on it now. The identified cells did this specifically and other, genetically unrelated cells residing also in the POA, did not change their activity, suggesting cell-type specificity. Moreover, inhibiting acclimation-induced activity in AANs by genetic means prevented mice to cope with heat. Additionally, artificially inducing neuronal activity in AANs chemogenetically or optogenetically was sufficient to induce heat acclimation. Thus within my ERC project Acclimatize we identified neurons that are important for heat acclimation. We are currently assessing the molecular mechanism that drives the adaptive changes in these neurons and have started to prepare a manuscript that describes these fascinating results to the research community. Again, the Covid pandemic slowed us down considerably, both by limiting access of researchers to the lab but, more dramatically, by limiting mouse breedings in our animal facility. We envision that we will submit the heat-acclimation manuscript to a reputable scientific journal towards the end of this year. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Progress to date:A. Identification of a Synaptic Temperature sensorThus far and since the discovery that warm-sensitive neurons (WSNs) exists in the hypothalamus more than 100 years ago (Barbour, Naunyn-Schmiedeberg’s Archives of Pharmacology, 1912) our discovery that a Trp channel can act as a deep brain synaptic temperature sensor is an entirely new concept. Interestingly, we find that strong deep brain heat stimulation triggers plasticity and long lasting hypothermia and this could be explained by a synaptic temperature sensor that mediates synaptic plasticity. Thus we are excited to promote a new concept of interoceptive synaptic temperature detection and we believe that these findings will stimulate new research concerning interoception.B. The identification of Acclimation-Activated Neurons (AANs)The identification of cells that mediate heat acclimation is, we believe, a breakthrough in acclimation research, particularly in times of global warming. These findings, we envision, will trigger future research on molecular mechanisms and neuronal pathways encompassing these cells. Likely, multiple homeostatic pathways will be integrated at the level of AANs and thus they will have considerable therapeutic potential. C. Development of miniature head-mounted deep brain temperature stimulator (thermode).We believe this novel piece of custom-made equipment will have a strong impact not only on basic thermoregulatory research but be alos relevant in dissecting pathways relevant for energy metabolism and obesity.Expected Progress until the end of the project:I expect that we will extend our knowledge on molecular, synaptic and circuit-level mechanisms of plasticity that accompany and drive acclimatization.Additionally I envision that we shed more light on the mechanism that mediates deep brain temperature detection. Last but not least I reckon that we will be able to provide proof-of-concept that deep brain temperature manipulation within the hypothalamus may modulate energy metabolism and obesity.