Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

The cold- and menthol-activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non-neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8-expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch-clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.Instituto de Salud Carlos III, Grant/Award Number: PI12/0058; National Institutes of Health, Grant/Award Number: ZIA DE000721-12; Ministerio de Ciencia e Innovación, Grant/Award Numbers: SAF2009-11175, SAF2010-14990-R, SAF2016-77233-R; Ministerio de Economía y Competitividad, Grant/Award Numbers: BES-2011-047063, BES-2017-080782; Severo Ochoa Programme for Centres of Excellence in R&D, Grant/Award Number: SEV-2017-0723 and cofinanced by the European Regional Development Fund; Generalitat Valenciana, Grant/Award Number: GRISOLIA/2008/02

The cold-sensing ion channel TRPM8 regulates central and peripheral clockwork and the circadian oscillations of body temperature

[Abstract] Aim: Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function. Methods: We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc). Results: TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8-/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8-/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues. Conclusion: Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc.Ministerio de Ciencia e Innovación (España); RT2018-099995-B100Ministerio de Ciencia e Innovación (España); AEI/10.13039/501100011033Generalitat Valenciana; PROMETEO/2021/031Ministerio de Asuntos Económicos y Transformación Digital (España); BES-2011-04706

Expression of the cold thermoreceptor TRPM8 in mouse brain circuits involved in thermal homeostasis

Resumen del trabajo presentado al VII Congreso Red Española Canales Iónicos, celebrado en Cáceres del 15 al 17 de mayo de 2019.The ion channel TRPM8 is the principal sensor of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in certain non-neuronal tissues. Little is known about the expression of this thermosensitive ion channel in the central nervous system. The objective of this work was to study the expression and anatomical distribution of TRPM8 channels in mouse brain. We used RT-PCR and “in situ” hybridization (ISH). Furthermore, GFP immunohistochemistry was carried out in two transgenic TRPM8 reporter mouse models: TRPM8-green fluorescent protein (GFP) knock-in mice, Trpm8EGFPf and TRPM8-yellow fluorescent protein (YFP) transgenic mice, Trpm8BAC-EYFP+. Finally, we performed patch-clamp recordings in Trpm8BAC-EYFP+ septal neurons. We found that TRPM8 is expressed in mouse central nervous system, although with much lower levels of expression than in peripheral sensory ganglia. Positive cells were mainly identified in the preoptic hypothalamus, septal area, reticular thalamic nucleus and limbic regions with projections widely distributed within the brain and brainstem. Electrophysiological recordings in brain slices revealed the functionality of these ion channels. Our results showing expression of TRPM8 in the central nervous system open a new window in TRPM8 physiology. Further experiments are required to fully understand the potential roles of this molecular sensor within the brain.The study was supported by the projects SAF2009-11175 and PI12/0058 (RS), SAF2010-14990-R and SAF2016-77233-R (FV, AG), co-financed by the European Regional Development Fund (ERDF), the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723) and the IRP of the National Institute of Dental and Craniofacial Research, NIH (MAH). CF-P, PO and PH held predoctoral fellowships of the Generalitat Valenciana and Spanish MINECO (GRISOLIA/2008/025, BES-2011-047063 and BES-2017-080782).Peer reviewe

Función de los canales iónicos TRPM8 y TRPA1 en la homeostasis térmica y metabólica

En este estudio, se evaluó el papel de los canales iónicos TRPM8 y TRPAl, principales sensores de frío, en la interrelación entre la percepción de la temperatura, la termorregulación y la homeostasis energética. Usando distintas líneas murinas deficientes en dichos canales, se determinaron la temperatura corporal interna y la actividad motora, así como sus principales respuestas autonómicas, comportamentales y metabólicas y finalmente se evaluó su impacto en la homeostasis energética. La deleción de TRPM8 indujo una termorregulación defectuosa en animales expuestos al frío o al ayuno, además de obesidad de aparición tardía. Por su parte, la deficiencia de TRPA1 provocó caídas de temperatura corporal, pero sin alteraciones en el fenotipo energético y metabólico

Mammalian cold TRP channels: impact on thermoregulation and energy homeostasis

Body temperature regulation is a fundamental homeostatic function in homeothermic animals. It is governed by the central nervous system that integrates temperature signals from internal body structures and the skin and provides efferent responses to adjust heat-exchange rates with the environment. Thermoregulation has a major influence on energy balance by regulating food intake as well as heat production and energy expenditure. Surprisingly, although almost 50% of our energy expenditure is dedicated to maintaining homeothermy, very little is yet known about the molecular aspects and the neural wiring involved in the intimate interrelationship between these two critical homeostatic systems. Some non-selective cation channels of the transient receptor potential (TRP) family work as molecular thermal sensors in sensory neurons and other cells. In this review, we discuss recent advances in our understanding of the basic mechanisms responsible for thermoregulation in the cold. We have focused our attention on the role of two cold-activated TRP channels (transient receptor potential melastatin 8 and transient receptor potential ankyrin 1) in body temperature regulation as well as their impact on energy balance and metabolism. A better understanding of the mechanisms coupling thermoregulation to energy homeostasis, including the involvement of thermosensitive TRPs, may uncover additional mechanisms underlying the pathogenesis of obesity and its metabolic consequences in humans, opening new strategies for the diagnosis, treatment, and prevention of this disease.The study was supported by projects MINECO SAF2009-11175 (RS) and SAF2016-77233-R (FV) and by the Instituto de Salud Carlos III PI12/0058 (RS) and was cofinanced by the European Regional Development Fund (ERDF) and the Severo Ochoa Programme for Centres of Excellence in R&D (ref. SEV-2013-0317). PO held a predoctoral fellowship MINECO (BES-2011-047063).Peer reviewe

Sex differences in thermoregulation in mammals: Implications for energy homeostasis

Thermal homeostasis is a fundamental process in mammals, which allows the maintenance of a constant internal body temperature to ensure an efficient function of cells despite changes in ambient temperature. Increasing evidence has revealed the great impact of thermoregulation on energy homeostasis. Homeothermy requires a fine regulation of food intake, heat production, conservation and dissipation and energy expenditure. A great interest on this field of research has re-emerged following the discovery of thermogenic brown adipose tissue and browning of white fat in adult humans, with a potential clinical relevance on obesity and metabolic comorbidities. However, most of our knowledge comes from male animal models or men, which introduces unwanted biases on the findings. In this review, we discuss how differences in sex-dependent characteristics (anthropometry, body composition, hormonal regulation, and other sexual factors) influence numerous aspects of thermal regulation, which impact on energy homeostasis. Individuals of both sexes should be used in the experimental paradigms, considering the ovarian cycles and sexual hormonal regulation as influential factors in these studies. Only by collecting data in both sexes on molecular, functional, and clinical aspects, we will be able to establish in a rigorous way the real impact of thermoregulation on energy homeostasis, opening new avenues in the understanding and treatment of obesity and metabolic associated diseases.This study was supported by the Projects RT2018-099995-B100 and PID2021-124785OB-100 (AEI/10.13039/501100011033/ ERDF A way of making Europe) to RS and PID2019-108194RB-I00 (AEI/10.13039/501100011033) to FV, (Spanish Ministry of Science and Innovation), co-financed by Generalitat Valenciana (PROMETEO/2021/031) and the Severo Ochoa Programme for Centers of Excellence in R&D (ref. CEX2021-001165). AR holds a Margarita Salas postdoctoral fellowship (Spanish Ministry of Universities).Peer reviewe

Deletion of the cold thermoreceptor TRPM8 increases heat loss and food intake leading to reduced body temperature and obesity in mice

The coupling of energy homeostasis to thermoregulation is essential to maintain homeothermy in changing external environments. We studied the role of the cold thermoreceptor TRPM8 in this interplay in mice of both sexes. We demonstrate that TRPM8 is required for a precise thermoregulation in response to cold, in fed and fasting. Trpm8−/− mice exhibited a fall of 0.7°C in core body temperature when housed at cold temperatures, and a deep hypothermia (<30°C) during food deprivation. In both situations, TRPM8 deficiency induced an increase in tail heat loss. This, together with the presence of TRPM8-sensory fibers innervating the main tail vessels, unveils a major role of this ion channel in tail vasomotor regulation. Finally, TRPM8 deficiency had a remarkable impact on energy balance. Trpm8−/− mice raised at mild cold temperatures developed late-onset obesity and metabolic dysfunction, with daytime hyperphagia and reduction of fat oxidation as plausible causal factors. In conclusion, TRPM8 fine-tunes eating behavior and fuel utilization during thermoregulatory adjustments to mild cold. Persistent imbalances in these responses result in obesity.This work was supported by Project SAF2009-11175 and PI12/0058 to R.S., SAF2010-14990-R and SAF2016-77233-R to F.V., (Ministry of Economy and Competitiveness). European Regional Development Fund, and Severo Ochoa Programme for Centres of Excellence in R&D SEV-2013-0317. C.F.-P. and P.O. held predoctoral fellowships of the Generalitat Valenciana (GRISOLIA/2008/025) and MINECO (BES-2011-047063), respectively. J.L.P.-V. received Human Frontier Science Program Long-Term Award LT000442/2012.Peer reviewe

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

In Press.The cold‐ and menthol‐activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non‐neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8‐expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch‐clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.Instituto de Salud Carlos III, Grant/Award Number: PI12/0058; National Institutes of Health, Grant/Award Number: ZIA DE000721‐12; Ministerio de Ciencia e Innovación, Grant/Award Numbers: SAF2009‐11175, SAF2010‐14990‐R, SAF2016‐77233‐R; Ministerio de Economía y Competitividad, Grant/Award Numbers: BES‐2011‐047063, BES‐2017‐080782; Severo Ochoa Programme for Centres of Excellence in R&D, Grant/Award Number: SEV‐2017‐0723 and cofinanced by the European Regional Development Fund; Generalitat Valenciana, Grant/Award Number: GRISOLIA/2008/02