title: Characterization of mouse preoptic area cellular populations  involved in thermoregulation
creator: Zuza, Kristina
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-570
subject: 570 Life sciences
description: Thermoregulation is a dynamic homeostatic process, tightly regulated by the autonomic nervous   system. How the brain coordinates maintenance of the body temperature within a narrow range of 37˚C,   a condition that is needed for the survival of most of the species, remains unclear. Among the brain regions   implicated in thermoregulation is the anterior portion of the hypothalamus, the preoptic area (POA). In   this region neurons that respond to direct temperature stimuli and to the skin and spinal cord warming   were found, suggesting that these warm-sensitive neurons (WSNs), are the cells that detect deep brain   temperature and integrate it with temperature information from the periphery. The limiting factor in   studying the WSNs of the POA and their role in thermoregulation is the lack of specific molecular markers   that identify them.   Therefore, the goal of this thesis work was to characterize WSNs of the POA at the molecular level   and to find their genetic marker(s). To achieve this goal I used a primary POA cell culture and performed   calcium imaging while applying a temperature stimulus of 45˚C in the presence and the absence of the   TRPM2 antagonist, 2-Aminoethoxydiphenyl borate (2-APB). I identified and hand-picked temperature   responding cells and temperature non-responding cells for the single-cell RNA-sequencing (scRNA-seq).   Analysis of the scRNA-seq data pointed to the limitations of the P9 cell culture used. The majority of the   temperature non-responding cells expressed glial marker genes together with neuronal markers, a   combination not found in vivo. I concluded that the genetic heterogeneity of the sequenced cells was too   large and putative WSNs’ molecular markers identified from cultured neurons would be ambiguous. In   addition, to find markers of WNSs one has to take another approach, such as Patch-seq to analyze these   neurons in more natural conditions.  One of the POA neuronal populations activated by a change in ambient temperature is expressing   leptin receptor (POALepR). These neurons also exhibit an increase in action potential firing frequency (AP   FF) during the process of chronic heat exposure to 36˚C that also leads to an increased heat endurance (at   39˚C). in mice. This intrinsic property of neurons (not affected by synaptic blockers), seems to be needed   for a mouse to endure heat, as the animals in which the firing of POALepR is abolished fail to do so.   To learn more about the role of the POALepR neurons in the heat acclimation process I used FACS   (Fluorescence Activated Cell Sorting) to isolate the POALepR neurons from POA of non-acclimated and   acclimated ( 5 and 30 days at 36 ˚C) LeprCreHTB mice and performed RNA sequencing. I identified three   genes Kcnq2, Kcnn2, and Kcnh2, all three coding for potassium ion channels, whose expression level   changed with the course of heat acclimation. I have tested the functionality of these ion channels in AP   viii  firing of the POALepR neurons, by employing electrophysiology and pharmacology in acute POA slices. Ion   channels Kv7.2 and Kv11.1, coded by Kcnq2, and Kcnh2, respectively, exhibited a role in shaping the AP   firing of POALepR neurons. Applying the antagonist of Kv7.2 disrupted harmonious AP firing of POALepR  neurons coming from acclimated mice, rendering their membrane potential unsteady and their firing   bursty. In addition, the application of the Kv11.1 antagonist increased the AP FF of POALepR neurons even   further in the long-term acclimated condition.   Heat acclimation is a naturally occurring process happening across mammalian species, including   humans. It is important for enduring physical burdens in hotter climates as it leads to the improved   function of the thermoregulatory system. It is forthright to hypothesize that the POA, the central regulator   of temperature homeostasis, plays a role in heat acclimation. However, knowledge about it is scarce.   Knowing which molecules change in POALepR neurons transcriptome to increase firing, and to which other   thermoregulatory relays these neurons project will help us understand their role and the role of POA in   heat acclimation.
date: 2023
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/32305/1/Doctoral_Thesis_Kristina_Zuza.pdf
identifier: DOI:10.11588/heidok.00032305
identifier: urn:nbn:de:bsz:16-heidok-323052
identifier:   Zuza, Kristina  (2023) Characterization of mouse preoptic area cellular populations involved in thermoregulation.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/32305/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng