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• W3133838079 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Maintaining stable body temperature through environmental thermal stressors requires detection of temperature changes, relay of information, and coordination of physiological and behavioral responses. Studies have implicated areas in the preoptic area of the hypothalamus (POA) and the parabrachial nucleus (PBN) as nodes in the thermosensory neural circuitry and indicate that the opioid system within the POA is vital in regulating body temperature. In the present study we identify neurons projecting to the POA from PBN expressing the opioid peptides dynorphin and enkephalin. Using mouse models, we determine that warm-activated PBN neuronal populations overlap with both prodynorphin (Pdyn) and proenkephalin (Penk) expressing PBN populations. Here we report that in the PBN Prodynorphin (Pdyn) and Proenkephalin (Penk) mRNA expressing neurons are partially overlapping subsets of a glutamatergic population expressing Solute carrier family 17 (Slc17a6) (VGLUT2). Using optogenetic approaches we selectively activate projections in the POA from PBN Pdyn, Penk, and VGLUT2 expressing neurons. Our findings demonstrate that Pdyn, Penk, and VGLUT2 expressing PBN neurons are critical for physiological and behavioral heat defense. Introduction Maintaining body temperature in the face of changing environmental conditions is a core attribute of mammals, including humans, and is critical for life. Achieving a stable body temperature requires information about the temperature of the periphery and environment to be integrated to drive physiological and behavioral programs to defend the core temperature (Jessen, 1985). Physiological parameters modulated to maintain temperature include thermogenesis (utilization of brown adipose tissue [BAT], shivering), changes in circulation (vasodilation and vasoconstriction), and evaporation (Cabanac, 1975). Behavioral modifications include selection, when possible, of ambient temperature, altering posture to alter heat loss, and modulation of physical activity level. Responding to thermal challenges involves perception of temperature, encoding the valence of the temperature (e.g. too hot), and evoking appropriate physiological responses (Tan and Knight, 2018). Perceptive, affective, and autoregulatory elements may be encoded by overlapping or discrete neuronal circuits. The preoptic area of the hypothalamus (POA) and the parabrachial nucleus (PBN) have been identified as key nodes within the neurocircuitry regulating body temperature. In the report presented here, we identify and delineate the unique roles of genetically defined neuronal populations in PBN projecting to the POA in responding to environmental warmth. The POA contains neurons critical for integration of information about body temperature and for coordination of responses to thermal challenges to maintain core temperature (Abbott and Saper, 2017; Abbott and Saper, 2018; Tan et al., 2016). Neurons in POA, identified by different genetic markers, can regulate BAT activation, drive vasodilation, and shift ambient temperature preferences (Tan et al., 2016; Yu et al., 2016). Prior evidence has suggested critical roles for inputs from the PBN to the POA in regulating temperature (Geerling et al., 2016; Miyaoka et al., 1998; Morrison, 2016). The PBN is, however, a highly heterogenous structure with subpopulations known to relay various sensory information from the periphery (thirst, salt-appetite, taste, pain, itch, temperature, etc.) and playing key roles in nocifensive responses, specifically escape and aversive learning (Chiang et al., 2020; Kim et al., 2020; Palmiter, 2018). The studies here examine the roles for parabrachial glutamatergic neurons expressing the opioid peptides dynorphin and enkephalin. Regulation of body temperature requires integration of homeostatic and environmental inputs across varying time scales creating opportunities for neuromodulatory signaling to play key roles. In vivo experiments suggest that opioid neuropeptides, as a neuromodulator, may play a critical role in thermal homeostasis. Pharmacologic manipulation of opioid systems induces changes in body temperature and can impair thermoregulatory control in humans, rats, and other animals (Chen et al., 2005; Ikeda et al., 1997; Spencer et al., 1990). Opioid receptor signaling within the POA has been implicated in modulating body temperature, but potential sources for native ligands remain to be identified (Baker and Meert, 2002; Clark, 1979). Activation of mu receptors in the POA can drive opposing effects on body temperature. A recent study indicated that neurons in PBN expressing prodynophin (Pdyn), which is processed to dynorphin, the endogenous ligand for the kappa opiate receptor (KOR), are activated by ambient warmth (Chavkin et al., 1982; Geerling et al., 2016). PBN neurons expressing the endogenous mu and delta opioid receptor ligand, enkephalin, have not been examined in relation to how they may regulate temperature. In this study we used a series of modern anterograde and retrograde viral approaches to determine the connection of PBN neurons expressing Pdyn (Pdyn+) and Penk (Penk+), to the POA (Henry et al., 2017). We delineate the overlap of the neuronal populations expressing these peptides with warm-activated PBN neurons. We identify subsets of Pdyn+ and Penk+ neurons that project to POA from the PBN. We then combine optogenetic and chemogenetic tools with Cre driver mouse lines to determine the causal roles of PBN neurons that project to the POA in mediating physiological and behavioral responses to thermal challenge. Here we also examine potential roles of opioid receptor mediated behaviors in both Pdyn+ and Penk+ PBN-POA projections. We report that glutamatergic, Pdyn+, and Penk+ neuronal populations projecting from PBN to POA initiate physiological and behavioral heat defensive behaviors. Chemogenetic inhibition of glutamatergic PBN neurons blocks vasomotor responses to thermal heat challenge. The studies reported here provide new insights into the thermoregulatory properties of parabrachial neuropeptide-containing projections to the hypothalamus in homeostatic and metabolic behavior. Results Ambient warmth activates Pdyn+ and Penk+ neurons in PBN Effects of mu and kappa receptor signaling on body temperature have been described and mRNA for Pdyn and Penk has been reported to be expressed in the PBN (Baker and Meert, 2002; Chen et al., 2005; Clark, 1979; Engström et al., 2001; Hermanson and Blomqvist, 1997; Hermanson et al., 1998). To examine if PBN neurons expressing dynorphin or enkephalin opioid neuropeptides are activated by ambient warmth, we exposed mice to ambient warmth (38°C) or room temperature (21–23°C) for 4 hr prior to preparation of brain for Fos staining. We performed immunohistochemistry (IHC) on collected brains sections containing the PBN with antibody directed against Fos (anti-Fos) to examine induction of Fos expression as a marker of neuronal activation (Sheng and Greenberg, 1990). Consistent with recent reports, we observed induction of Fos expression in the lateral PBN (LPBN) (Figure 1B,C; Geerling et al., 2016). In brain sections from warm exposed mice (n = 8) compared to room temperature controls (n = 4), Fos staining revealed a robust and significant (p=0.003) increase in mean ± SEM number of neurons positive for Fos expression in the LPBN per brain: 265.8 ± 41.9 in warm exposed mice compared to 23.2 ± 4.0 in room temperature controls (Figure 1C). Cells in LPBN, lateral to superior cerebellar peduncle, in sections corresponding −5.0 to −5.4 caudal to bregma were counted. In brains from recombinase reporter mice (Ai14) crossed to Pdyn-Cre (Ai14xPdyn-Cre) or Penk-Cre (Ai14xPenk-Cre) lines, tdTomato was robustly expressed in LPBN indicating expression of Pdyn (Al-Hasani et al., 2015; François et al., 2017; Krashes et al., 2014; Madisen et al., 2010) and Penk (François et al., 2017) in LPBN neurons (Figure 1D,E). Cells expressing tdTomato in Pdyn-Cre mice (Pdyn+) and Penk-Cre mice (Penk+) were most abundant in the caudal LPBN. To determine the overlap of warm-activated neurons with Pdyn+ or Penk+ cells in LPBN, we exposed mice, Ai14xPdyn-Cre and Ai14xPenk-Cre, to a warm (38°C) ambient temperature for 4 hr prior to harvesting brains and used IHC on sections with anti-Fos. In the LPBN of Ai14xPdyn-Cre mice, we found that a mean ± SEM of 81% ± 2.5 of the cells positive for Fos staining were also positive for tdTomato expression (n = 4 animals, 1017 cells) (Figure 1F). In the LPBN of Ai14xPenk-Cre mice, an average ± SEM of 54% ± 4.6 (n = 4 animals, 1109 cells) of Fos-positive cells in warm-exposed mice were also positive for tdTomato (Figure 1G). We blindly sampled tdTomato neurons in the LPBN and then quantified the number of cells also labeled for Fos. In samples from Ai14xPdyn-Cre mice we found 22% ± 4 (n = 3 animals, 150 cells) overlap and from Ai14xPenk-Cre 18% ± 4 (n = 3 animals, 150 cells). These data indicate that warmth activated neurons in LPBN may co-express the neuropeptides dynorphin and enkephalin. Figure 1 with 1 supplement see all Download asset Open asset Warm-activated neurons in parabrachial nucleus (PBN) overlap with Pdyn and Penk expression. (A) Schematized view of PBN regions analyzed for Fos expressing neurons and the genetic cross schemes of Ai14xPdyn-Cre/Ai14xPenk-Cre reporter mouse lines used. (B) Representative images of brain sections harvested from animals exposed to room temperature or 38°C and probed with anti-Fos. Brains from 38°C exposed mice had significantly more neurons in PBN positive for Fos staining. (C) Quantification of Fos positive LPBN neurons per brain. Data are presented as mean ± SEM; n = 4 animals in room temp group, n = 8 animals in warm exposed group; t-test, ∗∗p<0.01. (D) Representative images of Fos labeling (cyan) in Ai14 x Pdyn-Cre brains with Fos labeling of Pdyn+ (red) (filled arrows) neurons and Pdyn- (open arrows). (E) Representative images of Fos labeling in Ai14xPenk-Cre brains with Fos labeling of Penk+ (magenta) (filled arrows) and Penk- neurons (open arrows). (F and G) Quantification of the overlap of Fos staining in Ai14xPdyn-Cre and Ai14xPenk-Cre brains demonstrated 81% or 46% of Fos cells were also overlapped with tdTomato expression in Ai14xPdyn-Cre or Ai14xPenk-Cre brains, respectively. Pdyn+ and Penk+ LPBN neurons project to the ventral medial preoptic area in the POA and are VGLUT2+ Next, to delineate possible overlapping expression of the neuropeptides, we used fluorescent in situ hybridization (FISH) with targeted probes for Pdyn, Penk, and Slc17a6 and examined serial coronal brain sections encompassing the PBN. Based on previous studies implicating glutamate in LPBN thermosensory relay neurons (Nakamura and Morrison, 2007; Nakamura and Morrison, 2010), we hypothesized that the majority of Pdyn and Penk expressing (Pdyn+ and Penk+) LPBN neurons would also express Slc17a6, indicating they are glutamatergic. Consistent with expression patterns evident in the Ai14xPdyn-Cre and Ai14xPenk-Cre mice, Pdyn and Penk FISH probes labeled neurons in the LPBN (Figure 2F and G). Pdyn+ and Penk+ cells were most abundant in the caudal LPBN. Sections were also co-labeled with Slc17a6 probes with Pdyn or Penk probes. The overlap of cells in LPBN labeled with each probe was quantified. A mean ± SEM of 98% ± 0.9 (n = 760 cells, n = 4 mice) of Pdyn labeled cells were positive for Slc17a6 (Figure 2A,I). A mean ± SEM of 97% (n = 650 cells, n = 4 mice) of Penk labeled cells were positive for Slc17a6 (Figure 2B,J). Surprisingly, a mean ± SEM of 51% ± 6.6 (n = 760, n = 4 mice) of LPBN neurons positive for Pdyn were also positive for Penk labeling (Figure 2C,K). Reciprocally, a mean ± SEM of 58% ± 2.3 (n = 650 cells, n = 4 mice) of cells labeled by Penk probes were also labeled by Pdyn probes (Figure 2D,K). These FISH based experiments indicate that Pdyn+ and Penk+ cells in the LPBN express Slc17a6 and are partially overlapping subpopulations of glutamatergic LPBN cells. A recent report on PBN→POA neurons implicated cholecystokinin (Cck) expressing LPBN neurons in heat defense (Yang et al., 2020). We examined if Pdyn labeled neurons in LPBN were co-labeled by probes for Cck and found that 70% ± 0.7 (mean ± SEM, n = 150 cells, n = 3 mice) of LBP Pdyn labeled neurons were co-labeled by Cck probes (Supplemental Figure 2—figure supplement 2D–F) suggesting that mRNA for Cck and pDyn is expressed in overlapping neuronal populations. Figure 2 with 2 supplements see all Download asset Open asset Pdyn+ and Penk+ LPBN neuron populations overlap, express Slc17a6, and project to the POA. (A–D) Quantification of cells labeled with (A) Pdyn probe (Pdyn+) and Slc17a6 (VGLUT2+) probes, or (B) Penk (Penk+) and Slc17a6 (VGLUT2+) probe, or (C and D) Pdyn and Penk probes. (E) Illustration of area of parabrachial nucleus (PBN) depicted in F–K. (F–H) Representative FISH images of LPBN neurons expressing (F) Pdyn, (G) Penk, and (H) Slc17a6. (I–K) (similar results were obtained in n = 3 mice) Representative images of overlays of (I) Pdyn with Slc17a6 and Penk with Slc17a6 (J), and (K) Pdyn with Penk. Arrowheads mark examples of cells positive for co-labeling of two transcripts. 98% of neurons expressing Pdyn and 97% of neurons labeled for Penk were also labeled with probes for Slc17a6. Data are presented as mean ± SEM; n = 4 animals, 760 cells for Pdyn and n = 4 animals, 760 cells for Penk. Diagram of viral injections into wild-type mice. (M) Anatomical location of representative FISH images shown in (N and O) that show overlap of (N) Cre expression, mediated by retrovirus transduction, with (O) Pdyn and (P) Penk. Arrowheads mark cells expressing Cre, with filled arrowheads co-expressing (O) Pdyn or (P) Penk and open arrowheads only expressing Cre. Next, we examined the connection of Pdyn and Penk expressing neurons in the LPBN to the POA using retrograde AAVs and FISH. We injected AAV2-retro-Cre into POA of wild-type mice (Figure 2L) and collected brain sections containing LPBN. We probed these sections for viral induced Cre expression (Figure 2M). Using FISH, we observed retrograde viral induced expression of Cre in LPBN (Figure 2N) in cells also labeled with Pdyn (Figure 2O) and Penk (Figure 2P) indicating that neurons expressing these two opioid peptides project to the POA. To probe whether the PBN→POA neuronal population co-expresses Pdyn and/or Penk, we injected retro-AAV-Cre-GFP into the POA and probed LPBN containing brain sections with FISH probes for GFP, Pdyn, and Penk. We found that of GFP labeled cells in the LPBN, 49 ± 4% (mean ± SEM) were labeled by both Penk and Pdyn probes (Figure 2—figure supplement 2A–C). Of the remaining GFP labeled LPBN neurons, 26 ± 2% were labeled by Pdyn and 12 ± 1% by Penk (mean ± SEM). 13 ± 3% (mean ± SEM) of the quantified GFP labeled LPBN neurons were not labeled by either Pdyn or Penk probes (n = 3 mice, 169 cells). To further examine the projections of Pdyn+ and Penk+ LPBN neurons to the POA, we employed both retrograde AAV’s and anterograde tracing in Pdyn-Cre and Penk-Cre mice. To identify anterograde projections of LPBN neurons, we injected the Pdyn-Cre or Penk-Cre mice with AAV5-Ef1a-DIO-eYFP or AAV5-Ef1a-DIO-ChR2-eYFP into the LPBN. To retrogradely label POA projecting neurons we injected AAV2-retro-CAG-FLEX-tdTomato-WPRE into the POA of the same Pdyn-Cre or Penk-Cre animals (Figure 3A,F). In this experiment we observed anterograde labeling of processes with eYFP in the POA, from viral injections in the PBN, with dense projections in the ventral medial preoptic hypothalamus (VMPO) from both Pdyn-Cre (Figure 3C) and Penk-Cre (Figure 3H) mice. Retrograde labeling of LPBN neurons by Cre-dependent expression of tdTomato from retroAVV injected into the POA was evident in sections from both Pdyn-Cre (Figure 3E) and Penk-Cre (Figure 3J) brains. Double-labeled cells expressing both tdTomato (retrograde) and eYFP were present in the LPBN of both Pdyn-Cre and Penk-Cre mice (arrowheads in Figure 3E and J). In sagittal sections of brains taken from Pdyn-Cre mice injected with AAV-DIO-ChR2e-YFP in the PBN we also observed labeled projections to the POA among other brain areas (Figure 2—figure supplement 1J,K). Figure 3 Download asset Open asset Pdyn+ and Penk+ LPBN neurons project to VMPO. (A) Illustration of injection of retroAAV-DIO-tdTomato in POA and AAV5-DIO-eYFP in a Pdyn-Cre mouse. (B) Diagram of POA region depicted in (C) showing antero- (green) and retrograde (red) labeling of Pdyn+ neurons in POA. (D) Diagram of parabrachial nucleus (PBN) region depicted in (E) showing retrograde labeling from POA (red) and eYFP expression (green). Yellow cells in overlay image, marked with arrow heads, illustrate dual labeling by locally injected and retrograde viruses. (F) Illustration of injection of retroAAV-DIO-tdTomato in POA and AAV5-DIO-eYFP in an Penk-Cre mouse. (G) Diagram of POA region depicted in (H) show antero- (green) and retrograde (red) labeling of Penk+ neurons in POA. (I) Diagram of PBN region depicted in (J) showing retrograde labeling from POA (red) and eYFP expression (green). Yellow cells in overlay image, marked with arrow heads, illustrate dual labeling by locally injected and retrograde viruses. To examine which neurons comprise the PBN to POA projecting population, we injected mice expressing Cre under control of the VGLUT2 (Slc17a6) promoter (VGLUT2-Cre) (Vong et al., 2011) with AAV5-DIO-ChR2e-YFP bilaterally in the PBN, labeling VLGUT2 expressing PBN neurons (Figure 2—figure supplement 1E). We observed VGLUT2-Cre positive cells labeled by eYFP in the MPBN and LPBN after viral injection (Figure 2—figure supplement 1F,G). VGLUT2+ projections from the PBN to the POA including the VMPO and the median preoptic nucleus (MNPO) were labeled by AAV5-DIO-ChR2-eYFP injected in the PBN (Figure 2—figure supplement 1H,I). To determine whether Pdyn+ or VGLUT2+ cells represented the whole of the population of PBN to POA neurons, a retrograde recombinase dependent red-to-green (tdTomato to EGFP) Cre-switch virus (AAV-retro-DO_DIO-tdTomato_EGFP) was injected into the POA of Pdyn-Cre or VGLUT2-Cre mice (Figure 2—figure supplement 1A). In Pdyn=Cre mice, we observed cells in LPBN expressing tdTomato (Cre negative cells) and neurons expressing eGFP (Cre positive cells) (Figure 2—figure supplement 1C). In VGLUT2-Cre mice, we only observed eGFP expressing (Cre positive cells) neurons in LPBN (Figure 2—figure supplement 1D) indicating that the PBN to POA projection is composed entirely of VGLUT2+ cells. Taken together, results from FISH experiments and viral tracing studies indicate that Pdyn+ and Penk+ neurons in LPB project to the POA, particularly the VMPO, and that both Pdyn+ and Penk+ POA projecting neurons are subsets of the VGLUT2+ population of LPBN neurons that project to POA. Photostimulation of PdynPBN→POA, PenkPBN→POA, and VGLUT2PBN→POA generates rapid onset of hypothermia Using the respective Cre driver lines, we next examined the roles of POA-projecting Pdyn+, Penk+, and VGLUT2+ PBN neurons (circuits are denoted as PdynPBN→POA, PenkPBN→POA, and VGLUT2PBN→POA, respectively) in regulating body temperature. We injected AAV5-DIO-ChR2-eYFP bilaterally into the LPBN of Pdyn-Cre, Penk-Cre, and VGLUT2-Cre mice, and after 6 weeks, we implanted a single midline optic fiber above VMPO, where projections from PBN were observed (Figure 4A,B). We implanted mice with a subdermal wireless temperature transponder to enable touch free recording of body temperature. For each trial, we connected mice to an optic patch cable, and following a 1-hr period of habituation to the behavioral arena, we photostimulated PBN→POA terminals for 15 min with 10 ms light pluses at pulse frequencies of 2, 5, 10, and 15 Hz (Figure 4C). We recorded body temperature every 5 min for 65 min, beginning 5 min prior to photostimulation (Figure 4C). Figure 4 with 1 supplement see all Download asset Open asset Photostimulation of PdynPBN→POA, PenkPBN→POA, and VGLUT2PBN→POA causes acute hypothermia by evoking thermal heat defenses. (A) Illustration of viral injections in parabrachial nucleus (PBN) and fiber optic implantation over POA in Pdyn-Cre, Penk-Cre, or VGLUT2-Cre mice. (B) Illustration shows viral and fiber optic delivery in a Pdyn-Cre mouse along with representative expression of ChR2-eYFP (green) in PBN injection site and POA implantation site. (C) Diagram shows core body temperature measurement method and paradigm for photostimulation for 15 min and temperature recording for 65 min trials. (D–F) Body temperature vs. time graphs for 2 (yellow), 5 (orange), 10 (red), and 15 (dark red) Hz photostimulation of (D) PdynPBN→POA, (E) PenkPBN→POA, (F) VGLUT2PBN→POA, and controls for each. Photostimulation was delivered from t = 0 to t = 15 min and led to a frequency dependent reduction in body temperature in Pdyn-Cre, Penk-Cre, and VGLUT2-Cre mice. Body temperature of control animals was stable throughout the trials. Data are presented as mean ± SEM. For experimental animals, n = 6 (D and E) and n = 8 (F). For control animals, n = 8 (D) and n = 7 (E and F). (G) Representative quantitative thermal imaging from a representative trial showing a mouse before, during, and after 10 Hz photostimulation of PdynPBN→POA. Arrows show temperatures of eye, BAT, or tail. Eye and BAT temperature decreased as a result of stimulation; tail temperature increased as a result of stimulation. (H–L) Quantitative thermal imaging measurements of (H) eye, (I) tail, (J) eye minus tail, (K) BAT, and (L) BAT minus eye temperature vs. time graphs for 10 Hz photostimulation of PdynPBN→POA. Photostimulation was delivered from t = 0 to t = 15 min and led to decreases in eye and BAT temperatures, an increase in tail temperature. Tail and eye temperatures equilibrated in Cre+ animals. BAT thermogenesis was suppressed with a decline in the difference between eye and BAT temperatures during stimulation. Data are presented as mean ± SEM. See Figure 4—figure supplement 1 for data from Penk-Cre animals. Photostimulation of PdynPBN→POA neuron terminals caused rapid and significant reduction in body temperature in Pdyn-Cre mice (n = 6), with increasing magnitude of drop in body temperature corresponding to increasing photostimulation frequency up to 10 Hz (Figure 4D). 15 min of stimulation of PdynPBN→POA projections reduced the body temperature to 36.0 ± 0.1°C at 2 Hz (p=0.571 vs. control), 33.3 ± 0.6°C (p=0.0032) at 5 Hz, 31.9 ± 0.3°C, (p<0.0001) at 10 Hz, and 31.9 ± 0.4°C (p<0.0001) at 15 Hz compared to control. In control mice (n = 7), photostimulation did not cause significant changes in body temperature at any of the tested frequencies (Figure 4D). Photostimulation of PenkPBN→POA neuron terminals also caused a rapid reduction in body temperature (Figure 4E) in a stimulation frequency dependent manner. 15 min of stimulation of PenkPBN→POA projections in Penk-Cre mice reduced body temperature to 36.4 ± 0.3°C at 2 Hz (p=0.999 vs. control), 34.9 ± 0.7°C (p=0.495) at 5 Hz, 33.8 ± 0.3°C (p=0.0001) at 10 Hz, and 33.7 ± 0.4°C (p=0.0002) at 15 Hz, compared to a separate cohort of control mice (n = 7) which did not display altered body temperatures in response to photostimulation. In VGLUT2-Cre mice with AAV-DIO-ChR2-eYFP injected into PBN, stimulation of VGLUT2PBN→POA terminals in POA also caused a rapid and significant decrease in body temperature (Figure 4F). 15 min of photostimulation in VGLUT2-Cre mice (n = 8) significantly reduced the mean ± SEM body temperature to 36.5 ± 0.5°C at 2 Hz (p=0.257 vs. control) 34.0 ± 0.5°C at 5 Hz (p=0.0005), 32.8 ± 0.3°C (p<0.0001) at 10 Hz, and 32.9 ± 0.2°C (p<0.0001) at 15 Hz compared to control mice (n = 7). The average changes in body temperature that we measured in Pdyn-Cre and VGLUT2-Cre mice were not significantly different at any of the tested stimulation frequencies. The body temperature reduction evoked by photostimulation in Penk-Cre mice was smaller in magnitude than that in either Pdyn-Cre or VGLUT-Cre mice. The mean body temperature we measured in Penk-Cre mice after 15 min of simulation was significantly different than Pdyn-Cre at 10 Hz (p=0.02), with activation of the Penk+ terminals having less of an effect. These data demonstrate that activation of PBN→POA terminals causes rapid decreases in body temperature. Photostimulation of PdynPBN→POA and PenkPBN→POA terminals causes vasodilation and suppresses brown fat thermogenesis We sought to examine mechanisms causing core body temperature reduction in response to photostimulation of PBN→POA projections. We used thermal imaging to measure temperatures of eye, tail, and interscapular region, which covers BAT, in Pdyn-Cre mice (representative imaging in Figure 4G). Thermal imaging of the eye has previously been demonstrated as an accurate proxy for core body temperature (Vogel et al., 2016). We recorded eye temperatures every minute during a 10 Hz photostimulation paradigm, as described above. Recorded eye temperatures demonstrated a rapid reversible decrease after photostimulation (Figure 4H) and closely tracked values obtained using implanted wireless transponders. In Pdyn-Cre mice, mean ± SEM eye temperature dropped from 36.9°C ± 0.3 to 32.8°C ± 0.2 with 15 min of stimulation (Figure 4H). Thermal imaging to quantify tail temperature can be used to observe heat loss from vasodilation in response to warmth (Meyer et al., 2017). We obtained thermal imaging measurements of the tail temperatures approximately 1 cm from the base of the tail each minute. In Pdyn-Cre mice, tail temperature measurements demonstrated a very rapid increase following the onset of photostimulation, increasing a mean ± SEM of 4.2°C ± 0.5 after 2 min of photostimulation (Figure 4I). Increase in tail temperature preceded the decline in core body temperature. As core body temperature began to decrease, the tail temperature also began to decrease (Figure 4H and I). We examined the difference between the tail and eye temperatures (Figure 4H–J) to determine whether the gradient between core and peripheral temperature was maintained as body temperatures declined during stimulation. At baseline we observed a mean ± SEM difference 6.9 ± 0.29°C between the measured eye and tail temperatures. Eye–tail temperature difference significantly (p<0.0001) decreased compared to control to a mean ± SEM of 1.3 ± 0.3°C and remained stable during photostimulation even as body temperature declined. The difference in eye–tail temperature returned to baseline shortly after photostimulation was stopped (Figure 4J). Previous studies have implicated the POA in regulating BAT activation in response to cooling (Nakamura and Morrison, 2007; Tan et al., 2016). To simultaneously examine changes in BAT activity in response to the PBN→POA photostimulation-induced hypothermia, temperature measurements were also made of the interscapular BAT region temperature in mice with the fur removed from over the intrascapular region. In Pdyn-Cre mice, the temperature of the BAT region decreased rapidly following the onset of stimulation and returned to baseline post-stimulation in a pattern similar to body temperature (Figure 4K). If BAT activity responded to the decrease in body temperature by increasing metabolism, then the BAT–eye temperature difference would be expected to increase, reflecting the warming activity of BAT and the falling body temperature. The temperature difference between BAT and eye (BAT–eye) decreased during the period of stimulation but returned to baseline when stimulation was stopped (Figure 4L). We conducted similar experiments using thermal imaging in Penk-Cre and additional control animals (Figure 4—figure supplement 1). In the Penk-Cre mice, we found similar effects, but of smaller overall amplitude. Photostimulation of PenkPBN→POA terminals led to a decrease in eye temperature, a rapid increase in tail temperature, decrease in BAT temperature, and collapse of the eye–tail temperature gradient (Figure 4—figure supplement 1G–J). Together these results indicate that PBN to POA neurons can drive physiologic adaptation to lower body temperature by increasing heat dissipation and suppressing thermogenesis. Changes in body temperature evoked by photostimulation of PdynPBN→POA and PenkPBN→POA terminals are opioid peptide and receptor independent To test the potential role of endogenous opioids and their receptors in mediating the alterations in body temperature evoked by activation of PdynPBN→POA and PenkPBN→POA terminals, mice were treated with opioid receptor antagonists prior to photostimulation (Figure 4—figure supplement 1A). Pdyn-Cre (n = 7) and control mice (n = 7) were treated with the opioid receptor antagonist naltrexone (3 mg/kg) via intraperitoneal (IP) injection and then given a 10 Hz photostimulation paradigm as above (Figure 4—figure supplement 1B). The order of naltrexone and saline was varied between animals, and trials were run on separate days. 30 min after treatment with naltrexone, we did not observe a significant impact on photostimulation induced change in body temperature compared to saline treated animals. Naltrexone was paired with the Pdyn-Cre line because of the relatively higher affinity of naltrexone for kappa opioid receptors compared to naloxone (Meng et al., 1993). A distinct cohort of Pdyn-Cre mice (n = 4) was treated with saline and for subsequent trials with Norbinaltorphimine (norBNI) 10 mg/kg via IP injection 1 day prior and again 30 min prior to photostimulation. Pretreatment with norBNI did not significantly alter the decr" @default.
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• W3133838079 date "2020-12-23" @default.
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• W3133838079 title "Author response: Parabrachial opioidergic projections to preoptic hypothalamus mediate behavioral and physiological thermal defenses" @default.
• W3133838079 doi "https://doi.org/10.7554/elife.60779.sa2" @default.
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