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Parabrachial opioidergic projections to preoptic hypothalamus mediate behavioral and physiological thermal defenses

  1. Aaron J Norris  Is a corresponding author
  2. Jordan R Shaker
  3. Aaron L Cone
  4. Imeh B Ndiokho
  5. Michael R Bruchas  Is a corresponding author
  1. Department of Anesthesiology, Washington University School of Medicine, United States
  2. Medical Scientist Training Program, University of Washington, United States
  3. Center for the Neurobiology of Addiction, Pain and Emotion, Departments of Anesthesiology and Pharmacology, University of Washington, United States
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Cite this article as: eLife 2021;10:e60779 doi: 10.7554/eLife.60779

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,CGeerling 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
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
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).

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
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) PdynPBNPOA, (E) PenkPBNPOA, (F) VGLUT2PBNPOA, 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 PdynPBNPOA. 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 PdynPBNPOA. 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 decrease in body temperature induced by 10 Hz photostimulation of PdynPBN→POA terminals (Figure 4—figure supplement 1B). We confirmed that the doses and time courses of our naltrexone and norBNI administration were effective by examining the block of suppression of locomotion by the kappa receptor agonist U50 by the antagonists naltrexone or norBNI (Paris et al., 2011). Pretreatment with naltrexone or norBNI mitigated U50 mediated suppression of locomotor activity (Figure 4—figure supplement 1D–F). Penk-Cre mice (n = 5) injected with AAV5-DIO-ChR2eYFP in the PBN and control mice (n = 4) were treated with naloxone 5 mg/kg and saline, with the order of treatments varied between animals and trials conducted on separate days. 30 min after treatment with naloxone, PenkPBN→POA terminals were photostimulated at 10 Hz. No significant effect of naloxone on photostimulation-induced changes in body temperature was observed (Figure 4—figure supplement 1). These data suggest that the acute alterations in body temperature due to stimulation of PdynPBN→POA and PenkPBN→POA terminals are not driven by endogenous opioid release and subsequent opioid receptor signaling.

Glutamatergic PBN neuronal activity is necessary for heat-induced vasodilation

Glutamatergic signaling in the POA and in PBN has previously been implicated in heat defensive behaviors (Nakamura and Morrison, 2010), and our results, presented here, demonstrate sufficiency of PBN VGLUT2+ neurons in driving hypothermia (Figure 4F). To examine the necessity of VGLUT2+ PBN neurons in mediating heat defensive behaviors in awake behaving animals, AAVs encoding Gi coupled DREADDs in a Cre-dependent manner (AAV-DIO-hM4DGi) were injected into PBN bilaterally in VGLUT2-Cre mice (n = 5). Mice were treated with saline or clozapine-N-oxide (CNO) (2.5 mg/kg IP) 30 min prior to a heat challenge of 34°C for 15 min and were tested with the reciprocal during a subsequent trial more than 24 hr later. We used a custom small arena with floor and walls lined with a water jacket connected to circulating water baths at 20°C or 34°C to create a rapid change in temperature between two stable set points while allowing for continuous thermal imaging (Figure 5A,B). Using quantitative thermal imaging, we measured tail temperatures and arena floor temperatures (depicted by the yellow-orange shaded areas) every minute during chemogenetic inhibition of VGLUT2 activity (Figure 5B–D). In mice treated with saline, measurements of tail temperatures showed a rapid rise following the shift of arena temperature to 34°C and measured tail temperatures were higher than the arena floor temperature (Figure 5B,D, and F). In mice treated with CNO, which activated the inhibitory DREADD in VGLUT2+ PBN neurons, the mean ± SEM tail temperature after 15 min of exposure to 34°C was 34.8°C ± 0.5, significantly (p=0.01) lower than the corresponding average tail temperature after saline treatment, 37.1°C ± 0.5 (Figure 5D). The tail temperature in saline treated mice exceeded the temperature of the arena floor (Figure 5B and F), but in CNO treated mice, tail temperature rose only to the temperature of the floor (Figure 5B and E). Consistent with an effect of passive heating of the tail, as opposed to the active vasodilation evoked by the thermal challenge, the rate of increase in the tail temperature was also slower following CNO treatment compared to saline (Figure 5C). After return of the arena floor temperature to 20°C, tail temperatures returned to a baseline of approximately 22°C following both saline and CNO treatments. Similar experiments carried out in Pdyn-Cre mice demonstrated that Gi DREADD mediated inhibition of Pdyn+ PBN neurons is not sufficient to prevent vasodilation in response to thermal heat challenge (Figure 5—figure supplement 1). CNO treatment in WT mice had no significant effects on tail temperature changes compared to saline treatment (Figure 5—figure supplement 1). These results indicate that VGLUT2+ PBN neurons are required for heat defensive responses including physiological vasodilation.

Figure 5 with 1 supplement see all
VGLUT2+ parabrachial nucleus (PBN) neurons are necessary for heat-defensive tail vasodilation.

(A) Illustrations depict viral injections in VGLUT2-Cre mice and purpose-built heat challenge arena that allowed for rapid changing of environmental temperature between two stable set points. (B) Tail temperature as determined using quantitative thermal imaging vs. time graph for 34°C thermal heat challenge for mice expressing hM4D(Gi) DREADDs in VGLUT2+ PBN neurons treated either with CNO or saline. Heat challenge was delivered from t = 0 to t = 15 min, and arena temperature measured using thermal imaging during the trial is represented by the orange line. In mice injected with CNO 2.5 mg/kg, tail temperature passively equilibrated with arena temperature (34°C) over the 15 min heat challenge. In mice injected with saline, tail temperature rose above arena temperature after 5 min of heat challenge representing heat release through vasodilation. Data are presented as mean ± SEM. n = 5 animals, paired between CNO and saline conditions. (C) Tail temperature vs. time graph for 34°C heat challenge between t = 0 and t = 5 min. Note the separation between average tail temperatures of the saline condition vs. the CNO condition. Data are presented as mean ± SEM. n = 5 animals, paired between CNO and saline conditions. (D) Tail temperature at t = 15 min of 34°C heat challenge. Tail temperatures in the saline condition were an average of 2.3 ± 0.68°C higher than those in the CNO condition. (E) Representative thermal images of trials for mice treated with CNO and measurement of tail temperature showing tail temperatures remain close to the temperature of the area floor. (F) Representative thermal images of trials for mice treated with saline and tail temperature exceed floor temperature. Data are presented as mean ± SEM. n = 5 animals, paired between CNO and saline conditions. Student’s t-test, ∗p<0.05. See Figure 5—figure supplement 1 for data from the same assay in Pdyn-Cre mice.

Photostimulation of PBN→POA drives thermal defensive behaviors

The PBN has been found to play essential roles in driving escape and aversive learning to nociceptive stimuli. Previous studies have shown that the spinothalamic pathway is not required for behavioral thermoregulation and that muscimol mediated inhibition of PBN blocked thermal preference seeking (Yahiro et al., 2017). To test the sufficiency of PdynPBNPOA, PenkPBNPOA, and VGLUT2PBNPOA to drive avoidance behavior, we conducted real-time place aversion (RTPA) experiments using the respective Cre driver lines and photostimulation of terminals in the POA. Photostimulation of terminals was paired to entry into one compartment of a balanced two-compartment conditioning apparatus void of salient stimuli. Neurons that encode a negative valence will cause an aversion from the chamber paired with photostimulation, and those with a positive valence will drive a preference for it (Kim et al., 2013; Namburi et al., 2016; Siuda et al., 2015; Stamatakis and Stuber, 2012; Tan et al., 2012). As in experiments above, we injected AAV-DIO-ChR2-eYFP into the PBN bilaterally of Cre driver line mice and implanted optical fibers over the POA (Figure 6A,D,G). Photostimulation of PdynPBNPOA terminals drove aversion in a frequency dependent manner, with time spent in the stimulation side being significantly lower (p<0.0001) at 5, 10, and 20 Hz stimulation frequencies compared to control mice (Figure 6A–C). Results from parallel RTPA experiments using Penk-Cre mice demonstrated a similar effect of aversion seen at 5 (p=0.0002), 10 (p<0.0001), and 20 Hz (p<0.0001) stimulation frequencies compared to control mice (Figure 6D–F). Results we obtained using VGLUT2-Cre mice in RTPA experiments showed significant (p<0.0001) decreases in time spent on the stimulation side at 2, 5, 10, and 20 Hz compared to control animals (Figure 6G–I). For each genetic line, we examined the locomotor activity or distance traveled during the RTPA. In Pdyn-Cre mice, we observed a small but significant (p=0.009) difference in mean ± SEM total distance traveled only during trials using 20 Hz stimulation – 29 m ± 2 (n = 8) compared to control 48 m ± 5 (n = 7) – but not during trials using lower stimulation frequencies (Figure 6—figure supplement 1A). In Penk-Cre and VGLUT2-Cre mice, we observed no significant differences between Cre+ and control animals at any of the photostimulation frequencies (Figure 6—figure supplement 1B,C). Comparisons of male vs female mice did not reveal sex dependent effects in the acute hypothermic changes in body temperature evoked by photostimulation of PBN→POA terminals (Figure 6—figure supplement 1D,E).

Figure 6 with 1 supplement see all
Photostimulation of PdynPBN→POA, PenkPBN→POA, and VGLUT2PBN→POA terminals induces real time place aversion.

(A, D, and G) Illustrations of viral injections in parabrachial nucleus (PBN) and fiber optic implantations over POA in Pdyn-Cre mice, Penk-Cre, and VGLUT2-Cre mice, respectively. (B, E, and H) Representative heat maps showing spatial distribution of time-spent behavior resulting from side-conditional 10 Hz photostimulation of control or Pdyn-Cre, Penk-Cre, and VGLUT2-Cre mice, respectively. (C) For Pdyn-Cre vs control mice, frequency response of RTPP at 0 (baseline), 2, 5, 10, and 20 Hz. Data are presented as mean ± SEM; n = 6 Cre+, eight control; two-Way ANOVA, Bonferroni post hoc. (F) Penk-Cre frequency response of RTPP at 0 (baseline), 2, 5, 10, and 20 Hz. Data are presented as mean ± SEM; n = 6 Cre+, seven control; two-Way ANOVA, Bonferroni post hoc (5 Hz ChR2 vs. 5 Hz control ∗∗∗p<0.001, 10 Hz ChR2 vs. 10 Hz control ∗∗∗∗p<0.0001, 20 Hz ChR2 vs. 20 Hz control ∗∗∗∗p<0.0001). (I) VGLUT2-Cre frequency response of RTPP at 0 (baseline), 2, 5, 10, and 20 Hz. Data are presented as mean ± SEM; n = 8 Cre+, seven control; two-Way ANOVA, Bonferroni post hoc (2 Hz ChR2 vs. 20 Hz control ∗∗∗∗p<0.0001, 5 Hz ChR2 vs. 5 Hz control ∗∗∗∗p<0.0001, 10 Hz ChR2 vs. 10 Hz control ∗∗∗∗p<0.0001, 20 Hz ChR2 vs. 20 Hz control ∗∗∗∗p<0.0001). See also Figure 6—figure supplement 1.

Other thermoregulatory behaviors including posture, stance, and locomotion are altered by exposure to warm temperatures (Cabanac, 1975). Therefore, we next examined alterations in locomotion using 20 min open field-testing trials in Pdyn-Cre and control mice (Figure 7A–C). Stimulation of PdynPBN→POA terminals at 10 Hz resulted in a large and significant (p=0.0008) decrease in mean ± SEM distance traveled: 26.1 m ± 6.2 (n = 5) in Pdyn-Cre compared to 65.9 m ± 5.6 in control mice (n = 7) (Figure 7B,C). Postural extension, depicted in the photograph (Figure 7D), a heat evoked behavior in rodents that reduces heat production by postural tone and increases exposed body surface to promote thermal transfer (Roberts, 1988), was evoked by photostimulation of PdynPBN→POA terminals. Scoring of video recordings of trials of Pdyn-Cre (n = 7) and control mice (n = 4) revealed that Pdyn-Cre mice quickly transition to a sprawled posture after the onset of 10 Hz photostimulation, which also induces hypothermia. Following the end of photostimulation, mice transition and spend more time in a posture with their tail curled under their bodies to minimize exposed surface area (Figure 7E). We did not observe postural extension at any time in control mice during these trials.

Photostimulation of PdynPBN→POA suppresses locomotion, evokes postural extension but does not alter temperature preference.

(A) Illustration of injection in parabrachial nucleus (PBN) and fiber implantation over POA in Pdyn-Cre mice. (B) Representative heat maps show spatial distribution of time-spent behavior resulting from constant 20 min 10 Hz photostimulation of control or PdynPBNPOA. (C) Quantification of movement during open field testing. Control animals moved an average of 39.84 ± 8.33 meters more than Cre+ animals during open field trials. Data are presented as mean ± SEM; n = 5 Cre+, seven control; Student’s t test, ∗∗∗p<0.001 (D) 10 Hz photostimulation of PdynPBNPOA leads to postural extension behavior as shown. Representative images of a mouse pre stimulation and during 10 Hz photostimulation of PdynPBNPOA. (E) Quantification of percent time spent in time spent engaged in postural extension in Pdyn-Cre mice in two min time bins. Following onset of photostimulation mice engaged in postural extension (red). With termination of stimulation mice, we noted to switch to a posture with their tails curled under their bodies (grey). Postural extension was not observed in any control mice. (F) Overview of paradigm with three epochs: 40 min of pre-stim, 10 Hz photostimulation for 20 min, and post-stim for 20 min in an arena with aluminum floor held at 20°C and 26°C on opposing sides. (G) Quantification of time spent in each temperature area showed non-significant changes in percent time spent in each area during delivery of stimulation, with a strong preference for the 26°C side during all epochs. Data presented as mean ± SEM with individual values, n = 9 Pdyn-Cre (ANOVA ns = 0.7341 for Pdyn-Cre mice across epochs) and (t-test ns p>0.99 for Pdyn-Cre vs Control during stimulation epoch).

Temperature selection is an important complex thermal defense behavior. Moving to an area with cooler environmental temperature, when possible, is a way to defend against excessive heat. Available studies indicate thermal selection requires the engagement of multiple poorly understood neural circuits. We next tested whether photoactivation of PdynPBN→POA terminals is sufficient to induce a shift to slightly cooler temperature preference. To examine temperature preference, we placed mice in an arena with an aluminum floor in which each side is held at a set temperature of 20°C or 26°C (Figure 7F). Mice were habituated to the arena prior to the start of the trial to familiarize the animals to area. Trials consisted of a 40 min pre-stimulation period, a 20 min stimulation period, and a 20 min post-stimulation period. As expected, at baseline, mice spent a greater amount of time on the 26°C side (Figure 7G). In Pdyn-Cre mice (n = 6), photostimulation of the POA at 10 Hz did not alter animals’ thermal preference to the cooler side of arena (Figure 7G), despite this photostimulation paradigm evoking hypothermia (Figure 4D) and driving other thermal defense behaviors. This result indicates that the PdynPBN→POA neurons are not sufficient to drive cool seeking behavior when activated, suggesting that other neural pathways are also likely required to drive this behavior.

Discussion

In the present study we demonstrate that warm-activated neurons within the PBN overlap with neural populations (Pdyn+ and Penk+) marked by Cre reporters for expression of Pdyn and Penk (Figure 1 and Figure 2—figure supplement 2). Employing FISH, we found that Pdyn and Penk expressing neuronal populations are glutamatergic (express Slc17a6) and partially overlap with each other (Figure 2). Using anterograde and retrograde viral tools, we demonstrate that Pdyn+, Penk+, and VGLUT2+ PBN neurons project to the POA (Figure 3 and Figure 2—figure supplement 1). We found that photoactivation activation of terminals from Pdyn+ or Penk+ or VGLUT2+ PBN→POA drove physiological and behavioral heat defense behaviors (Figure 8).

Graphical summary.

The presented studies focused on parabrachial nucleus (PBN)→POA projecting cells by photostimulating terminals in the POA. We identified warm-activated neurons (red circle) in the lateral PBN that incompletely overlap with Penk+ and Pdyn+ PBN neuronal populations. Further, we found that these Penk (green circle) and Pdyn (blue circle) neurons express VGLUT2 (gray) and partially overlap with each other. Photostimulation of PBN→POA projections revealed that PBN VGLUT2+, Pdyn+, or Penk+ projections drive physiological and behavioral heat defenses including vasodilation to promote heat loss, avoidance, suppression of BAT thermogenesis, and postal extension to promote heat loss.

Overlapping populations of warm-activated PBN neurons express Penk and Pdyn, are glutamatergic, and project to the POA

We report that PBN neurons are activated following exposure to warmth. The majority of warm-activated PBN neurons are Pdyn+ and, surprisingly, a smaller population of these warm-activated PBN cells are Penk+ (Figure 1). 81% and 54% of cFos+ cells are Pdyn+ or Penk+, respectively, suggesting that Pdyn+ and Penk+ populations overlap, as the sum of the Fos+ cells that are Pdyn+ and Penk+ exceeds 100%. Fos labeled, warm-activated, neurons are a subset of total Pdyn+ and Penk+ PBN neurons. These findings are consistent with previous reports that implicated glutamatergic FoxP2+ and Pdyn+ neurons in the dorsal lateral PBN in responding to warmth (Geerling et al., 2016).

VGLUT2+, Pdyn+, and Penk+ neurons from the PBN project to POA including the VMPO (Figure 3 and Figure 2—figure supplement 1K). Pdyn+PBN→POA neurons represent a subset of the VGLUT2+PBN→POA population. Results obtained with retro-AAV Cre-switch, red (tdTomato) to green (GFP), injections into the POA revealed that all of the projecting PBN neurons are VGLUT2+ and a subset are Pdyn+ (Figure 2—figure supplement 1A–D). Gi DREADD mediated inhibition of PBN VGLUT2+ neurons but not of Pdyn+ PBN neurons (Figure 5 and Figure 4—figure supplement 1) blocks vasodilation is response to thermal challenge. Further, results examining the expression of Pdyn and Penk in neurons retrogradely labeled with GFP from the POA (Figure 2—figure supplement 2) showed partially overlapping expression of GFP with both Pdyn and Penk. GFP+ cells that not labeled by either Pdyn or Penk were also evident. Taken together these results indicated that Pdyn+ and Penk+ PBN→POA neurons represent subsets of the glutamatergic warm-activated PBN neurons. A recent report presented data indicating the Pdyn vs Cck expression in the PBN marked a functional division of neurons driving vasodilation and BAT regulation (Yang et al., 2020). In contrast to those results, we found that Pdyn and Cck expression overlap in many lateral PBN neurons and that activation of Pdyn+PBN→POA neurons induces tail vasodilation. To examine Cck and Pdyn expressing PBN populations, Yang et al. combined IHC for DynA with AAV based recombines reporter in Cck-Cre mice as a proxy expression of Cck. Based on this hybrid approach they reported minimal overlap of Dyn immunoreactivity and reporter expression. In contrast, we used FISH for mRNA for both peptides and found 70% overlap of Pdyn and Cck labeled LPBN neurons. The discordance of the two observation may rest in part with differing techniques (FISH vs IHC) used to examine peptide expression, the use of the Cck-Cre mouse line, underlying biological factors, such as variable peptide expression levels under different conditions, and known challenges in IHC staining vs mRNA labeling for cell identification. Future studies will need to employ RNAseq or other high resolution genetic methods to more clearly define PBN-POA cell identities, as is largely now accepted as a more thorough way, together with in situ to cluster and quantify neuronal groups within a given brain region.

The PBN projects to multiple areas in the POA and regulates other homeostatic processes including water balance and arousal (Gizowski and Bourque, 2018; Qiu et al., 2016). The studies presented here show PBN projections to the VMPO, which contains warm-activated neurons involved in regulating body temperature (Tan et al., 2016). Although we implanted fiberoptics above the VMPO light likely reach immediately adjacent regions of the POA, such as the MnPO, and PBN→POA projections were also observed in these areas. PBN to POA projections may be important in an array of homeostatic process. Recently, a connection of warm-activated neurons in the POA to promotion of sleep state has been described, and a role for temperature information from the PBN to the POA in promoting sleep has been suggested (Harding et al., 2020; Harding et al., 2018). Also, activation of neurons in the ventral lateral preoptic can also induce sleep and hypothermia (Kroeger et al., 2018). A potential role of PBN→POA projections in promoting sleep would suggest a bidirectional relationship of sleep by the PBN because PBN neurons are also able to cause arousal. A key to resolving the many roles of PBN neurons may lie in further understanding potential anatomical segregation of functionally discrete PBN circuit pathways. Anatomic segregation of pathways has been described for thermal information conveyed by the PBN, with cold responsive PBN separated from warm responsive neurons, which are located relatively caudal in the PBN (Geerling et al., 2016).

Likely postsynaptic targets in POA for warm-activated PBN cells include the recently identified warmth activated neurons in the POA that express the neuropeptides brain-derived neurotrophic factor (BDNF) and pituitary adenylate cyclase-activating polypeptide (PACAP) (Tan et al., 2016) as well as neurons in nearby areas implicated in mediating various homeostatic functions. Yang et al. show that blocking glutamatergic neurons in the POA blocked the effects on body temperature of activating PBN→POA (Yang et al., 2020). Also, prior studies have shown that hM3-Gq-DREADD induced stimulation of glutamatergic VMPO neurons (expressing the receptor for the hormone leptin) causes a reduction in core body temperature similar in magnitude to the effect seen by activation of PdynPBN→POA terminals we observed in the present study. Further, activation of leptin receptor expressing VMPO neurons also causes mice to display similar postural extension behavior as we observed following activation of PdynPBN→POA terminals (Figure 7Yu et al., 2016). Glutamatergic neurons in MnPO can drive vasodilation and may also be targets of the PBN warm-activated cells (Abbott and Saper, 2018).

Experiments presented here indicate that opioid signaling is not required for the rapid change in body temperature in response to activation of PBN neurons; however, evidence suggests that opioid systems may have important roles in regulation of body temperature and metabolism. Injections of opioid receptor agonists into the POA have been shown to alter body temperature indicating that opioid receptors, either pre- or post-synaptic to the PBN terminals, may have important functional neuromodulatory roles in thermoregulation (Xin et al., 1997). Further, POA KOR signaling was found to modulate body temperature and weight loss during calorie restriction (Cintron-Colon et al., 2019). Further supporting roles for opioid signaling in linking body temperature and metabolism, deletion of the KOR gene alters weight gain induced by a high fat diet by modulating metabolism (Czyzyk et al., 2010).

PBN to POA projections regulate body temperature by evoking physiological and behavioral responses

Here we report results obtained in awake freely behaving mice that demonstrate the role of PBN to POA projecting neurons in driving physiological and behavioral responses to warm thermal challenge. Selective photostimulation of PBN→POA terminals in the three Cre lines (Pdyn, Penk, and VGLUT2) caused a robust and rapid decrease in body temperature (Figure 4). Thermal imaging paired with photoactivation of terminals revealed that the decrease in body temperature was due to heat loss via rapid vasodilation and suppression of BAT thermogenesis (Figure 4G–L). We found that hM4-Gi-DREADD mediated inhibition of VGLUT2+ (Figure 5), which encompasses both the Penk and Pdyn positive cells, but not Pdyn+ PBN neurons (Figure 5—figure supplement 1) alone blocked vasodilation in response to warm thermal challenge in awake animals. Activation of Penk+ or Pdyn+ PBN→POA terminals leads to rapid vasodilation and hypothermia (Figure 4 and Figure 4—figure supplement 1) indicating that a subset of the VGLUT2+PBN→POA population is sufficient to mediate vasodilation and suppress BAT activation. Taken together, our results demonstrate the necessity and sufficiency of transmission from VGLUT2+ PBN neurons to the POA for physiological responses to thermal heat challenge. Dyn peptide expression as marker of a separation of PBN neurons regulating BAT form those regulating heat loss by vasodilation as suggested by Yang et al. was not supported by the results in our experiments. In the presented report we did not examine the functional roles of Cck expressing neurons but did examine Penk+ PBN neurons. We found differing magnitudes of responses to activation of Pdyn+ or Penk+ terminals in the POA rather than categorical differences in the responses for the parameters examined. The results reported by Yang et al. are, overall, highly consistent with the results we present here, and there is divergence on Pdyn as a marker of functional separation in thermal defense circuits in the PBN. Future studies may help resolve if heat defense circuitry bifurcates at the level of the PBN using more genetically defined cell types, or through downstream activity in neurons in the POA mediated via specific neurotransmitters.

In rodents, thermal heat stress evokes behavioral changes including grooming, suppression of physical activity, postural changes (postural extension), and thermal seeking (Roberts, 1988). We found that activation of PdynPBN→POA terminals can mediate these behaviors, including markedly suppressed locomotor activity and postural extension (Figure 7). Lesions of POA have been shown to block postural extension in response to warmth (Roberts and Martin, 1977), and selective activation of subpopulations of POA neurons evokes postural extension behavior (Yu et al., 2016). Many of the behavioral defenses appear to be due to activation of cells in the POA by PBN terminals.

Activation of PBN to POA projecting neurons drives avoidance but does not promote thermal cool seeking

The PBN and the POA have been found to play important roles in thermal seeking behaviors, but the neural circuitry involved remains poorly understood. Warmth activated neurons within the POA have previously been found to drive a temperature preference Tan et al., 2016; however, the role of the POA in driving thermal seeking behaviors remains unclear. In contrast, prior studies using lesion approaches in the POA did not block thermal seeking behaviors (Almeida et al., 2006; Almeida et al., 2015; Matsuzaki et al., 2015). Studies examining the role of PBN in other aversive stimuli have found roles for the PBN in encoding valence and engaging motivational systems to drive avoidance without disruption of behaviors driven by sensory input. For example, functional silencing of LPBN Calcitonin gene-related peptide expressing neurons suppressed pain escape behavior; however, sensory reflex responses (paw withdrawal latency) remained intact (Han et al., 2015). In this example, disruption of PBN circuit activity blocks the expression of avoidance behaviors but not the transmission of sensory input.

The PBN may play a similar role, driving avoidance/escape behavior without altering sensation, in thermal defense. Muscimol mediated inhibition of PBN blocks temperature preference behavior, and a spinothalamic pathway independently conveys temperature information (Yahiro et al., 2017). We found that stimulation of PBN→POA terminals engages affective and motivational circuitry driving avoidance (Figure 6). Photoactivation of PdynPBN→POA terminals did not, however, induce a change in thermal preference for cooler temperatures (Figure 7F,G). In the context of previous studies, we interpret this to suggest that the coolness of the arena (20°C) as transduced by sensory pathways remains aversive despite the decrease in body temperatures evoked by the same photostimulation. Taken together with the literature, the results presented here support the conclusion that PBN neurons are necessary, but activation of this pathway (PBN to POA) alone is not sufficient for expression of cold seeking behaviors. Thermal seeking may also require information from additional neural circuits, with the PBN encoding valence. An alternative is that additional targets of PBN neurons outside the POA may be required to engage thermal cool seeking behaviors, and those targets were not affected by our experimental photostimulation of POA terminal fields. Supporting the notion that areas outside of the POA are required for thermal seeking, animals with POA lesion display amplification of motivated behaviors relating to thermal regulation due to impaired ability to defend core body temperature, and thus dependence on ambient temperature (Lipton, 1968; Satinoff et al., 1976). Future efforts will be necessary to understand the roles of POA and PBN circuits in modulating thermal motivated behaviors more fully.

The endogenous opioid system is not required for acute effects of PBN neuron activation on body temperature

Opioid receptor modulation by agonists and antagonists has effects on body temperature regulation, acting at both central and peripheral sites through mu, kappa, and delta receptors (Baker and Meert, 2002). Specific effects of centrally administered mu and kappa antagonists on body temperature suggested a tonic balance between mu and kappa systems in maintaining body temperature (Chen et al., 2005). Here we examined the potential roles of the endogenous opioid system in the acute hypothermic response evoked by activation of PBN→POA terminals in the POA by blocking opioid signaling with naloxone, naltrexone, or norBNI (Figure 4—figure supplement 1). None of the selective opioid antagonists we used here significantly altered the response to acute stimulation of PBN terminals in the POA. One explanation for this lack of effect is that the PBN neuronal populations we examined are glutamatergic, and glutamate is known to be a key neurotransmitter for thermal regulation in the POA (Nakamura and Morrison, 2010). A role for the opioid system may be evoked by sustained changes in environmental temperature and may play a role in maintaining thermal set point in a modulatory capacity or play roles in context of altered metabolism or sleep. Additionally, our photo-activation paradigm might not be sufficient to produce endogenous opioid peptide release in these neurons. This is unlikely, however, given that our recent efforts in another region have shown that comparable photostimulation was sufficient to evoke both endogenous dynorphin and enkephalin release in vivo (Al-Hasani et al., 2018). Future studies with additional approaches and more sensitive peptide sensors may reveal further insights regarding the role of endogenous opioids in this circuitry.

Conclusions and future directions

Previous studies have found that prior application of opioid receptor agonists affects the response of body temperature to opioid antagonists (Baker and Meert, 2002) and that environmental temperature, warm or cold, can dramatically alter the responses to centrally administered opioid peptides (Handler et al., 1994). Here we identified a potential source for multiple opioid peptides in the thermoregulatory neurocircuitry and delineated a role for the neurons expressing Pdyn and Penk in regulating body temperature. How these neuromodulators are involved in regulating body temperature and the target neurons will require further experimentation to delineate. How opioidergic circuits and signaling contribute to processes involving thermal regulation and dysregulations, such as during opiate withdrawal and alterations in calorie intake, merit further study. In sum, we report here that Pdyn+, Penk+, and VGLUT2+ PBN neurons project to the POA, mediate physiological (vasodilation, suppression of thermogenesis) thermal defenses, drive behavioral thermal response behaviors (suppression of locomotion, postural changes), and drive aversion. The presented results will enable further studies to understand how homeostatic thermal regulation interacts with the motivational circuitry to drive behavior, provide targets for experiments testing the roles of neuromodulation of thermosensory pathways to regulate energy expenditure in balance with environmental factors, and help inform our understanding of how organisms balance competing interests, such as food intake, physical activity, and environmental conditions when selecting behaviors.

An area of future investigation will be to examine if subpopulations of POA cells may independently drive individual behavioral and physiological components of thermal regulation such as suppression of locomotion, postural extension, vasodilation, and alterations in metabolism. Yang et al. report that functional division of the circuits mediating aspects of physiological responses to warmth defense at the level of the PBN (Yang et al., 2020).

Although a functional division in the PBN is plausible, our results (particularly based on Pdyn expression) suggest that this conclusion warrants further study using high resolution approaches. It is also important to note that we recently observed that modulation of Pdyn PBN neurons can replicate behavioral effects that were once attributed solely to CGRP neurons in the PBN, and while these two populations are genetically distinct, similar behavioral effects were observed (Bhatti et al., 2020). Interestingly, Yang et al. observe hyperthermia in response activation of VGLUT2+ PBN neurons in some animals but with activation VGLUT2 PBN→POA projecting cells raising questions for future study about hypothermia activated PBN neurons and what their projection targets are. Taken together, the results we present demonstrate that PBN neurons, expressing VGLUT2 also express Penk, and/or Pdyn, project to the POA, and drive behavioral and physiological thermal heat coping behaviors.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyAlexa Fluor 633 goat polyclonal anti-rabbit IgGInvitrogenCat# A-21070, RRID:AB_25357311:1000
AntibodyPhospho-c-Fos (Ser32) Rabbit monoclonalCell Signaling TechnologyCat# 5348S
RRID:AB_10557109
1:500
Chemical compound, drugClozapine N-oxide dihydrochlorideHellobioCat# HB6149
Chemical compound, drugNaloxone hydrochlorideTocrisCat# 0599
Chemical compound, drugNaltrexone hydrochlorideSigma-AldrichCat# N3136
Chemical compound, drugNorbinaltorphimine (norBN)Sigma-AldrichCat# N1771
Chemical compound, drugU50,488 (U50)Sigma-AldrichCat# D8040
Strain, strain background (Mus musculus)Pdyntm1.1(Cre)MjkrGift from Brad Lowell, HarvardRRID:MGI:5562890
Strain, strain background (Mus musculus)B6;129S-Penktm2(Cre)Hze/JThe Jackson LaboratoryRRID: IMSR_JAX: 025112
Strain, strain background (Mus musculus)Slc17a6tm2(cre)Lowl/JGift from Brad Lowell, HarvardRRID: IMSR_JAX: 028863
Strain, strain background (Mus musculus)B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JThe Jackson LaboratoryRRID: IMSR_JAX: 007914
Strain, strain background (Mus musculus)C57BL/6JThe Jackson LaboratoryRRID: IMSR_JAX:000664
Strain, strain background (AAV5)AAV5-EF1a-DIO-hChR2(H134R)-EYFPWashington University Hope Center Viral Vector CoreN/A(2.5 × 1013 vg/ml)
Strain, strain background (AAV5)AAV5-hSyn-DIO-hM4D(Gi)-mCherryAddgeneAddgene_44362-AAV5(7 × 10¹² vg/ml)
Strain, strain background (rAAV2-retro)AAV2-retro-DIO-ChR2-eYFPWashington University Hope Center Viral Vector CoreN/A(2.8 × 1012 vg/ml)
Strain, strain background (AAV)AAV-retro-CAG-FLEX-tdTomato-WPREAddgeneAddgene_51503-AAVrg(1 × 1013 vg/ml)
Strain, strain background (AAV2)AAV2-retro-DO_DIO-tdTomato_EGFP-WPRE-pAAddgeneRRID:Addgene_37120(8 × 10^12 GC/ml)
Strain, strain background (AAV5)AAV5-Ef1a-DIO-eYFPWashington University Hope Center Viral Vector CoreN/A(1.4 × 1013 vg/ml)
Strain, strain background (rAAV2-retro)AAV2-retro-GFP-CreWashington University Hope Center Viral Vector CoreN/A(3 × 1013 vg/ml)
Strain, strain background (AAV5)AAV5/hSyn-dio-hm4D(Gi)-mcherryAddgeneRRID:Addgene_44362(7.8 × 10^12 vg/ml)
Sequence-based reagentRNAscope probe PdynAdvanced Cell Diagnosticsaccession number NM_018863.3probe region 33–700
Sequence-based reagentRNAscope probe PenkAdvanced Cell Diagnosticsaccession number NM_001002927.2probe region 106–1332
Sequence-based reagentRNAscope probe Slc17a6Advanced Cell Diagnosticsaccession number NM_080853.3probe region 1986–2998
Sequence-based reagentRNAscope probe GFPAdvanced Cell Diagnosticsaccession numberAF275953.1probe region 12–686
Sequence-based reagentRNAscope probe CreAdvanced Cell Diagnosticsaccession number KC845567.1probe region 1058–2032
Sequence-based reagentRNAscope probe CckAdvanced Cell Diagnosticsaccession number NM_031161.3probe region 23–679
Software, algorithmImageJNIHRRID: SCR_003070
Software, algorithmResearchIRFLIR Systems IncN/A
Software, algorithmLeica Application Suite Advanced FluorescenceLeica MicrosystemsN/A
Software, algorithmBonsaiBonsai-rx.orgN/A
Software, algorithmDeepLabCutMathis Lab (Mathis et al., 2018)N/A

Contact for reagent and resource sharing

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Further information regarding reagents and resources may be directed to Aaron Norris, norrisa@wustl.edu, or Michael Bruchas, mbruchas@uw.edu.

Experimental model and subject details

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Adult (25–35 g, older than 8 weeks of age during experiments) male and female Pdyn-Cre (RRID:MGI:5562890) (Krashes et al., 2014), Penk-Cre (RRID: IMSR_JAX: 025112) (Harris et al., 2014), Ai14-tdTomato (Madisen et al., 2010), and VGLUT2-Cre (Vong et al., 2011) mice (species Mus musculus) were group housed (no more than five littermates per cage) and allowed food and water ad libitum. Mice were maintained on a 12 hr:12 hr light:dark cycle (lights on at 7:00 am). All procedures were approved by the Animal Care and Use Committee of Washington University and adhered to NIH guidelines. The mice were bred at Washington University in Saint Louis by crossing the Pdyn-Cre, Penk-Cre, Ai14-tdTomato, and VGLUT2-Cre mice with C57BL/6 (RRID: IMSR_JAX:000664) wild-type mice and backcrossed for seven generations. Additionally, where needed, Pdyn-Cre and Penk-Cre mice were then crossed to Ai14-tdTomato mice on C57BL/6 background. Male and female mice were included and analyzed together.

Stereotaxic surgery

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Mice were anesthetized in an induction chamber (4% isoflurane), placed in a stereotaxic frame (Kopf Instruments), and anesthesia was maintained with 2% isoflurane. Mice were then injected bilaterally using a blunt needle Neuros Syringe (65457–01, Hamilton Com.) and syringe pump (World Precision Instruments) according to the injection schemes in the table below. The animal was kept in a warmed recovery chamber until recovery from anesthesia before being returned to its home cage.

VirusVirus volumeBrain region/coordinates
AAV5-EF1a-DIO-hChR2(H134R)-EYFP (Hope Center Viral Vector Core, viral titer 2.5 × 1013 vg/ml)150 nlPBN, bilateral, (AP −5.00, ML ± 1.35, DV −3.50)
AAV5-hSyn-DIO-hM4D(Gi)-mCherry (Addgene, viral titer 7 × 10¹² vg/ml)150 nlPBN, bilateral, (AP −5.00, ML +1.35, DV −3.50)
AAV2-retro-DIO-ChR2-eYFP (Hope Center Viral Vector Core, viral titer 2.8 × 1012 vg/ml)100 nlPOA, unilateral, (+0.45 AP, +0.25 ML, −4.90 DV)
AAV2-retro-CAG-FLEX-tdTomato-WPRE (Addgene, viral titer 1 × 1013 vg/ml)100 nlPOA, unilateral, (+0.45 AP, +0.25 ML, −4.90 DV)
AAV5-EF1a-DIO-eYFP (Hope Center Viral Vector Core, viral titer 1.4 × 1013 vg/ml)150 nlPBN, bilateral, (AP −5.00, ML +1.35, DV −3.50)
AAV2-retro-GFP-Cre (Hope Center Viral Vector Core, viral titer 3 × 1013 vg/ml)100 nlPOA, unilateral, (+0.45 AP, +0.25 ML, −4.90 DV)
AAV-retro-DO_DIO-tdTomato_EGFP-WPRE-pA (Addgene, viral titer 8 × 1012 GC/ml)100 nlPOA, unilateral, (+0.45 AP, +0.25 ML, −4.90 DV)
AV5/hSyn-dio-hm4D(Gi)-mcherry (7.8 × 10^12 vg/ml)150 nlPBN, bilateral, (AP −5.00, ML ± 1.35, DV −3.50)

150 nl injections were injected at a rate of 30 nl/min, while 100 nl injections were injected at a rate of 20 nl/min. The injection needle was withdrawn 5 min after the end of the infusion. For anatomic experiments, mice that received unilateral or bilateral injections did not undergo further surgical procedures. For all behavioral experiments, mice underwent bilateral injections, implantations of a fiber optic for photostimulation over POA, and were implanted with a wireless IPTT-300 temperature transponder (Bio Medic Data Systems) subdermally directly rostral to right hindleg.

For photostimulation of PBN to POA projections, mice were injected with AAV5-EF1a-DIO-hChR2(H134R)-EYFP and were allowed 6 weeks for sufficient proteins to reach distal axons. Mice were then implanted with mono fiber optic cannulas (ChR2 mice: Thor Labs, 1.25 mm OD ceramic ferrule, 5 mm cannula with 200 μm OD, 0.22 NA) in the VMPO (+0.45 AP, +0.25 ML, and −4.60 DV for ChR2 mice). The fiber optic implants were affixed using Metabond (Parkell). Mice were allowed 7 days of recovery before the start of behavioral experiments. Viral injection coverage and optical fiber placements were confirmed in all animals using fluorescent microscopy in coronal sections (30 μm) to examine injection and implantation sites. Data from mice with incomplete viral coverage (i.e. unilateral expression of ChR2-eYFP in the PBN) or inaccurate optical fiber placement were excluded. Data from mice with bilateral PBN viral coverage and optical fiber placements near midline position over the POA were included in the study.

Anatomical tracing

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For anterograde viral tracing experiments, virus (AAV5-EF1a-DIO-hChR2(H134R)-EYFP or AAV5-EF1a-DIO-eYFP were used in our experiments) was injected at least 6 weeks prior to transcardial perfusions with 4% paraformaldehyde to allow for anterograde transport of the fluorophore. For retrograde viral tracing experiments, after the virus (AAV2-retro-DIO-ChR2-eYFP, AAV2-retro-CAG-FLEX-tdTomato-WPRE, AAV2-retro-EF1a-DO_DIO-TdTomato_EGFP-WPRE-pA, or AAV2-retro-GFP-Cre) was injected, there was a 3-week wait prior to perfusion to allow sufficient time for retrograde transport of the virus.

Warm temperature exposure

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Ai14xPdyn-Cre and Ai14xPenk-Cre mice in the warm condition were placed in a clean cage wrapped by a circulating water blanket which was set to 38°C. Mice in the room temperature condition were placed in a clean cage in a 22–23°C room. Water was supplied ad libitum in all cages. Cages in the warm condition were given enough time to reach the target temperature as confirmed by a thermometer before mice were placed inside of them. Temperature exposures lasted for 4 hr, after which mice were immediately anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde in phosphate buffer, and brains were subsequently collected.

Immunohistochemistry

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IHC was performed as previously described by Al-Hasani et al., 2013, Kim et al., 2013; McCall et al., 2015. In brief, mice were intracardially perfused with 4% PFA and then brains were sectioned (30 microns) and placed in 1× PB until immunostaining. Free-floating sections were washed in 1× PBS for 3 × 10 min intervals. Sections were then placed in blocking buffer (0.5% Triton X-100% and 5% natural goat serum in 1× PBS) for 1 hr at room temperature. After blocking buffer, sections were placed in primary antibody rabbit Phospho-c-Fos (Ser32) antibody (RRID:AB_10557109, 1:500 Cell Signaling Technology) overnight at room temperature. After 3 × 10 min 1× PBS washes, sections were incubated in secondary antibody goat anti-rabbit Alexa Fluor 633 (RRID:AB_2535731, 1:1000, Invitrogen) for 2 hr at room temperature, followed by subsequent washes (3 × 10 min in 1× PBS then 3 × 10 min 1× PB washes). After immunostaining, sections were mounted on Super Frost Plus slides (Fisher) and covered with Vectashield Hard set mounting medium with DAPI (RRID:AB_2336788, Vector Laboratories) and cover glass prior to being imaged on a Leica DM6 B microscope.

Alexa fluor
633 anti-rabbit IgG
Goat1:1000InvitrogenRRID:AB_2535731
Phospho-c-Fos
(Ser32) Rabbit mAb
Rabbit1:500Cell SignalingRRID:AB_10557109

Imaging and cell quantification

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Brain sections in figures are labeled relative to bregma using landmarks and neuroanatomical nomenclature as described in ‘The Mouse Brain in Stereotaxic Coordinates’ (Franklin and Paxinos, 2013).

To quantify the number of cells expressing cFos, dynorphin, and/or enkephalin, cFos was labeled by Alexa Fluor 633, a fluorophore with emission in 610–800 nm (max 650 nm) range and preproenkephalin/prodynorphin were labeled by tdTomato with emission in the 540–700 nm (max 581 nm) range. All sections were imaged on a Leica DM6 B epifluorescent microscope using a Texas Red Filter Cube (Excitation: BP 560/40, Dichroic: LP 585, Emission: BP 630/75) for tdTomato visualization and a CY5 Filter Cube (Excitation: BP 620/60, Dichroic: LP 660, Emission: BP 700/75) for Alexa Fluor 633. Images were obtained for each 30 μm section that contained neurons in the PBN.

We defined the boundaries of LPBN as follows. Sections between −5.0 and −5.4 rostral to bregma were imaged for LPBN exclusively. The superior cerebellar peduncle marked the medial and ventral boundaries of LPBN. The lateral boundary was marked by the ventral spinocerebellar tract, and the dorsal boundary was marked by the cuneiform nucleus.

All image groups were processed in parallel using ImageJ (RRID: SCR_003070, v1.50i) software. IHC was quantified as previously described (Al-Hasani et al., 2013; Kim et al., 2013). Briefly, channels were separated, an exclusive threshold was set, and positive staining for each channel was counted in a blind-to-treatment fashion using ImageJ. The counts from each channel were then overlaid and percent of co-labeled cells were reported.

Fluorescent in situ hybridization (FISH)

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Following rapid decapitation of mice, brains were flash frozen in −50°C 2-methylbutane and stored at −80°C for further processing. Coronal sections containing the PBN region, corresponding to the injection plane used in the behavioral experiments, were cut at 20 μM at −20°C and thaw-mounted onto Super Frost Plus slides (Fisher). Slides were stored at −80°C until further processing. FISH was performed according to the RNAScope 2.0 Fluorescent Multiple Kit User Manual for Fresh Frozen Tissue (Advanced Cell Diagnostics, Inc) as described by Wang, 2012 – see below. Slides containing the specified coronal brain sections were fixed in 4% paraformaldehyde, dehydrated, and pretreated with protease IV solution for 30 min. Sections were then incubated for target probes for mouse Pdyn (Pdyn, accession number NM_018863.3, probe region 33–700), Penk (Penk, accession number NM_001002927.2, probe region 106–1332), VGLUT2 (Slc17a6, accession number NM_080853.3, probe region 1986–2998), GFP (GFP, accession number AF275953.1, probe region 12–686), and/or Cre (Cre, accession number KC845567.1, probe region 1058–2032) for 2 hr. All target probes consisted of 20 ZZ oligonucleotides and were obtained from Advanced Cell Diagnostics. Following probe hybridization, sections underwent a series of probe signal amplification steps (AMP1–4) including a final incubation of fluorescently labeled probes (Alexa 488, Atto 550, Atto 647), designed to target the specified channel (C1–C3 depending on assay) associated with the probes. Slides were counterstained with DAPI and coverslips were mounted with Vectashield Hard Set mounting medium (Vector Laboratories). Alternatively, mice transcardially perfused with cold PBS and PFA with fixed brain tissue collected and sectioned at 30 µM as described previously were processed for FISH as above.

Images were obtained on a Leica DM6 B upright microscope (Leica), and Application Suite Advanced Fluorescence (LAS AF) and ImageJ software were used for analyses. To analyze images for quantification of Pdyn/Penk/VGLUT2 coexpression, each image was opened in ImageJ software, channels were separated, and an exclusive fluorescence threshold was set. We counted total pixels of the fluorescent signal within the radius of DAPI nuclear staining, assuming that each pixel represents a single molecule of RNA as per manufacturer guidelines (RNAscope). A positive cell consisted of an area within the radius of a DAPI nuclear staining that measured at least five total positive pixels. Positive staining for each channel was counted in a blind-to-condition fashion using ImageJ or natively in LAX software (Leica).

Behavior

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All behaviors were performed within a sound-attenuated room maintained at 23°C at least 1 week after the final surgery. For open field assays, lighting was stabilized at ∼250 lux for aversion behaviors (Figures 6 and 7, and Figure 6—figure supplement 1A–C) and ~200 lux for body temperature change recordings and heat challenges (Figures 4 and 5, Figure 4—figure supplement 1, Figure 5—figure supplement 1, and Figure 6—figure supplement 1D,E). Movements were video recorded and analyzed using Ethovision XT 10 (Noldus Information Technologies). For all optogenetic experiments, a 473 nm laser (Shanghai Lasers) was used and set to a power of ~15 mW from the tip of the patch cable ferrule sleeve. All patch cables used had a core diameter of 200 μm and a numerical aperture of 0.22 (Doric Lenses Inc). At the end of each study, mice were perfused with 4% paraformaldehyde followed by anatomical analysis to confirm viral injection sites, optic fiber implant sites, and cell-type-specific expression.

Real-time place aversion testing

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We used four copies of a custom-made, unbiased, balanced two-compartment conditioning apparatus (52.5 × 25.5 × 25.5 cm) as described previously (Jennings et al., 2013; Stamatakis and Stuber, 2012). Mice were tethered to a patch cable and allowed to freely roam the entire apparatus for 30 min. Entry into one compartment triggered constant photostimulation at either 0 Hz (baseline trial), 2 Hz, 5 Hz, 10 Hz, or 20 Hz (473 nm, 10 ms pulse width) while the mouse remained in the light paired chamber. Entry into the other chamber ended the photostimulation. The side paired with photostimulation was counterbalanced across mice. Ordering was counterbalanced with respect to stimulation frequency and placement in each of four of the copies of behavior apparatus. Bedding in all copies of the behavior apparatus was replaced between every trial, and the floors and walls of the apparatus were wiped down with 70% ethanol. Time spent in each chamber and total distance traveled for the entire 30 min trial were measured using Ethovision 10 (Noldus Information Technologies).

Core body temperature, vasodilation, and BAT thermogenesis recordings

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We used transparent circular behavioral arenas (diameter = 14.5 inches, wall height = 21 cm) for experiments measuring core body temperature changes, vasodilation, and BAT thermogenesis suppression corresponding to optogenetic stimulation. Mice were tethered to a patch cable and allowed to habituate to the arena for 1 hr. Core body temperature measurements were made every 5 min beginning 5 min prior to turning the laser on. Laser frequencies of 2 Hz, 5 Hz, 10 Hz, and 15 Hz were used. Core body temperature measurements were made using a DAS-8007 Reader (Bio Medic Data Systems) which wirelessly read the temperature from a subdermally implanted IPTT-300 temperature transponder in each mouse (previously validated by Langer and Fietz, 2014).

Thermal imaging of mice was carried out using a FLIR E53 thermal imaging camera (FLIR Systems Inc) to record the 65 min trial. Fur over the intrascapular region was shaven to facilitate temperature readings of the interscapular BAT (Crane et al., 2014). Thermal imaging videos were scored in a blind-to-genotype/condition fashion using ResearchIR software (FLIR Systems Inc). Eye, tail, and BAT temperatures were read every minute for the first 35 min of each trial and every 5 min for the final 30 min. Tail temperature readings were taken ~1 mm away from the base of the tail. BAT temperature readings were taken at the warmest point of the intrascapular region. Eye temperature readings were taken at the warmest point of the eye. To quantify the postural extension during these experiments, an investigator reviewed each video and quantified in two-minute bins the percent time the mice were in an extended posture (sprawled on the bedding) and time in huddled position with their tail tucked under their bodies.

Real-time place aversion testing

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We used four copies of a custom-made, unbiased, balanced two-compartment conditioning apparatus (52.5 × 25.5 × 25.5 cm) as described previously (McCall et al., 2015; Parker et al., 2019). Mice were tethered to a patch cable and allowed to freely roam the entire apparatus for 30 min. Entry into one compartment triggered constant photostimulation at either 0 Hz (baseline trial), 2 Hz, 5 Hz, 10 Hz, or 20 Hz (473 nm, 10 ms pulse width) while the mouse remained in the light paired chamber. Entry into the other chamber ended the photostimulation. The side paired with photostimulation was counterbalanced across mice. Ordering was counterbalanced with respect to stimulation frequency and placement in each of four of the copies of behavior apparatus. Bedding in all copies of the behavior apparatus was replaced between every trial, and the floors and walls of the apparatus were wiped down with 70% ethanol. Time spent in each chamber and total distance traveled for the entire 30 min trial were measured using Ethovision 10 (Noldus Information Technologies).

Locomotion changes with U50 and antagonists

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Mice were habituated in a clear chamber (Cambro 18SFSCW135 CamSquare 18 Qt., Cambro City of Industry, Huntington Beach, CA, USA) and then placed on a plywood platform. Two LED lights were placed above the chamber to provide adequate lighting. Room temperature and lighting intensity remained consistent (22.4°C, 132 lux). Cameras (Camera body: ELP-USBFHD01M-SFV, 2.8–12 mm lens) recorded videos for 45 min at 60fps at a resolution of 1920 × 1080 p. Cameras were mounted directly above the chamber and placed on a tripod perpendicular to the chamber. Video recording was controlled through a custom Bonsai Program (Lopes et al., 2015) to allow simultaneous video recording. Mice were allowed to roam the chamber freely upon being injected.

Mice locomotion was analyzed using DeepLabCut, a markerless pose estimator (Mathis et al., 2018). A mouse model was trained using Resnet-50 and k-means clustering on 75 frames from three videos. The model was trained to 250,000 iterations with an average test error of pixel error 5.71 pixels, a calculation of the distance between human labels versus labels predicted by DeepLabCut to determine the accuracy of the trained model. Videos were trained on a Dell workstation on Windows 10 Enterprise with 4.10 GHz Intel Xenon processor with 32 GB RAM and a NVIDIA Quadro RTX 5000 GPU.

A custom Python script (version 3.8) was created to quantify mice locomotion in terms of velocity in the form of pixels per second (NorrisLab 2020). Top-down videos were analyzed to determine cumulative moving averages for each condition; results were then averaged for 10 s. Results were compared to determine the effects of drug administration on locomotion to ensure appropriate CNS targeting.

Drug administration

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Clozapine N-oxide dihydrochloride (Hellobio) was made in sterilized distilled water and mice received an intraperitoneal (i.p) injection of water (vehicle) or CNO (2.5 mg/kg) and were placed in the heat challenge apparatus or thermal plate preference apparatus for 30 min of habituation prior to beginning the assay. Naloxone hydrochloride (Tocris) and naltrexone hydrochloride (Sigma-Aldrich) were dissolved in 0.9% saline. Penk-Cre and Pdyn-Cre mice received an i.p. injection of naloxone (5 mg/kg), naltrexone (3 mg/kg), or saline (vehicle) respectively and were placed back in their home cages for 30 min before being placed into behavioral arena. In experiments using norBN, norBNI dissolved in DMSO (10 mg/kg) was given IP approximately 24 hr prior to start of experiments and again 30 min immediately prior to start of the assay.

Heat challenge

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For chemogenetic inhibition experiments exposing mice to a heat challenge (Figure 5 and Figure 5—figure supplement 1), we used a purpose-built, two-temperature water circulation apparatus to rapidly change the floor and wall temperatures of a square, transparent behavioral arena (15.25 × 15.25 × 19 cm). After drug or saline administration, mice were habituated to the arena at 20°C for 30 min. The water flow to the arena was changed to 34°C, and the temperature of floor and walls rose quickly, reaching steady state in the first 4 min (time course of ambient temperature change can be seen in Figure 5B). The water flow to the arena was switched back to 20°C after 15 min. Thermal imaging recording was obtained beginning 5 min prior to heat challenge and for 10 min post heat challenge for a total of 30 min. Thermal imaging videos were used to measure eye temperature, tail temperature, BAT temperature, and the temperature of the behavioral arena every minute throughout the 30 min heat challenge trial. Thermal imaging videos were scored in a blind-to-genotype/condition fashion.

Thermal preference

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For experiments presenting mice with a choice between two floor plate temperatures (Figure 7F,G), we used a purpose-built apparatus consisting of two fused Cold/Hot Plate Analgesia Meters (Columbus Instruments International) with plastic walls surrounding and dividing the plates to create two behavioral arenas with 4-inch width, 19.5-inch length, and 9-inch height of walls. One side of the behavioral arena was set to 26°C and the other to 20°C. The side set to 20°C was counterbalanced across mice. Pdyn-Cre mice were tethered to a patch cable and placed into a behavioral arena. Mice were allowed to roam the arena for 40 min before photostimulation. The laser frequency was set to 10 Hz and was left on for 20 min. Mice were kept in the behavioral arena for an additional 20 min post-stimulation. Time spent on each side for the entire 80 min trial was quantified using Ethovision 10.

Open field test

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For experiments quantifying distance moved upon photostimulation of PdynPBNPOA (Figure 7A–C), we used a purpose-built 20in square behavior arena. Pdyn-Cre mice were tethered to a patch cable and placed into the behavioral arena. The laser frequency was set to 10 Hz and was left on for 20 min. Distance moved for the 20 min trial was quantified using Ethovision 10. Bedding in the arena was replaced between every trial, and the floors and walls of the arena were wiped down with 70% ethanol.

Statistical analyses

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All data are expressed as mean ± SEM. Statistical significance was taken as p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, and ∗∗∗∗p<0.0001 as determined by Student’s t-test, one-way ANOVA, or a two-way repeated measures ANOVA followed by a Bonferroni post hoc tests as appropriate. Statistical analyses were performed in GraphPad Prism 7.0. For each experiment, control groups and statistics are described in the main text. All ‘n’ values represent the number of animals in a particular group for an experiment.

Experiments involving optogenetic stimulation of PBN inputs to POA using Pdyn-Cre, Penk-Cre, and VGLUT2-Cre mice (Figures 4, 6, and 7, Figure 4—figure supplement 1, and Figure 6—figure supplement 1) were replicated in three separate cohorts for each genotype. Chemogenetic inhibition experiments (Figure 5 and Figure 5—figure supplement 1) were replicated in two separate cohorts of VGLUT2-Cre mice and two separate cohorts of wild-type mice. Warm temperature exposure with cFos immunohistochemical staining experiments (Figure 1) were performed in four separate iterations. Each iteration replicated the results of those prior to it, and data from each iteration was included in the overall statistical analysis of the experiment.

An investigator was blinded to allocation of groups in experiments whose data is shown in Figure 1 (warm-induced cFos+ cell quantification), Figure 1—figure supplement 1/Figure 2/Figure 2—figure supplement 2 (in situ hybridization quantification), and Figure 4/Figure 4—figure supplement 1/Figure 5/Figure 5—figure supplement 1/Figure 7 (thermal video scoring).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

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    4. Adler MW
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    Effect of ambient temperature on the ability of mu-, kappa- and delta-selective opioid agonists to modulate thermoregulatory mechanisms in the rat
    The Journal of Pharmacology and Experimental Therapeutics 268:847–855.
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    3. Burks TF
    (1990)
    Alteration of thermoregulatory set point with opioid agonists
    The Journal of Pharmacology and Experimental Therapeutics 252:696–705.
    1. Wang F
    (2012)
    RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues
    The Journal of Molecular Diagnostics : JMD 14:.
    1. Xin L
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Decision letter

  1. Rebecca Seal
    Reviewing Editor; University of Pittsburgh School of Medicine, United States
  2. Ronald L Calabrese
    Senior Editor; Emory University, United States
  3. Jan Siemens
    Reviewer; University of Heidelberg, Germany
  4. William Wisden
    Reviewer; Imperial College London, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Work by Norris et al. uses circuit-tracing and optogenetic and chemogenetic manipulations to examine the role of a parabrachial nucleus to preoptic area (POA) circuit in thermal defense behaviors. Specifically, the authors show that a population of dynorphin neurons (some also express enkephalin) that project to the POA and were previously shown to induce expression of c-Fos in response to warming (Geerling et al., 2016) drive a decrease in BAT and core body temperature and an increase in tail vasodilation as well as behavioral changes such as decreased locomotor activity and induction of extended body posture. The study fills in some of the important and still unresolved knowledge gaps in the identity of central circuits that direct thermal homeostasis.

Decision letter after peer review:

Thank you for submitting your article "Parabrachial Opioidergic Projections to Preoptic Hypothalamus Mediate Behavioral and Physiological Thermal Defenses" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Ronald Calabrese as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jan Siemens (Reviewer #2); William Wisden (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

Work by Norris et al. use circuit-tracing and optogenetic and chemogenetic manipulations to examine the role of a parabrachial nucleus (PBN) to preoptic area (POA) circuit in thermal defense behaviors. Specifically, the authors show that a population of dynorphin neurons (some also express enkephalin) that project to the POA and were previously shown to induce expression of c-Fos in response to warming (Geerling et al., 2016) drive a decrease in BAT and core body temperature and an increase in tail vasodilation as well as behavioral changes such as decreased locomotor activity and induction of extended body posture. The study fills in some of the important and still unresolved knowledge gaps in the identity of central circuits that direct thermal homeostasis.

Essential revisions:

1) This PBN-POA circuit is also the subject of a similar study by Yang et al., currently released on bioRxiv (https://doi.org/10.1101/2020.06.11.138370). The studies are complementary and reinforcing, but also show differences that the authors should discuss including the role of PBN to POA neuronal populations in tail vasodilation. This function is assigned by Yang et al. to a population of PBN neurons that instead express cholecystokinin.

2) With respect to defining the PBN aspect of the circuit, the authors should report more precisely the scope of the c-Fos expression within the dynorphin, enkephalin and VGLUT2 populations. For example, they should express the c-Fos positive population as a fraction of Dyn- and Enk-positive PBN populations. This would reveal the possibility that some dynorphin neurons in this region may project to other areas and have other functions. The authors should mention/discuss the potential heterogeneity of this population. Related to this, are all of the EnK+ neurons that project to the POA Dyn+? A Venn diagram could help clarify the relationship of the four markers (Dyn, VGLUT2, c-Fos, Enk). Finally, the authors should include whether Dyn or Enk expression in PBN changes over development. Concerns relate to whether tomato expression differs from Dyn, Enk, or actual Cre expression in the adult.

3) With respect to defining the POA aspect of the circuit, the authors are focused on VMPO, but based on the data presented, it appears that other nearby regions may also have been targeted in their experiments (Figure 3C)? If so, what happens to body temperature when optogenetically activating those extra-VMPO Dyn-fiber terminals? MnPO/AVPe and OVLT have been implicated in osmo- and thirst regulation and may modulate heart rate (as described in cited papers and reviews), which could affect body temperature. Did the authors measure heart rate change upon optogenetic stimulation of PBN (Dyn/Enk) fibers terminating in the VMPO?

4) Data demonstrating the requirement for the circuit is weakened by an apparent lack of specificity in chemogenetically inhibiting the VGLUT2 population. Do VGLUT2+ neurons in this area project elsewhere? Specific inhibition of the Dyn+ neuron-POA circuit would be more appropriate.

5) The authors frequently mention a role for opioids in the POA in thermoregulation and perform an experiment to test whether dynorphin and enkephalin in this particular PBN-POA circuit has a role. It may be the case that the peptides released by these projections do not have a role, but the authors need to demonstrate positive controls for the antagonists. Are they able to cross the blood brain barrier and was there sufficient time for the drugs to enter the brain before optogenetic stimulation? The authors could also consider trying other antagonists particularly for KOR such as DIPPA (Cintron-Colon et al., Current Biology, 2019).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Parabrachial Opioidergic Projections to Preoptic Hypothalamus Mediate Behavioral and Physiological Thermal Defenses" for further consideration by eLife. Your revised article has been evaluated by Ronald Calabrese (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The reviewers appreciate the importance of this work to understanding the neural circuitry of thermal defense behaviors and some of the revisions that were made by the authors did help clarify the manuscript, as outlined in attached reviews. On the other hand, a couple of the major issues raised by the reviewers were not adequately addressed (#1 and #2 in previous review). 1) The authors have advanced rather than resolved a discrepancy between their new data and that of Yang et al. (Science Advances) regarding Cck and Pdyn expression, with the authors claiming considerable overlap and Yang et al. claiming distinct populations. It is important to clarify this point and also with respect to the physiological mechanisms of thermal defense behavior. As it stands now the authors have presented a contradiction. 2) The percentage of Pdyn+ neurons that expressed c-Fos with warming and the percentage of Pdyn+ neurons that project to POA that expressed c-Fos with warming should be reported. The same should be reported for PenK+ neurons. Results from this analysis could alter the representations shown in Figure 8. 3) It should be noted that the new data in Figure 2—figure supplement 2 in theory provide important new information, but the specific signals within the images are not robust making interpretations of them difficult. i.e. There is one GFP cell clearly visible in panel B and what is background for panel E?

Reviewer #1:

The study by Norris et al. uses optogenetic /chemogenetic manipulations with physiological measurements and behavior as well as circuit tracing to demonstrate that excitatory neurons in the LPBN, some of which express Pdyn and Penk (partially overlapping), and that project to the POA are important for heat defense behaviors.

1) Explain how their work fits with work by Yang et al., which is now published in Science Advances.

Yang et al. claim that CcK+ and Pdyn+ neurons are distinct populations. The new in situ in Figure 2—figure supplement 2 presumably indicates 70% of Pdyn+ cells are CcK+, but the image shown is not entirely convincing. Whether the signal is over background in the area where Pdyn+ cells are located is unclear even based on the inset. Nevertheless, the finding does not entirely reconcile the differences with the Yang et al. study. Yang et al. claim that Pdyn+ cells are involved in a defensive heat response via an inhibition of iBAT while CCK neurons are involved in a defensive heat response involving vasodilation. Activation of Pdyn neurons does not evoke tail vasodilation while activation of Cck cells does. Yang et al., also show that TeNT inhibition of Pdyn neurons affects core temp and iBAT temp and very modestly tail temp, while inhibition of Cck neurons had effects on core temp and fever, but not tail temp. This suggests Pdyn are required for IBAT but Cck cells are involved but not required for tail vasodilation. Norris et al. show here that activation of the Pdyn and Penk to POA projections cause changes in core, BAT and tail temp. They examine only tail temp with inhibition of Pdyn or Penk neurons, which they show does not change in either case. In sum, the two studies differ in whether the Pdyn neurons are sufficient to evoke tail temp change and it is unclear how the Yang et al. studied a population of Pdyn neurons distinct from Cck while Norris et al. studied Pdyn neurons that largely express Cck.

2) Illustrate better the overlap of c-Fos within the three populations, whether all PenK+ neurons that project to POA are also Pdyn+ and whether Pdyn or Penk expression from the tomato mouse cross differs from adult in situ or adult virus injection (developmental change).

In Figure 1, the authors cross Ai14 tomato mouse to the Cre lines and look at c-Fos with warmth exposure. However, in the Figure 1—figure supplement 1 they do not address whether Ai14 tomato distribution differs from adult Penk or Pdyn rather they instead compare to an in situ probe for Cre in adult. This control is useful for comparing viral expression, but not for addressing overlap of c-Fos shown in Figure 1. To address development, it would been good to perform in situ for tomato with Penk/ Pdyn and c-fos in adult assuming they still have the animals.

In Figure 2—figure supplement 2 the GFP signal in panel B is surprisingly not very robust assuming this is representative. Very few neurons in the slice appear to be GFP+ and many are not where the arrows are clustered. It would be helpful if the reviewers showed on the image the areas that were used in the analyses. Was it only where the arrows were clustered?

Reviewer #2:

The authors have not adequately addressed the reviewers' comments and concerns. This is not to say that we, as reviewers, expect the authors to do all the experiments --or for that matter even change all the wording in the text-- as per reviewers' suggestion. But authors should at least address in the point-by-point rebuttal letter and state why they disagree with the suggestions and/or (i) did not need to or (ii) couldn't or (iii) did not want to implement the changes the reviewers suggested.

1) The authors suggest that Pdyn neurons in the PBN specifically relay temperature information to the POA/VMPO. If Pdyn is labeling neurons that are warm-activated, I would expect a substantial fraction of cfos-positive cells to overlap with pdyn. To assess this requires to calculate the cFos-positive population as a fraction of Dyn- and Enk-positive populations (e.g. what % of Dyn neurons is cfos positive upon warming) instead of expressing the cfos-fraction that is positive for dyn or enk (as has been done by the authors).

In the response the authors vaguely argue that this information can be found in Geerling et al., 2016. We looked it up and Geerling et al. suggest that around 20% of pdyn neurons are specifically induced to express cfos upon warming. Comparing the figure in Geerling et al., 2016 (Figure 7) with the equivalent one in this manuscript (Figure 1D) my best guess is that in the hands of the authors this fraction is even lower. And thus this fraction is misrepresented in the summary Figure 8: here in the cartoon it looks like the majority of warm activated cells are pdyn positive! This also renders the following sentence in the Discussion questionable "...A subset (Pdyn+ or PenK+) of the VGLUT2+ PBN→POA population is likely sufficient to mediate vasodilation and suppress BAT activation.." We disagree, in our view this means that the largest fraction of pdyn neurons is likely doing something else and not relaying temperature information. This possibly explains also the new data in Figure 5—figure supplement 1: blocking the pdyn population by Gi-DREADD does not have an effect (different to blocking vglut2-Cells).

Reversely, does this mean that their Gq-DREADD data (Figure 4I) is wrong and activating the pdyn population should also not activate tail vasodilation as suggested by Yang et al., biRxv or Science Advances 2020? Again, I like to reiterate that the authors don't need to do all what the reviewers suggest, but glossing over important points and not addressing them properly in the letter (and the manuscript) is not a good practice. This brings us to the next point:

2) The authors now do dual-color in situs to test what fraction of CCK neurons overlap with pdyn neurons to conclude that "..Findings from these experiments indicate 70% of Pdyn labeled neurons are also labeled by Cck probes. This congruency helps to resolve any discrepancy that a majority of these two neuronal populations are indeed overlapping…". We beg to differ: the authors Yang et al., 2020 use --as far as we can see-- the same pdyn-Cre mouse line as the authors and they don't see any tail vasodilation when activating these neurons (Figure 5B in the Science Advances publication) and this has nothing to do with any CCK expression. Again, this discrepancy may not be easily resolvable, but to hide it and to say CCK is a subset of these neurons and this resolves the discrepancy is not correct! This functional difference may --at least for some researchers- be important and thus should be spelled out.

3) The manuscript is very difficult to read with all the edits, "invalid citations" etc.

– The authors should read the manuscript carefully and correct before submission.

In summary, we still believe that the manuscript is valuable and adds to the Yang et al. study, but we feel the above aspects should be addressed, at least in the response letter, and the manuscript carefully edited before publication.

Reviewer #3:

It's been known for many years that warm receptors on the skin pass on information through the parabrachial relay glutamate neurons in the brainstem, and that these glutamate neurons project onwards to the preoptic hypothalamic area. The current paper is a functional anatomical study exploring in more detail the role of glutamate neurons in the parabrachial nucleus that project to the preoptic area to regulate defensive control of body temperature. The authors find that dynorphin and enkephalin release in the preoptic area are not needed, but glutamate, is for external warmth induced body cooling. Although not explicitly shown by the authors, this is probably one type of cell in the parabrachial that co-releases glutamate, dynorphin and enkephalin. The standard of work was high and detailed. A lot of technically excellent work is present tin the paper and it is beautifully presented.

The authors have adequately revised the manuscript and I have no further concerns

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Parabrachial Opioidergic Projections to Preoptic Hypothalamus Mediate Behavioral and Physiological Thermal Defenses" for further consideration by eLife. Your revised article has been evaluated by Ronald Calabrese (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) The comparison of the Pdyn staining and the Pdyn-Cre line shown in Yang et al. Figure 2A indicates a high level of co-localization. Norris et al. have used the same Pdyn-Cre line in the present manuscript. Comparison of Pdyn and Cck in Yang et al. Figure 2L was done by staining the Cck-Cre mouse with the Pdyn antibody used in Figure 2A. The difference between Yang et al. and Norris et al. is therefore the difference between the Cck-Cre line (Yang et al) and the use of RNAScope (current manuscript). The authors should correct how they discuss this in the manuscript, as it now is stated as being due strictly to antibody staining by Yang et al. vs RNAScope used by the authors of the current manuscript.

2) As for showing the percentage of Pdyn and Penk neurons that express c-Fos based on data shown in Figure 1, an additional limitation (on top of those noted by the authors) to the interpretation of reporting cell counts as a percentage of either c-Fos or the marker would also be if the number of tomato+ cells observed using the Cre lines crossed to the reporter is not the same as the Pdyn or Penk population manipulated by the viruses. The authors addressed this concern in an earlier version in their response to the reviewers, but direct evidence in the paper is lacking. Nevertheless, providing the readers with some sense of the percentage of Pdyn or Penk neurons that were c-Fos positive from the images that they quantified in Figure 1 would be similarly useful.

https://doi.org/10.7554/eLife.60779.sa1

Author response

Essential revisions:

1) This PBN-POA circuit is also the subject of a similar study by Yang et al., currently released on bioRxiv (https://doi.org/10.1101/2020.06.11.138370). The studies are complementary and reinforcing, but also show differences that the authors should discuss including the role of PBN to POA neuronal populations in tail vasodilation. This function is assigned by Yang et al. to a population of PBN neurons that instead express cholecystokinin.

We thank the reviewers for this insight. We agree that there are some important considerations in that regard, and now address them here and in the revised manuscript.

In the work from Yang et al., the authors report findings largely consistent with those we present, including the central finding that glutamatergic PBN neurons expressing Pdyn drive heat defense (Yang, Du et al., 2020). Yang et al. show that photostimulation of parabrachial Cck or Pdyn expressing PBN neurons drives a decrease in body core temperature. They observe increasing tail temperatures indicating of vasodilation in response to photostimulation of Cck but not Pdyn expressing PBN→POA terminals. Surprisingly, they do not report a change in tail temperature evoked by activation of PBN Pdyn+ neurons despite decreases in core body temperature greater than those seen by activation of Cck expressing terminals. Here, we report rapid rise in tail temperature in response to photostimulation of VGLUT2+, Pdyn+, and PenK+ PBN→POA terminals. In response to reviewers’ suggestion, we examined the overlap of Pdyn and Cck expression in the PBN using in situ hybridization. In Pdyn labeled cells, we found frequent co-labeling by Cck probes (Figure 2—figure supplement 2). Findings from these experiments indicate 70% of Pdyn labeled neurons are also labeled by Cck probes. This congruency helps to resolve any discrepancy that a majority of these two neuronal populations are indeed overlapping.

2) With respect to defining the PBN aspect of the circuit, the authors should report more precisely the scope of the c-Fos expression within the dynorphin, enkephalin and VGLUT2 populations. For example, they should express the c-Fos positive population as a fraction of Dyn- and Enk-positive PBN populations. This would reveal the possibility that some dynorphin neurons in this region may project to other areas and have other functions. The authors should mention/discuss the potential heterogeneity of this population. Related to this, are all of the EnK+ neurons that project to the POA Dyn+? A Venn diagram could help clarify the relationship of the four markers (Dyn, VGLUT2, c-Fos, Enk). Finally, the authors should include whether Dyn or Enk expression in PBN changes over development. Concerns relate to whether tomato expression differs from Dyn, Enk, or actual Cre expression in the adult.

This is an important point and clarification strengthens the manuscript’s findings. In agreement with the reviewers’ suggestion, the warm-activated (Fos positive) PBN neural population is a subset of the larger Pdyn expressing PBN neuron population. Our findings showing that subsets of PenK+ and Pdyn+ neurons are positive for Fos after warmth exposure are consistent with results reported by Geerling et al. (Geerling, et al., 2016) and findings indicating a diversity of roles for PBN Pdyn expressing neurons. Chiang et al. recent reported that Pdyn cells represent a quarter of the excitatory PBN neurons (Chiang et al., 2020). The presented sagittal image of Pdyn+ PBN neuronal projections (Figure 2—figure supplement 1K) shows likely projections of PBN Pdyn+ neurons to multiple brain regions including accumbens, regions of the thalamus, bed nucleus of the stria terminalis, and hypothalamic areas. Recent anatomic studies also found diverse projection targets for Pdyn expressing PBN neurons (Huang, Grady et al. 2020). Forthcoming work from the Bruchas lab implicates PBN neurons in modulating eating behaviors (Bhatti, Luskin et al., 2020).

We have expanded our discussion of functional diversity of PBN Pdyn expressing neurons including recent work indicating a role in aversive learning (Chiang, Nguyen et al., 2020) and in regulating feeding (Kim, Heo et al., 2020). To improve the overall clarity of paper we have added an additional figure (Figure 8) outlining the relationships of PBN neural populations and the behavioral and physiologic outputs of circuit activation. We think this makes it more clear how these various systems are integrated across the circuit, along with what we reported in this study.

To examine the question of whether all PenK+ PBN neurons projecting to the POA are positive for Dyn, we used in situ hybridization to examine the expression of Pdyn and Penk in POA projecting PBN neurons. We found that of cells in LPBN labeled by retroAAV-eGFP (POA projecting), 49 ±4% (mean ± SEM) were also labeled by Penk and Pdyn probes (Figure 2—figure supplement 2). Of the remaining GFP labeled LPBN neurons, 26 ±2% were labeled by either Pdyn or 12 ±1% (mean ± SEM) by Penk, but not both. 13 ±3% (mean ± SEM) of GFP labeled LPBN neurons were not labeled by either (Figure 2—figure supplement 2C).

With respect to the possible change in the expression of Pdyn in the PBN during development, this is a potential confound to defining the Pdyn expressing population in PBN using a permanent recombination marker line like the Ai14 line used in the present study to generate the results pertaining to warmth activation of PBN neural populations presented in Figure 1. This particular mouse line was previously well characterized using in situ by our group. We found in those experiments, that the line was largely protected from leaky A14 expression, and that Pdyn expression with Ai14 colabeling, aligns with wild-type in situ from our group and the Allen Institute(Al-Hasani, McCall et al., 2015). Finally, there is also limited evidence for dynorphin gene expression during critical development and cell fate decisions, which could create non-selective Ai14 reporting. Nevertheless, we are aware of this limitation and have added discussion to acknowledge the possibility.

The results here showing an overlap of Pdyn expression and activation by warmth are similar to earlier reports (Geerling, Kim et al., 2016) and results obtained by RNA sequencing in the work by Yang et al. (Yang, Du et al., 2020). The remainder of the studies presented here utilize viral injections into adult animals, and concerns regarding changing expression of Pdyn during development are not applicable.

3) With respect to defining the POA aspect of the circuit, the authors are focused on VMPO, but based on the data presented, it appears that other nearby regions may also have been targeted in their experiments (Figure 3C)? If so, what happens to body temperature when optogenetically activating those extra-VMPO Dyn-fiber terminals? MnPO/AVPe and OVLT have been implicated in osmo- and thirst regulation and may modulate heart rate (as described in cited papers and reviews), which could affect body temperature. Did the authors measure heart rate change upon optogenetic stimulation of PBN (Dyn/Enk) fibers terminating in the VMPO?

We appreciate this concern. To clarify further, the median preoptic nucleus (MnPO) , anteroventral periventricular nucleus (AVPe) and vascular organ of the lamina terminalis (OVLT) are located medial to the VMPO, and ventral portions on MNPO and portions of AVPe and OVLT were likely covered in the light cone from the midline placement of optical fibers as illustrated in Figure 4B. To further aid in clarity, we added outlines demarcating MnPO and OVLT to Figure 3.

Considering the additional physiological impact of these manipulations, increased heart rate occurs in response to cold as part of increased metabolism to support thermogenesis (Nakamura and Morrison, 2007). Heart rate may change in response to activation of the PBN→POA projections but whether this is a primary or secondary effect due to the hypothermia would require substantial carefully designed experiments targeted specifically at this interesting question. In the presented studies on heat defense, we did not include an examination of heart rate change in our studies on thermal defensive behaviors and body temperature regulation. However, the hypothesis that activation of Pdyn+PBN→POA projections inhibits cold evoked increases in HR is an exciting avenue for future studies.

4) Data demonstrating the requirement for the circuit is weakened by an apparent lack of specificity in chemogenetically inhibiting the VGLUT2 population. Do VGLUT2+ neurons in this area project elsewhere? Specific inhibition of the Dyn+ neuron-POA circuit would be more appropriate.

This is an important point the reviewers make regarding specificity of the chemogenetic manipulations. Therefore, in response to reviewer comments, we undertook new experiments using heat challenge in Pdyn-Cre mice expressing inhibitory Gi DREADDs in the Pdyn+ PBN neurons to address this question. Activation of Gi DREADDs by CNO did not significantly alter tail vasodilation is response to thermal challenge (Figure 5—figure supplement 1). Gi DREADD mediated inhibition of Pdyn+ PBN neurons was not sufficient to block thermal challenge evoked tail vasodilation. We interpret these findings to suggest that, as represented in Figure 8, the entirety of the heat defensive neural population in PBN is VGLUT2+, whereas only a subset of this population is Pdyn+ (Figure 1F). The remaining warm-activated LPBN neurons not blocked by inhibition of the Pdyn+ population may be enough to drive heat defensive tail vasodilation. We have added more to the Discussion to address these new findings in a scholarly manner.

5) The authors frequently mention a role for opioids in the POA in thermoregulation and perform an experiment to test whether dynorphin and enkephalin in this particular PBN-POA circuit has a role. It may be the case that the peptides released by these projections do not have a role, but the authors need to demonstrate positive controls for the antagonists. Are they able to cross the blood brain barrier and was there sufficient time for the drugs to enter the brain before optogenetic stimulation? The authors could also consider trying other antagonists particularly for KOR such as DIPPA (Cintron-Colon et al., Current Biology, 2019).

In the presented studies we employed three separate opioid receptor antagonists with different selectivity for opioid receptors. All have been previously used in vivo, well studied and established to cross into the CNS. Our laboratory has substantial expertise in opioid neuropharmacology and have used and published with of all these ligands across a host of behavioral assays. We chose these ligands based on the consensus in the field. We note the following:

Naloxone is a non-selective opioid receptor antagonist with higher affinity for the μ-receptor and crosses(Goldstein and Naidu 1989, Dean, Bilsky et al., 2009). Naltrexone is a more potent opioid receptor antagonist and has a higher relative affinity for κ-receptors over μ-receptors. It crosses into the brain rapidly with high levels(Wang, Raehal et al., 2004). Norbinaltorphimine (norBNI) is a long-acting k-receptor selective antagonist that actively penetrates the CNS (Bruchas, Yang et al., 2007). To further validate that the doses and time courses we used were effective, we examined the block of suppression of locomotion by the kappa receptor agonist U50,488 by naltrexone and norBNI (Paris, Reilley et al., 2011).

To show a positive control for norBNI’s selective action at KORs under the conditions we used here, we did an experiment, whereby pretreatment with naltrexone or norBNI was effective in reducing U50 (selective KOR agonist) mediated suppression of locomotor activity (Figure 4—figure supplement 1D-F). The presented pharmacology data indicate that acute heat defense behaviors are not mediated by engagement of opioid signaling. Opioid signaling may be implicated in balancing system tone and matching thermal demands, metabolism, and calorie intake, and is an area of active ongoing investigation.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The reviewers appreciate the importance of this work to understanding the neural circuitry of thermal defense behaviors and some of the revisions that were made by the authors did help clarify the manuscript, as outlined in attached reviews. On the other hand, a couple of the major issues raised by the reviewers were not adequately addressed (#1 and #2 in previous review). 1) The authors have advanced rather than resolved a discrepancy between their new data and that of Yang et al. (Science Advances) regarding Cck and Pdyn expression, with the authors claiming considerable overlap and Yang et al. claiming distinct populations. It is important to clarify this point and also with respect to the physiological mechanisms of thermal defense behavior. As it stands now the authors have presented a contradiction. 2) The percentage of Pdyn+ neurons that expressed c-Fos with warming and the percentage of Pdyn+ neurons that project to POA that expressed c-Fos with warming should be reported. The same should be reported for PenK+ neurons. Results from this analysis could alter the representations shown in Figure 8. 3) It should be noted that the new data in Figure 2—figure supplement 2 in theory provide important new information, but the specific signals within the images are not robust making interpretations of them difficult. i.e. There is one GFP cell clearly visible in panel B and what is background for panel E?

1) In sum, the results we report are highly consistent with results from a parallel, now published, studies by Yang et al(Yang et al., 2020). This is particularly noteworthy considering the studies were conducted by independent groups working in parallel located in separate countries and the results are highly consistent including the magnitude of changes in body temperature. Differences in results are, however, present between the two studies. The key differences are most relevant to one of the conclusions drawn by Yang el al. that our findings do not support.

The first notable difference in results is regarding populations of neurons expressing Pdyn and Cck. Yang et al. use immunohistochemistry (IHC) to localize Cck and Pdyn protein expression in the PBN. Their reported data shows minimal cell by cell overlap of labeling for Pdyn and Cck. Neurons labeled for either peptide are present in the lateral PBN with more broad expression of Cck in the PBN. The use of IHC to identify peptide expression is well established to be technically challenging for numerous neuropeptides. As a laboratory focused on them, we’ve ultimately decided in the past 5 years to forgo using IHC and antibodies to classify peptide-containing neurons due to our inability to be consistently confident in what the method tells us. Selectivity of antibodies, expression levels, protein-peptide levels changing under various condition, etc..all of these reasons are reported confounds of the techniques required (Fritschy, 2008; Gautron, 2019; Nassel and Ekstrom, 1997). Yang et al., used colchicine treatment to disrupt normal peptide trafficking, concentrating the peptide in the soma, and this is often required for IHC of peptides, such as dynorphin.

In contrast, we employed fluorescent in situ hybridization (FISH) to examine Cck and Pdyn transcript expression in PBN to avoid challenges with IHC, and for higher resolution of genetic markers of cell identity. Even in RNAseq sutides, in situ is ofen required as a post-hoc validation and more closely represents “ground truth” for identification of a particularly genetic cellular identity. One thing we also mention, is that even though we used in situ, our findings are largely consistent with those reported by Yang et al. with respect to distribution and anatomic localization. We did, however, identify that Cck is co-expressed in 70% of Pdyn expressing PBN neurons (Figure 2—figure supplement 2) A further factor relevant to addressing this specific discrepancy in results is potential changes in expression levels of the peptides. The amount of protein produced and stored in the cell for a given peptide may vary with time and conditions. Therefore, we think it is certainly reasonable to presume that a potential source of the difference in finding overlapping vs separate Cck and Pdyn PBN neural populations results may rest with biological variables or differing techniques used to examine Pdyn and Cck expression in each study. We have now noted these caveats in the Discussion.

A second point where the results in Yang et al. and our results differ is in vasodilation and suppression of BAT in response to activation/suppression of Pdyn+ or CcK+ PBN→POA neurons, respectively. They report that activation of Pdyn+ PBN neurons induces hypothermia (similar to our results) but does not lead to vasodilation when done with Gq DREADDs or activation of POA terminals with photostimulation. They reported photostimulation for examining tail vasodilation used is by Yang et al. is the 20Hz alternating on and off at 2 seconds intervals and they used a completely different opsin (ChIEF vs hChR2(H134R)) construct to mediate photoactivation. The differences in these opsins are important because they could drive release of different transmitters and thus impact down-stream effects in the POA with various outcomes. Yang et al. report that tail vasodilation in response to activation of CcK+ PBN neurons by DREADDs and photoactivation of PBN→POA terminals. They further report that blocking transmission of Pdyn+ neurons lead to BAT activation but blocking CcK+ neurons did not have the same effect. We found hypothermia, vasodilation, and suppression of BAT activity in response to activation of VGLUT2+, Pdyn+, or PenK+ terminals in response to 10hz stimulation of PBN→POA terminals.

We agree with the reviewers that implications of these differences are worth discussing. The Yang el al. study did not examine distinct roles for Cck or Dyn peptides in neuromodulation. The expression of the peptides served only as markers to narrow the involved PBN→POA neural populations. From their data they argue for separation of the neural circuits involved in heat defense by modulating BAT and vasomotor tone at the level of the PBN but not separate roles for the peptides themselves. We found no roles for Dyn or Enk signaling in mediating the acute effects of PBN→POA neural activation. Our data indicate instead, that neither Pdyn nor Penk expression mark a functional separation in heat defense neural circuitry at the level of the PBN. Thus, the data we report does not support one conclusion from Yang et al., specifically, that Pdyn expression in PBN→POA neurons marks a functional (or categorical) separation in thermal defense circuitry. Our studies do not address if such separation may or may not exist beyond Pdyn+ and PenK+ PBN →POA neurons. Taken together, the two reports can be used together make predictions to be tested in future studies and further delineate thermal regulatory neural circuits. For example, identification of separate neuronal population in the POA that project differentially to distinct nuclei and received inputs from separable PBN populations would support the functionally distinct pathways that are separate at the level of the PBN. We also note that the authors of the Yang paper contacted us unsolicited and were scholarly and accepting of the potential differences and similarities in our studies, and results.

Regarding the conclusions we draw in our report, the data presented in Yang et al. are only discordant with respect two points. First, that Dyn and Cck expressing PBN populations are distinct. We see overlapping expression at the level of transcripts by FISH and they however indicate separation at the cellular level in protein expression using IHC. Second, we observe vasodilation of the tail in response to activation of Pdyn+ PBN→POA neurons and they do not. Because they do not observe tail vasodilation in response to activation of PBN→POA Pdyn+ neurons it is unclear how the persistence of warmth induced tail vasodilation despite toxin-based blockade of Pdyn+ POA neurons should be interpreted within this context but it does fit with our data. We found that the Gi DREADD based suppression of Pdyn+ PBN neurons did not block warmth induced tail vasodilation.

Our report and the paper from Yang et al. are remarkably consistent for parallel studies from completely independent groups. The differences as we note above, can be attributed to experimental details, different techniques, or underling biological variables. We have now added further consideration of these points to the discussion to ensure that the field is aware of these differences, and provide ideas for further exploration using high resolution approaches including RNAseq in PBN-POA projection neurons – a massive, albeit, fascinating potential future direction, which will be more definitive in regard to cell type segregation in this region. While we cannot completely resolve the focal difference in the data in our report and those in the Yang et al. manuscript, we did expand the discussion of this difference in the Discussion section. The differing results do lead to testable hypotheses that can be examined in future studies on thermal circuits.

2) The reviewers ask that “the percentage of Pdyn+ neurons that expressed c-Fos with warming and the percentage of Pdyn+ neurons that project to POA that expressed c-Fos with warming should be reported.” As we detail below, the experiments we can undertake to address this question would yield a quantification, but experimental factors present confounds that make a meaningful interpretation which would answer the underlying question highly unlikely.

The goal of quantifying the percentage Pdyn+ or PenK+ PBN cells and/or Pdyn+ or PenK+ PBN→POA neurons activated by warmth is highly unlikely to be meaningfully answered due to technical confounds of available methods and limitations inherent to interpretation of Fos staining as a marker of neuronal activity. If we understand the reviewers concerns correctly, they are asking, do all (or nearly all) Pdyn+ or PenK+ PBN→POA projecting neurons respond to environmental warmth? The option for attempting to address this question that we see as tenable would is injection of retroAAV-DIO-eFYP (or other marker) into the POA of Dyn-Cre and Penk-Cre mice to label Pdyn+ or PenK+ expressing PBN→POA neurons, exposing the animals to environmental warmth, and then probe PBN containing sections for Fos expression. Although this experiment would yield a relative quantification, meaningful interpretation of potential results would be confounded by multiple variables. For example, retrogradely labeled PBN neurons not positive for Fos staining may be considered not warmth responsive, but it would remain unknown if treatment with environmental warmth at a higher temperature or a more prolonged time would induce Fos expression in the cells. The time course and changes therein are not trivial to work out in a careful manner within the scope of this report. Further, cells positive for Fos labeling but not labeled by the retrograde virus could be interpreted as not projecting to the POA. However, such cells may simply have not been labeled by the retrograde virus but do in fact project to better POA. As an additional example, cells not directly involved in thermal heat defense could also be activated in response to environmental warmth due the effects of warmth on multiple physiological parameters. In the context of the PBN and POA, which drive a wide range of homeostatic and behavioral processes, this is particularly relevant. As these example illustrates, clear quantification to address what we understand to be the underlying question from the reviewer is likely to yield a number, but the meaning of the quantification would remain unclear. It is likely that projection-specific RNAseq and other high resolution physiological measures like in vivo imaging would be better suited for this experiment, yet as the reviewers might imagine, these are beyond the scope of this initial report.

The requested quantification of Fos positive cells from the Pdyn-Cre crossed to a recombination reporter line (Fos+/Pdyn+) has been performed previously in published articles (Geerling et al., 2016). Repeating that analysis here where add little total value and such experiments would be confounded by similar concerns as those identified above regarding Fos staining as a marker. As a further barrier, we no longer have the require mouse lines and the original slides are now many years old.

The reviews indicate that these results might alter the graphic illustration in Figure 8. We apologize for confusion derived from this cartoon. The illustration is meant as a Venn diagram to illustrate, in a logical framework, our findings of overlapping expression without making overt statements regarding quantification. In response to reviewers concerns we have edited the amount the circle representing the Pdyn+ neurons overlap with warm-activated cells to help avoid confusion about what may be implied.

3) With respect to questions about data shown in Figure 2—figure supplement 2, we appreciate the feedback regarding how the images are displayed. To address this we have now revised the figure by adding additional panels showing three areas of the PBN in greater detail.

We found these four-color FISH experiments, examining expression of two neuropeptides and retrogradely labeled neurons be technically challenging. The question regarding the single GFP positive cell, perhaps reflects concerns about overall displayed figure. The puncta from the FISH labeling for GFP can be seen in many of the cells and we have revised this figure to help make that more evident by displaying images at two levels of detail to convey anatomic and cellular level information. It is worth noting that we find the function of the fluorophore (GFP) is disrupted by the hybridization process and thus, the fluorescent signal is derived from the tags on the probes targeting GFP mRNA. We feel the additional panels demonstrate the background labeling was quite low in this experiment was low. This experiment was undertaken to address reviewer question about overlap of Pdyn and Penk expression in PBN→POA neurons.

Reviewer #1:

The study by Norris et al. uses optogenetic /chemogenetic manipulations with physiological measurements and behavior as well as circuit tracing to demonstrate that excitatory neurons in the LPBN, some of which express Pdyn and Penk (partially overlapping), and that project to the POA are important for heat defense behaviors.

1) Explain how their work fits with work by Yang et al., which is now published in Science Advances.

2) Illustrate better the overlap of c-Fos within the three populations, whether all PenK+ neurons that project to POA are also Pdyn+ and whether Pdyn or Penk expression from the tomato mouse cross differs from adult in situ or adult virus injection (developmental change).

We agree with the reviewer’s points, and they make important comments with respect to our study as compared to the Yang report, that weren’t addressed as clearly in the original resubmission.

1) With respect to the overall findings in Yang et al. report compared to the data that we present; the results are very similar. Reviewer one has summed up nicely much of how the results are different. As we discussed above in our reply to summary comments, there are technical and experimental details which may account for some portion of the difference in findings; however, the results we obtained with respect to differential modulation of BAT activity and vasomotor tone by Pdyn+ neurons do not match those reported by Yang et al. Future studies designed to further delineate potential separation of neural circuits regulating aspects of thermal heat defense behaviors at the level of the PBN and POA may help resolve this narrow difference in the conclusions drawn by the two studies. Our study does not support a conclusion drawn in Yang et al., that expression of Pdyn is a marker of this functional division in the neural circuitry at the level of the PBN. We have greatly expanded our Discussion of these differences and possibly implications in the revised manuscript.

2) In previous studies expression of tdTomato in Ai14 x Pdyn-Cre mice has been carried out in in the nucleus accumbens are high level of concordance was found between expression Pdyn and tdTomato (Al-Hasani et al., 2015). We have not had the cross of Ai14 to Pdyn or Penk mice for several years now and cannot perform the requested in situ hybridization in the parabrachial nucleus without a substantial delay required to breed and allow the required animals to come to adulthood. Review of the Allen Brain Developing Brain Atlas shows expression of Pdyn in the external PBN at P28. Experiments examining overlap of tdTomato expression with Pdyn transcripts in the PBN would one time of information about the stability through development we do not feel that results may be obtained would substantially alter interpretations of the results presented. Only data in figure one, which is largely consistent with Geerling et al., 2016, is impacted by this concern (Geerling et al., 2016). We feel we offer a conservative interpretation of this data. The remainder of the experiments are not impacted by concerns about changes in Pdyn during development.

Reviewer #2:

The authors have not adequately addressed the reviewers' comments and concerns. This is not to say that we, as reviewers, expect the authors to do all the experiments --or for that matter even change all the wording in the text-- as per reviewers' suggestion. But authors should at least address in the point-by-point rebuttal letter and state why they disagree with the suggestions and/or (i) did not need to or (ii) couldn't or (iii) did not want to implement the changes the reviewers suggested.

1) The authors suggest that Pdyn neurons in the PBN specifically relay temperature information to the POA/VMPO. If Pdyn is labeling neurons that are warm-activated, I would expect a substantial fraction of cfos-positive cells to overlap with pdyn. To assess this requires to calculate the cFos-positive population as a fraction of Dyn- and Enk-positive populations (e.g. what % of Dyn neurons is cfos positive upon warming) instead of expressing the cfos-fraction that is positive for dyn or enk (as has been done by the authors).

In the response the authors vaguely argue that this information can be found in Geerling at el 2016. We looked it up and Geerling et al. suggest that around 20% of pdyn neurons are specifically induced to express cfos upon warming. Comparing the figure in Geerling et al., 2016 (Figure 7) with the equivalent one in this manuscript (Figure 1D) my best guess is that in the hands of the authors this fraction is even lower. And thus this fraction is misrepresented in the summary Figure 8: here in the cartoon it looks like the majority of warm activated cells are pdyn positive! This also renders the following sentence in the Discussion questionable "...A subset (Pdyn+ or PenK+) of the VGLUT2+ PBN→POA population is likely sufficient to mediate vasodilation and suppress BAT activation.." We disagree, in our view this means that the largest fraction of pdyn neurons is likely doing something else and not relaying temperature information. This possibly explains also the new data in Figure 5—figure supplement 1: blocking the pdyn population by Gi-DREADD does not have an effect (different to blocking vglut2-Cells).

Reversely, does this mean that their Gq-DREADD data (Figure 4I) is wrong and activating the pdyn population should also not activate tail vasodilation as suggested by Yang et al., biRxv or Science Advances 2020? Again, I like to reiterate that the authors don't need to do all what the reviewers suggest, but glossing over important points and not addressing them properly in the letter (and the manuscript) is not a good practice. This brings us to the next point:

We understand that two points of concern are raised in first comment. One addresses the presentation in the cartoon in Figure 8 of the potential for implied percentage of Pdyn+ POA neurons that are positive for Fos expression following warmth exposure(warm-activated). We agree with the findings from Geerling et al. that a minority of total Pdyn+ PBN neurons are positive for Fos following warmth exposure. Further, analysis of these experiments would, we feel, produce data which that be difficult to interpret for reasons detailed above. We have revised Figure 8 to reduce potential for confusion about the extent of the overlap of Pdyn and Fos staining after warm exposure.

With respect to the second point, we are less clear on what the reviewer is pointing out but would like to review a few of the points with the hope clarify. We find that photoactivation Pdyn+ and PenK+ PBN→POA terminals in the POA leads to tail vasodilation, hypothermia, BAT suppression, and aversion. We showed by FISH that neurons labeled by Penk and Pdyn probes also express Slc17a6 (VGLUT2). From these findings taken together, we conclude that activation of PenK+ or Pdyn+ PBN→POA neurons, which are subpopulations of the VGLUT2+ PBN neuron population, is sufficient to drive tail vasodilation, BAT suppression, hypothermia, and aversion. Our studies focused on the PBN→POA neurons. In all activation experiments we applied light via fiber optic implanted over the VMPO. This was done to avoid photoactivation of PBN neurons that do not project to the POA. The data and conclusions we present make no claims to the functional roles of all or most PBN Pdyn+ neurons. None of the presented experiments were done with Gq-DREADDs as the reviewer suggests. The data in Figure 4I was obtained from experiments using 10Hz photostimulation in the POA of Pdyn+ POA→POA neurons.

We found that Gi-DREADD mediated inhibition of VGLUT2+ but Pdyn+ PBN neurons blocked tail vasodilation is response to thermal challenge. We interpret this finding to suggest that the Pdyn+ PBN→POA projecting population is a subset of the VGLUT+ PBN→POA population. As a technical point, this could be also due to a limited effect of the Gi signaling mediated inhibition.

2) The authors now do dual-color in situs to test what fraction of CCK neurons overlap with pdyn neurons to conclude that "..Findings from these experiments indicate 70% of Pdyn labeled neurons are also labeled by Cck probes. This congruency helps to resolve any discrepancy that a majority of these two neuronal populations are indeed overlapping…". We beg to differ: the authors Yang et al., 2020 use --as far as we can see-- the same pdyn-Cre mouse line as the authors and they don't see any tail vasodilation when activating these neurons (Figure 5B in the Science Advances publication) and this has nothing to do with any CCK expression. Again, this discrepancy may not be easily resolvable, but to hide it and to say CCK is a subset of these neurons and this resolves the discrepancy is not correct! This functional difference may --at least for some researchers- be important and thus should be spelled out.

The differences between the results we show and those from Yang et al. focus on the effect of activation of Pdyn PBN→POA neurons on tail vasodilation and if Pdyn and Cck expression are markers for functional division in the heat defense circuitry at the level of the PBN. We find that activation of Pdyn+ PBN→POA terminals results in tail vasodilation and suppression of BAT thermogenesis and leads to rapid hypothermia. Yang et al. do not report tail vasodilation in response to activation of Pdyn+ PBN→POA but do observe similar rapid onset in hypothermia despite the lack of active heat shedding via vasodilation. They do see tail vasodilation in response to activation of Cck PBN neurons and report that Cck and Pdyn expressing neurons are sperate populations based on IHC. Considering the technical challenges with IHC for Pdyn, we used FISH to examine expression of Pdyn and Cck in the PBN and found these two populations had substantial overlap. Thus, our data does not indicate there is a functional separation in heat defense circuity at the level PBN demarcated by Pdyn expression. Despite this narrow difference in the results overall, both reports are quite consistent. We have expanded the Discussion in the manuscript of these the divergent results in the manuscript and addressed the points further in the reply to the summary comments.

3) The manuscript is very difficult to read with all the edits, "invalid citations" etc.

The authors should read the manuscript carefully and correct before submission.

Thank you for highlighting these concerns. We worked to correct and refine the manuscript and have minimized the edits evident in the uploaded version.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) The comparison of the Pdyn staining and the Pdyn-Cre line shown in Yang et al. Figure 2A indicates a high level of co-localization. Norris et al. have used the same Pdyn-Cre line in the present manuscript. Comparison of Pdyn and Cck in Yang et al. Figure 2L was done by staining the Cck-Cre mouse with the Pdyn antibody used in Figure 2A. The difference between Yang et al. and Norris et al. is therefore the difference between the Cck-Cre line (Yang et al) and the use of RNAScope (current manuscript). The authors should correct how they discuss this in the manuscript, as it now is stated as being due strictly to antibody staining by Yang et al. vs RNAScope used by the authors of the current manuscript.

We have edited the text of the manuscript to further clarify the differences between how Yang et al. and we assayed for expression of Cck and Pdyn. As highlighted by the comments, we used RNAScope and Yang et al. used a combination if IHC (for Dyn) and viral Cre dependent reporter in the CCk-Cre mice.

2) As for showing the percentage of Pdyn and Penk neurons that express c-Fos based on data shown in Figure 1, an additional limitation (on top of those noted by the authors) to the interpretation of reporting cell counts as a percentage of either c-Fos or the marker would also be if the number of tomato+ cells observed using the Cre lines crossed to the reporter is not the same as the Pdyn or Penk population manipulated by the viruses. The authors addressed this concern in an earlier version in their response to the reviewers, but direct evidence in the paper is lacking. Nevertheless, providing the readers with some sense of the percentage of Pdyn or Penk neurons that were c-Fos positive from the images that they quantified in Figure 1 would be similarly useful.

We blindly sampled tdTomato expressing cells in the LPBN in brain sections from Ai14xPdyn-Cre or Ai14xPenk-Cre mice and then quantified the number of those cells that were also labeled for Fos induction after warm exposure. For Pydn+ neurons we found 22% of the tdTomato cells were also Fos+, similar to the finding of 27% by Geerling et al., 2016 in similar experiments. For PenK+ we found 18% of tdTomato cells were labeled for Fos. These values are now reported in the text.

https://doi.org/10.7554/eLife.60779.sa2

Article and author information

Author details

  1. Aaron J Norris

    Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Contributed equally with
    Jordan R Shaker
    For correspondence
    norrisa@wustl.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7825-1756
  2. Jordan R Shaker

    Medical Scientist Training Program, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing
    Contributed equally with
    Aaron J Norris
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4496-3904
  3. Aaron L Cone

    Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4411-6673
  4. Imeh B Ndiokho

    Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1924-1368
  5. Michael R Bruchas

    Center for the Neurobiology of Addiction, Pain and Emotion, Departments of Anesthesiology and Pharmacology, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Writing - review and editing
    For correspondence
    mbruchas@uw.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4713-7816

Funding

National Institute of Mental Health (K08MH119538)

  • Aaron J Norris

National Institute of Mental Health (R37DA033396)

  • Michael R Bruchas

Hope Center for Neurological Disorders

  • Aaron J Norris
  • Michael R Bruchas

National Eye Institute (R21EY031269)

  • Aaron J Norris

National Institute of Mental Health

  • Michael R Bruchas

National Institute of Mental Health (R37DA03339607)

  • Michael R Bruchas

National Institute of Mental Health (P30DA048736)

  • Michael R Bruchas

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank Megan Votoupal for her technical assistance in mouse husbandry. This work was supported by a Foundation for Anesthesia Education and Research (FAER) Grant, and National Institute for Mental Health (NIMH) grant K08MH119538 and R21EY031269 to AJN, by R01MH11235505 and R37DA03339607 to MRB, P30DA048736, and by a Pilot Project Award from the Hope Center for Neurological Disorders at Washington University to AJN and MRB. The Mallinkrodt Foundation (MRB Professorship). The graphic summary illustration (Figure 8) was created by Percy Griffin with Astrid Rodriguez Velez in association with InPrint at Washington University School of Medicine.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of Washington University (#19-0835).

Senior Editor

  1. Ronald L Calabrese, Emory University, United States

Reviewing Editor

  1. Rebecca Seal, University of Pittsburgh School of Medicine, United States

Reviewers

  1. Jan Siemens, University of Heidelberg, Germany
  2. William Wisden, Imperial College London, United Kingdom

Publication history

  1. Received: July 7, 2020
  2. Accepted: February 24, 2021
  3. Version of Record published: March 5, 2021 (version 1)

Copyright

© 2021, Norris et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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