Inhibition of the hypothalamic ventromedial periventricular area activates a dynorphin pathway-dependent thermoregulatory inversion in rats

Shaun F Morrison; Georgina Cano; Shelby L Hernan; Pierfrancesco Chiavetta; Domenico Tupone

doi:10.1016/j.cub.2024.11.006

. Author manuscript; available in PMC: 2026 Jan 6.

Published in final edited form as: Curr Biol. 2024 Dec 2;35(1):59–76.e4. doi: 10.1016/j.cub.2024.11.006

Inhibition of the hypothalamic ventromedial periventricular area activates a dynorphin pathway-dependent thermoregulatory inversion in rats

Shaun F Morrison ¹, Georgina Cano ², Shelby L Hernan ², Pierfrancesco Chiavetta ¹, Domenico Tupone ^1,^3,^4,^5,^6,^*

PMCID: PMC11706707 NIHMSID: NIHMS2035266 PMID: 39626667

Summary

To maintain core body temperature in mammals, CNS thermoregulatory networks respond to cold exposure by increasing brown adipose tissue and shivering thermogenesis. However, in hibernation or torpor, this canonical thermoregulatory response is replaced by a new, emerging paradigm, thermoregulatory inversion (TI), an alternative homeostatic state in which cold exposure inhibits thermogenesis and warm exposure stimulates thermogenesis. Here we demonstrate that in the non-torpid rat, either exclusion of the canonical thermoregulatory integrator in the preoptic hypothalamus or inhibition of neurons in the ventromedial periventricular area (VMPeA) induces the TI state through an alternative thermoregulatory pathway. Within this pathway, we have identified a dynorphinergic input to the dorsomedial hypothalamus from the dorsolateral parabrachial nucleus that plays a critical role in mediating the cold-evoked inhibition of thermogenesis during TI. Our results reveal a novel thermosensory reflex circuit within the mammalian CNS thermoregulatory pathways and support the potential for pharmacologically inducing the TI state to elicit a therapeutic hypothermia in non-hibernating species, including humans.

Keywords: Thermoregulation, Thermoregulatory Inversion, Brown Adipose Tissue, Shivering, Vasomotion, Dynorphin, Parabrachial, Preoptic Area, Hibernation, Torpor

Graphical Abstract

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eTOC Blurb

Morrison et al. show that the inhibition of ventromedial periventricular area (VMPeA) induces a state of thermoregulatory inversion (TI), in which thermogenic responses to cold ambient exposure are actively inhibited by an alternative thermoregulatory pathway, via a dynorphinergic system. VMPeA is a switch for this novel thermoregulatory paradigm.

Introduction

The core body temperature (T_CORE) of mammals is normally maintained within a narrow range that is optimal for enzymatic reactions and cellular function. The neural circuitry for normal thermoregulation alters both behavior and the neural outflows to thermoeffector organs in response to changes in skin and core temperatures to minimize deviations in T_CORE¹. In particular, brown adipose tissue (BAT) and skeletal muscle (shivering) thermogenesis are increased during exposure to cold ambient temperatures (T_AMB) and inhibited in a warm T_AMB^2-6. In normal thermoregulation¹, cold and warm signals from the skin thermoreceptors are transmitted via the dorsal horn to the parabrachial nuclei (PBN). Cold-responsive neurons in the external lateral PBN (elPBN)^4,7,8 and warm-responsive neurons in the dorsolateral PBN (dlPBN)^3,7,8 relay thermosensory signals to the POA. Responding to these cutaneous thermal signals, POA outputs regulate the balance of inhibitory^1,9 and excitatory^1,10 inputs to thermogenesis-promoting neurons in the dorsomedial hypothalamus (DMH), that in turn excite thermogenesis premotor neurons in the medullary rostral raphe pallidus (rRPa)^1,11. During skin cooling, excitation of thermogenesis-promoting DMH neurons prevails and thermogenesis is augmented. Conversely, in a warm environment, net inhibition of these DMH neurons reduces the excitatory drive to thermogenesis premotor neurons in the rRPa, and thermogenesis is reduced.

When certain mammals enter torpor or hibernation^12-14, their brain circuits for thermoregulation switch from a normal to an inverted state in which they respond to a cold T_AMB by inhibiting thermogenesis, which leads to hypothermia and a reduction in energy expenditure. For nearly a century, the hypothalamic preoptic area (POA) has been considered the integrative center for the CNS control of body temperature and thermogenesis. More recently, the POA has become a focus for understanding the ability of mice to go into torpor^13,15-17. Although many studies have explored thermoregulatory changes in response to POA manipulations^9,13,15-26, this report is the first to characterize the responses of thermogenic thermoeffectors to cutaneous thermal stimuli following exclusion of the POA, or of a neuronal subpopulation in the ventromedial periventricular area (VMPeA), a region adjacent to the third ventricle and extending for approximately −0.4 mm caudal from bregma, that includes the anteroventral periventricular nucleus AVPe, the ventromedial preoptic nucleus (VMPO) and the most medial portion of the medial preoptic areas (MPA) of the hypothalamus. Here, we demonstrate that exclusion of the POA or inhibition of neurons in VMPeA recruits a non-canonical thermosensory reflex pathway through which skin thermal stimuli regulate metabolic thermogenesis and body temperature in an inverted manner. This alternative pathway for the skin thermoreceptor control of thermogenesis induces a torpor-like hypothermia in cold-exposed rats.

Central administration of an A1-adenosine receptor agonist induces hypothermia^27,28, however, we provided the first demonstration that this hypothermic state in rats is accompanied by physiological alterations that mimic several of those occurring during natural torpor^29,30. The hypothermia and hypometabolism that characterize this torpor-like state arise from the induction of a novel thermoregulatory control paradigm that we have called thermoregulatory inversion (TI)^29,31. In the TI state, as in natural torpor/hibernation, the CNS control of thermogenesis is inverted, such that thermogenesis is inhibited in a cold T_AMB, and stimulated in response to a warm T_AMB. Inspired by the parallels between the TI state and the control of T_CORE in natural torpor/hibernation wherein skin cooling inhibits thermogenesis and T_CORE falls^12,32, we have sought to understand how the TI state might be achieved within the overall framework of the classic neural circuit supporting normal thermoregulation¹. Our study demonstrates that inhibition of neuronal activity within VMPeA unveils an alternative thermosensory reflex pathway between the PBN and the DMH that controls thermogenesis and T_CORE in the TI state. We hypothesize that this alternative circuit represents the neural basis for the skin cooling-evoked hypothermia and hypometabolic state observed during natural torpor/hibernation, and that it could facilitate the induction of a TI state for therapeutic hypothermia^29,31,33 in species that do not have an endogenous ability to enter torpor/hibernation.

Results

Inhibition of neurons in the VMPeA subregion of the POA induces the TI state

Descending projections from the POA, including those to the DMH and rRPa, mediate the cutaneous and core temperature-evoked changes in BAT thermogenesis during normal thermoregulatory reflex responses^{3,4,6,9,10,31,34-37}. Neurons in the POA also play a critical role in the induction of torpor in mice^13,15,16. A brain transection through the medial hypothalamus (i.e., a pre-DMH transX) establishes the TI state³¹. To determine if a pre-DMH transX induced the TI state by eliminating a critical descending input from a population of POA neurons to the DMH, we examined the relationship between skin temperature (T_SKIN) and BAT sympathetic nerve activity (SNA) following inhibition of neuronal activity in a large portion of the POA (see Figure S1B) with multiple large nanoinjections (180 nl) of muscimol in medial and median POA. Inhibition of neurons in POA induced the TI state, in which skin cooling inhibited BAT SNA (see Figure S1A) in a manner exactly paralleling the effects of a pre-DMH transX³¹. Subsequent skin warming activated BAT thermogenic variables indicating the induction of a true TI state rather than a simple inhibition of neurons that drive thermogenesis^38,39.

To more precisely localize a population of neurons within the medial POA whose pharmacological inhibition would also induce the TI state, we determined that selective inhibition of neurons in the VMPeA with small (30-60 nl), bilateral nanoinjections of muscimol elicited BAT SNA and BAT thermogenic responses to skin cooling and to skin warming that were characteristic of the TI state. In naïve, anesthetized rats, the normal thermogenic response to skin cooling is a prompt increase in BAT SNA (ΔT_SKIN = −4.4 ± 0.5 °C from a baseline of 36.4 ± 0.4 °C; ΔBAT SNA: + 1053.8 ± 244.4 % of precooling control; n = 8, p = 0.0018; Figures 1A, 1Ba) and BAT thermogenesis (ΔT_BAT = +0.3 ± 0.1 °C from a baseline of 36.3 ± 0.3 °C, n = 8, p = 0.0106). These changes in BAT activation were accompanied by parallel increases in HR (ΔHR = +47.12 ± 8.7 bpm from a baseline of 352.7 ± 12.3 bpm, n = 8, p < 0.0005). Conversely, skin rewarming produced a strong inhibition of BAT SNA and thermogenesis (Figure 1A). Bilateral nanoinjections of isotonic saline (vehicle) in the VMPeA did not affect basal levels of BAT SNA (pre-saline in VMPeA: 216.6 ± 74.9 %BL; post-saline in VMPeA: 238.8 ± 122.6 %BL; n = 3, p = 0.7262, Figure 1Bb), and skin cooling produced the stereotypic increase in BAT SNA (Δ T_SKIN = −5.1 ± 2.3°C; Δ BAT SNA: +1305.3 ± 166.6 % of pre-saline control; n = 3, p = 0.0159; Figure 1Bc) characteristic of a normal cold-defense response.

Bilateral nanoinjections of muscimol into the VMPeA (Figure 1C) slightly reduced the level of BAT SNA (ΔT_SKIN = −0.05 ± 0.10 °C; Δ BAT SNA: −115.9 ± 46.57 % of pre-muscimol control; n = 8, p = 0.0208; Figures 1A, 1Bd) and BAT thermogenesis (ΔT_BAT = −0.26 ± 0.06 °C from a baseline of 36.9 ± 0.4 °C, n = 8, p = 0.0038), but did not change HR (ΔHR = +6.2 ± 4.7 bpm from a baseline of 355.7 ± 18.0 bpm, n = 8, p = 0.1121). In marked contrast to the effect of saline nanoinjections in the VMPeA (Figure 1Bc), nanoinjections of muscimol into the VMPeA (Figure 1C) induced the TI state in which skin cooling reduced BAT SNA (ΔT_SKIN = −6.5 ± 0.6 °C from a baseline of 38.7 ± 0.3 °C; Δ BAT SNA: −216.98 ± 30.15 % of precooling control; n = 8, p < 0.0001; Figures 1A, 1Be), BAT thermogenesis (ΔT_BAT = −0.34 ± 0.07 °C from a baseline of 36.9 ± 0.5°C, n = 8, p = 0.0011), and HR (ΔHR = −12.6 ± 5.4 bpm from a baseline of 358.5 ± 18.2 bpm, n = 8, p = 0.0256). Also characteristic of the TI state³¹, skin rewarming following muscimol injection in the VMPeA led to an increase in BAT SNA (Figures 1A, 1Be) that reversed the previous skin cooling-induced inhibition of BAT SNA. Bilateral nanoinjections of muscimol positioned at 1.5 mm lateral to the midline (Figure 1C, lower right panel) did not elicit TI (n=3).

Pre-DMH transX inverts the normal thermoregulatory shivering response to cold

Since skeletal muscle shivering is the most significant source of thermoregulatory thermogenesis in humans⁴⁰, it is important to determine if the thermoregulatory circuitry controlling shivering³⁶ can also be transitioned to the TI state by a pre-DMH transX. To test the hypothesis that cold-evoked shivering, like BAT thermogenesis³¹, would be inhibited in the process of eliciting a torpor/hibernation-like hypothermia, we employed our original approach to induce the TI state with a pre-DMH transX³¹ because the effects are long lasting in comparison to those of muscimol injections in VMPeA, which recover as the muscimol is metabolized.

In naïve, anesthetized rats, the normal thermoregulatory response to skin and core cooling is a prompt increase in shivering^36,41, registered as an increase in nuchal muscle EMG (nEMG) (ΔT_SKIN = −8.3 ± 1.1°C from a baseline of 35.56 ± 0.7°C; ΔnEMG: +526.6 ± 195.0 % of pre-cooling control; n = 9, p = 0.002; Figures 2A, 2Da). Conversely, skin rewarming produces a strong inhibition of nuchal muscle shivering (Figure 2A), returning the nEMG to the low levels observed in warm rats.

Figure 2. — A. During normal thermoregulation (NT) in an anesthetized, naïve rat, episodes of skin and core cooling (reductions in skin temperature (T_SKIN), dotted blue lines) produced an increase in the neck muscle EMG (nEMG), indicative of shivering. Skin rewarming reversed the increases in nEMG. Complete pre-DMH TransX to −10 mm produced an immediate increase in nEMG. Subsequent skin cooling inhibited nEMG, indicating the TI state and an inverted regulation of shivering thermogenesis by T_SKIN. With the rat in the TI state, bilateral nanoinjection of AP5/CNQX into the DMH completely reversed the warm-evoked increases in nEMG.

B. Histological section through the DMH illustrating the deposits of blue fluorescent beads indicating the location of bilateral nanoinjection sites of AP5/CNQX in DMH.

C. Schematic of the observed locations of AP5/CNQX nanoinjections into the DMH (n = 6).

D. Group data representing the levels of nEMG (%BL: % baseline) at warm or cold levels of T_SKIN. a. Skin cooling in naïve rats increased nEMG; b. in rats with a warm skin, pre-DMH TransX (schematic to the right of bar graphs) (n = 9) elicited an increase in nEMG; c. after pre-DMH TransX, nEMG was greater during skin warming than during skin cooling; d. in the TI state after pre-DMH TransX, AP5/CNQX nanoinjections into the DMH (n = 6) eliminated the warm-evoked activation of nEMG. t-test *p < 0.05; T_CORE: core temperature; T_SKIN: skin temperature. See also Figures S2, S6.

With T_CORE and T_SKIN in a warm condition and nEMG at a low, non-shivering level, a complete pre-DMH transX produced an immediate increase in nEMG and nuchal muscle shivering (T_SKIN = 35.7 ± 0.1°C; ΔnEMG: +347.1 ± 145.7% of pre-transX control; n = 6, p = 0.0156; Figures 2A, 2Db). Paralleling the pre-DMH transX-evoked activation of BAT SNA in warm conditions³¹, the pre-DMH transX-induced activation of shivering nEMG in rats with a warm T_SKIN is indicative of the TI state. The TI state for shivering was confirmed by the demonstration that skin cooling inhibited these warming-evoked shivering nEMG responses. Following a pre-DMH transX, skin cooling consistently decreased nEMG (ΔT_SKIN = −10.3 ± 1.4°C from a baseline of 38.6 ± 0.9°C; ΔnEMG: −366.65 ± 212.3% of pre-cooling nEMG; n = 5, p = 0.0313; Figures 2A, 2Dc). Subsequent skin rewarming consistently increased shivering nEMG (Figure 2A). This paradigm-shifting demonstration that the principal human thermogenic response to a cold environment⁴⁰ can be inverted could lead to a pharmacological intervention for the significant clinical problem of post-surgical shivering in a warm environment⁴².

A glutamatergic excitation of DMH neurons is necessary for the skin warming-evoked increases in BAT SNA and BAT thermogenesis, and in shivering EMG during TI

The CNS circuits for the normal cold-defensive activation of BAT and shivering thermogenesis require a glutamatergic activation of neurons in the DMH that project to thermogenic premotor neurons in the rRPa^{1,6,10,36,43,44}. In the TI state, skin warming activates DMH neurons that project to rRPa (see Figure S2). Is a glutamatergic excitation of thermogenesis-promoting neurons in the DMH also required for the skin warming-induced activation of BAT and shivering thermogenesis in the TI state (Figures 1, 2)?

Following a pre-DMH transX, the TI state was validated by demonstrating that skin warming (T_SKIN = 38.2 ± 0.6 °C) resulted in an activation of BAT SNA (BAT SNA: 878.8 ± 184.3 %BL; n = 6, Figure 3A). Subsequent bilateral nanoinjections of AP5/CNQX in the DMH (Figure 3C) eliminated the warm-evoked increase in BAT SNA (ΔBAT SNA: −836.0 ± 194.4 %BL; n = 6, p = 0.0039; Figures 3A, 3B). The abrupt fall in BAT SNA resulted in a decrease in T_BAT (−0.7± 0.1 °C, n=6, p= 0.0018) and in expired CO₂ (−0.4 ± 0.1 %, n=6, p= 0.0044; Figure 3B).

In rats in the TI state after a pre-DMH transX and with a warm skin (T_SKIN = 38.5 ± 1.1 °C) and an active shivering nEMG (243.7± 114.2 %BL; Figure 2A), bilateral nanoinjections of AP5/CNQX in the DMH (Figures 2B, 2C) reversed the warm-evoked activation of shivering nEMG (ΔnEMG: −217.0 ± 104.1 %BL; n = 6, p = 0.045; Figures 2A, 2Dd).

In the TI state after pre-DMH transX, bilateral nanoinjection of saline (vehicle) in the DMH (Figure 3F) had no effect on the skin warming-evoked activation of BAT SNA (pre-saline BAT SNA: 750.2 ± 306.1 %BL, post-saline BAT SNA: 700.9 ± 230.4 %BL; n = 4, p = 0.3260; Figures 3D, 3E). Additionally, the cold-evoked inhibition of BAT SNA characteristic of the TI state was unaffected by saline nanoinjections in the DMH (ΔT_SKIN = −11.7 ± 0.8°C from a baseline of 38.9 ± 0.5 °C; ΔBAT SNA: −513.2 ± 108.3 % of pre-cooling control; n = 4, p = 0.0089; Figures 3D, 3E).

These results indicate that increases in BAT and shivering thermogenesis in the TI state are dependent on a glutamatergic excitation of thermogenesis-promoting neurons in the DMH. Since a complete pre-DMH transX (Figure 2D) or a nanoinjection of muscimol in VMPeA (Figure 1C) induces a robust TI state, it seems unlikely that the source of the glutamatergic input to the DMH required for the skin warming-evoked activation of BAT and shivering thermogenesis in the TI state is located within the POA, as it is for normal thermoregulation ¹⁰, but rather from neurons located caudal to the pre-DMH transX.

Glutamatergic neurons in the DMH that project to the rRPa mediate the cold-evoked activation of thermogenesis in normal thermoregulation^4,45. To provide evidence that the DMH neurons that mediate the warm-evoked activation of thermogenesis in the TI state also project to rRPa, we examined the Fos expression (Fos-ir) in DMH neurons that were retrogradely-labeled with FluoroGold (FG) injected into the rRPa in warm-exposed rats after a pre-DMH transX (Warm-T rats). A significantly higher percentage of rRPa-projecting (FG-ir) neurons in the DMH were double-labeled (FGFos) in Warm-T rats than in Cold-T rats (Warm-T: 22.38 ± 4.01 % FGFos/FG vs. Cold-T: 11.34 ± 1.3 % FGFos/FG, n=4, p=0.02319, see Figure S2).

A glutamatergic excitation of PBN neurons is necessary for the skin warming-evoked increase in BAT SNA during TI

In the normal thermoregulatory state, cutaneous thermoreceptor activity influences thermogenesis via projections from tertiary thermosensory neurons in the dlPBN and the elPBN to POA neurons^3,4,7,8 that, in turn, control the activity of thermogenesis-promoting neurons in the DMH^1,46. The level of activation of thermogenesis is still modulated by T_SKIN, albeit in an inverted manner, in the TI state elicited by a brain transection between the POA and the DMH³¹ (Figure 2) that likely eliminates direct projections from POA to DMH. Based on this finding, we hypothesize that in the TI state, skin thermosensory information influences the activity of thermogenesis-promoting neurons in the DMH through a previously unrecognized, tertiary thermosensory pathway from the PBN that targets DMH neurons rather than those in the POA. To begin to understand this key aspect of the thermosensory circuit for TI, we sought to confirm that a glutamatergic excitation of PBN neurons is essential for the inverted skin thermoreceptor regulation of thermogenesis in the TI state.

In the TI state following a pre-DMH transX, skin cooling consistently decreased BAT SNA (ΔT_SKIN = −7.0 ± 1.1°C from a baseline of 37.4 ± 0.7°C; ΔBAT SNA: −411.4 ± 98.4 % of pre-cooling control; n = 5, p = 0.0007; Figures 4A, 4C) and skin rewarming consistently increased BAT SNA (ΔT_SKIN = +6.6 ± 1.3°C from a baseline of 30.4 ± 0.8°C; ΔBAT SNA: +359.1 ± 89.3% of pre-cooling control; n = 5, p = 0.0012; Figures 4A, 4C). During skin warming (T_SKIN = 40.6 ± 1.0°C, n=5) and an elevated BAT SNA (515.11 ± 130.7 %BL; n = 5), bilateral nanoinjections of AP5/CNQX into the PBN (Figure 4D) promptly decreased BAT SNA (ΔBAT SNA: −329.6 ± 112.5 %BL, n = 5, p = 0.0214; Figures 4A, 4B). The long-lasting (>1 hr) inhibition of the skin warming-evoked activation of BAT SNA resulted in a decrease in T_BAT (−0.6 ± 0.2 °C, n = 3, p = 0.0355) and in expired CO₂ (−0.2 ± 0.1 %, n = 5, p = 0.0387; Figure 4B). Thus, in the TI state, the inverted control of thermogenesis by skin thermoreceptors requires glutamatergic activation of neurons in the PBN.

Figure 4. — A. In the TI state following a complete pre-DMH TransX, episodes of skin cooling (reductions in T_SKIN, dotted blue lines) produced the typical cold-evoked inhibition of BAT SNA and reductions in T_BAT and expired CO₂ (EXP CO₂). Skin rewarming activated BAT SNA and reversed the effects of skin cooling. Bilateral nanoinjections of saline (SAL) into the PBN did not alter either the ongoing level of BAT SNA or the inhibitions of BAT SNA and reductions in T_BAT and EXP CO₂ during skin cooling. Subsequent nanoinjections of AP5/CNQX into the PBN produced a prompt and long-lasting inhibition of the warm-evoked activation of BAT SNA and reduced T_BAT and EXP CO₂.

B. Group data (n = 5) representing the time course of the reductions in BAT SNA (% BL: % baseline) and EXP CO₂ in response to nanoinjections of AP5/CNQX into the PBN. One way-ANOVA Bonferroni (data point −300 vs 600s) *p < 0.05; T_CORE: core temperature; T_BAT: BAT temperature.

C. Group data (n = 5) representing the levels of BAT SNA (% BL: % baseline) vs. T_SKIN. Following a complete pre-DMH TransX, BAT SNA was elevated during skin warming, typical of the TI state. BAT SNA remained higher during skin warming than during skin cooling after nanoinjections of SAL into PBN. t-test *p < 0.05.

**D. a.** An example of a histological section through the PBN illustrating the deposits of red fluorescent beads indicating the locations of the bilateral nanoinjection sites of SAL and of AP5/CNQX in the PBN; b. schematic of the locations of the bilateral nanoinjections of SAL and of AP5/CNQX into the PBN.

In the TI state and with the skin kept warm, bilateral nanoinjections of isotonic saline (vehicle) in the PBN had no effect on the warm-evoked activation of BAT SNA (pre-saline in PBN BAT SNA: 333.5 ± 134.4 %BL, post-saline in PBN BAT SNA: 281.33± 80.5 %BL; n = 5, p = 0.5231; Figures 4A, 4C). In the TI state, nanoinjection of saline in the PBN also had no effect on the characteristic cold-evoked inhibition of BAT SNA (ΔT_SKIN = −9.9 ± 1.8°C from a baseline of 38.7 ± 0.6°C; ΔBAT SNA: −212.2 ± 52.6 % of pre-cooling BAT SNA; n = 4, p = 0.0078; Figures 4A, 4C).

PBN neurons projecting to DMH are activated during the inverted thermogenic responses in the TI state

The skin thermoreceptor modulation of thermogenesis in the TI state requires both the descending thermogenesis-promoting pathways from the DMH to the rRPa (Figures 2, 3, also see Figure S2), as well as a glutamatergic excitation of neurons in the specific regions of the PBN (Figure 4) receiving thermosensory signals from second-order thermosensory neurons in the spinal dorsal horn. Since the TI state occurs in the likely absence of direct POA inputs to the DMH (Figures 1, 2, 3), we tested the hypothesis that PBN neurons with direct projections to the DMH are activated during skin thermoreceptor stimulation in the TI state.

Localization of DMH-projecting PBN neurons activated during skin warming in anesthetized, naïve rats and in anesthetized, pre-DMH transX rats

We compared anatomical assessments of PBN neuronal activation (Fos-ir) in 4 groups of anesthetized rats: naïve rats and pre-DMH transX rats during skin warming and during skin cooling. Our injections of the retrograde tracer, cholera toxin subunit b (CTb), in the DMH overlapped with DMH neurons retrogradely-labeled following injections of another retrograde tracer, Fluorogold (FG), in the rRPa (see Figures S3A, S3B). We observed CTb retrograde labeling of neurons in the elPBN and dlPBN (see Figure S3C). These anatomical results are consistent with the potential for PBN neurons to directly influence the activity of thermogenesis-promoting neurons in the DMH. There was no difference between the number of CTb-ir neurons in the elPBN and in the dlPBN in the 4 treatment groups (p>0.05, Fig 5E). To analyze the extent of Fos expression in dlPBN and elPBN neurons (Figure 5A) that were retrogradely labeled from CTb injections in DMH (Figure 5D), we calculated the percentage of CTb-labeled neurons in elPBN and in dlPBN that were also Fos-ir (% CTbFos/CTb; Figure 5C) in PBN sections at 4 consecutive rostro-caudal levels separated by approximately 100 μm.

Figure 5. — A. An example of the distribution of neurons double-labeled for warm-evoked Fos (red nucleus) and for CTb retrogradely transported from DMH (green cytoplasm) in the PBN of anesthetized naïve rats (Warm-N) and anesthetized pre-DMH TransX rats (Warm-T). The dotted yellow circles in dlPBN and elPBN represent the counting boxes used for the quantitative analysis reported in panel C.

B. An example of the distribution of neurons double-labeled for warm-evoked Fos (red nucleus) and retrogradely transported CTb from DMH (green cytoplasm) in the parabrachial nuclei (PBN) of anesthetized naïve rats (Cold-N) and anesthetized pre-DMH TransX rats (Cold-T). The dotted yellow circles in dlPBN and in elPBN represent the counting boxes used for the quantitative analysis reported in panel C.

C. Group data representing the percentage of retrogradely-labeled neurons (% CTbFos/CTb) in dlPBN and elPBN at the different rostrocaudal levels of PBN that also exhibited Fos in response to the four different treatments: Warm-N (n = 5), Warm-T (n = 5), Cold-N (n = 4), and Cold-T (n = 7). t-test *p < 0.05. Rostral: Bregma −8.90 mm; Intermediate-1: Bregma −9.00 mm; Intermediate-2: Bregma −9.10 mm; Caudal: Bregma −9.20 mm.

D. Schematic of the location and observed diffusion of unilateral (right) nanoinjection of the retrograde tracer, cholera toxin subunit B (CTb), into the DMH of the four treatment groups: Warm-N, Warm-T, Cold-N, and Cold-T.

E. Group data indicating the counts (#CTb) of dlPBN and elPBN neurons retrogradely-labeled from CTb injections in the DMH in the different treatment groups: Warm-N (n = 5), Warm-T (n = 5), Cold-N (n = 4), and Cold-T (n = 7). There were no differences in CTb counts among the 4 groups.

Scale bar in all images = 100 μm.

F. Experimental timeline from the initial surgery for CTb injection to the perfusion.

See also Figures S3, S4.

During skin warming in anesthetized, naive (Warm-N) rats, we observed Fos-ir in both dlPBN (total: 16.60 ± 2.06 %) and elPBN (total: 17.75 ± 3.58 %) neurons that projected to DMH (Figures 5A, 5C). In anesthetized, pre-DMH transX rats with a warm skin (Warm-T), we also found Fos-ir DMH-projecting neurons in both dlPBN (total: 16.93 ± 2.19 %) and elPBN (total: 13.89 ± 2.50 %) (Figures 5A, 5C), and at comparable levels to those observed in the Warm-N rats.

Localization of DMH-projecting PBN neurons activated during cold exposure in anesthetized, naive rats and in anesthetized, pre-DMH transX rats

Skin cooling in anesthetized naïve rats (Cold-N), a condition in which thermogenesis is strongly stimulated, activated 27 ± 0.95 % of the total elPBN neurons that project to DMH (Figures 5B, 5C). Skin cooling in anesthetized, pre-DMH transX rats (Cold-T), a condition in which thermogenesis is inhibited, activated significantly fewer DMH-projecting neurons in elPBN (14.73 ± 2.31%; n=4, p=0.003; Figs 5B, 5C) than in the Cold-N group. This reduction was most prominent at the Intermediate-1 level of the elPBN (Cold-N: 33.09 ± 3.65 % vs. Cold-T: 14.52 ± 4.27 %, n=7, p=0.0151; Figure 5C). Skin cooling in anesthetized, naive rats (Cold-N) also activated DMH-projecting neurons in dlPBN (11.79 ± 0.81 %). A similar fraction (12.98 ± 0.91 %) of the DMH-projecting neurons in dlPBN was also activated by skin cooling in anesthetized, pre-DMH transX rats (Cold-T) (Figures 5B, 5C). These data support the idea that in anesthetized, naive rats, activation of DMH-projecting neurons in both the elPBN and the dlPBN contributes to the regulation of the discharge of thermogenesis-promoting neurons in DMH during normal thermoregulatory cold defense. In addition, our finding that the activation of DMH-projecting neurons in elPBN was lower in Cold-T than in Cold-N rats would be consistent with a reduced excitation of DMH neurons from their elPBN inputs in the TI state, when skin cooling reduces thermogenesis³¹ (Figs 1, 2, 3).

PBN neurons with direct projections to the DMH are activated during warm and cold exposure in naïve, free-behaving rats

Since a thermoregulatory function of PBN neurons projecting to the DMH has not been investigated previously in rats⁴⁷, we sought to determine if such PBN neurons are also activated during skin thermoreceptor stimulation in free-behaving rats. Naïve, free-behaving rats, previously injected with CTb in the DMH (and FG in the rRPa), were exposed to either a warm or a cold T_AMB, and Fos expression was quantified in DMH-projecting PBN neurons. Since there was no difference between the number of elPBN and dlPBN CTb-retrogradely neurons in warm- or cold-exposed free-behaving rats (elPBN: 140.0 ± 12.93 vs. 175.8 ± 7.56, n= 5, p=0.06; dlPBN: 114.0 ± 10.63 vs. 146.6 ± 22.06, n= 5, p=0.2275), we expressed the double-labeled PBN neuron counts as a percentage of the number of retrogradely-labeled PBN neurons (% CTbFos/CTb) throughout the dlPBN and elPBN subdivisions of the PBN.

During warm exposure in naïve, free-behaving rats, a condition characterized by low levels of thermogenesis, we observed similar percentages of CTbFos/CTb in DMH-projecting neurons in the dlPBN (7.68 ± 1.74 %) and within elPBN (5.67 ± 0.95 %) (see Figures S4A, S4C). During cold exposure, when thermogenesis is activated in naïve rats, Fos-ir was also observed in DMH-projecting neurons within dlPBN (3.78 ± 0.93 %) and within elPBN (19.41 ± 3.64 %) (see Figures S4B, S4C). A significantly greater percentage of the dlPBN neurons projecting to DMH expressed Fos in warm-exposed rats than in cold-exposed rats (n = 5 per group; p = 0.0416; see Figure S4C). In contrast, a significantly greater percentage of the elPBN neurons projecting to the DMH expressed Fos in cold-exposed rats than in warm-exposed rats (n = 5 per group; p = 0.0032; see Figure S4C). In these same rats, we observed FG-ir neurons in the DMH that expressed Fos after cold exposure (see Figure S2A). These anatomical data indicate that neurons in the dlPBN and in the elPBN that project to the region of the DMH containing thermogenesis-promoting neurons (see Figure S3C) are activated during changes in T_AMB. This finding is consistent with the potential for these populations of PBN neurons to take part in the control of DMH thermoregulatory neurons during normal thermoregulation in naïve, free-behaving rats.

Dynorphinergic neurons in PBN project to DMH

The region of the PBN containing DMH-projecting neurons (Figures 5A, 5B, also see Figure S4A, S4B) also contains dynorphin (Dyn) neurons^7,8,48. DMH-projecting neurons in dlPBN at the intermediate-1 level are activated during skin cooling in pre-DMH transX rats (Figure 5C), a TI state in which skin cooling inhibits thermogenesis by reducing the discharge of DMH neurons. These findings, coupled with Dyn exerting an inhibitory influence on neuronal activation (through activation of κ-opioid receptors^49-51), prompted us to test the hypothesis that Dyn, potentially released from terminals of PBN neurons projecting to the DMH, plays a role in the cooling-evoked inhibition of thermogenesis characteristic of the TI state (Figures 1, 2).

We first sought to determine if any of the Dyn neurons in the PBN project to the region of the DMH containing thermogenesis-promoting neurons, and if such a neuronal population is activated during normal thermoregulation and/or in the TI state. Dyn neurons were observed in several PBN subdivisions along its entire rostro-caudal extent (Figure 6A), but the strongest transcript labeling was in dense clusters located in the dlPBN at the intermediate-1 and -2 levels (Figure 6A). All Dyn neurons in these clusters express VGluT2 transcripts (in a sample of 375 Dyn neurons in 2 sections counted bilaterally, all neurons colocalized with VGluT2). None of the Dyn neurons in PBN express VGAT transcripts (Figure 6A). Following CTb injections in the DMH and intracerebroventricular (ICV) colchicine treatment, we observed colocalization of CTb and Dyn mainly in the dense clusters of Dyn neurons in the dlPBN (Figure 6B). Thus, there is a concentration of VGluT2-expressing Dyn neurons in the dlPBN region, and many of these Dyn neurons project to the region of the DMH that contains thermogenesis-promoting neurons.

Figure 6. — A. Dynorphinergic neurons (containing pDyn transcripts; red) were observed in several PBN subdivisions along its rostro-caudal extent, but the strongest labeling was found in dense clusters located in the dlPBN at the intermediate-1 and −2 levels. All Dyn neurons in these clusters express VGluT2 transcripts (blue), but none expresses VGAT transcripts (green, n=2). Neurons expressing both pDyn and VGluT2 transcripts display pink fluorescence (n=4). Samples were processed for ISH with RNAScope.

B. A subset of DMH-projecting neurons in the dlPBN, retrogradely labeled with CTb (green), colocalize Dyn (red) in a rat (n=1) treated with colchicine to concentrate Dyn in the cell bodies. Double-labeled neurons display yellow fluorescence. Cell counts for each label are shown in parentheses.

C. Example of DMH-projecting (CTb labeling with IHC; green) Dyn (pDyn transcripts with ISH; red) neurons in the dlPBN activated (c-fos transcripts with ISH; blue) during skin warming in an anesthetized naïve rat (Warm-N, n=1), a condition in which thermogenesis is inhibited. Triple-labeled neurons (CTb-pDyn-c-fos) display light brown fluorescence. Double-labeled CTb-pDyn neurons appear yellow; double-labeled CTb-c-fos neurons appear cyan; double-labeled pDyn-c-fos neurons appear pink. Cell counts for each label are shown in parentheses.

D. Example of Dyn neurons (pDyn transcripts with ISH; blue) in the dlPBN activated (c-fos transcripts with ISH; red) in anesthetized, naïve rats exposed to skin cooling (Cold-N, n=1), or to skin warming (Warm-N, n=1), and in a pre-DMH TransX rat subjected to skin cooling (Cold-T, n=1). There are Dyn neurons in dlPBN that are activated during warm exposure in naïve rats and during cold exposure in pre-DMH TransX rats, both conditions in which thermosensory afferents drive an inhibition of thermogenesis. Cell counts for each label are shown in parentheses. Scale bar in all images = 100 μm.

See also Figure S3.

To determine whether the DMH-projecting Dyn neurons in dlPBN are active, and thus could be influencing the level of thermogenesis in normal thermoregulation or in the TI state, we performed a co-detection procedure to identify DMH-projecting Dyn neurons in PBN that were activated by cutaneous thermal stimuli in naïve and pre-DMH transX rats. We observed many DMH-projecting (CTb-ir) Dyn neurons in dlPBN that were activated (c-fos) when thermogenesis is inhibited during skin warming in anesthetized, naïve (Warm-N) rats (Figure 6C). We also observed many Dyn neurons clustered in dlPBN that were activated (c-fos) when thermogenesis is inhibited during skin cooling in pre-DMH transX (Cold-T) rats (Figure 6D). Our finding that many DMH-projecting Dyn neurons in the dlPBN are activated in Warm-N rats and in Cold-T rats, when inhibitory influences on the discharge of thermogenesis-promoting neurons in DMH predominate, is consistent with a thermogenesis-inhibiting role for these DMH-projecting Dyn neurons in the dlPBN in both normal thermoregulation and during TI. Such a role for Dyn neurons in the dlPBN is also supported by our finding that very few of them are activated in Cold-N rats, when thermogenesis is active (Figure 6D).

Dynorphin in DMH inhibits normal, cold-evoked BAT SNA and BAT thermogenesis

Having identified a population of Dyn neurons in dlPBN that project to the region of the DMH containing thermogenesis-promoting neurons and that are active when thermogenesis is inhibited, we sought to determine if Dyn in the DMH would inhibit normal, cold-evoked BAT SNA and BAT thermogenesis in naïve, anesthetized rats. Since the degradation products of exogenous Dyn by extracellular peptidases lead to non-specific activation of NMDA receptors⁵², we pretreated the DMH with the peptidase inhibitor Amastatin.

In anesthetized naïve rats with a cold skin (T_SKIN = 35.2 ± 0.4°C) and an activated BAT SNA, bilateral nanoinjections of Amastatin into the DMH (Figure 7C) did not affect BAT SNA (pre-Amastatin: 787.5 ± 33.0% BL, post-Amastatin: 816.7 ± 163.06% BL, n= 4, p= 0.8459; Figures 7A, 7B) or T_BAT (pre-Amastatin: 36.4 ± 0.7 °C, post-Amastatin: 36.6 ± 0.6 °C; n = 4, p = 0.0546; Figures 7A, 7B). Subsequent nanoinjections of Dyn in the same site promptly inhibited normal, cold-defensive BAT SNA (pre-Dyn: 613.0 ± 116.6 %BL, post-Dyn: 61.7 ± 39.4 %BL; n = 4, p = 0.0142), which caused a significant decrease in T_BAT (pre-Dyn: 36.2 ± 0.6 °C, post-Dyn: 36.8 ± 0.7 °C; n = 4, p = 0.0469; Figures 7A, 7B). Following Dyn nanoinjection in the DMH, subsequent skin cooling no longer activated BAT SNA (Figure 7A). Additionally, skin warming had no effect on the complete inhibition of BAT SNA following Dyn nanoinjection (Figure 7A), indicating that Dyn nanoinjection in the DMH does not, by itself, induce the TI state in which skin warming activates BAT SNA (cf. Figures 1, 3).

We explored whether Dyn in the DMH might contribute to the maintenance of warm-evoked inhibition of BAT SNA in normal thermoregulation. In anesthetized naïve rats with a warm skin (T_SKIN = 36.38± 0.5°C) and an inhibited BAT SNA, bilateral nanoinjection of the κ-opioid receptor antagonist, nor-BNI, into the DMH (Figure 7C), elicited a prompt increase in BAT SNA (pre-nor-BNI: 235.68 ± 220.1 %BL, post-nor-BNI: 860.61 ± 263.89 %BL; n = 3, p = 0.012, see Figure S5), and strongly potentiated the cold-evoked increase in BAT SNA (cooling naïve: 1760.1 ± 815.1 %BL, cooling post-nor-BNI: 3950.8 ± 1111.4 %BL; n = 3, p = 0.0089, see Figure S5). These data indicate that κ-opioid receptor activation, potentially by Dyn, contributes tonically to the inhibition of DMH thermogenesis-promoting neurons during warm exposure in naïve rats.

A κ-opioid receptor antagonist in the DMH prevents the cold-evoked inhibition of BAT thermogenesis during TI

Since a population of Dyn-expressing neurons in PBN projects to the DMH, and Dyn nanoinjection into the DMH inhibits normal, cold-evoked BAT SNA and reduces BAT thermogenesis, we tested the hypothesis that activation of κ-opioid receptors⁵³ in the DMH, contributes to the cold-evoked inhibition of BAT SNA in the TI state.

In the TI state following pre-DMH transX, skin cooling consistently decreased BAT SNA (ΔT_SKIN = −8.0 ± 1.5 °C from a baseline of 37.3 ± 0.3°C; ΔBAT SNA: −1139.9 ± 323.9 %BL; n = 6, p = 0.0085; Figures 7D, 7E). With a warm T_SKIN (39.0 ± 0.7 °C) and an active BAT SNA (1426.9 ± 277.0 %BL), bilateral nanoinjections of the κ-opioid receptor antagonist, nor-BNI, in the DMH (Figure 7F) prevented any subsequent cold-evoked inhibitions of BAT SNA (ΔT_SKIN = −9.3 ± 2.2 °C from a baseline of 39.0 ± 0.7 °C; ΔBAT SNA: −280.4 ± 172.6 %BL; n = 6, p = 0.0825; Figures 7D, 7E). Thus, in the TI state, nor-BNI administration into the DMH resulted in an 82.1 ± 19.2 % reduction in the cold-evoked inhibition of BAT SNA, indicating that κ-opioid receptor activation in the DMH is necessary for the skin cooling-evoked reduction in BAT thermogenesis in the TI state.

Blockade of central κ-opioid receptors reduces the hypothermic response to ICV administration of an adenosine 1A receptor agonist in free-behaving rats

Central administration of the adenosine 1A receptor (A1A-R) agonist, CHA, in free-behaving rats produces a TI state and the characteristic cold-evoked inhibition of thermogenesis that leads to a progressive hypothermia³¹. Since Dyn release in the DMH is required for the skin cooling-evoked inhibition of thermogenesis in the TI state in anesthetized rats (Figures 7D, 7E), we sought to determine if blockade of central κ-opioid receptors with ICV nor-BNI would reduce the cooling-evoked hypothermia during the TI state induced by the central administration of CHA in free-behaving rats.

One hour after reducing the T_AMB from 25°C to 15°C, free-behaving rats chronically instrumented for T_CORE recording, received an ICV (5 μl) pretreatment of either 0.9% saline vehicle or nor-BNI, followed after 10 minutes by an ICV injection of CHA (1 mM, 5μl). During the 1 h exposure to the T_AMB of 15°C prior to pretreatment, the rats maintained a normal T_CORE of 36.7 ± 0.1°C (n=3; Figure 7G), reflecting a normal thermoregulatory cold-defense response. Following saline pretreatment, administration of CHA elicited a prompt reduction in T_CORE (Figure 7G), which reached a minimum of 22.4 ± 0.2 °C (ΔT_CORE = −14.2 ± 0.2 °C from a baseline of 36.7 ± 0.2 °C, n=3) at 7 h:36 min ± 7 min following CHA injection (Figure 7G). Following pretreatment with nor-BNI, injection of CHA also elicited a rapid reduction in T_CORE (Figure 7G). However, the fall in T_CORE (ΔT_CORE = −7.2 ± 0.8 °C from a baseline of 36.7 ± 0.3 °C, n=3) following CHA administration was significantly less after pretreatment with nor-BNI than after saline pretreatment (p < 0.001, Bonferroni post-hoc test). Additionally, because the rate of decline in T_CORE was the same in both saline and nor-BNI pretreatment conditions, the minimum T_CORE of 29.5 ± 0.8 °C after CHA administration was reached at a shorter time (4 h:56 min ± 50 min, p = 0.0448) after the nor-BNI pretreatment than after the saline pretreatment. No changes in T_CORE were observed following control treatments, in which rats received an ICV (5 μl) pretreatment of either 0.9% saline vehicle or nor-BNI, followed after 10 minutes by an ICV injection of 0.9% saline vehicle (Fig. 7G). The finding that during the TI state in CHA-treated, free-behaving rats²⁹, pretreatment with nor-BNI reduced the maximum hypothermia but not the rate of decline in T_CORE suggests that central κ-opioid receptor activation, potentially by Dyn, plays a permissive role in sustaining the skin cooling-induced inhibition of thermogenesis that is a hallmark of the TI state³¹.

Discussion

In sharp contrast to the normal thermosensory reflex control of thermogenesis and its obligatory energy expenditure, during natural torpor/hibernation the CNS regulation of thermogenesis is inverted, such that thermogenesis is inhibited and T_CORE and metabolism fall in a cold environment, and thermogenesis, energy expenditure and T_CORE increase when the ambient temperature increases^12,32,54,55. The TI state is a recently discovered thermoregulatory paradigm³¹ in which the thermogenic responses to cold and warm environments are inverted compared to those during normal thermoregulation, mimicking the regulation of T_CORE seen in natural torpor. Although a subthermoneutral T_AMB (i.e., cold exposure) may not be an essential requirement for torpor entrance in all torpid species⁵⁶, it is sufficient to induce torpor in laboratory mice^13,15,16 and is an essential aspect of clinical approaches to induce therapeutic hypothermia⁵⁷. Although the potential role of TI in the natural torpor induced at a subthermoneutral T_AMB remains to be tested, we speculate that the TI state mimics the CNS regulation of thermogenesis observed in such natural torpor/hibernation, and that the neural circuitry underlying TI represents a fundamental brain pathway for the modulation of T_CORE during such natural torpor/hibernation. The TI state and the CNS circuitry underlying this thermoregulatory paradigm represent both a promising new model for understanding the neural mechanisms underlying the hypothermia and hypometabolism accompanying natural torpor/hibernation, and, importantly, a potential target for the development of pharmacological approaches for the induction of therapeutic hypothermia in non-hibernating species. Our study reveals several of the principal components of the efferent and afferent neural mechanisms underlying the TI state in rats. We demonstrate that the inverted skin thermoreceptor regulation of thermogenesis during the TI state is effective for shivering, the principal human thermogenic mechanism, as it is for BAT³¹, the principal source of facultative thermogenesis in rat and in naturally torpid/hibernating species (e.g., mouse and arctic ground squirrel). The pathways mediating the inverted thermosensory modulation of thermogenesis in the TI state includes a glutamatergic excitation of third-order thermosensory neurons in the PBN, and a glutamate receptor driven excitation of neurons in the DMH for the skin warming-evoked activation of thermogenesis. Most importantly, we describe a previously unrecognized, thermosensory pathway between the PBN and the DMH which contributes to the skin thermoreceptor control of thermogenesis in both the normal and the TI states. Of particular significance is a dynorphinergic input to DMH neurons from the dlPBN that is required for the cold-evoked inhibition of thermogenesis in the TI state.

We discovered that a GABA-A receptor mediated inhibition of neuronal activity in the VMPeA subregion of the POA induces the same transition from a normal thermoregulatory state to the TI state as we found with a pre-DMH transX³¹ or with large multiple injections of muscimol in the medial and median POA (see Figure S1). We propose that the VMPeA region contains neurons that function as a “torpor switch” for the control of thermogenesis in rats: a certain level of activity of these VMPeA neurons is required to maintain the normal thermoregulatory control of thermogenesis, but when VMPeA neuronal activity falls, thermoregulation flips to the TI state with an inverted thermosensory control of thermogenesis (see Figure S6). The existence in a non-torpid species of both torpor switch neurons and the neural circuitry for an inverted thermosensory control of thermogenesis raises the questions of the identity of the signals that normally control the activity of torpor switch neurons, and why the TI circuitry appears to be functional in torpid/hibernating mammals such as mice and arctic ground squirrels⁵⁸, but cannot be naturally activated in non-torpid mammals such as rats and humans.

Recent studies to identify ‘torpor’ neurons in mice^13,15,16 have also concluded that neurons in the POA are essential for establishing the hypothermic torpor state in mice, including those Q neurons that project to the DMH¹³ and estrogen-sensitive neurons that project to medial hypothalamic areas¹⁶. Although a population of VMPeA neurons may be among those many populations of ‘torpor’ neurons described in mice, none of the studies in mice has provided enough insight into the physiological regulation of thermogenesis during natural or artificially-induced torpor to determine if mouse torpor and the accompanying hypothermia involves the induction of a TI state⁵⁹. In particular, it is important to emphasize that the cold-induced hypothermia characteristic of the torpid state and of the TI state is not due to a simple inhibition of thermogenesis-promoting neurons (e.g., in DMH or RPa^38,39), nor is it due solely to activation of thermogenesis inhibiting inputs to these areas from the POA^13,15,16. Although such manipulations of the CNS circuits for normal thermosensory reflex control of thermogenesis would elicit a long-lasting hypothermia and hypometabolism in a cold environment, rewarming the animal would not promptly activate thermogenesis to return the animal to a normal T_CORE, but rather would be dependent on a passive heat accumulation from the warm environment to return to a normal T_CORE. Torpor/hibernation is a multifaceted behavior that puts many brain-regulated functions into an energy-conserving mode. Further studies are required to determine if inhibition of VMPeA neurons in our rat model is selective for the torpor-mimicking control of thermogenesis, or if it will elicit a more extensive response, similar to ICV CHA³¹, that is a more complete mimic of the natural torpor state. Supporting the latter possibility is our observation that induction of the TI state by inhibition of VMPeA elicited Fos expression in brain regions outside the PBN (e.g., infralimbic and prelimbic cortices), consistent with non-T_CORE-related features (e.g., cognitive function) of natural torpor/hibernation. Both BAT and shivering thermogenesis are dependent on the activation of populations of thermogenesis-promoting neurons in the DMH that project to the rRPa^36,43. We show that POA exclusion inverts the skin thermoreceptor-mediated regulation of shivering (Figure 2), just as it does for BAT thermogenesis (Figure 3). The activation of BAT and shivering thermogenesis that occurs during rewarming in the TI state requires a glutamatergic excitation of DMH neurons (Figures 2, 3), paralleling the DMH dependence of the cold-induced activation of BAT and shivering in the normal thermoregulatory state^3,43. These findings strongly support our conclusion that in both the normal thermoregulatory and the TI states, activation or inhibition of neurons in the DMH controls both BAT and shivering thermogenesis. It is also clear from these results that the principal thermoregulatory mechanism underlying the cooling-induced hypothermia in the TI state is a widespread inhibition of thermogenesis at the level of the thermogenesis-promoting neurons in the DMH (see Figures S2, S6). It will be of significant interest to determine if this is also the principal physiological basis for torpor hypothermia in mice.

In both the rat^3,4 and mouse^7,8, warm and cold thermosensory signaling, mediated by ascending glutamatergic inputs from the spinal and trigeminal dorsal horns to PBN neurons, is essential for the normal skin thermoreceptor-mediated control of thermogenesis¹. Our finding that blockade of glutamate receptors in the PBN prevented the warm-induced activation of BAT thermogenesis in the TI state is consistent with the same thermosensory inputs to PBN regulating thermogenesis in the TI state. However, our demonstration that skin thermoreceptor afferents can still regulate the level of thermogenesis from BAT³¹ and from shivering (Figure 2) after either a pre-DMH transX (Figures 2, 3, 4, 7) or multiple large muscimol injections in the medial POA (see Figure S1), indicates the existence of a neuronal substrate through which cold and warm stimuli from the skin can modulate thermogenesis in the TI state, and that these thermosensory reflex pathways are anatomically independent of the thermosensory integrative circuits between the PBN and the POA controlling thermogenesis during normal thermoregulation^1,3,36.

We provide compelling evidence that this alternative thermosensory integrative system mediating the inverted thermoreceptor control of thermogenesis in the TI state involves direct inputs from third-order thermosensory neurons in the dlPBN and the elPBN to thermogenesis-promoting neurons in the DMH. Although projections from PBN to the DMH have been described in both rat and mouse^4,47,60-63, their potential roles in normal thermoregulation or during torpor-like states remains unclear. Our anatomical data (Figures 5, 6, also see Figure S3) demonstrate the presence of connections between neurons in the rat dlPBN and elPBN and neurons in the region of the DMH that contains thermogenesis-promoting neurons that project to the rRPa. We determined, in both free-behaving and anesthetized naïve rats, that many of these DMH-projecting PBN neurons are active during normal thermoregulatory responses to skin cooling (in agreement with data in rat⁴ and mice⁴⁷) and to skin warming (Figures 5, 6, also see Figure S4), consistent with a potential role for these pathways in normal thermoregulation in rats.

We provide the first observation of a population of DMH-projecting Dyn neurons in the dlPBN region that is activated during skin warming in naïve rats (Figure 6C, D), a condition in which the inhibitory influences on the discharge of thermogenesis-promoting neurons in DMH predominate. Antagonism of κ-opioid receptors in the DMH of warm-exposed naïve rats is sufficient to activate BAT SNA and to potentiate BAT SNA responses to skin cooling, indicating that tonically activated κ-opioid receptors in the DMH have a potent inhibitory influence on thermogenesis-promoting neurons in the DMH during normal thermoregulation. These observations are consistent with Dyn neurons, including those in the dlPBN, having a significant thermogenesis-inhibiting role during normal thermoregulation.

Many Dyn neurons in the dlPBN were activated during the skin cooling-induced inhibition of thermogenesis in the TI state (Figure 6D). Blockade of the κ-opioid receptors for Dyn in the DMH markedly reduced the cold-evoked inhibition of BAT SNA in the TI state, and significantly reduced the maximum hypothermia induced in our torpor-mimicking CHA model of TI in free-behaving rats³¹. These results are consistent with Dyn acting via κ-opioid receptors in the DMH to play a necessary, permissive role in sustaining the skin cooling-induced inhibition of thermogenesis that is a hallmark of TI. However, it is important to note that activation of Dyn receptors in the DMH is not sufficient to induce the TI state. If the VGluT2-expressing Dyn neurons in dlPBN are glutamatergic, their role in inhibiting thermogenesis could be explained by their targeting of GABAergic interneurons in the DMH⁶⁴. Alternatively, on the basis of the recent demonstration that axon terminals within DMH from VGluT2-expressing neurons in the POA express VGAT⁹, a marker for GABAergic terminals, we propose that the VGluT2-expressing Dyn neurons in the dlPBN might release both Dyn and GABA from their terminals in the DMH to provide a direct inhibitory regulation of thermogenesis-promoting neurons in the DMH. Together, these results provide strong support for our discovery of a novel thermoregulatory role for the dynorphinergic pathway from the dlPBN to the DMH in the inhibitory regulation of thermogenesis, both in the normal thermoregulatory state when skin warming inhibits thermogenesis, as well as in the TI state when skin cooling inhibits thermogenesis.

We propose that the cold-evoked inhibition of thermogenesis, which is a prominent characteristic of the TI state and the physiological driver of torpor/hibernation hypothermia, employs a previously undescribed, direct dynorphinergic pathway between the dlPBN and the DMH (Figures 6B, 6C), and that Dyn activation of κ-opioid receptors in the DMH is required for the cold-evoked inhibition of thermogenesis in the TI state (Figures 7D, 7E). This novel Dyn pathway appears to act in concert with those from the POA to the DMH to contribute to the normal thermoregulatory control of thermogenesis (Figures 6, also see Figure S5). Overall, our results are consistent with a model (see Figure S6) in which the activity of a population of torpor switch neurons in VMPeA modulates the balance between the normal thermoregulatory state and the TI state by altering the relative influence of the two thermosensory output pathways from the PBN: the canonical one to the POA^1,3,36 and the newly recognized one to the DMH (Figures 5, 6, also see Figure S4). The transition from normal thermoregulation to the TI state occurs when torpor switch neurons in the VMPeA, which normally inhibit the cold-activated Dyn input from dlPBN to DMH, are themselves inhibited, thereby allowing cold-activated Dyn neurons in dlPBN to inhibit DMH neurons and reduce thermogenesis (see Figure S6).

We have demonstrated that CHA can induce a hypothermic state that appears to mimic TI and that the nucleus of the solitary tract (NTS) may play a role in this response²⁹. We here demonstrated that the cold-induced inhibition of thermogenesis following ICV administration of CHA is attenuated by the central antagonism of κ-opioid receptors (Figure 7G). This result is consistent with the potential for the neural circuit mediating the CHA-induced TI state to share some common elements with the pathway mediating the TI state following either the inhibition of VMPeA or a pre-DMH transection. Whether CHA might directly⁶⁵ or indirectly (e.g., via NTS²⁹) produce the necessary inhibition of VMPeA to trigger TI remains to be determined.

In conclusion, we demonstrate the existence, in a non-torpid/hibernating species, of previously unrecognized, secondary thermosensory pathways between the PBN and the DMH that are independent of the POA. Although these pathways may be minimally active in naïve rats, they become the predominant regulators of thermogenesis in the torpor-mimicking TI state induced by inhibition of torpor switch neurons in the VMPeA or by section of rostrally-derived inputs, possibly from POA, to the DMH. We speculate that the level of activity of torpor switch neurons in the VMPeA determines the thermoregulatory state along a spectrum from normal thermoregulation with higher VMPeA activity to a predominance of the TI state as VMPeA activity decreases. In this manner, reduced activity of such VMPeA neurons, including in non-torpid mammals, could shift the balance toward the TI state³¹ during dysregulated thermoregulation (e.g., post anesthetic shivering in which patients shiver in a warm environment⁶⁶) or in chronic metabolic dysregulation (e.g. obesity, in which cold fails to activate thermogenesis^67-69). It will be of significant interest to determine if the neural circuits described here to mediate the TI state are also the neuronal substrates underlying the inverted thermosensory control of thermogenesis that leads to the hypothermia in natural torpor/hibernation. Since this neuronal network is present in non-torpid/hibernating mammals (e.g., rats), it could represent an avenue to develop pharmacological approaches to induce clinical hypothermia for a variety of therapeutic applications.

EXPERIMENTAL MODEL

Male Sprague Dawley rats (300–400 g, Charles River Laboratories) were maintained in a standard 12 hr/12 hr, light/dark cycle (lights on at 0900) with ad libitum access to standard chow and water. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8^th Edition (National Research Council, National Academies Press, 2010) and protocols were approved by the Institutional Animal Care and Use Committee of Oregon Health and Science University.

METHOD DETAILS

BAT nerve and muscle EMG recordings

Rats were anesthetized initially with 3% isoflurane in 100% O₂ and transitioned to urethane (0.8 g/kg) and chloralose (80mg/kg) following cannulation of a femoral artery and vein. Heart rate (HR) was derived from the femoral arterial pressure (AP) signal. Rats were positioned in a stereotaxic frame with the incisor bar at −4 mm below interaural zero and a spinal clamp installed on the T10 vertebra used to maintain the spine in a rigid and elevated position (detailed in ³¹). Rats were paralyzed with D-tubocurarine (0.3 mg initial dose, 0.1 mg/h supplements) and artificially ventilated with 100% O₂ (60-70 cycles/min, tidal volume 3–3.5 ml). Thermocouples (Physitemp Instruments with Sable Systems International meter) were placed (a) on the shaved abdominal skin to measure the skin temperature (T_SKIN) beneath a water-perfused blanket wrapped around the rat’s trunk, (b) 6 cm into the rectum to measure body temperature (T_CORE), and (c) into the medial aspect of the left interscapular BAT pad to measure BAT temperature (T_BAT). T_CORE was normally maintained at ~37°C by perfusing the water blanket with warm water. As required for specific experimental protocols, T_SKIN and T_CORE were adjusted by changing the temperature of the water perfusing the thermal blanket.

Postganglionic BAT sympathetic nerve activity (SNA) was recorded from the central cut end of a small nerve bundle dissected from the ventral surface of the right interscapular BAT pad after dividing the fat pad along the midline and reflecting it laterally. BAT SNA was recorded with bipolar hook electrodes, filtered (1–300 Hz), and amplified (20,000x; Cyberamp 380, Axon Instruments). The viability and correct identification of the isolated BAT nerve were verified by significant increases in BAT SNA evoked by skin cooling.

A similar surgical preparation was used for experiments in which shivering EMG was recorded with a bipolar electrode inserted into a nuchal (neck) muscle. Nuchal EMG (nEMG) recordings were performed in rats anesthetized initially with 3% isoflurane in 100% O2, and subsequently transitioned to a continuous intravenous infusion of inactin (85 mg/ml at 0.2 ml/h). Rats were artificially ventilated but were not paralyzed.

Pre-DMH transection (pre-DMH transX)

A cranial window (~4x4 mm) was made just behind the bregma and centered on the sagittal suture. The dura mater was carefully dissected from the superior sagittal sinus and removed throughout the cranial window to allow transection of the brain without damage to the sinus or the major vessels converging on it. A transection knife (15 mm long, 2 mm wide, and 0.1 mm thick) was mounted vertically in a stereotaxic manipulator and positioned perpendicular to the sagittal sinus at −1.5 mm caudal to bregma and with the medial edge on the midline. After a slight lateral retraction of the sagittal sinus, the knife was inserted into the brain sequentially on the left and right sides of the superior sagittal sinus to a depth of approximately −10 mm to make a complete transection.

Drug nanoinjection procedures

Intraparenchymal brain nanoinjections of drugs were performed as previously described^10,29,64 via glass micropipettes, using a pressure injection system (Toohey model IIe). For repeated nanoinjections at the same site, the micropipette was retracted vertically, emptied, rinsed with saline, refilled, and repositioned at the original dorsoventral coordinate. The injection sites were marked with fluorescent polystyrene microspheres (1:10 dilution of FluoroSpheres F8797, F8801, or F8803, Invitrogen).

With the incisor bar positioned at −4 mm, bilateral nanoinjections (120 nl each) in the PBN were performed at −8.9 mm caudal to bregma, 2.2 mm from the midline, and 5.5 mm below the brain surface; and in the DMH at −3.2 mm caudal to bregma, 0.4 mm from the midline, and 7.8 mm below the brain surface. Bilateral nanoinjections (120-180 nl each) were performed in the medial preoptic area (MPA; −0.4 mm caudal to bregma, ±0.4 mm from the midline, and −7.5 mm below the brain surface), and in the median preoptic area (MnPO; at bregma on the midline, −6.5 mm below the brain surface). Bilateral nanoinjections (30-60 nl each) were performed in the VMPeA area at 0.2 mm caudal to bregma, 0.2 mm from the midline, and 8 mm below the brain surface. In a few rats (e.g., Figure 1C, lower right panel), anatomical control nanoinjections of muscimol were made at 0.2 mm caudal to bregma, 1.5 mm from the midline, and 8 mm below the brain surface several hours after the TI-producing effect of muscimol nanoinjections in VMPeA had disappeared and normal thermoregulatory responses had returned.

Following experimental procedures, rats were perfused transcardially with isotonic saline, followed by 4% paraformaldehyde (PFA) in 10 mM sodium phosphate buffered saline (PBS; pH 7.4). The brains were removed, postfixed in 4% PFA (2 h), equilibrated overnight in 30% sucrose, and sectioned (60 μm coronal sections) to localize the fluorescent spots indicating the approximate centers of the injection sites. The coordinates used for the brain intraparenchymal injections were adapted from a rat brain atlas ⁷⁰ and from our previous studies involving these brain regions^1,10,31.

Drugs

The A1 adenosine receptor (A1AR) agonist, N6-cyclohexyladenosine (CHA, 1mM, Sigma Aldrich), which also binds to peripheral A3AR^71,72 , the NMDA receptor antagonist, (2R)-amino-5-phosphonopentanoate (AP5, 5 mM, Tocris), the AMPA/kainate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt hydrate (CNQX, 5 mM, Tocris), the peptidase inhibitor, (2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl-Val-Val-Asp hydrochloride hydrate (Amastatin, 1 mM, Sigma Aldrich), Dynorphin A (100 μM, Tocris), and the κ-opioid receptor antagonist, nor-Binaltorphimine dihydrochloride (nor-BNI, 27 μM intraparenchymal or 1.6 mM ICV, Tocris), were dissolved in isotonic saline.

Experiments in free-behaving rats

Central administration of the A1AR agonist, CHA, produces the TI state, featuring a dramatic fall in T_CORE due to an inhibition of thermogenesis in a cold T_AMB²⁹. Rats were anesthetized with 2% isoflurane in 100% O₂ and instrumented for chronic recording of physiological variables as previously described ²⁹. Rats were implanted with an intraperitoneal implantable temperature probe (Anipill^®) for recording of T_CORE. A guide cannula (C315G-26GA, PlasticsOne) was stereotaxically positioned in the lateral ventricle for intracerebroventricular (ICV) injection of drugs. The cannula was secured to the skull with screws and dental acrylic. Following the surgical procedure, rats were treated with buprenorphine (0.1 mg/kg), penicillin G (40,000 units/kg) and hydrated with isotonic saline (5 ml, subcutaneous). Each rat recovered for 7 days in a temperature-controlled recording chamber at an ambient temperature (T_AMB) of 25°C and received daily meloxicam (1 mg/kg orally) for the first 3 days to reduce post-surgical inflammation. For ICV injections of CHA and nor-BNI, 5 μl of drug solution were injected over 2 minutes through an internal cannula connected to a 25 μl Hamilton syringe. Rats were briefly removed from the recording chamber, the ICV injection procedure was performed in less than 5 minutes, and the rats were immediately returned to the recording chamber for continued data acquisition.

Neuroanatomy

For anatomical tracing experiments, adult male Sprague Dawley rats (240–400 g) were anesthetized with 2-3% isoflurane in 100% O2, and stereotaxically injected with cholera toxin subunit b (CTb) conjugated with Alexa-488 (1 mg/ml, 120 nl) into the right DMH (bregma: 3.2 mm caudal, 0.4 mm lateral, 7.8 mm ventral to the brain surface; incisor bar at −4 mm), and FluoroGold (FG, 2%, 30 nl) into the rRPa (relative to lambda: 3.0 mm caudal, 0.0 mm lateral, 9.2 mm ventral to the brain surface; incisor bar at −4 mm). Rats were pretreated with intramuscular injections of an antibiotic (40,000 units/kg penicillin G) and an analgesic (1 mg/kg meloxicam), and subcutaneous injection of isotonic saline (3 ml). One week after tracer injections, free-behaving rats were exposed to a cold ambient (T_AMB: 10 °C) or to a warm ambient (T_AMB: 30 °C) for 2 h to elicit Fos expression as an indicator of neuronal activation. To maximize Fos expression, rats were maintained for 24 h at a T_AMB of 30 °C prior to the 2 h cold exposure or maintained for 24 h at a T_AMB of 10 °C prior to the 2 h warm exposure.

We employed retrograde transport, combined with Fos expression, to identify DMH-projecting neurons in PBN that are activated following induction of TI in anesthetized rats. Seven days prior to the terminal experiment, rats were injected with non-conjugated CTb into the right DMH as detailed above, with some rats also receiving an injection of FG into the rRPa to identify the DMH region containing thermogenesis-promoting neurons ^5,64. Following a 7-day recovery period for retrograde transport of CTb, rats were anesthetized with 2-3% isoflurane, placed in the stereotaxic frame, and prepared for pre-DMH transX surgery. Four groups were studied: (a) cold naïve (Cold-N) rats were maintained with a cold skin (T_SKIN < 35 °C) for 1 h of baseline recordings with a stable T_SKIN and T_CORE, after which they received a sham brain transection surgery (transection knife lowered into the cortex) and were then maintained with a cold skin for 2 h; (b) warm naïve (Warm-N) rats were treated similarly, but with a skin warming (T_SKIN > 35 °C); (c) Cold-T rats were maintained with a cold skin for 1 h of baseline recording and then received a complete pre-DMH transX, followed by 2 h with a cold skin; (d) Warm-T rats were treated similarly, but with skin warming throughout the experiment. Following the sham or complete transX, rats were anesthetized with pentobarbital (80 mg/kg i.p.) and transcardially perfused with saline followed by 4% PFA. The brains were removed and postfixed in 4% PFA for 12 h and equilibrated overnight in 30% sucrose in PBS. Serial coronal sections (20 μm and 40 μm) were cut RNAse free in a microtome, collected sequentially in 6 sets (4 x 40 um, 2 x 20 um), and stored in cryoprotectant at −20 °C.

An immunofluorescence procedure was performed to label DMH-projecting PBN neurons and rRPa-projecting DMH neurons that express Fos in response to cold or warm exposure (from free-behaving and from anesthetized rats). Sections (40 μm) containing the PBN or the DMH were washed in PBS, blocked for 1 h in PBS with 0.3% Triton-X 100 (PBST) containing 3% normal donkey serum, and incubated overnight at room temperature in PBST containing the primary antibodies for Fos (rabbit anti-c-Fos, 1:2K; Encor, cat#: RPCA-c-Fos) and CTb (goat anti-CTb, 1:5K; Calbiochem, cat#: 227040). After several washes in PBS, sections were incubated for 2 h in PBST containing the secondary species-specific antibodies Alexa Fluor-594 donkey anti-rabbit IgG (1:500) and Alexa Fluor-488 donkey anti-goat IgG (1:250), both from Jackson Immuno Research. After incubation, sections were washed in PBS, mounted on Superfrost Plus slides, air-dried and coverslipped. DMH-projecting neurons in the PBN activated by cold or warm exposure displayed green cytoplasmic (CTb) and red nuclear (c-Fos) fluorescence. rRPa-projecting neurons in DMH (from rats injected with FG into the rRPa) displayed cytoplasmic FG and red nuclei when activated.

To identify the phenotypes of PBN neurons, mRNA transcripts for pro-Dynorphin (pDyn, Advanced Cell Diagnostics, Rn-Pdyn #417441), vesicular glutamate transporter 2 (VGluT2, Advanced Cell Diagnostics, Rn-Slc17a6-C2, #317011-C2), and vesicular GABA transporter (VGAT, Advanced Cell Diagnostics, Rn-Slc32a1-C3, #424541-C3) were detected by in situ hybridization (ISH) using the RNAScope Multiplex Fluorescent v2 assay (Advanced Cell Diagnostics) in 20 μm-sections containing the PBN, following manufacturer instructions. mRNA transcripts were labeled with Opal fluorophores (Akoya Biosciences): Opal-520 (1:500, green) for VGAT, Opal-570 (1:2K, red) for pDyn, and Opal-690 (1:500, far-red; assigned blue color in captured images) for VGluT2. Slides were coverslipped with anti-fade mounting medium (Pro-Long Gold, Invitrogen).

To characterize DMH-projecting (CTb-labeled) PBN neurons that express c-fos in response to cold or warm exposure in the 4 groups of isoflurane-anesthetized rats, we used the RNA-Protein Co-detection ancillary kit (Advanced Cell Diagnostics). c-fos and pDyn mRNA transcripts were detected by ISH using the RNAScope procedure, whereas CTb was labeled with immunofluorescence. Sections (20 μm) containing the PBN were first incubated in the primary goat anti-CTb antibody (1:500) overnight at 4°C, then washed in PBST and subjected to the RNAscope Multiplex Fluorescent v2 assay following manufacturer instructions (with minor modifications). mRNA transcripts were labeled with Opal fluorophores (Opal-570, red) and (Opal-690, far-red assigned blue color). After RNAScope procedure, slides were washed in PBST and incubated for 2 h in the species-specific secondary antibody for CTb (Alexa-488 donkey anti-goat, 1:200). Slides were washed in PBST and coverslipped with anti-fade mounting medium. CTb-labeled neurons displayed cytoplasmic green fluorescence, whereas c-fos and pDyn mRNA transcripts were labeled as red and far-red (blue) punctate, respectively. In some cases, the colors were reversed for c-fos and pDyn transcripts.

All slides, processed for immunohistochemistry (IHC) and ISH, were visualized at an Olympus BX-51 fluorescence microscope, and images were captured using Simple PCI software (C-Imaging Systems) and used to perform cell count. Brightness and contrast were adjusted using Adobe Photoshop.

The CTb antigenicity is severely impaired by the protease step of the ISH procedure, causing a substantial decrease in CTb labeling. In addition, the pDyn and c-fos transcript signals are very strong; therefore, the number of triple-labeled neurons (CTb-pDyn-c-fos) was significantly underestimated with this co-detection procedure.

To further visualize Dyn-containing neurons in the PBN, a group of rats (n=3) received, after 7 days recovery from previous injection of CTb in DMH, an ICV injection of colchicine, which disrupts axonal transport and concentrates the neuropeptide in the soma. Rats were injected, under general anesthesia, with 10 μl of colchicine (25 mM in 10% DMSO saline) ICV. Rats were allowed to survive for 24-36 h after colchicine injection and were perfused with PFA. Brains were removed and processed for IHC as described above, using the primary antibodies for CTb and Dyn (rabbit anti-Dyn A, 1:2K; Peninsula, cat #: T-4268).

Data acquisition

Physiological variables were digitized (Micro 1401 MKII; Cambridge Electronic Design) at the following rates: BAT SNA (1 kHz), T_BAT (5 Hz), T_CORE (5 Hz), T_SKIN (5 Hz), T_PAW (5 Hz), expired CO₂ (200 Hz), AP (200 Hz), EKG (10–300 Hz, 1 kHz), EMG (10–300 Hz, 5 kHz) and recorded into a computer hard drive for subsequent analysis (Spike 2, CED). Continuous measures (4 s bins) of BAT SNA and nEMG amplitudes were calculated as the root mean square (rms, square root of the total power in the 0.1 to 20 Hz band for BAT SNA, and in the 0.1–500 Hz band for EMG) value of sequential 4-s segments of the BAT SNA and nEMG signals ^31,36.

QUANTIFICATION AND STATISTICAL ANALYSIS

Data analysis

For analysis of the physiological variables, the data were averaged into 30s bins, and group data were reported as mean ± standard error of the mean (SEM). To account for slight differences in BAT SNA and nEMG recording characteristics (e.g., tissue-electrode contact, amplifier noise, etc.) among experiments, values for BAT SNA and nEMG throughout each experiment were expressed as ‘percent baseline’ (%BL), where baseline values for BAT SNA and for nEMG were the low levels recorded under warm T_SKIN and warm T_CORE conditions when BAT SNA and nEMG are at their minimum levels in naïve rats. Treatment effects on BAT SNA or nEMG are quantified as the difference between pre-treatment (control) and post-treatment levels of BAT SNA and in nEMG.

For analysis of anatomical data, neurons expressing Fos, CTb, and CTbFos were counted in the PBN in the side ipsilateral to the CTb injection in DMH. Counting was performed in the dlPBN and elPBN in 4 sections (separated 100 μm) that were labeled as “rostral” (Bregma: −8.90 mm), “intermediate-1” (Bregma: −9.00 mm), “intermediate-2” (Bregma: −9.10 mm), and “caudal” (Bregma: −9.20 mm), based on the Paxinos rat brain atlas ⁷⁰. Counts at each rostro-caudal level or total counts (sum of counts from the 4 levels) were used in different analysis. The counts of double-labeled (CTbFos) neurons were normalized to the number of CTb-labeled neurons (% CTbFos/CTb) to counteract variability among CTb injections.

Neurons expressing Fos, FG, and FGFos were counted in one DMH hemi-section, located contralateral to the CTb injection in DMH, at the level where the main FG-ir cluster is located (i.e., neurons projecting to the rRPa). The counts of double-labeled (FGFos) neurons were normalized to the number of FG-labeled neurons (% FGFos/FG) to counteract variability among FG injections. Data are reported as mean ± SEM.

Statistics

All statistics were performed using Prism software (version 10, GraphPad Software Inc.). Paired one- or two-tailed t-tests were used to assess the effect pre vs. post skin cooling, by comparing one data point taken 30s before and at the nadir of the skin cooling for each analyzed variable. One-way ANOVA followed by post-hoc Bonferroni correction was used to assess the effect pre vs. post injections, by comparing one data point taken 300s before and 600s after the injection. Two-way ANOVA followed by post-hoc Bonferroni correction was used for group treatments statistical comparisons. Tests used are described in figure legends. Statistical results with p < 0.05 were considered significant.

Supplementary Material

NIHMS2035266-supplement-1.pdf^{(962.5KB, pdf)}

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Goat anti-Cholera Toxin b	Calbiochem (now Millipore)	Cat #: 227040; Lot #: 39427-49 RRID:AB_211712
Rabbit anti-c-Fos	Encor	Cat #: RPCA-c-Fos-AP; Lot #: 1732-041-422 RRID:AB_2572236
Alexa Fluor-594 donkey anti-rabbit IgG	Jackson Immuno Research	711-585-152; Lot #: 163459
Alexa Fluor-488 donkey anti-goat IgG	Jackson Immuno Research	705-545-147; Lot #: 153047
Rabbit anti-Dynorphin A	Peninsula	Cat #: T-4268; Lot #: A11121 RRID:AB_518294
Bacterial and virus strains
N/A
Biological samples
N/A
Chemicals, peptides, and recombinant proteins
n6-cyclohexyladenosine (CHA)	Sigma Aldrich	N/A
(2R)-amino-5-phosphonopentanoate (AP5)	Tocris	Cat. No. 3693
6-cyano-7-nitroquinoxaline-2,3-dione disodium salt hydrate (CNQX)	Tocris	Cat. No. 1045
(2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl-Val-Val-Asp hydrochloride hydrate (Amastatin)	Sigma Aldrich	A1276
nor-Binaltorphimine dihydrochloride (nor-BNI).	Tocris	N/A
Dynorphin A (100 μM)	Tocris	Cat. No. 3195
FluoroGold	Fluorochrome	N/A
Critical commercial assays
RNAScope Multiplex Fluorescent Detection Reagents V2	Advanced Cell Diagnostics (ACD)	Cat #: 323110 Lot #: 2016014
RNAScope probe for rat pro-Dynorphin	ACD	Rn-Pdyn (417441)
RNAScope probe for rat c-Fos	ACD	Rn-Fos-C3 (403591-C3)
RNAScope probe for rat VGluT2	ACD	Rn-Slc17a6-C2 (317011-C2)
RNAScope probe for rat VGAT	ACD	Rn-Slc32a1-C3 (424541-C3)
Deposited data
N/A
Experimental models: Cell lines
N/A
Experimental models: Organisms/strains
Male Sprague Dawley rats	Charles River Laboratories	001
Oligonucleotides
N/A
Recombinant DNA
N/A
Software and algorithms
Simple PCI software	C-Imaging Systems	N/A
Prism software (version 10)	GraphPad Software Inc.	N/A
Other
N/A

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Highlights:

Ventromedial periventricular area controls thermoregulatory inversion (TI)
Dynorphinergic pathways are required for TI inhibition of thermogenesis
The neural circuitry mediating TI may mediate torpor hypothermia
TI could be a novel approach to managing therapeutic hypothermia

Acknowledgments

We thank Rubing Xing for excellent histological assistance. This study was supported by grants from the US National Institutes of Health: NS099234, NS091066, OD010996.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of interests

The authors declare no competing interests.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Domenico Tupone (tupone@ohsu.edu)

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

All data reported in this paper will be shared by the lead contact upon reasonable request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available upon request.

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PERMALINK

Inhibition of the hypothalamic ventromedial periventricular area activates a dynorphin pathway-dependent thermoregulatory inversion in rats

Shaun F Morrison

Georgina Cano

Shelby L Hernan

Pierfrancesco Chiavetta

Domenico Tupone

Summary

Graphical Abstract

eTOC Blurb

Introduction

Results

Inhibition of neurons in the VMPeA subregion of the POA induces the TI state

Figure 1. Inhibition of neurons in the ventromedial periventricular area (VMPeA) region induces thermoregulatory inversion (TI) of brown adipose tissue (BAT) thermogenesis.

Pre-DMH transX inverts the normal thermoregulatory shivering response to cold

Figure 2. Pre-DMH TransX induces thermoregulatory inversion (TI) of shivering EMGs.

A glutamatergic excitation of DMH neurons is necessary for the skin warming-evoked increases in BAT SNA and BAT thermogenesis, and in shivering EMG during TI

Figure 3. Following pre-DMH TransX, bilateral nanoinjection of AP5/CNQX in the DMH blocks the warm-evoked increase in BAT SNA.

A glutamatergic excitation of PBN neurons is necessary for the skin warming-evoked increase in BAT SNA during TI

Figure 4. Bilateral nanoinjections of AP5/CNQX in the parabrachial nuclei (PBN) blocked the warm-evoked BAT SNA characteristic of TI.

PBN neurons projecting to DMH are activated during the inverted thermogenic responses in the TI state

Localization of DMH-projecting PBN neurons activated during skin warming in anesthetized, naïve rats and in anesthetized, pre-DMH transX rats

Figure 5. Effect of warm and cold exposure on the activation of DMH-projecting neurons in the PBN in anesthetized naïve and pre-DMH TransXed rats.

Localization of DMH-projecting PBN neurons activated during cold exposure in anesthetized, naive rats and in anesthetized, pre-DMH transX rats

PBN neurons with direct projections to the DMH are activated during warm and cold exposure in naïve, free-behaving rats

Dynorphinergic neurons in PBN project to DMH

Figure 6. VGluT2-expressing, dynorphinergic neurons in dlPBN project to DMH and are activated during cold exposure in the TI state.

Dynorphin in DMH inhibits normal, cold-evoked BAT SNA and BAT thermogenesis

Figure 7. Activation of κ-opioid receptors is necessary for the cold-evoked inhibition of BAT SNA in the TI and torpor-like state.

A κ-opioid receptor antagonist in the DMH prevents the cold-evoked inhibition of BAT thermogenesis during TI

Blockade of central κ-opioid receptors reduces the hypothermic response to ICV administration of an adenosine 1A receptor agonist in free-behaving rats

Discussion

EXPERIMENTAL MODEL

METHOD DETAILS

BAT nerve and muscle EMG recordings

Pre-DMH transection (pre-DMH transX)

Drug nanoinjection procedures

Drugs

Experiments in free-behaving rats

Neuroanatomy

Data acquisition

QUANTIFICATION AND STATISTICAL ANALYSIS

Data analysis

Statistics

Supplementary Material

Highlights:

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

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