Inhibition of 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 responses3, 4, 13, 15, 16, 20–23. Neurons in the POA play a critical role in the induction of torpor in mice8, 24, 25. Transection of pathways between the POA and the DMH (pre-DMH transX) establishes the novel thermoregulatory state of TI, in which the normal BAT thermogenic responses to skin cooling and skin warming are inverted13. To test the hypothesis that POA neuronal activity is required for the induction of the TI state, we determined the BAT sympathetic nerve activity (SNA) and thermogenic responses to skin cooling and to skin warming after injecting muscimol into the POA to inhibit local neurons.
In naïve, anesthetized rats, the normal thermoregulatory response to skin cooling is a prompt increase in BAT SNA (ΔTSKIN = -5.6 ± 0.5°C from a baseline of 36.7 ± 0.3°C; ΔBAT SNA: + 983.9 ± 152.1% of precooling control; n = 10, p = 0.0001; Figs. 1A, 1Ba). Conversely, skin warming produces a strong inhibition of BAT SNA and thermogenesis (Fig. 1A). Bilateral nanoinjections of isotonic saline (vehicle) in the POA did not affect basal levels of BAT SNA (pre-saline in POA: 216.6 ± 74.9%BL; post-saline in POA: 238.8 ± 122.6%BL; n = 3, p = 0.7262, Fig. 1Bb), and skin cooling produced the stereotypic increase in BAT SNA (Δ TSKIN = -5.1 ± 2.3°C; Δ BAT SNA: +1305.3 ± 166.6% of pre-saline control; n = 3, p = 0.0159; Fig. 1Bc) characteristic of a normal cold-defense response.
Bilateral nanoinjections of muscimol into the POA (Fig. 1C) did not alter the level of BAT SNA (ΔTSKIN = -0.1 ± 0.2°C; Δ BAT SNA: +0.1 ± 83.7% of pre-muscimol control; n = 10, p = 0.4996; Figs. 1A, 1Bd). In marked contrast to saline nanoinjections in the POA (Fig. 1Bc), nanoinjections of muscimol into the POA induced the TI state in which skin cooling reduced BAT SNA (ΔTSKIN = -8.9 ± 0.8°C from a baseline of 39.4 ± 0.6°C; Δ BAT SNA: 438.08 ± 103.0% of precooling control; n = 10, p = 0.0011; Figs. 1A, 1Be). Also characteristic of the TI state13, skin rewarming following muscimol injection in the POA increased BAT SNA and reversed the cooling-induced inhibition of BAT SNA (Figs. 1A, 1Be).
Pre-dmh Transx Inverts The Normal Thermoregulatory Shivering Response
Since skeletal muscle shivering is the most significant source of thermoregulatory thermogenesis in humans26, it is important to determine if the thermoregulatory circuitry controlling shivering22 can also be manipulated to transition to the TI state in which cooling would inhibit shivering and allow TCORE to fall as it does in torpor/hibernation.
Partial pre-DMH transX eliminates cold-evoked shivering
In naïve, anesthetized rats, the normal thermoregulatory response to skin and core cooling is a prompt increase in shivering, registered as an increase in nuchal muscle EMG (nEMG) (ΔTSKIN = -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; Figs. 2A, 2Da). Conversely, skin rewarming produces a strong inhibition of nuchal muscle shivering (Fig. 2A), returning the nEMG to the low levels observed in warm rats.
A pre-DMH transX to -9 mm from the dorsal surface of the brain did not produce any change in nEMG in warm rats (TSKIN = 36.4 ± 0.2°C; ΔnEMG: -99.9 ± 88.4% of pre-transX control; n = 5, p = 0.5; Figs. 2A, 2Db). However, as with BAT SNA13, this partial pre-DMH transX completely prevented the increase in shivering nEMG in response to skin cooling (ΔTSKIN = -10.8 ± 1.2°C from a baseline of 36.7 ± 0.5°C; ΔnEMG: +57.1 ± 17.8% of pre-cooling control; n = 5, p = 0.125; Figs. 2A, 2Dc).
Complete pre-DMH transX inverts the thermoregulatory shivering response
With TCORE and TSKIN in a warm condition and nEMG at a low, non-shivering level, a complete pre-DMH transX to -10 mm from the dorsal brain surface produced an immediate and remarkable increase in nEMG and nuchal muscle shivering (TSKIN = 35.7 ± 0.1°C; ΔnEMG: +347.1 ± 145.7% of pre-transX control; n = 6, p = 0.0156; Figs. 2A, 2Dd). Paralleling the pre-DMH transX-evoked activation of BAT SNA in warm conditions13, the pre-DMH transX-induced activation of shivering nEMG in rats with a warm TSKIN 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 (ΔTSKIN = -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; Figs. 2A, 2De). Subsequent skin rewarming consistently increased shivering nEMG (Fig. 2A).
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 rRPa1, 6, 16, 22, 27. In the TI state, skin warming activates DMH neurons that project to rRPa (Extended Data Fig. 2). 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 (Figs. 1, 2)?
Following a pre-DMH transX, the TI state was validated by demonstrating that skin warming (TSKIN = 38.2 ± 0.6°C) resulted in an activation of BAT SNA (BAT SNA: 878.8 ± 184.3%BL; Fig. 3A). Subsequent bilateral nanoinjections of AP5/CNQX in the DMH (Fig. 3C) eliminated the warm-evoked increase in BAT SNA (ΔBAT SNA: -836.0 ± 194.4%BL; n = 6, p = 0.0039; Figs. 3A, 3B). The abrupt fall in BAT SNA resulted in a decrease in TBAT (-0.7 ± 0.1°C, n = 6, p = 0.0018) and in expired CO2 (-0.4 ± 0.1%, n = 6, p = 0.0044; Fig. 3B).
In rats in the TI state after a pre-DMH transX and with a warm skin (TSKIN = 38.5 ± 1.1°C) and an actively shivering nEMG (243.7 ± 114.2%BL; Fig. 2A), bilateral nanoinjections of AP5/CNQX in the DMH (Figs. 2B, 2C) reversed the warm-evoked activation of shivering nEMG (ΔnEMG: -217.0 ± 104.1%BL; n = 6, p = 0.0313; Figs. 2A, 2Df).
In the TI state after pre-DMH transX, bilateral nanoinjection of saline (vehicle) in the DMH (Fig. 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; Figs. 3D, 3E). Additionally, the cold-evoked inhibition of BAT SNA characteristic of the TI state was unaffected by saline nanoinjections in the DMH (ΔTSKIN = -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; Figs. 3D, 3E).
These results indicate that in the TI state increases in BAT and shivering thermogenesis are dependent on a glutamatergic excitation of thermogenesis-promoting neurons in the DMH. Since a complete pre-DMH transX (Fig. 2D) or a nanoinjection of muscimol in POA (Fig. 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 16, but rather from neurons located caudal to the pre-DMH transX.
To provide evidence that in the TI state skin warming activates DMH neurons that 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, Extended Data Fig. 2).
A glutamatergic excitation of PBN neurons is necessary for the skin warming-evoked increase in BAT SNA during TI
The central afferent pathways from cutaneous thermoreceptors, which drive a glutamatergic excitation of neurons in two subnuclei of the PBN (dlPBN and elPBN3, 4, 14), comprise the sensory components for the normal thermoregulatory reflex control of thermogenesis. Is a glutamatergic excitation of PBN neurons also 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 (ΔTSKIN = -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; Figs. 4A, 4C) and skin rewarming consistently increased BAT SNA (ΔTSKIN = + 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; Figs. 4A, 4C). During skin warming (TSKIN = 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 (Fig. 4D) promptly decreased BAT SNA (ΔBAT SNA: -329.6 ± 112.5%BL, n = 5, p = 0.0313; Figs. 4A, 4B). The long-lasting (> 1 hr) inhibition of the skin warming-evoked activation of BAT SNA resulted in a decrease in TBAT (-0.6 ± 0.2°C, n = 3, p = 0.0355) and in expired CO2 (-0.2 ± 0.1%, n = 5, p = 0.0387; Fig. 4B). Thus, in the TI state, the inverted control of thermogenesis by skin thermoreceptors requires a glutamatergic activation of neurons in 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; Figs. 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 (ΔTSKIN = -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 = 0.0089; Figs. 4A, 4C).
PBN neurons projecting to DMH are activated during the inverted thermoregulatory thermogenic responses in the TI state
The skin thermoreceptor-mediated modulation of thermogenesis in the TI state requires both the descending thermogenesis-promoting pathways from DMH to the rRPa (Figs. 2,3; Extended Data Fig. 2), as well as an ionotropic glutamate receptor-mediated excitation of neurons in the specific regions of the PBN (Fig. 4) receiving thermosensory signals from second-order thermosensory neurons in the spinal dorsal horn. Since the inverted regulation of thermogenesis in the TI state occurs in the absence of direct POA inputs to the DMH (Figs. 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.
DMH-projecting neurons in the PBN are 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 (Extended Data Figs. 1A, 1B), and resulted in CTb retrograde labeling of neurons in the elPBN and dlPBN (Extended Data Fig. 1C). This basic anatomical result is 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 (Fig. 5A) that were retrogradely labeled from CTb injections in DMH (Fig. 5D), we calculated the percent of CTb-labeled neurons in elPBN and in dlPBN that were also Fos-ir (% CTbFos/CTb; Fig. 5C) in PBN sections at 4 consecutive rostro-caudal levels separated by approximately 100 µm.
During skin warming in anesthetized, naive rats (Warm-N), we observed Fos-ir in both dlPBN (total: 16.60 ± 2.06%) and elPBN (total: 17.75 ± 3.58%) neurons that projected to DMH (Figs. 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%) (Figs. 5A, 5C), and at comparable levels to those observed in the Warm-N rats.
Pattern of activation of DMH-projecting neurons in the PBN in anesthetized, naive rats and in anesthetized, pre-DMH transX rats after cold exposure
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 (Figs. 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 = 5, 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 = 5, p = 0.0151; Fig. 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) (Figs. 5B, 5C). These data support the idea that in anesthetized, naive rats, activation of DMH-projecting neurons in elPBN and in 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 thermogenesis (Figs. 1, 2, 3).
PBN neurons with direct projections to the DMH are activated during warm and cold exposure in naïve, free-behaving rats
We sought to establish that PBN neurons with projections to the DMH were also activated during skin thermoreceptor stimulation in free-behaving rats. One week following CTb injections in the DMH, these naïve, free-behaving rats were exposed to either a warm or a cold TAMB, 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 in which we expect 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%) (Figs. 6A, 6C). During cold exposure, when thermogenesis should be activated, Fos-ir was also observed in DMH-projecting neurons within dlPBN (3.78 ± 0.93%) and within elPBN (19.41 ± 3.64%) (Figs. 6B, 6C). Of the dlPBN neurons that projected to DMH, a significantly greater percentage expressed Fos-ir in warm-exposed rats than in cold-exposed rats (n = 5 per group; p = 0.0416; Fig. 6C). In contrast, for elPBN neurons that projected to the DMH, a significantly greater percentage expressed Fos-ir in cold-exposed rats than in warm-exposed rats (n = 5 per group; p = 0.0032; Fig. 6C). In these same rats, we observed FG-ir neurons in the DMH that expressed Fos-ir after cold exposure (Extended Data Fig. 2A). These anatomical data indicate that neurons in both the dlPBN and the elPBN project to the region of the DMH containing thermogenesis-promoting neurons (Fig. S1C). These populations of PBN neurons 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 (Figs. 5A, 6A, 6B) also contains Dynorphin (Dyn) neurons14, 28. The intermediate-1 level subpopulation of DMH-projecting neurons in dlPBN is activated during skin cooling in pre-DMH transX rats (Fig. 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 receptors29–31), prompted us to test the hypothesis that Dyn, potentially released from terminals of the PBN neurons projecting to the DMH, plays a role in the cooling-evoked inhibition of thermogenesis characteristic of the TI state (Figs. 1, 2). Initially, we 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.
To identify Dyn neurons in the PBN and whether they express vesicular glutamate transporter 2 (VGluT2) or vesicular GABA transporter (VGAT), we performed in situ hybridization (ISH) with RNAScope on sequential brain sections containing the PBN. Dyn neurons were observed in several PBN subdivisions along its entire rostro-caudal extent (Fig. 7A), but the strongest transcript labeling was in dense clusters located in the dlPBN at the intermediate-1 and − 2 levels (Fig. 7A). All Dyn neurons in these clusters express VGluT2 transcripts (in a sample of 375 Dyn neurons in 2 sections counted bilaterally, all neurons colocalized VGluT2). None of the Dyn neurons in PBN express VGAT transcripts (Fig. 7A).
To determine if the Dyn neurons in the PBN project to the DMH, we performed immunohistochemistry (IHC) for Dyn and CTb in brains from rats that had been injected with CTb in DMH and treated with intracerebroventricular (ICV) colchicine. We observed colocalization of CTb and Dyn mainly in the dense clusters of Dyn neurons in the dlPBN (Fig. 7B). 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.
To determine whether the activity of DMH-projecting Dyn neurons in dlPBN could influence the level of thermogenesis in normal thermoregulation or in the TI state, we performed a co-detection procedure to identify DMH-projecting (CTb labeling with IHC) Dyn (pDyn transcripts with ISH) neurons in PBN that were activated (c-fos with ISH) by cutaneous thermal stimuli in naïve and pre-DMH transX rats. We observed DMH-projecting (CTb) Dyn neurons in dlPBN that were activated (c-fos) during skin warming in anesthetized naïve (Warm-N) rats (Fig. 7C), a condition in which we expect thermogenesis to be inhibited. Noticeably, fewer Dyn neurons in the dlPBN were activated in Cold-N rats than in either Warm-N or Cold-T rats (Fig. 7D). Our observation that DMH-projecting Dyn neurons in the dlPBN are activated in Warm-N rats, when inhibitory influences on the discharge of thermogenesis-promoting neurons in DMH predominate, is consistent with a significant thermogenesis-inhibiting role for these DMH-projecting Dyn neurons in the dlPBN. Such a role for Dyn neurons in the dlPBN is also supported by our finding that more of them are activated in Warm-N and Cold-T rats when thermogenesis is inhibited than in Cold-N rats, when thermogenesis is active (Fig. 7D).
Dynorphin In Dmh Inhibits Normal, Cold-evoked Bat Sna And Bat Thermogenesis
Having identified a dynorphinergic projection from the PBN to the region of the DMH containing thermogenesis-promoting neurons, we sought to determine if Dyn in the DMH would affect normal, cold-evoked BAT SNA and BAT thermogenesis. Since the degradation products of exogenous Dyn by extracellular peptidases lead to non-specific activation of NMDA receptors32, we pretreated the DMH with the peptidase inhibitor Amastatin.
In anesthetized naïve rats with a cool skin (TSKIN = 35.2 ± 0.4°C) and an activated BAT SNA, bilateral nanoinjections of Amastatin into the DMH (Fig. 8C) did not affect BAT SNA (pre-Amastatin: 787.5 ± 33.0% BL, post-Amastatin: 816.7 ± 163.06% BL, n = 4, p = 0.8459; Figs. 8A, 8B) or TBAT (pre-Amastatin: 36.4 ± 0.7°C, post-Amastatin: 36.6 ± 0.6°C; n = 4, p = 0.0546; Figs. 8A, 8B). 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 TBAT (pre-Dyn: 36.2 ± 0.6°C, post-Dyn: 36.8 ± 0.7°C; n = 4, p = 0.0469; Figs. 8A, 8B). Following Dyn nanoinjection in the DMH, subsequent skin cooling no longer activated BAT SNA (Fig. 8A). Additionally, skin warming had no effect on the post-Dyn completely inhibited level of BAT SNA (Fig. 8A), indicating that Dyn nanoinjection in the DMH does not induce the TI state in which skin warming activates BAT SNA (cf. Figures 1, 3).
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 Dyn, acting via k-opioid33 receptors 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 (ΔTSKIN = -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; Figs. 8D, 8E). With a warm TSKIN (39.0 ± 0.7°C) and an active BAT SNA (1426.9 ± 277.0%BL), bilateral nanoinjections of nor-BNI in the DMH (Fig. 8F) prevented any subsequent cold-evoked inhibitions of BAT SNA (ΔTSKIN = -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; Fig. 8D, 8E). 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 Dyn release in the DMH is necessary for the skin cooling-evoked reduction in 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
In free-behaving rats exposed to a cool TAMB, central administration of the adenosine 1A receptor (A1A-R) agonist, CHA, produces a progressive hypothermia consistent with an inverted regulation of thermogenesis, characteristic of the TI state13. Since Dyn release in the DMH is required for the skin cooling-evoked inhibition of thermogenesis in the TI state in anesthetized rats (Figs. 8D, 8E), we determined if blockade of central κ-opioid receptors with ICV nor-BNI would affect the cooling-evoked hypothermia during the TI state induced by the central administration of CHA in free-behaving rats.
One hour after reducing the TAMB from 25°C to 15°C, free-behaving rats chronically instrumented for TCORE recording, received an ICV pretreatment of either 0.9% saline vehicle (5 µl) or nor-BNI, followed after 10 minutes by an ICV injection of CHA (1 mM, 5µl). During the 1 h exposure to the TAMB of 15°C prior to pretreatment, the rats maintained a normal TCORE of 36.7 ± 0.1°C (n = 3; Fig. 8G), reflecting a normal thermoregulatory cold-defense response. Following saline pretreatment, administration of CHA elicited a prompt reduction in TCORE (Fig. 8G), which reached a minimum of 22.4 ± 0.2°C (ΔTCORE = -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 (Fig. 8G). Following pretreatment with nor-BNI, injection of CHA also elicited a rapid reduction in TCORE (Fig. 8G). However, the fall in TCORE (ΔTCORE = -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 TCORE was the same in both saline and nor-BNI pretreatment conditions, the minimum TCORE 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. The finding that during the TI state in free-behaving rats10 pretreatment with nor-BNI reduced the maximum hypothermia but not the rate of decline in TCORE suggests that Dyn, acting via central κ-opioid receptors, plays a permissive role in sustaining the skin cooling-induced inhibition of thermogenesis that is a hallmark of the TI state13.