Tonic inhibition of brown adipose tissue sympathetic nerve activity via muscarinic acetylcholine receptors in the rostral raphe pallidus

Abstract

Key points

• A tonically active, muscarinic cholinergic inhibition of rostral raphe pallidus (rRPa) neurons influences thermogenesis of brown adipose tissue (BAT) independent of ambient temperature conditions.

• The tonically active cholinergic input to rRPa originates caudal to the hypothalamus.

• Muscarinic acetylcholine receptor (mAChR) activation in rRPa contributes to the inhibition of BAT sympathetic nerve activity (SNA) evoked by activation of neurons in the rostral ventrolateral medulla (RVLM).

• The RVLM is not the sole source of the muscarinic cholinergic input to rRPa.

• Activation of GABA receptors in rRPa does not mediate the cholinergic inhibition of BAT SNA.

Abstract

We sought to determine if body temperature and energy expenditure are influenced by a cholinergic input to neurons in the rostral raphe pallidus (rRPa), the site of sympathetic premotor neurons controlling thermogenesis of brown adipose tissue (BAT). Nanoinjections of the muscarinic acetylcholine receptor (mAChR) agonist, oxotremorine, or the cholinesterase inhibitor, neostigmine (NEOS), in the rRPa of anaesthetized rats decreased cold‐evoked BAT sympathetic nerve activity (SNA, nadirs: −72 and −95%), BAT temperature (Tbat, −0.5 and −0.6°C), expired CO₂ (Exp. CO₂, −0.3 and −0.5%) and heart rate (HR, −22 and −41 bpm). NEOS into rRPa reversed the increase in BAT SNA evoked by blockade of GABA receptors in rRPa. Nanoinjections of the mAChR antagonist, scopolamine (SCOP), in the rRPa of warm rats increased BAT SNA (peak: +1087%), Tbat (+1.8°C), Exp. CO₂ (+0.7%), core temperature (Tcore, +0.5°C) and HR (+54 bpm). SCOP nanoinjections in rRPa produced similar activations of BAT during cold exposure, following a brain transection caudal to the hypothalamus, and during the blockade of glutamate receptors in rRPa. We conclude that a tonically active cholinergic input to the rRPa inhibits BAT SNA via activation of local mAChR. The inhibition of BAT SNA mediated by mAChR in rRPa does not depend on activation of GABA receptors in rRPa. The increase in BAT SNA following mAChR blockade in rRPa does not depend on the activity of neurons in the hypothalamus or on glutamate receptor activation in rRPa.

Key points

• A tonically active, muscarinic cholinergic inhibition of rostral raphe pallidus (rRPa) neurons influences thermogenesis of brown adipose tissue (BAT) independent of ambient temperature conditions.

• The tonically active cholinergic input to rRPa originates caudal to the hypothalamus.

• Muscarinic acetylcholine receptor (mAChR) activation in rRPa contributes to the inhibition of BAT sympathetic nerve activity (SNA) evoked by activation of neurons in the rostral ventrolateral medulla (RVLM).

• The RVLM is not the sole source of the muscarinic cholinergic input to rRPa.

• Activation of GABA receptors in rRPa does not mediate the cholinergic inhibition of BAT SNA.

Introduction

Brown adipose tissue (BAT) is a significant source of thermogenesis for thermoregulation and febrile responses (Nakamura & Morrison, 2011). BAT thermogenic activity is primarily determined by its sympathetic input. Understanding the complex neural pathways involved in the sympathetic regulation of BAT will contribute to the development of improved therapeutic approaches to suppress life‐threating elevations in body temperature, to induce therapeutic hypothermia for neuroprotection and cardioprotection during ischaemic insults, and to harness BAT activation to ameliorate metabolic disorders.

The rostral raphe pallidus area (rRPa) contains the sympathetic premotor neurons that regulate BAT thermogenic activity (Morrison et al. 1999; Nakamura et al. 2002), and their activity is determined by the balance of a multitude of inhibitory and excitatory inputs to the rRPa (Morrison & Madden, 2014; Conceicao et al. 2017). Cold‐evoked and febrile activations of BAT thermogenesis require a glutamatergic excitation of BAT sympathetic premotor neurons in the rRPa from the dorsomedial hypothalamus (DMH) (Nakamura et al. 2002, 2004; Cao & Morrison, 2003), although a glutamatergic input to rRPa also originates from a hindbrain location (Conceicao et al. 2017). Orexinergic inputs from the perifornical lateral hypothalamus stimulate the sympathetic premotor neurons in rRPa to activate BAT thermogenesis (Tupone et al. 2011). The rRPa also receives GABAergic inhibitory inputs from neurons in the intermediate and parvicellular reticular nuclei that contribute to the regulation of BAT sympathetic nerve activity (SNA) during negative energy balance (Nakamura et al. 2017). Glycinergic inhibitory inputs can reduce the activation of rRPa neurons, decreasing BAT thermogenesis (Conceicao et al. 2017). Activation of neurons in the rostral ventrolateral medulla (RVLM) inhibits BAT SNA, independent of GABA receptor (GABAR) activation in the rRPa (Cao et al. 2010) and activation of neurons in the caudal ventrolateral medulla inhibits BAT SNA, in part via activation of alpha2‐adrenergic receptors in the rRPa (Madden et al. 2013).

Cholinergic neurotransmission also plays a role in thermoregulation. Intracerebroventricular (i.c.v.) injections of the muscarinic acetylcholine receptor (mAChR) agonists, oxotremorine or McN‐A‐343, reduce body temperature in rats (Sen & Bhattacharya, 1991), and the hypothermic effect of cholinergic receptor agonists is blocked by mAChR antagonists (Lin et al. 1980; Unal et al. 1998). In mice, the activation of cholinergic neurons in DMH was suggested to play a role in the inhibition of BAT thermogenesis during skin warming, via an inhibition of serotoninergic neurons in rRPa (Jeong et al. 2015). Clinically, enhanced thermogenesis could contribute to the life‐threatening hyperthermias occurring during blockade of mAChR following atropine overdose (McCarron et al. 1991).

In this study, we evaluated the effects of modulating mAChR in the rRPa on BAT thermogenesis and cardiovascular parameters in anaesthetized rats. Our results indicate that a tonically active cholinergic input from a region caudal to the hypothalamus onto mAChR on neurons in the rRPa drives an inhibition of BAT SNA under both warm and cold ambient conditions. The inhibition of BAT SNA evoked by mAChR activation in the rRPa does not require the activation of local GABAergic receptors and the activation of BAT following blockade of the tonically active cholinergic input to the rRPa is driven by a non‐glutamatergic input to BAT sympathetic premotor neurons in rRPa.

Methods

Ethical approval

The experiments reported herein were approved by the Animal Care and Use Committee of the Oregon Health and Science University. They also conformed to the regulations detailed in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and to the principles and regulations as described by Grundy (2015). Male Sprague‐Dawley rats (350–450 g, n = 41, Charles River, Indianapolis, IN, USA) were housed with ad libitum access to food and water, at a vivarium temperature of 22–23°C and under a 12‐h:12‐h light/dark cycle.

General procedures

Male rats, breathing spontaneously, were initially anaesthetized with isoflurane (2–3% in 100% O₂). Adequacy of anaesthesia was verified by the lack of motor responses to strong tail pinch. The femoral artery was cannulated for monitoring mean arterial pressure (MAP) and the femoral vein was cannulated for drug administration. Following cannulation, the rats were transitioned to urethane (750 mg kg⁻¹ i.v.) and α‐chloralose (60 mg kg⁻¹ i.v.) anaesthesia. Adequacy of anaesthesia was assessed hourly and verified by the lack of cardiovascular or motor responses to strong tail pinch. The trachea was cannulated for artificial ventilation. Following the remaining surgical procedures and prior to recording data, the rats were subject to neuromuscular blockade with d‐tubocurarine (initially 0.6 mg per rat i.v., supplemented with 0.3 mg h⁻¹ i.v.) and artificially ventilated with 100% O₂ at a minute volume of 180–240 ml, such that the end‐expired CO₂ remained between 3.5 and 5.0%. During data acquisition, expired (Exp) CO₂ was treated as a dependent variable and no adjustments were made to ventilatory volume or rate. Subsequent to the initial paralysis, the adequacy of anaesthesia was assessed hourly, just prior to the d‐tubocurarine supplementation, and verified by the lack of cardiovascular responses to strong tail pinch. Supplements (10% of initial dose) of the anaesthetic drugs were administered when necessary. This regime usually resulted in anaesthetic supplementation every 2 h beginning approximately 6 h after the initial dose. The rats were positioned prone in a stereotaxic frame and thermocouples (Physitemp, Clifton, NJ, USA) were inserted into the rectum to measure core body temperature (Tcore), into the left interscapular BAT pad to measure BAT temperature (Tbat), and onto the hindquarter skin under the thermal blanket to measure skin temperature (Tskin) (TC‐1000 thermocouple reader, Sable Systems, Las Vegas, NV, USA). Tcore was maintained between 36.5 and 37.5°C with a thermostatically controlled heating lamp or a water‐perfused thermal blanket, except as noted for cold‐evoked increases in BAT SNA when the water blanket was perfused with water at 20 ± 3°C (Nakamura & Morrison, 2011).

BAT sympathetic nerve recording

The right postganglionic BAT SNA was recorded from a small nerve bundle dissected from the ventral surface of the right interscapular BAT pad. The nerve was placed on a bipolar hook recording electrode under mineral oil. Nerve activity was differentially amplified (10 000–50 000 times; CyberAmp 380, Axon Instruments, Union City, CA, USA), filtered (1–300 Hz), digitized and recorded onto a hard drive using Spike 2 software [Cambridge Electronic Design (CED), Cambridge, UK] along with all other variables.

Administration of drugs

Micropipettes (∼20 μm tip diameter) were stereotaxically positioned into selected brain sites, and drug nanoinjections (60 nl) were accomplished with a pressure‐injection apparatus (Toohey, Fairfield, NJ, USA) and the injection volumes were measured using a calibrated microscope reticule to observe the displacement of the fluid meniscus in the glass micropipette. Nanoinjections were made into rRPa (∼12.0 mm caudal to bregma, 0.0 mm lateral, −9.0 to −9.5 mm ventral to dural surface) at the dorso‐ventral location of the lowest microstimulation threshold (<20 μA) for evoking an excitatory BAT SNA potential with twin pulses (1‐ms duration, 6‐ms interpulse interval). All drugs for nanoinjections were obtained from Sigma‐Aldrich (St Louis, MO, USA) and diluted in 0.9% saline: oxotremorine (OXO, 10 mm), neostigmine (NEOS, 50 mm), scopolamine (SCOP, 50 mm), NMDA (0.2 mm), muscimol (1.2 mm), bicuculine (BIC, 250 μm), saclofen (25 mm), (2R)‐amino‐5‐phosphonovaleric acid (AP5, 5 mm), 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 5 mm) and dl‐2‐amino‐3‐phosphonopropionic acid (dl‐AP3, 5 mm). Nanoinjection of the isotonic saline vehicle into rRPa had no effects on either BAT thermogenic or cardiovascular variables. Up to three of the experiments described below were performed in some rats, with at least 1 h allowed between different experimental protocols.

Brain transection

With the aim of severing the caudally projecting axons of thermogenesis‐promoting neurons in the DMH (Morrison et al. 2004), a blade (4 mm wide, angled 5° from the midline) was inserted caudal to the hypothalamus (−5.0 mm caudal to bregma; n = 4). The transection blade was positioned perpendicular to the sagittal sinus and with its medial edge just lateral to the sagittal sinus, and then it was lowered ∼10.0 mm below the dural surface. This procedure was performed bilaterally, with the transection knife sequentially positioned on each side of the sagittal sinus.

Histological localization of injection sites

The microinjection sites were marked by pressure nanoinjection of fluorescent polystyrene microspheres (FluoSpheres, F8797, F8801 or F8803; Molecular Probes, Eugene, OR, USA) included in the injectate (1:100 dilution of FluoSpheres). After the physiological recordings, rats were perfused (5% paraformaldehyde) transcardially under anaesthesia (750 mg kg⁻¹ i.v. urethane and 60 mg kg⁻¹ i.v. α‐chloralose), and brains were removed and postfixed for 2–12 h. Coronal sections (60 μm) were mounted on slides to localize the nanoinjection sites (Madden & Morrison, 2006). The position and completeness of the brain transections were assessed from coronal sections of fixed tissue through the region of the transection.

Data analysis

For analysis of BAT SNA, a continuous measure (4 s bins) of BAT SNA amplitude was obtained as the root mean square (rms) value of the BAT SNA, calculated (Spike 2, CED) as the square root of the total power in the 0.1–20 Hz frequency band of the autospectra of sequential 4 s segments of BAT SNA. To normalize BAT SNA measurements among rats and to take into account inter‐experiment variability in amplifier noise, nerve–electrode contact, and the variable, but small, cardiovascular component in BAT nerve bundles, BAT SNA values are expressed as % of baseline (BL), where the BL value of BAT SNA is the minimal value of BAT SNA when the rat is warm enough so that there is no cold‐evoked BAT SNA. Pretreatment control values of BAT SNA (expressed as % of BL) and other variables were the mean values during the 60‐s period before the treatment. Pretreatment control conditions were either with a high Tskin (∼37.0°C) and Tcore (∼37.0°C,) which resulted in low levels of BAT SNA, or with a low Tskin (∼35.0°C) and Tcore (∼35.0°C), which led to a modestly elevated level of pretreatment BAT SNA. The amplitudes of treatment‐evoked responses were calculated from the mean levels of the variables during the 30 s period of the maximum response (either increase or decrease) occurring within 10 min of the treatment. The data are expressed as mean ± SEM and compared with Student's t test, with P ≤ 0.05 considered significant.

Results

OXO in rRPa during cold exposure inhibits BAT SNA

Since activation of brain mAChR elicits hypothermia (Sen & Bhattacharya, 1991), we tested the effect of activating mAChR in rRPa on the elevated BAT SNA during cold exposure. Nanoinjection of the broad spectrum mAChR agonist, OXO (10 mm, 60 nL) in rRPa (Fig. 1 F) during cold exposure (Tskin: 33.8 ± 0.9°C and Tcore: 35.7 ± 0.3°C, n = 5) inhibited cold‐evoked BAT SNA (Fig. 1 A; 72 ± 9% inhibition of the cold‐evoked SNA, P = 0.02), and decreased Tbat (Fig. 1 A; −0.5 ± 0.1°C from 34.5 ± 0.5°C, P = 0.01), Exp CO₂ (Fig. 1 A; −0.3 ± 0.1% from 4.3 ± 0.3%, P = 0.04), Tcore (Fig. 1 A; −0.2 ± 0.1°C from 35.7 ± 0.3°C, P = 0.04) and heart rate (HR; Fig. 1 A; −22 ± 9 bpm from 391 ± 28 bpm, P = 0.04). MAP was not significantly changed (Fig. 1 A; P = 0.1) by OXO nanoinjections in rRPa.

Figure 1. Effect of nanoinjection of OXO, NEOS or SCOP in rRPa.

A, nanoinjection of OXO in rRPa (dashed line) in a cold‐exposed rat decreased brown adipose tissue (BAT) sympathetic nerve activity (SNA), BAT temperature (Tbat), core temperature (Tcore), expired CO₂ (Exp CO₂) and heart rate (HR). Histograms of the levels of BAT SNA, Tbat, Exp CO₂, Tcore, mean arterial pressure (MAP) and HR prior to (open bars) and following (solid bars) OXO nanoinjection into rRPa. ^*Significant difference (P < 0.05). B, nanoinjection of NEOS in rRPa (dashed line) in a cold‐exposed rat decreased BAT SNA, Tbat, Tcore, Exp CO₂ and HR. Histograms of the levels of BAT SNA, Tbat, Exp CO₂, Tcore, MAP and HR prior to (open bars) and following (solid bars) NEOS nanoinjection into rRPa. ^*Significant difference (P < 0.05). C, nanoinjection of SCOP in rRPa (dashed line) in a warm‐exposed rat increased BAT SNA, Tbat and Exp CO₂. Histograms of the levels of BAT SNA, Tbat, Exp CO₂, Tcore, MAP and HR prior to (open bars) and following (solid bars) SCOP nanoinjection into rRPa. ^*Significant difference (P < 0.05). D, nanoinjection of SCOP in rRPa (dashed line) in a cold‐exposed rat increased BAT SNA, Tbat, Tcore, Exp CO₂, MAP and HR. Histograms of the levels of BAT SNA, Tbat, Exp CO₂, Tcore, MAP and HR prior to (open bars) and following (solid bars) SCOP nanoinjection into rRPa. ^*Significant difference (P < 0.05). E, partial histological coronal section containing blue fluorescent beads marking a representative OXO nanoinjection site in rRPa. Composite maps of the nanoinjection sites of OXO (asterisk, n = 5), NEOS (stars, n = 6), SCOP in warm‐exposed rats (circles, n = 6) and SCOP in cold‐exposed rats (@, n = 6) plotted on a schematic coronal drawing through the rRPa at −11.3 mm caudal to bregma. [Color figure can be viewed at wileyonlinelibrary.com]

NEOS in rRPa inhibits cold‐evoked BAT SNA

To determine if increasing the level of endogenous acetylcholine (ACh) within rRPa would inhibit the enhanced BAT SNA during cold exposure, we nanoinjected the reversible acetylcholinesterase inhibitor, NEOS (10 mm, 60 nl), in the rRPa during cooling (Tskin: 33.4 ± 1.0°C and Tcore: 36.5 ± 0.4°C). Nanoinjection of NEOS in the rRPa (Fig. 1 F, n = 6) of cold‐exposed rats reduced the BAT SNA (Fig. 1 B; 95 ± 4% inhibition of the cold‐evoked BAT SNA, P = 0.02), Tbat (−1.3 ± 0.2°C from 36.3 ± 0.4°C, P = 0.0004), Exp CO₂ (−0.7 ± 0.1% from 4.3 ± 0.2%, P = 0.0001), Tcore (−0.4 ± 0.1°C from 36.5 ± 0.4°C, P = 0.001) and HR (− 66 ± 18 bpm from 444 ± 27 bpm, P = 0.01). Nanoinjection of NEOS in the rRPa did not change MAP (Fig. 1 B; P = 0.1).

SCOP in rRPa during normothermia increases BAT SNA

To determine whether BAT thermogenesis is modulated by an endogenous muscarinic cholinergic input to rRPa, the non‐selective mAChR antagonist, SCOP (50 mm, 60 nL), was nanoinjected in the rRPa (Fig. 1 F) of anaesthetized rats during warm conditions (Tcore: 36.9 ± 0.3°C, n = 6), when basal BAT SNA was low (Fig. 1 C, n = 6). The antagonism of mAChR in rRPa with SCOP increased BAT SNA [Fig. 1 C; peak: +1087 ± 220% from baseline (BL), P = 0.002], Tbat (+1.8 ± 0.3°C from 34.7 ± 0.3°C, P = 0.001), Exp CO₂ (+0.7 ± 0.1% from 3.6 ± 0.3%, P = 0.0005), Tcore (+0.5 ± 0.1°C from 36.9 ± 0.3°C, P = 0.004) and HR (+ 54 ± 6 bpm from 414 ± 13 bpm, P = 0.0001), with no change in MAP (Fig. 1 C; P = 0.1).

SCOP in rRPa during cold exposure increases BAT SNA

To determine whether the BAT sympathoinhibitory, cholinergic input to rRPa is active during cold conditions, we nanoinjected SCOP into rRPa (Fig. 1 F) when Tskin was 34.8 ± 0.3°C and Tcore was 35.6 ± 0.3°C (n = 6). Similar to the effect of SCOP in rRPa during skin warming, SCOP increased BAT SNA (Fig. 1 D; from a cold‐evoked level of 754 ± 126% of BL to +1750 ± 451% of BL, P = 0.01), Tbat (+0.4 ± 0.1°C from a cold‐evoked peak of 35.4 ± 0.4°C, P = 0.003), Exp CO₂ (+0.4 ± 0.1% from a cold‐evoked peak of 4.6 ± 0.4%, P = 0.001), Tcore (+0.2 ± 0.1°C from a cold‐evoked peak of 35.8 ± 0.3°C, P = 0.04), MAP (+8 ± 3 mmHg from a cold‐evoked peak of 100 ± 4 mmHg, P = 0.01) and HR (+22 ± 5 bpm from a cold‐evoked peak of 443 ± 12 bpm, P = 0.002).

SCOP in rRPa activates BAT SNA following a transection of the neuraxis caudal to the hypothalamus

To determine if the tonically active, cholinergic input to the rRPa that inhibits BAT thermogenesis arises from the DMH, as reported in mice (Jeong et al. 2015), we nanoinjected SCOP in rRPa after transecting the brain just caudal to the hypothalamus (−5 mm caudal to bregma, Fig. 2 B, n = 4). We reasoned that if the DMH was the source of a tonically active, cholinergic, BAT sympathoinhibitory input to the rRPa, then a brain transection caudal to DMH would interrupt this pathway and the activation of BAT SNA produced by SCOP injection in rRPa would be significantly reduced. We functionally verified the efficacy of our brain transections in eliminating the axonal connections between the DMH and the rRPa (Hermann et al. 1997; Samuels et al. 2002) by observing that these transections (Fig. 2 B) eliminated the elevated BAT SNA during cold exposure (Fig. 2 A), which is dependent on the activity of DMH neurons projecting to rRPa (Nakamura & Morrison, 2007). One hour after the brain transection, and now under warm conditions, nanoinjection of SCOP in rRPa (Fig. 2 C) increased BAT SNA (Fig. 2 A; peak: +953 ± 158% from the pre‐SCOP BL, P = 0.01), Tbat (+1.8 ± 0.1°C from 34.1 ± 0.3°C, P = 0.001), Exp CO₂ (+0.5 ± 0.1% from 3.4 ± 0.2%, P = 0.005), MAP (+13 ± 3 mmHg from 102 ± 8 mmHg, P = 0.02) and HR (+61 ± 8 bpm from 364 ± 10 bpm, P = 0.004), with no change in Tcore (Fig. 2 A; P = 0.1). There were no statistical differences between the SCOP‐induced increases in BAT thermogenic or in cardiovascular parameters [BAT SNA (P = 0.2), Tbat (P = 0.4), Exp CO₂ (P = 0.2), Tcore (P = 0.3), MAP (P = 0.1) and HR (P = 0.3)] in intact versus brain‐transected rats.

Figure 2. Nanoinjection of SCOP in rRPa following a brain transection caudal to DMH increased BAT SNA and BAT temperature.

A, data traces during bilateral transections of the neuraxis caudal to the DMH (continuous lines; left side, L and right side, R), performed in a rat with a cold‐evoked elevation in BAT SNA. Note that the transections eliminated the cold‐evoked BAT SNA. The grey bar represents an approximately 1.0 h interval between the transection and the SCOP nanoinjection in rRPa. Nanoinjection of SCOP in rRPa (dashed line) increased BAT SNA, Tbat, Exp CO₂, MAP and HR. Histograms of the levels of BAT SNA, Tbat, Exp CO₂, Tcore, MAP and HR prior to (open bars) and following (solid bars) SCOP nanoinjection into rRPa following brain transection. ^*Significant difference (P < 0.05). B, schematic drawing (top panel) of a sagittal section through the rat brain indicating the DMH (dashed oval) and the approximate level (continuous line) of the transection; also shown is a representative histological section (lower panel) at the level of a transection superimposed on a camera lucida drawing of a coronal brain section at −5.0 mm caudal to bregma. C, a composite map of the sites (circles, n = 4) of SCOP nanoinjections plotted on a schematic coronal drawing through the rRPa at −11.3 mm caudal to bregma. [Color figure can be viewed at wileyonlinelibrary.com]

Effect of mAChR antagonism in rRPa on the RVLM‐evoked inhibition of BAT SNA and BAT thermogenesis

Having identified a BAT sympathoinhibitory, muscarinic cholinergic input to rRPa that arises from a region caudal to the hypothalamus, we sought to determine if activation of mAChR in rRPa plays a role in the inhibition of BAT SNA and BAT thermogenesis evoked by activation of neurons in the RVLM (Cao et al. 2010). We reasoned that if the RVLM‐evoked inhibition of BAT SNA is mediated by mAChR in rRPa, then SCOP in rRPa should prevent the inhibition of BAT SNA evoked by activation of RVLM neurons. During cold exposure (Tskin: 34.7 ± 0.4°C and Tcore: 35.6 ± 0.2°C), which elevates BAT SNA (Fig. 3 A, n = 5), activation of neurons in the RVLM (Fig. 3 C) with a local nanoinjection of NMDA (0.2 mm, 60 nL) completely inhibited the cold‐evoked activation of BAT SNA (Fig. 3 A; nadir: 142 ± 32% of BL from a cold‐evoked BAT SNA of 1844 ± 378% of BL, P = 0.01), Tbat (−0.5 ± 0.2°C from 35.4 ± 0.4°C, P = 0.03), Exp CO₂ (−0.3 ± 0.1% from 4.8 ± 0.3%, P = 0.02) and HR (−35 ± 11 bpm from 466 ± 4 bpm, P = 0.02).

Figure 3. Nanoinjection of SCOP in rRPa attenuated the inhibition of BAT SNA evoked by activation of neurons in RVLM, but inhibition of neurons in RVLM did not block the sympathoinhibitory response to NEOS nanoinjection in rRPa.

A, nanoinjection of NMDA in RVLM (1^st continuous line) inhibits the cold‐evoked activation of BAT SNA and reduces Tbat, Tcore, Exp CO₂ and HR. SCOP nanoinjection in rRPa (dashed line) increased BAT SNA, Tbat, Tcore, Exp CO₂, MAP and HR. Nanoinjection of NMDA (2^nd continuous line) in RVLM after the SCOP nanoinjection in rRPa modestly decreased BAT SNA and Tbat. The effect of NMDA nanoinjection in the naïve RVLM recovered ∼35 min (3^rd continuous line) after the SCOP nanoinjection. Histograms illustrating the levels of BAT SNA, Tbat, Exp CO₂ and HR prior to (open bars) and following (solid bars) each of the three nanoinjections of NMDA into RVLM. ^*Significant difference (P < 0.05). B, bilateral nanoinjections of muscimol in RVLM (continuous lines; L, left side and R, right side) did not block the inhibitory effect of nanoinjection of NEOS in rRPa (dashed line) on BAT SNA and Tbat, Tcore, Exp CO₂, MAP or HR. Histograms showing the levels of BAT SNA, Tbat, Exp CO₂ and HR prior to (open bars) and following (solid bars) NEOS nanoinjection into rRPa. ^*Significant difference (P < 0.05). Composite maps of the nanoinjection sites of NMDA in RVLM (C, circles, n = 5), SCOP in rRPa (D, circles, n = 5), bilateral nanoinjections of muscimol (E, circles, n = 5) in RVLM, and NEOS (F, circles, n = 5) in rRPa plotted on schematic drawings through the rRPa at −11.3 mm caudal to bregma and the RVLM at −12.2 mm caudal to bregma.

After BAT SNA recovered to the original cold‐evoked level, nanoinjection of SCOP in the rRPa (Fig. 3 D) increased BAT SNA (Fig. 3 A; from a cold‐evoked level of 1572 ± 326% of BL, to a post‐SCOP peak of +2534 ± 613% of BL, P = 0.02), Exp CO₂ (+0.1 ± 0.02% from 3.8 ± 0.3%, P = 0.002) and HR (+12 ± 3 bpm from 462 ± 7 bpm, P = 0.01). Ten minutes after the SCOP nanoinjection, when the increase in BAT SNA had reached a plateau, nanoinjection of NMDA in the RVLM (Fig. 3 C) still decreased BAT SNA (Fig. 3 A; nadir: 1951 ± 631% of BL from a SCOP‐evoked level of 3203 ± 1024% of BL, P = 0.02) and HR (− 23 ± 10 bpm from 474 ± 7 bpm, P = 0.04). The change in BAT SNA evoked by activating neurons in the RVLM was smaller following SCOP than that evoked prior to SCOP: BAT SNA (−1252 ± 437% of BL after SCOP vs. −1702 ± 352% of BL before SCOP, P = 0.001). The NMDA nanoinjection in the RVLM after SCOP in the rRPa did not change Tbat (P = 0.2), Exp CO₂ (P = 0.1) or Tcore (P = 0.2), probably because the RVLM‐evoked inhibition of BAT SNA was smaller and shorter than that evoked prior to SCOP in the rRPa. The RVLM‐evoked reductions in HR were not affected (P = 0.1) by the nanoinjection of SCOP in the rRPa.

A third NMDA nanoinjection in RVLM (Fig. 3 C), performed approximately 35 min after the SCOP nanoinjection in rRPa to test recovery of the RVLM‐evoked reductions in BAT thermogenesis, reduced BAT SNA (nadir: 220 ± 73% of BL from a cold‐activated level of 2427 ± 859% of BL, P = 0.047), Tbat (−0.6 ± 0.1°C from 35.5 ± 0.5°C, P = 0.02), Exp CO₂ (−0.2 ± 0.1% from 4.8 ± 0.4%, P = 0.03) and HR (−26 ± 2 bpm from 463 ± 8 bpm, P = 0.001). These decreases did not differ from those evoked by the first NMDA nanoinjection in RVLM: BAT SNA (P = 0.3), Tbat (P = 0.3), Exp CO₂ (P = 0.1) and HR (P = 0.2). The reduction in the amplitude of the RVLM‐evoked inhibition of BAT thermogenic parameters during blockade of mAChR in rRPa suggests that although activation of mAChR in rRPa plays a role in the RVLM‐evoked inhibition of BAT SNA, the latter is not mediated entirely by mAChR in rRPa.

NEOS nanoinjections in rRPa inhibit BAT SNA after inhibition of RVLM neurons

To determine whether the activity of neurons in the RVLM is required for the tonic activity of the BAT sympathoinhibitory, muscarinic cholinergic input to rRPa, we nanoinjected NEOS in the rRPa after inhibiting neurons in the RVLM (Fig. 3 B, n = 5). After bilateral nanoinjections of the GABA_AR agonist, muscimol (1.2 mm, 100 nL), in the RVLM (Fig. 3 E), nanoinjection of NEOS in rRPa (Fig. 3 F) decreased BAT SNA (Fig. 3 B; nadir: 118 ± 7% from the pre‐NEOS level of 724 ± 106% of BL, P = 0.002), Tbat (−0.6 ± 0.1°C from 35.2 ± 0.3°C, P = 0.0002) and Exp CO₂ (−0.5 ± 0.1% from 4.6 ± 0.4%, P = 0.003). The NEOS‐evoked changes following muscimol injections in RVLM were not different (P > 0.05 for all changes) from those evoked by NEOS in rRPa under control conditions (compare Figs 1 B and 3 B). These results indicate that the activity of neurons in the RVLM is not required for the tonic release of ACh onto mAChR in rRPa. These data are consistent with the tonically active, cholinergic input to rRPa arising from neurons outside of the RVLM.

Inhibition of BAT thermogenesis by NEOS in rRPa is independent of GABA_AR and GABA_BR in rRPa

To determine if the BAT sympathoinhibition evoked by mAChR activation in rRPa involves local GABA release onto BAT sympathetic premotor neurons in rRPa, we tested the ability of NEOS in rRPa to inhibit BAT SNA after blocking either GABA_AR or GABA_BR in rRPa. Disinhibition of BAT sympathetic premotor neurons in the rRPa with local injections of the GABA_AR antagonist, bicuculline (BIC, 250 μm, 60 nL; n = 5), increased BAT SNA (Fig. 4 A and B), as previously described (Morrison et al. 1999). Subsequent NEOS, but not saline vehicle (Fig. 4 A), nanoinjections in rRPa reduced the BIC‐evoked increase in BAT SNA (Fig. 4 B; nadir: 340 ± 29% of BL from the BIC‐evoked level of 1972 ± 431% of BL, P = 0.01), and decreased Tbat (−1.3 ± 0.1°C from 37.7 ± 0.3°C, P = 0.0001) and Exp CO₂ (−0.9 ± 0.2% from a pre‐BIC value of 6.0 ± 0.2%, P = 0.003).

Figure 4. NEOS nanoinjection in rRPa inhibits the increased BAT SNA evoked by local nanoinjection of BIC or saclofen.

A, nanoinjection of saline vehicle (dotted line) in rRPa did not affect the increases in BAT SNA, Tbat or Exp CO₂ evoked by BIC nanoinjection in rRPa (continuous line). B, example of a nanoinjection of NEOS (short‐dashed line) in rRPa inhibiting the BIC‐evoked activation of BAT SNA and reduced Tbat and Exp CO₂. Composite map of the nanoinjection sites for saline after BIC (asterisk, n = 4) and for NEOS after BIC (circles, n = 5) plotted on a schematic drawing through the rRPa at −11.30 mm caudal to bregma. Histograms of the levels of BAT SNA, Tbat and Exp CO₂ prior to (open bars) and following (solid bars) NEOS nanoinjection into rRPa. ^*Significant difference (P < 0.05). C, nanoinjection of saline vehicle (dotted line) in rRPa did not affect the activation of BAT SNA or the increases in Tbat and Exp CO₂ evoked by saclofen nanoinjection in rRPa (long‐dashed line). D, example of a nanoinjection of NEOS (short‐dashed line) in rRPa, which decreased the saclofen‐evoked (long‐dashed line) increase in BAT SNA and reduced Tbat and Exp CO₂. Composite map of the nanoinjection sites for saline after saclofen (asterisk, n = 5) and for NEOS after saclofen (circles, n = 4) plotted on schematic drawing through the rRPa at −11.30 mm caudal to bregma. Histograms of the levels of BAT SNA, Tbat and Exp CO₂ prior to (open bars) and following (solid bars) NEOS nanoinjection into rRPa. ^*Significant difference (P < 0.05).

Nanoinjection of the GABA_BR antagonist, saclofen (25 mm, 60 nL; n = 4) in the rRPa increased BAT SNA (Fig. 4 C and D; peak: 678 ± 130% of BL, P = 0.006), Tbat (+1.7 ± 0.5°C from 35.2 ± 0.2°C, P = 0.01) and Exp CO₂ (+0.8 ± 0.3% from 4.9 ± 0.2%, P = 0.02). Subsequent nanoinjection of NEOS, but not saline vehicle (Fig. 4 C), in rRPa decreased the elevated level of BAT SNA following saclofen nanoinjection in rRPa (Fig. 4 D; nadir: 99 ± 24% of BL from the saclofen‐evoked level of 360 ± 30% of BL, P = 0.004), Tbat (−1.3 ± 0.2°C from 37.1 ± 0.1°C, P = 0.003) and Exp CO₂ (−0.6 ± 0.2% from 5.6 ± 0.1%, P = 0.02). The finding that blockade of neither GABA_AR nor GABA_BR in rRPa prevented the BAT sympathoinhibition evoked by increasing endogenous ACh in the rRPa is consistent with the absence of a role for either GABA_AR or GABA_BR in rRPa in the BAT sympathoinhibition mediated by ACh release in rRPa.

Glutamate receptor blockade in rRPa did not reduce activation of BAT evoked by SCOP nanoinjection in rRPa

To determine if the activation of BAT following the nanoinjection of SCOP in rRPa requires the activation of local glutamate receptors (Cao & Morrison, 2006; Conceicao et al. 2017), we nanoinjected glutamate receptor antagonists in rRPa during the peak activation of BAT SNA following SCOP injection in rRPa. Nanoinjection of the ionotropic glutamate receptor antagonists, AP5 and CNQX (60 nL of a cocktail of 5 mm AP5 and 5 mm CNQX; n = 5), into rRPa (Fig. 5 A) did not affect the elevated levels (Fig. 5 A) of BAT SNA (from 327 ± 49% of BL before AP5/CNQX to 336 ± 50% of BL at 3 min after AP5/CNQX, P = 0.4), Tbat (from 35.9 ± 0.4°C before AP5/CNQX to 36.0 ± 0.5°C at 3 min after AP5/CNQX, P = 0.3) or Exp. CO₂ (from 4.2 ± 0.3% before AP5/CNQX to 4.2 ± 0.3% at 3 min after AP5/CNQX, P = 0.1) evoked by SCOP nanoinjections into rRPa. Nanoinjection of the group I metabotropic glutamate receptor antagonist, dl‐AP3 (60 nL, 5 mm; n = 5) in rRPa (Fig. 5 B) did not affect the increases in BAT thermogenic parameters evoked by SCOP nanoinjections into rRPa (Fig. 5 B): BAT SNA (from 357 ± 43% of BL before dl‐AP3 to 359 ± 50% of BL at 3 min after dl‐AP3, P = 0.5), Tbat (from 36.3 ± 0.4°C before dl‐AP3 to 36.4 ± 0.4°C at 3 min after dl‐AP3, P = 0.1) and Exp CO₂ (from 4.9 ± 0.2% before dl‐AP3 to 5.0 ± 0.2% at 3 min after dl‐AP3, P = 0.1).

Figure 5. SCOP‐evoked increase in BAT SNA does not require glutamate receptor activation in rRPa.

A, typical data traces showing that nanoinjection of the ionotropic glutamate receptor antagonists, AP5 and CNQX, in rRPa (continuous line) does not reverse the increases in BAT SNA, Tbat and Exp CO₂ evoked by prior nanoinjection of SCOP in rRPa (dashed line). Composite map of the nanoinjection sites of AP5 and CNQX after SCOP (circles, n = 5), plotted on a schematic drawing through the rRPa at −11.30 mm caudal to bregma. Histograms of the levels of BAT SNA, Tbat and Exp CO₂ prior to (open bars) and following (solid bars) AP5/CNQX nanoinjection into rRPa. ^*Significant difference (P < 0.05). B, example data traces showing that nanoinjection of the metabotropic glutamate receptor antagonist, dl‐AP3, in rRPa (short‐dashed line) does not reverse the increases in BAT SNA, Tbat and Exp CO₂ evoked by prior nanoinjection of SCOP into rRPa (long‐dashed line). Composite map of the nanoinjection sites of dl‐AP3 after SCOP (circles, n = 5) plotted on schematic drawings through the rRPa at −11.30 mm caudal to bregma.

Discussion

The major finding of the present study is the demonstration of a tonically active cholinergic inhibitory input to the rRPa under both cold and warm conditions. This tonically active cholinergic input probably originates from a site located caudal to the hypothalamus since transections of the neuraxis at the caudal hypothalamus did not prevent the increase in BAT SNA that was evoked by blockade of mAChR in the rRPa. Furthermore, the cholinergic inhibition occurs through the activation of mAChR and is not mediated indirectly by the activation of GABARs in the rRPa. Since the increase in BAT SNA following blockade of the mAChR in the rRPa is not dependent on glutamate receptor activation in the rRPa, it is also unlikely that the mAChR‐mediated inhibition of BAT SNA occurs by inhibition of a glutamatergic input to the BAT sympathetic premotor neurons. The cholinergic inhibition could occur by directly inhibiting BAT sympathetic premotor neurons or by suppression of a non‐glutamatergic excitatory input. In addition, although mAChR activation in the rRPa contributes to the inhibition of BAT SNA evoked by activation of neurons in the RVLM, the cholinergic input to the rRPa that is activated by stimulation of neurons in the RVLM is not tonically active in anaesthetized rats.

Organophosphorus pesticide poisoning causes a broad inhibition of acetylcholinesterase activity, resulting in hypothermia due to the activation of mAChR (Moffatt et al. 2010). The hypothermic effect of ACh administrated into the CNS is well documented (Kirkpatrick & Lomax, 1970; Lin et al. 1980; Unal et al. 1998; Jeong et al. 2015). Intracerebroventricular microinjections of AChR agonists produce a dose‐dependent hypothermia, which can be prevented by pretreatment with atropine, an mAChR antagonist (Lin et al. 1980; Unal et al. 1998). Conversely, a primary characteristic of anticholinergic toxicity includes elevated body temperature; however, the underlying aetiology of this effect has been poorly understood. Our findings suggest that increased BAT thermogenesis driven by blockade of mAChR in the rRPa contributes significantly to the increase in body temperature associated with anticholinergic toxicity.

The mAChRs are G protein‐coupled receptors: M1, M3 and M5 receptors are coupled to G_q/11 proteins, and the M2 and M4 receptors to G_i/0 proteins (Brown, 2010). The data concerning the identity of the mAChR isoform(s) that mediate the hypothermic effect of ACh are conflicting. Studies employing i.c.v. administration of drugs indicate that the activation of M1 and M3 (Unal et al. 1998), and M2 (Jeong et al. 2015) isoforms cause hypothermia. Our results using a broad‐spectrum mAChR agonist and a broad‐spectrum mAChR antagonist, and a cholinesterase inhibitor, do not differentiate among the mAChR isoforms that mediate the thermoregulatory responses evoked by the cholinergic input to the BAT sympathetic premotor neurons in the rat rRPa.

Heat defence mechanisms activated during skin or core warming include an inhibition of thermogenesis. Recent data, obtained exclusively in mice, suggest that a cholinergic input to the rRPa from ACh neurons in the DMH activates mAChR in the rRPa to mediate the heat defensive inhibition of thermogenesis during skin warming (Jeong et al. 2015). This interpretation implies that a cholinergic input to rRPa would be more active during warm exposure. However, in the rat, we found that blockade of mAChR in the rRPa increased BAT SNA equally well during both cold and warm conditions, and that endogenous acetylcholine is released in rRPa during cold exposure, as evidenced by the inhibition of cold‐evoked BAT SNA following NEOS injection into rRPa. Furthermore, in seeking to determine the source of the tonically active, cholinergic input to rRPa, we demonstrated that a brain transection caudal to the hypothalamus did not affect the magnitude of the increase in BAT thermogenesis following blockade of mAChR in the rRPa. Brain transections in this position are expected to sever the descending axons of DMH neurons projecting to rRPa, since they eliminate the increases in BAT SNA evoked by microinjection of prostaglandin E2 into the medial preoptic area (Morrison et al. 2004), and by cold exposure (Fig. 2; Conceicao et al. 2017), both of which are dependent on a glutamatergic, BAT sympathoexcitatory input from the DMH to the rRPa (Madden & Morrison, 2003; Cao & Morrison, 2006; Nakamura & Morrison, 2007). Therefore, it seems unlikely that the tonic cholinergic inhibition of BAT sympathetic premotor neurons in rRPa that we have demonstrated in the rat is derived from ACh neurons in the DMH. Nonetheless, since our transections spared the most lateral regions of the neuraxis (Fig. 2 B), we cannot exclude the possibility that a descending cholinergic input from the DMH to rRPa traverses these lateral regions and remained intact after our transections. Similarly, although it is possible that ACh continues to be released from the terminals of severed DMH axons at 1 h after their transection, this seems unlikely to account for the SCOP‐evoked increase in BAT SNA, since the cessation of cold‐evoked BAT SNA following brain transection indicates that glutamate does not continue to be released from the rRPa terminals of severed DMH axons.

The RVLM contains cholinergic neurons, but these cholinergic RVLM neurons regulate sensory information and do not project directly to midline raphe regions including the rRPa (Stornetta et al. 2013). Activation of neurons in the RVLM inhibits the increases in BAT SNA and BAT thermogenesis evoked by GABA_AR blockade into rRPa (Cao et al. 2010). Here, we demonstrate that blockade of mAChR in the rRPa attenuated the inhibition of cold‐evoked BAT SNA and BAT thermogenesis evoked by activation of neurons in the RVLM. These data suggest that RVLM neurons drive an unidentified, cholinergic inhibitory input to rRPa or drive release of ACh from local cholinergic neurons in the rRPa. If the tonic activity of neurons in the RVLM contributed to the tonic release of ACh in the rRPa then inhibition of the RVLM neurons should decrease the effectiveness of the acetylcholinesterase inhibitor, NEOS, to inhibit BAT SNA. However, inhibition of RVLM neurons did not attenuate the inhibitory effect of NEOS in the rRPa on BAT SNA. This implies that areas other than the RVLM contribute to the tonic activation of mAChR on BAT sympathetic premotor neurons. Further studies are necessary to elucidate the source(s) of the cholinergic inputs to the rRPa that tonically inhibit BAT thermogenesis.

Muscarinic receptors can excite or inhibit neuronal pathways by modulating the release of other neurotransmitters, including GABA (Wang et al. 2006), glycine (Wang et al. 2006), glutamate (Zhang et al. 2007) and ACh (Re, 1999; Brown, 2010). We conclude that the tonically active cholinergic input to rRPa does not inhibit sympathetically driven BAT thermogenesis by increasing GABA release in rRPa because increasing endogenous ACh in rRPa with local NEOS injection still inhibited BAT SNA after blockade of local GABA_AR with bicuculline, and after blockade of GABA_BR in rRPa with saclofen. Interestingly, nanoinjection of saclofen in rRPa elicited an increase in BAT SNA, consistent with a previously unreported, tonic activation of GABA_BR in rRPa to inhibit BAT thermogenesis.

Since activation of glutamate receptors in rRPa elicits increases in BAT thermogenesis and is required for cold‐evoked and febrile activations of BAT (Madden & Morrison, 2003; Cao & Morrison, 2006; Nakamura & Morrison, 2007), we tested whether an inhibition of glutamate release (Zhang et al. 2007) in rRPa might play a role in the inhibition of BAT thermogenesis by activation of mAChR in rRPa. If mAChR activation in rRPa contributed to a tonic inhibition of local glutamate release, then blockade of mAChR in rRPa would increase local glutamate release and the increase in BAT SNA following blockade of mAChR in rRPa would be reduced by blockade of local glutamate receptors. Since the increase in BAT SNA evoked by blockade of mAChR in the rRPa was unaffected by blockade of glutamate receptors in the rRPa, we conclude that inhibition of local glutamate release is not the mechanism through which activation of mAChR in rRPa inhibits BAT thermogenesis. As a corollary, the increased discharge of BAT sympathetic premotor neurons responsible for the elevated level of BAT SNA following SCOP injection in rRPa must be mediated by an excitatory neurotransmitter other than glutamate.

Alternatively, mAChR activation could directly inhibit BAT sympathetic premotor neurons in rRPa. This possibility is supported by the finding that the m2AChR agonist, methoctramine, hyperpolarized serotoninergic neurons in the nucleus raphe magnus (Pan & Williams, 1994) and that single cell quantitative RT‐PCR analysis of BAT‐regulating neurons in the mouse rRPa showed that all m2AChR‐expressing neurons are serotonergic (Jeong et al. 2015). Serotoninergic BAT sympathetic premotor neurons in rRPa (Cano et al. 2003) provide a critical excitatory drive to BAT sympathetic preganglionic neurons (Morrison, 1993, 2016; Madden & Morrison, 2010). Together, these data are consistent with a model in which a tonically active cholinergic input to the rRPa synapses directly on serotonergic, mAChR‐expressing BAT sympathetic premotor neurons, exerting a hyperpolarizing effect that reduces their excitability and curtails the activation of BAT SNA and BAT thermogenesis.

The findings of the current study demonstrate that a tonically active (under both warm and cold conditions) cholinergic input to neurons in the rRPa, probably originating caudal to the hypothalamus, inhibits BAT SNA via activation of mAChR. Moreover, neither the activation of GABARs in rRPa nor the inhibition of local glutamate release is necessary for the mAChR‐mediated inhibition of BAT SNA. Furthermore, mAChR activation in the rRPa contributes to the RVLM‐evoked inhibition of BAT SNA. These data provide novel insights into neural pathways that are likely to be involved in the pathological changes in thermoregulation during organophosphate poisoning and anticholinergic toxicity.

Additional information

Competing interests

The authors declare no conflicts of interest.

Author contributions

E.P.S.C., C.J.M. and S.F.M. conceived and designed the study, analysed the data, interpreted the results and approved the final version of the manuscript; E.P.S.C. performed experiments, prepared figures and drafted the manuscript; E.P.S.C, C.J.M. and S.F.M. edited and revised the manuscript.

Funding

This work was supported by a National Institutes of Health grant, R01‐NS091066 (S.F.M.).