Thermogenesis activated by central melanocortin signaling is dependent on neurons in the rostral raphe pallidus (rRPa) area

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Thermogenesis activated by central melanocortin signaling is dependent on neurons in the rostral raphe pallidus (rRPa) area Wei Fan a,⁎, Shaun F. Morrison b , Wei-Hua Cao b , Pinxuan Yu a a

Center for the Study of Weight Regulation and Associated Disorders and Vollum Institute, Oregon Health and Science University Portland, OR 97239-3098, USA b Neurological Sciences Institute, Oregon Health and Science University Portland, OR 97239-3098, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

The central melanocortin system plays a critical role in regulation of energy balance, including

Accepted 4 April 2007

thermogenesis in brown adipose tissue (BAT). Activation of the hypothalamic melanocortin

Available online 6 April 2007

signaling stimulates sympathetically-mediated interscapular BAT (IBAT) thermogenesis. The rostral raphe pallidus (rRPa) and adjacent area have been proposed as the location of

Keywords:

sympathetic premotor neurons for the central nervous system (CNS) control of IBAT

Melanocortin

thermogenesis. To determine if neuronal activity in rRPa area is required for the central

Thermogenesis

melanocortin-induced thermogenesis, we studied the effects of inhibition of the activity of

O2 consumption

neurons in the rRPa area on the sympathetic nerve activity (SNA) to IBAT evoked by lateral

Sympathetic nerve activity

ventricular injection of the melanocortin 3/4 receptor (MC3/4R) agonist, MTII, in urethane-

Raphe pallidus

chloralose-anesthetized rats and the effects on O2 consumption induced by third or fourth

Brown adipose tissue

ventricular injection of MTII in conscious freely moving mice. Icv injection of MTII (1 nmol) significantly increased rat IBAT SNA (+741% of control). Both third and fourth ventricular injections of MTII (1 nmol) significantly increased O2 consumption in conscious C57BL/6J mice (45% higher than that of saline control for third ventricular injection and 44% higher than that of saline control for fourth ventricular injection). The increases in IBAT SNA and in O2 consumption were reversed by inhibition of neurons in the rRPa and adjacent area with microinjections of glycine or muscimol into rRPa. These results suggest that the neurons in the RPa and its immediate vicinity play an essential role in mediating the increase in IBAT thermogenesis induced by activation of central melanocortin signaling. © 2007 Published by Elsevier B.V.

1.

Introduction

In both rodent and man, the central melanocortin system, including neurons containing the melanocortins, α-melanocyte-stimulating-hormone (α-MSH) and/or β-MSH, the endogenous melanocortin receptor antagonist, agouti-related protein (AGRP), or the melanocortin receptors, MC4R and/or MC3R, plays a critical role in energy homeostasis, including

⁎ Corresponding author. Fax: +1 503 494 5235. E-mail address: [email protected] (W. Fan). 0006-8993/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.brainres.2007.04.006

the regulation of thermogenesis (Butler and Cone, 2002; Coll et al., 2004; Cone, 1999; Harrold et al., 2003). The neurons that contain pro-opiomelanocortin (POMC), the precursor of the melanocortin agonists, are exclusively located in the hypothalamic arcuate nucleus (ARC) and in the caudal half of the commissural NTS (cNTS) of the brain stem and MC4R mRNA is widely expressed in hypothalamic and brainstem nuclei which are associated with energy homeostasis. Central

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administration of MTII, a selective agonist of the MC3R and MC4R, increases energy expenditure indexed by increased O2 consumption (Cowley et al., 1999; Hwa et al., 2001; Jonsson et al., 2001; Li et al., 2004), increased SNA to IBAT, increased IBAT temperature or increased uncoupling protein 1 (UCP1) mRNA expression in IBAT (Fan et al., 2004; Haynes et al., 1999; Li et al., 2003, 2004; Williams et al., 2003; Yasuda et al., 2004). In addition to hypothalamic melanocortin signaling, brainstem melanocortin signaling has also been implicated in the regulation of thermogenesis. Application of MTII into the fourth ventricle can increase body temperature (Zheng et al., 2005) and stimulate IBAT UCP1 mRNA expression, which is dependent on the sympathetic outflow to BAT (Williams et al., 2003). In contrast, administration of antagonists for the MC3/ 4R decreases body temperature, locomotor activity and O2 consumption (Adage et al., 2001; Small et al., 2003). Intracerebroventricular (icv) injection of the MC3/4R antagonist, AGRP, reduces IBAT SNA and IBAT temperature (Yasuda et al., 2004) and UCP1 mRNA expression (Baran et al., 2002; Small et al., 2001). MC4R signaling plays a critical role in the regulation of BAT thermogenesis. For example, MC4R −/− animals consume 20% less O2 than weight-matched, wild type

(WT) mice and the locomotor activity of young, nonobese MC4R −/− males is reduced compared with that of WT males (Ste Marie et al., 2000). Pair-feeding studies show that MC4R −/− females become significantly heavier than their WT counterparts when given the same amount of food as the WT, suggesting that a defective regulation of energy expenditure by MC4R signaling contributes to the obesity in MC4R −/− mice and that they are metabolically less efficient than WT mice (Ste Marie et al., 2000). Furthermore, placing MC4R −/− mice on a high fat diet shows that MC4R −/− mice develop accelerated, marked obesity due to an inability to increase O2 consumption and motor activity as well as a failure to reduce food intake, compared with WT and MC3R −/− mice (Butler et al., 2001). Despite the critical role of MC4R signaling plays in the regulation of thermogenesis, the exact underlying neuronal circuitry remains to be further delineated. Activation of central MC4R increases energy expenditure, at least in part through stimulation of sympathetically-mediated BAT thermogenesis, a common mechanism for the CNS to regulate adaptive thermogenesis in response to thermogenic stimuli such as leptin and pyrogens. Growing evidence suggests that the rRPa and its adjacent area contain the sympathetic premotor neurons

Fig. 1 – Inhibition of icv MTII-induced increase in rat BAT SNA and BAT thermogenesis by nanoinjection of glycine into rostral raphe pallidus (rRPa). Icv administration of MTII (MTII icv) elicits increases in brown adipose tissue sympathetic nerve activity (IBAT SNA; top trace: rms power, 4 s bins, peak increase is 911% of control level; second trace: oscillographic record), IBAT temperature (BAT TEMP), expired CO2, heart rate (HR). Arterial pressure (AP) was not consistently affected by icv MTII. Nanoinjection of glycine into rRPa (GLY rRPa, 30 nmol in 60 nl) reversed the icv MTII-evoked IBAT sympathoexcitation and the increases in IBAT temperature, expired CO2 and heart rate. A second nanoinjection of glycine into rRPa was equally effective in reversing the MTII-driven increases in sympathetic thermogenic variables that returned after the effect of the initial glycine-mediated inhibition of rRPa neurons dissipated. Vertical calibration is 150 μV for IBAT SNA (second trace).

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critical to the control of the sympathetic outflow to IBAT and that the rRPa acts as an important relaying and integrating station within the CNS circuitry controlling IBAT thermogenesis (Morrison, 2001b, 2003, 2004a; Nakamura et al., 2004). Functional inhibition of the rRPa neurons reverses the IBAT sympathetic nerve firing, thermogenesis and fever induced by central application of PGE2 or leptin (Morrison, 2001a, 2004b; Nakamura et al., 2002). In this study, we sought to test the hypothesis that neurons in rRPa and its adjacent area are also important in mediating the thermogenesis induced by central melanocortin administration. Some of these results have been presented in abstract form (Fan et al., 2004; Morrison et al., 2003).

2.

Results

2.1. Effect of inhibition of rRPa neurons on rat IBAT SNA responses to icv injection of the melanocortin agonist, MTII Icv administration of the melanocortin agonist, MTII (1 nmol in 5 μl), stimulated IBAT energy expenditure and thermogenesis

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through an activation of IBAT SNA from the normally quiescent levels seen in anesthetized rats maintained at a thermoneutral temperature (Fig. 1). In 8 rats, icv MTII produced a peak increase in IBAT SNA of 741 ±118% of control, which resulted in a peak rise in IBAT temperature of 1.5 ± 0.12 °C from a control level of 35.4 ± 0.22 °C, accompanied by a peak increase in expired CO2 of 1.3 ± 0.22% from a control level of 3.9 ± 0.16% and a maximal tachycardia of 75± 17 bpm from a resting level of 365± 15 bpm. Administration of 5 μl of saline vehicle into the lateral ventricle was without effect on any of the measured variables. During the peak of the response to icv MTII, microinjection of glycine (30 nmol in 60 nl), but not saline vehicle, into the rRPa to inhibit local neurons produced an immediate reversal of the MTIIevoked increase in IBAT SNA (−103 ± 3% of the MTII-evoked peak level of BAT SNA) and falls in BAT temperature (−1.5± 0.10 °C), expired CO2 (−1.3 ± 0.22%) and HR (−89 ± 31 bpm) to levels not different from their control values prior to MTII administration (Fig. 1). The histological localization of the glycine microinjection sites indicated that they were concentrated in the rRPa (Fig. 2), between the pyramids at the level of the caudal third of the facial nerve nucleus.

Fig. 2 – Localization of glycine nanoinjection sites in rat rostral raphe pallidus (rRPa). The medullary sites of glycine nanoinjection are shown on a representative histological section through the rostral medulla (panel A) and a composite mapping on a modified atlas drawing (panel B) approximately 11.3 mm caudal to bregma. Arrow in panel A indicates location of fast green dye spot in rRPa/RMg area. Filled circles in panel B indicate the locations of dye spots marking the injection sites in the rRPa region of the ventromedial medulla in 8 rats. Abbreviations: LPGi, lateral paragigantocellularis; py, pyramid; rRPa, rostral raphe pallidus; RMg, raphe magnus; SpV, trigeminal spinal nucleus; PrH, prepositus hypoglossi; Sol, nucleus of the solitary tract; 7n, facial nucleus.

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2.2. Effects of functional inhibition of rRPa neurons on the O2 consumption induced by 3rd ventricle injection of the melanocortin agonist, MTII, in C57BL/6J mice To assay the potential role of rRPa neurons in mediating the thermogenesis following activation of hypothalamic MC4R signaling, we determined the effect on the increase in O2 consumption induced by 3rd ventricle injection of MTII (1 nmol in 2 μl) of inhibiting neuronal activity in the rRPa and adjacent area by microinjection of muscimol (60 pmol in 0.3 μl) into rRPa and adjacent area in conscious C57BL/6J mice. As shown in Fig. 3, within 1 h of the treatments, the average O2 consumption in the 3rd ventricle saline/rRPa saline-treated control group was 3258 ± 69 ml/kg/h, while in the 3rd ventricle MTII/rRPa salinetreated group, the average O2 consumption was 4740.0 ± 67 ml/ kg/h — an increase (p < 0.0001) of 45% above that observed in control. The average O2 consumption in the 3rd ventricle MTII/ rRPa muscimol group was 2141 ± 214 ml/kg/h (p < 0.0001 compared with that in the 3rd ventricle MTII/rRPa saline group). These data indicate that 3rd ventricle injection of MTII significantly increases O2 consumption in conscious mice, which is completely reversed by inhibition of activity of neurons in the rRPa and adjacent region, suggesting that the functional integrity of the neurons in the rRPa and adjacent area is critically required for the enhanced thermogenesis following activation of hypothalamic MC3/4R signaling.

2.3. Effects of inhibiting rRPa neurons on the O2 consumption induced by 4th ventricle injection of MTII in C57BL/6J mice To determine if activation of brainstem MC3/4R signaling stimulates thermogenesis and if the activity of neurons in the rRPa

Fig. 3 – Effect of functional inhibition of neurons in the rRPa and adjacent area on O2 consumption induced by 3rd ventricle injection of the melanocortin agonist, MTII, in C57BL/6J mice. The average O2 consumption measured within 1 h following 3rd ventricle injection of the MC3/4 R agonist, MTII (1 nmol/ 2 μl), was significantly increased by 45% (***p < 0.0001, saline/ saline group vs MTII/saline group), and this increase in O2 consumption was reversed by microinjection of muscimol (60 pmol in 0.3 μl) into the rRPa area (###p < 0.0001, MTII/saline group vs MTII/muscimol group). The average O2 consumption within 1 h following rRPa microinjection of muscimol (60 pmol in 0.3 μl) was also significantly reduced compared with the saline/saline group (&&p < 0.01).

Fig. 4 – Effect of functional inhibition of neurons in the rRPa and adjacent area on O2 consumption induced by 4th ventricle injection of the melanocortin agonist, MTII, in C57BL/6J mice. The average O2 consumption measured within 1 h following 4th ventricle injection of the MC3/4 R agonist, MTII (1 nmol/2 μl), was significantly increased by 44% (**p < 0.01, saline/saline group vs MTII/saline group) and this increase in O2 consumption was reversed by microinjection of muscimol (60 pmol in 0.3 μl) into the rRPa area (##p <0.01, MTII/saline group vs MTII/muscimol group). The average O2 consumption measured within 1 h following rRPa microinjection of muscimol was reduced compared with the saline/saline group (p =0.0535, saline/muscimol group vs saline/saline group, p =0.0572, MTII/ muscimol group vs saline/saline group).

and adjacent area is also required for brainstem melanocortinmediated thermogenesis, we tested the effects of 4th ventricle injection of MTII (1 nmol in 2 μl) on O2 consumption and the effect of microinjecting muscimol (60 pmol in 0.3 μl) into rRPa and adjacent areas on the 4th ventricle MTII-evoked changes in O2 consumption in conscious C57BL/6J mice. As shown in Fig. 4, within 1 h of the treatments, the average of O2 consumption in 4th ventricle saline/rRPa saline-treated control group was 2879± 89.2 ml/kg/h and in the 4th ventricle MTII/rRPa saline-treated group was 4143.0 ± 235.8 ml/kg/h, 44% higher than that observed in control (p < 0.01); however, the average of O2 consumption in the 4th ventricle MTII/rRPa muscimol group was 2155± 295.2 ml/ kg/h (p < 0.01, comparing with the 4th ventricle MTII/rRPa saline group). These data indicate that 4th ventricle injection of MTII, potentially activating brainstem MC3/4R signaling, can also significantly increase O2 consumption in conscious mice and this increase in O2 consumption is also completely reversed by inhibition of activity of neurons in the rRPa and adjacent area. These data suggest that the integral functional activity of neurons in the rRPa and its adjacent area is also critically required for the increase in O2 consumption evoked by activation of brainstem MC3/4R signaling.

3.

Discussion

The present studies show that lateral ventricle injection of melanocortin agonist, MTII, stimulates IBAT SNA, IBAT temperature, expired CO2 and heart rate in anesthetized and artificially ventilated rats and that 3rd or 4th ventricular injection of

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MTII increases O2 consumption in conscious C57Bl/6J mice. The increased IBAT SNA, IBAT temperature, expired CO2 and heart rate in rats and the increased O2 consumption in mice were abolished by inhibition of neurons in the rRPa and its adjacent area with microinjections of either glycine or muscimol. These data are consistent with previous observations that there are independent sites within the hypothalamus and within the caudal brainstem at which MC3/4R stimulation can evoke increases in IBAT metabolism and thermogenesis (Cowley et al., 1999; Haynes et al., 1999; Williams et al., 2003; Yasuda et al., 2004; Zheng et al., 2005) and provide direct evidence that activation of neurons in the rostral ventromedial medulla, including the rRPa/RMg, is essential for the increases in IBAT SNA, IBAT metabolism and IBAT thermogenesis and for the augmented O2 consumption that accompanies stimulation of BAT metabolism induced by activation of either hypothalamic or brainstem MC3/4R signaling. The critical role played by rostral ventromedial medullary neurons in mediating these MTIIevoked stimulations of BAT thermogenesis likely derives from the existence of sympathetic premotor neurons for IBAT within the rRPa area, providing the essential excitatory input to IBAT sympathetic preganglionic neurons controlling the level of IBAT thermogenesis (Cano et al., 2003; Morrison, 2001b). Although the microinjection volume (0.3 μl) used in the conscious mice was relatively large, potentially allowing muscimol to diffuse to areas outside the rRPa, the complimentary results obtained in the anesthetized rat with a much smaller microinjection volume (60 nl) provide support for our overall conclusion that the neurons in the rRPa area are required for the melanocortininduced BAT thermogenesis. In fact, besides the rRPa, neurons in immediately adjacent areas, including the RMg, part of GiA and the parapyramidal region also project to T3 of the IML (Sasek and Helke, 1989; Sasek et al., 1990) and polysynaptically influence IBAT function (Bamshad et al., 1999; Cano et al., 2003; Voss-Andreae et al., 2007). Therefore, the rRPa and its adjacent area may comprise a functional complex in which the neurons are necessary to relay the melanocortin-mediated thermogenesis. MC4R mRNA is widely expressed in the brain, including areas overlapping with the central circuits that control the sympathetic regulation of IBAT thermogenesis. These include (a) hypothalamic sites in the paraventricular nucleus of the hypothalamus (PVH), lateral hypothalamic area (LH), dorsomedial hypothalamic nucleus (DMH) and ventral medial hypothalamic (VMH) nucleus, (b) brainstem sites such as RPa, lateral parabrachial nucleus (LPB), locus coeruleus (LC), lateral paragigantocellular reticular nucleus (PGL) and nucleus of the solitary tract (NTS) and (c) spinal sites in the intermediolateral cell column of the spinal cord (IML) (Kishi et al., 2003; Liu et al., 2003; Mountjoy et al., 1994). Using retrograde trans-synaptic tracing with attenuated pseudorabies virus (PRV) injected into the IBAT of MC4R-GFP transgenic mice (Liu et al., 2003) coupled with dual-label immunohistochemical analysis, we have recently demonstrated that specific subsets of MC4-R-expressing neurons in multiple nuclei of the CNS known to regulate sympathetic outflow are poly-synaptically connected to IBAT. The specific subset of dual-labeled MC4R-GFP/PRV-immunoreactive (IR) neurons include neurons in the IML in the thoracic spinal cord, in the rRPa, gigantocellular reticular nucleus, LC and NTS

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in the brainstem and in the PVH and DMH in the hypothalamus (Voss-Andreae et al., 2007). These studies, delineating both the neuroanatomical substrates of the specific circuit regulating IBAT thermogenesis and the potential sites of action of the central melanocortins indicate a distribution of MC4R that is consistent with a role for the central melanocortin system in regulating the sympathetic outflow to IBAT. Indeed, as in the present and other studies, lateral or third ventricular administration of either MTII or α-MSH, which would be expected to mainly activate hypothalamic MC3/4R, but could also diffuse through the cerebrospinal fluid to act at sites accessible from the fourth ventricle, increases oxygen consumption or produces IBAT sympathoexcitation and increases in IBAT temperature and UCP1 mRNA expression (Haynes et al., 1999; Li et al., 2003, 2004; Williams et al., 2003; Yasuda et al., 2004). MTII injected into the 4th ventricle, which would be expected to activate mainly brainstem MC3/4R, but could also act at sites in the spinal cord, can also stimulate oxygen consumption, consistent with the observation that 4th ventricular application of MTII stimulates IBAT UCP1 mRNA expression in chronically decerebrate rats where it would not have access to 3rd ventricular sites (Williams et al., 2003) and that 4th ventricle injection of MTII increases body temperature and heart rate (Zheng et al., 2005). Thus, although we can not determine the specific locations of MC3/4R responsible for the IBAT sympathoexcitation and augmented O2 consumption following ventricular administrations of MTII, the metabolic stimulation following 4th ventricular injection of MT II supports the hypothesis that the brainstem contains sites sensitive to α-MSH which can regulate the level of IBAT energy expenditure through activation of IBAT sympathetic premotor neurons in the rRPa and adjacent area. The finding of dual-labeled MC4-R-GFP/PRV-IR neurons in multiple brainstem nuclei, including the RPa, gigantocellular reticular nucleus, LC and NTS following PRV injections into IBAT (Voss-Andreae et al., 2007), raises the possibility that all these sites are potential targets for 4th ventricle injection of MTII to stimulate thermogenesis, and IBAT sympathetic premotor neurons in rRPa may be directly responsive to melanocortin signaling. It should be noted that BAT is a major thermogenic organ in rodents and it is estimated that thermogenesis in BAT may contribute to more than one-half of all oxygen consumption in a small animal in the cold (Cannon and Nedergaard, 2004), however, increased metabolism in other oxygen-consuming tissues besides BAT, including alteration of cardiac activity, could have contributed to the VO2 determinations that were made in the mouse experiments. Handling- or stress-induced thermogenesis also could have increased the level of oxygen consumption measured in both control and treatment groups, however, it could not have accounted for the differences between them. Previous studies have shown that icv co-administration of the MC3/4R antagonist, SHU9119, with MTII completely blocks the suppression of feeding induced by icv injection of MTII (Fan et al., 1997) and that SHU9119 completely blocked the increase in IBAT SNA produced by MTII (Haynes et al., 1999). Furthermore, MTII inhibits food intake and stimulates metabolic rate and oxygen consumption in wild-type mice but not in MC4R knockout (KO) mice (Balthasar et al., 2005; Chen et al., 2000; Marsh et al., 1999). Our unpublished data also showed that 3rd ventricle injection of 1 nmol MTII significantly increased VO2

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in WT and MC3R KO mice, but not in MC4R KO mice. These data indicate that MTII acts on the MC4R to exert its anorexic effects. Neuroanatomical studies have shown that the rRPa receives direct projections from both hypothalamic nuclei, including preoptic area, arcuate nucleus, PVH, LH, and DMH (Hermann et al., 1997; Hosoya, 1985), as well as from brainstem nuclei, including periaqueductal gray, retrorubral field, KF, parabrachial nuclei, subcoeruleus, gigantocellular reticular nucleus, LPG and raphe magnus (Bacon et al., 1990; Hermann et al., 1997; Nogueira et al., 2000). As described above, MC4R mRNA is also expressed in some of these nuclei. In turn, neurons in rRPa project directly to spinal sympathetic preganglionic neurons and to interneurons in the IML (Bacon et al., 1990; Bowker and Abbott, 1990; Bowker et al., 1983; Leanza et al., 1995; Loewy et al., 1981; Nevin et al., 1994; Sasek and Helke, 1989; Sasek et al., 1990). These data provide a potential anatomical substrate for IBAT sympathetic premotor neurons in the rRPa area to excite IBAT sympathetic preganglionic neurons and IBAT thermogenesis in response to direct inputs from the hypothalamus, some of which may be activated by melanocortin receptor agonists. Functional studies in anesthetized rats indicate that the rRPa neurons controlling IBAT thermogenesis receive a strong, tonic inhibition (Morrison et al., 1999), as well as tonic excitatory synaptic inputs which can be reversed by rRPa microinjection of the GABAA receptor antagonist, bicuculline, or the GABAA receptor agonist, muscimol, respectively (Cao et al., 2004; Morrison et al., 1999; Nakamura et al., 2002; Taniguchi et al., 2003; Zaretskaia et al., 2002). In addition, the following findings are consistent with the existence of IBAT sympathetic premotor neurons in rRPa (Morrison, 2001b): The sympathetically-mediated increase in IBAT thermogenesis evoked by central administration of PGE2 is prevented by blockade of excitatory amino acid (EAA) receptors within the rRPa (Madden and Morrison, 2003), the leptin-evoked increase in IBAT thermogenesis is inhibited by activation of 5-HT1A receptors in rRPa (Morrison, 2001a) and microinjection of muscimol in the area of rRPa reduces body temperature in conscious freely moving rats (Zaretsky et al., 2003). Our results, demonstrating that inhibition of the activity of neurons in the rRPa and its adjacent area abolishes the increases in IBAT SNA and in O2 consumption induced by either stimulation of hypothalamic or brainstem MC3/4R, indicate that the functional integrity of neurons in the rRPa and its immediate vicinity is critically required for activation of both hypothalamic and brainstem MC3/4R signaling-induced increases in IBAT thermogenesis. These data further support the role of IBAT sympathetic premotor neurons in the rRPa and its adjacent area in regulating thermogenic energy expenditure by the CNS, including the central melanocortin system. It is noteworthy that the average O2 consumption within 1 h following rRPa microinjection of muscimol is significantly reduced compared with the saline/saline group (Fig. 3), suggesting that activity of neurons in the rRPa and adjacent region also may also play an important role in the maintenance of basal metabolism. Additionally, it is noteworthy that in the rat, MT3/4R receptor activation by MTII administration elicited a marked increase in heart rate that was reversed by inhibition of neurons in the rRPa area. These results are consistent with the simultaneous, sympathetically-mediated, rRPa neurondependent increases in BAT metabolism and heart rate ob-

served in several other studies (Cao et al., 2004; Madden and Morrison, 2003; Morrison, 2001a, 2004b; Morrison et al., 1999), which may be understood from the need to increase cardiac output in order to appropriately distribute the increased heat generated by BAT stimulation. A likely substrate for the cardiac stimulation resides in the existence of cardiac sympathetic premotor neurons in the rRPa (Cao and Morrison, 2003). The present data indicate that the neuronal activity in the rRPa and adjacent area is critical to the melanocortin-induced increase in IBAT thermogenesis. However, other brain regions with direct projections to the IML, including the PVH in the hypothalamus and the rostral ventrolateral medulla (RVLM) in the brainstem exhibit early retrograde infection following PRV injections in IBAT and thus may contain putative IBAT sympathetic premotor neurons (Bamshad et al., 1999; Cano et al., 2003; Voss-Andreae et al., 2007). Although the potential roles of the PVH-IML and RVLM-IML pathways in the sympatheticallymediated IBAT thermogenesis induced by activation of the hypothalamic or brainstem MC3/4R signaling were not examined in this study, the finding that inhibition of neurons in the rRPa and adjacent area completely reverses the MTII-evoked stimulation of thermogenesis suggests that the rRPa-IML pathway may be predominant in mediating the thermogenic responses induced by activation of hypothalamic or brainstem MC3/4R signaling. Thus, any functional role of the PVH-IML or RVLM-IML pathways in the regulation of thermogenesis would depend on the integrity of the BAT sympathetic premotor neurons in the rRPa and adjacent area.

4.

Experimental procedures

4.1.

Animals and procedures

Male Sprague–Dawley rats (Charles River, Indianapolis, IN, USA) weighing 300–500 g and male C57BL/6J mice (Jackson Labs) weighing 22–30 g were used in these studies. The animals were housed upon arrival on a 12-h light:12-h dark cycle (lights off at 18:00 h) with free access to standard pellet diet and water. All procedures were performed according to the regulations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals under the protocols approved by the Animal Care and Use Committee of the Oregon Health and Science University. Animal numbers and experimental procedures were selected to minimize the number of animals used and animal suffering.

4.2.

Sympathetic nerve recording in rats

Rats were initially anesthetized with 3% isoflurane in 100% O2, allowing cannulation of the femoral artery for monitoring arterial pressure and the femoral vein for drug administration. Subsequently, the animals were transitioned to urethane (1 g/kg, iv) and α-chloralose (80 mg/kg, iv). The trachea was cannulated for artificial ventilation. The animals were paralyzed with D-tubocurarine (initially 0.6 mg/rat, iv, thereafter 0.3 mg/h iv) and artificially ventilated with 100% O2 at a minute volume of 180–300 ml. Endtidal (expired) CO2 was continuously monitored and maintained between 3.5% and 4.5% by adjusting ventilation volume and rate. The rats were positioned prone in a stereotaxic frame with the

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incisor bar approximately −3.8 mm below the interaural line (Paxinos and Watson, 1998). Colonic temperature was maintained at 37 °C with a thermostatically controlled heating pad and lamp. IBAT temperature was continuously monitored (Sable Systems TC-1000 thermocouple reader, Las Vegas, NV, USA) with a thermocouple (Physitemp, Clifton, NJ, USA) inserted into the left IBAT pad. Right postganglionic IBAT SNA was recorded from a small nerve bundle dissected from the ventral surface of the right IBAT pad. The central cut end of the IBAT nerve was placed on a bipolar hook recording electrode and immersed in mineral oil to avoid drying. Nerve activity was differentially amplified (10,000 to 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, UK).

4.3.

Intracerebroventricular (icv) and rRPa cannulation in mice

Mice were anesthetized by intraperitoneal injection of 0.1 cc mouse cocktail (Rompun, 20 mg/ml; Acepromazine, 10 mg/ml; Ketamine, 100 mg/ml) prepared by the Oregon Health and Sciences University veterinary staff. After the scalp fur was clipped, the animals were placed into a stereotaxic device (Cartesian Instruments, Bend OR). A small midline incision was made over the dorsal scalp to provide access to the cranium under aseptic conditions. The cranial surface was cleaned with hydrogen peroxide and two small holes were drilled for stereotaxic implantation of sterile 25 gauge stainless steel guide cannulae with obdurator stylets. The first was in the 3rd ventricle (coordinates: 0.82 mm posterior to the bregma, on the midline, and 4.8–4.82 mm below the bregma) or the 4th ventricle (coordinates: 5.85–6.0 mm posterior to bregma, on the midline, and 3.9–4.1 mm below bregma). In the same mouse, the second was targeted to the rRPa (coordinates: 6.5–6.64 mm posterior to and 4.85–4.88 mm below bregma, according to the Atlas of the Mouse Brain; Franklin and Paxinos, 1997). The cannulae were fixed into place with cyanoacrylate and dental cement. Mice were housed individually following the procedure and allowed 1 week for recovery before experiments.

4.4.

Indirect O2 consumption (VO2) measurement

Cannulated mice were allowed to adapt to the experimental conditions by being placed in the chambers of an indirect open-circuit calorimeter (Oxymax, Columbus Instruments) for the experimental time period each day for at least 2–3 days. On the day of the experiment, mice were placed in the measurement chambers for about 2 h (10:00–12:00 h) for acclimatization, then briefly removed from the calorimeter for icv injection of MTII (1 nmol/2 μl) or saline (2 μl), performed first, and then rRPa microinjection of either muscimol (0.2 mM/ 0.3 μl) or saline (0.3 μl), after which the mice were returned to the chambers for measurement of O2 consumption. Each experiment consisted of 4 groups of mice: icv saline/rRPa saline; icv MTII/rRPa saline; icv MTII/rRPa muscimol; icv saline/ rRPa muscimol. The measurements began at around 12– 13:00 h and the O2 consumption was recorded every 15 min

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with an inlet fresh air flow rate of 0.6 l/min and a sample flow rate of 0.5 l/min. Each chamber was sampled for 60 s, with a resettling time of 120 s.

4.5.

Administration of drugs

For icv injection in rat, a small hole was drilled in the parietal bone and a 30 gauge stainless steel injection tube connected with PE 10 tubing to a 10 μl syringe was stereotaxically inserted into the lateral ventricle (coordinates: 1.5 mm lateral, 1.0 mm posterior to bregma and −3.4 to −3.6 mm below the skull surface (Paxinos and Watson, 1998)). Drug or saline was infused within 2 min. For rRPa nanoinjection (60 nl) of saline or glycine in the rat, initially, a tungsten microstimulating electrode (30-μm exposed tip) and, subsequently, a nanoinjection pipette (tip outside diameter, 20 μm) were positioned stereotaxically in the rRPa. Relative to lambda, the coordinates for the rRPa were approximately anteroposterior −3.0 mm, mediolateral 0.0 mm, dorsoventral −9.5 mm below the dural surface (Paxinos and Watson, 1998). The optimal dorsoventral site for nanoinjection into rRPa was determined as that yielding the lowest microstimulation threshold (< 10 μA) for evoking an excitatory potential on the BAT sympathetic nerve with twin pulses (1-ms duration, 6-ms interpulse interval, 0.4 Hz, <50μA) applied to rRPa. The animals were then transcardially perfused with formalin and 40 μm brain sections were cut using a cryostat for histological localization of the nanoinjection sites which were plotted on camera lucida drawings of sections through the rostral medulla (Paxinos and Watson, 1998). For mouse icv injection, the injection cannulae were made of 32 gauge stainless steel tube, 0.4 mm longer than the guide cannula with a beveled tip and 2 μl of saline or drugs was injected within 1 min. For mouse rRPa microinjection, the injection tube protruded 1 mm beyond the guide cannula and 0.3 μl of saline or drug was injected within 1–2 min with a manual microsyringe driver (Stoelting). The dose of 1 nmol MTII was used because it significantly increased the IBAT SNA in anesthetized rat and VO2 in freely-moving mouse in our preliminary experiments, thus allowing us to be able to assess the role of the neurons in the rRPa area in the MTII-induced thermogenic energy expenditure. The potential damage from the microinjector was accounted for since we compared the effects of rRPa area microinjection of vehicle with that of muscimol.

4.6.

Data analysis

For analysis of rat BAT SNA, Spike 2 software was used to obtain a continuous measure (4-s bins) of SNA amplitude by calculating the root mean square amplitude of the SNA (square root of the total power in the 0–20 Hz band) from the autospectra of sequential 4-s segments of SNA (Fig. 1, upper trace). The control value of BAT SNA was the average of the BAT SNA amplitudes during the 60-s period immediately prior to icv MTII injection. The peak increase in BAT SNA evoked by icv MTII was calculated as the difference between the average of the BAT SNA values during the 60-s period of maximal change in BAT SNA following icv MTII and the control value of BAT SNA and is expressed as % of the pre-injection control

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value. The change in BAT SNA evoked by nanoinjection of glycine into rRPa was calculated as the difference between the peak level of BAT SNA and the average of the BAT SNA values during the 60-s period of minimal BAT SNA following nanoinjection of glycine into rRPa and is expressed as % of the peak increase evoked by icv MTII. Data are expressed as the mean ± S.E.M. Statistical comparisons were made with paired t tests with p < 0.05 indicating a significant difference. Data from mice are expressed as the mean ± S.E.M. Statistical comparisons were made with one-way ANOVA followed by the Dunnett's multiple comparison or an unpaired t test with p < 0.05 indicating a significant difference.

Acknowledgments This work is supported by the US National Institutes of Health Grants: NIDDK 62179 (WF) and NIDDK 57838 (SFM).

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