To elucidate the central neural pathways contributing to the thermogenic component of the autonomic response to intravenous administration of leptin, experiments were conducted in urethane-chloralose-anesthetized, ventilated rats to address 1) the role of neurons in the rostral ventromedial medulla, including raphe pallidus (RPa), in the leptin-evoked stimulation of brown adipose tissue (BAT) sympathetic nerve activity (SNA); and 2) the potential thermolytic effect of 5-hydroxytryptamine1A (5-HT1A) receptors on RPa neurons that influence BAT thermogenesis. Leptin (1 mg/kg) administration increased BAT SNA by 1,219% of control, BAT temperature by 2.8°C, expired CO2 by 1.8%, heart rate by 90 beats/min, and mean arterial pressure by 12 mmHg. Microinjection of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) into RPa resulted in a prompt and sustained reversal of the leptin-evoked stimulation of BAT SNA, BAT thermogenesis, and heart rate, with these variables returning to their pre-leptin control levels. Subsequent microinjection of the selective 5-HT1A receptor antagonist WAY-100635 into RPa reversed the BAT thermolytic effects of 8-OH-DPAT, returning BAT SNA and BAT temperature to the elevated levels after leptin. In conclusion, activation of neurons in RPa, possibly BAT sympathetic premotor neurons, is essential for the increases in BAT SNA, BAT thermogenesis, and heart rate stimulated by intravenous administration of leptin. Neurons in RPa express 5-HT1A receptors whose activation leads to reversal of the BAT thermogenic and the cardiovascular responses to intravenous leptin, possibly through hyperpolarization of local sympathetic premotor neurons. These results contribute to our understanding of central neural substrates for the augmented energy expenditure stimulated by leptin.
- sympathetic nerve activity
- temperature regulation
- ventromedial medulla
- energy expenditure
leptin, an anorexigenic hormone produced by adipose tissue, binds to specific receptors within the central nervous system to influence neuronal networks that govern energy balance as well as those that determine sympathetic tone to cardiovascular targets. In addition to a reduction in appetite and food intake, activation of central leptin receptors leads to an increase in energy expenditure, which, in small mammals and the young of larger mammals, occurs, at least in part, through an increase in the sympathetic outflow to brown adipose tissue (BAT) (17) and the resulting stimulation of BAT metabolism and BAT thermogenesis. Although the neural circuits through which leptin alters energy balance remain to be defined, considerable evidence indicates that neurons within the arcuate nucleus of the hypothalamus play a key role in transducing the blood-borne leptin signal (11, 12, 16). Because the neural circuits between the hypothalamic neurons directly influenced by leptin and the spinal sympathetic preganglionic neurons controlling BAT thermogenesis are not fully understood, the goal of this study was to provide information on the medullary neuronal populations mediating the leptin-evoked increase in BAT SNA and thermogenesis.
The rostral medullary raphe nuclei, including raphe pallidus (RPa) and the surrounding ventromedial medulla, have been identified as containing neurons essential for the sympathetic regulation of target tissues, including BAT, that are involved in thermoregulation and energy metabolism. Several lines of evidence converge to suggest that the RPa contains sympathetic premotor neurons for BAT (32): neurons in this region project to the thoracic intermediolateral nucleus (23), they are among the earliest medullary neurons infected after pseudorabies virus injections into interscapular BAT (2, 9), their activation leads to large increases in BAT sympathetic nerve activity (SNA) and thermogenesis (25, 32), and they are essential for the increase in BAT SNA and BAT thermogenesis that contributes to the febrile response elicited by PGE2 (31, 33). In the present study, the hypothesis that RPa neurons play an essential role in mediating the leptin-evoked stimulation of BAT SNA and BAT thermogenesis was tested by determining the effect of an 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT)-mediated inhibition of neurons in RPa on the increase in BAT SNA and BAT temperature after leptin administration. Some of these results have been presented in abstract form (29).
MATERIALS AND METHODS
Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996) and under protocols approved by the Institutional Animal Care and Use Committee of Oregon Health and Science University. Sprague-Dawley rats (n = 8, 240-400 g) were obtained from Charles River. Animals were anesthetized intravenously with urethane (0.8 g/kg) and chloralose (80 mg/kg) after induction with 3% isoflurane in 100% O2. A femoral artery, a femoral vein, and the trachea were cannulated for measurement of arterial pressure, drug injection, and artificial ventilation, respectively. Heart rate (HR) was derived from the arterial pressure signal. After the animals were positioned prone in a stereotaxic frame with the incisor bar at -3.8 mm and with a spinal clamp on the T10 vertebra, they were paralyzed with d-tubocurarine (0.3 mg initial dose, 0.1 mg/h supplements) and artificially ventilated with 100% O2 (50 cycles/min, tidal volume 3-4.5 ml). Small adjustments in minute ventilation were made as necessary to maintain basal mixed-expired CO2 levels between 3.5 and 4.5%. Colonic temperature was maintained at 37.5°C with a thermostatically regulated heat lamp and heating plate beneath the animal.
Postganglionic SNA to BAT was recorded from the central cut end of a small nerve bundle dissected from the ventral surface of the right interscapular BAT pad after dividing the fat pad along the midline and reflecting it laterally. Nerve activity was recorded with bipolar hook electrodes in a monopolar configuration, filtered (1-300 Hz), and amplified (50,000, Cyberamp 380, Axon Instruments). BAT temperature was measured by placing a thermistor (Physitemp) beneath the left half of the interscapular BAT pad, which was left intact. BAT SNA, BAT temperature, colonic temperature, expired CO2, arterial pressure, and stimulus trigger pulses were digitized (1 kHz) and recorded on VCR tape (Neurodata) and computer hard drive (Axoscope, Axon Instruments).
Initially, a tungsten microstimulating electrode (30-μm exposed tip) and, subsequently, a microinjection pipette (tip outside diameter, 20 μm) were positioned stereotaxically in the RPa. Relative to lambda, the coordinates for the RPa were approximately anteroposterior -3.0 mm, mediolateral 0.0 mm, dorsoventral -9.5 mm below the dural surface. The optimal dorsoventral site for microinjection into RPa 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) applied to RPa. At the end of each experiment, the microinjection pipette was retracted vertically from the RPa, refilled with a 4% solution (pH 8.0) of fast green dye, and lowered to the site of microinjection, and dye was electrophoretically deposited (15-μA anodal direct current for 15 min). After perfusion and histological processing, the locations of the microinjection sites in the RPa (Fig. 1) were plotted on camera lucida drawings of sections through the rostral medulla (38).
During each experiment, BAT SNA, BAT and colonic temperatures, expired CO2, arterial pressure, and HR were recorded during a control period of at least 30 min and after subsequent intravenous administration of murine leptin [Amgen, 1 mg/kg given as an initial bolus of 0.5 mg/kg in 0.5 ml saline followed by an infusion of 0.5 mg/kg in 5 ml over 1 h (17)]. The dose of leptin used in this study has been shown to produce a large and sustained increase in BAT SNA (17) on which the effects of altering neuronal discharge in RPa could be tested. The effects on leptin-evoked responses of microinjecting the following agents into RPa were determined: 1) saline vehicle (60 nl), applied 10-20 min after the onset of the leptin-evoked increase in BAT SNA; 2) the 5-hydroxytryptamine1A (5-HT1A) receptor agonist 8-OH-DPAT (Sigma, 10 mM, 60 nl), applied 30-60 min after the onset of the leptin-evoked increase in BAT SNA; and 3) the selective, competitive 5-HT1A receptor antagonist WAY-100635 (Sigma, 10 mM, 60 nl), applied 10-20 min after the microinjection of 8-OH-DPAT.
Spectral analysis was used to determine the amplitude of BAT SNA (42). Throughout each experiment, the autospectra of sequential 4-s segments of BAT SNA were calculated and the amplitude of BAT SNA in these 4-s bins was determined as the square root of the sum of the power values (rms power) in the 0- to 20-Hz frequency range. For control or drug treatment conditions, the level of BAT SNA was the average of the eight 4-s BAT SNA values in the 32 s of BAT SNA before a treatment or in the 32 s surrounding the maximal or minimal BAT SNA produced by the treatment.
For determination of plasma catecholamine levels, 1-ml arterial blood samples were taken 10 min before the onset of the intravenous leptin administration and at the peak of the increase in BAT SNA. Blood was replaced with an equal volume of a 30% solution of rat plasma (Sigma Chemical). Blood samples were centrifuged at 3,000 rpm for 20 min at 4°C, and the plasma was stored at -80°C until the catecholamine assay was performed. Catecholamine levels in plasma were assayed by liquid chromatography with electrochemical detection. The method combined liquid:liquid extraction of catecholamines from plasma with reversed-phase chromatography incorporating a cation-exchange reagent (Plasma Catecholamine Kit, Bioanalytical Systems, West Lafayette, IN) (24, 27). For replicate determinations of a 1.0-ml plasma sample containing 0.25 pmol epinephrine and 1.6 pmol norepinephrine, intra-assay coefficients of variation were 6 and 3%, respectively, and the interassay coefficients of variation were 6 and 4%, respectively.
All data are presented as means ± SE, unless otherwise noted. Statistical differences were assessed with Student's two-tailed paired t-test and Bonferroni correction (P < 0.05).
Leptin (1 mg/kg iv) was administered to eight urethane-chloralose-anesthetized rats whose average mean arterial pressure (MAP) and HR were 110 ± 4 mmHg and 359 ± 18 beats/min, respectively. Under control conditions, with the rat's core temperature maintained between 36.5 and 37.5°C, the activity on the sympathetic postganglionic nerve to the interscapular BAT was very low, exhibiting only occasional, low-amplitude bursts and reflecting the absence of either thermoregulatory or metabolic drives for BAT thermogenesis. As illustrated in the example in Fig. 2, leptin administration produced increases in the sympathetic outflow to interscapular BAT (peak, +1,475% of control), in BAT temperature (peak, +3.3°C), in expired CO2 (peak, +1.9%), in core temperature (peak, +0.5°C), in HR (peak, +148 beats/min), and in MAP (peak, +29 mmHg). The mean peak values of the BAT thermogenic and cardiovascular parameters after leptin administration are presented in Table 1. Leptin evoked a mean peak increase of +1,219 ± 298% (P < 0.001) in BAT SNA, which produced a mean peak increase in BAT temperature of +2.8 ± 0.5°C (P < 0.001). These were accompanied by a mean peak increase in expired CO2 of +1.8 ± 0.3% (P < 0.001), a mean maximum rise in core temperature of +0.4 ± 0.08°C (P < 0.01), a mean peak tachycardia of +90 ± 18 beats/min (P < 0.01), and a mean pressor response of +12 ± 4 mmHg (P < 0.05).
In addition to BAT thermogenesis, leptin administration also stimulated adrenal catecholamine release. Plasma epinephrine, measured at 50 ± 5 min after beginning the intravenous leptin infusion, was increased by 101 ± 65% (P < 0.05, n = 6) from a control level of 1.35 ± 0.40 pmol/ml. Plasma norepinephrine, reflecting both adrenal release and spillover from sympathetic nerve terminals, rose by 179 ± 48% (P < 0.05) from a control level of 1.82 ± 0.23 pmol/ml.
Microinjection of the 5-HT1A receptor agonist 8-OH-DPAT into RPa shortly after the leptin-evoked increase in BAT SNA reached peak levels produced a prompt reversal (Fig. 2) of the leptin-stimulated rise in BAT SNA (maximum reduction, -101% of the leptin-evoked increase), in BAT temperature (peak reduction, -2.2°C), in expired CO2 (maximum fall, -1.4%), and in HR (peak bradycardia, -129 beats/min). Microinjection of 8-OH-DPAT into RPa also resulted in small reductions in MAP (maximum fall, -7 mmHg) and core temperature (decline, -0.1°C), but these were less than the increases after leptin administration. In the absence of further treatment, the lowered levels of BAT thermogenesis and HR elicited by microinjection of 8-OH-DPAT into RPa were sustained for at least 30 min. The mean nadir values of the BAT thermogenic and cardiovascular parameters after 8-OH-DPAT microinjection into RPa are presented in Table 1. Application of 8-OH-DPAT to RPa neurons during the sustained thermogenesis stimulated by intravenous leptin resulted in a mean maximum fall in BAT SNA of -96 ± 15% (P < 0.01, n = 6) of the leptin-evoked increase in BAT SNA, resulting in a mean maximum fall in BAT temperature of -2.1 ± 0.3°C (P < 0.005) from the peak BAT temperature levels evoked by leptin administration. These declines in sympathetically mediated BAT thermogenesis were accompanied by maximal decreases in expired CO2 of -1.6 ± 0.2% (P < 0.001), in HR of -109 ± 12 beats/min (P < 0.001), and in MAP of -15 ± 7 mmHg (P < 0.05) from the peak levels evoked by leptin. The mean minimum values of BAT SNA, BAT temperature, HR, and expired CO2 after microinjection of 8-OH-DPAT into RPa were not significantly different from the control values before leptin administration. Microinjection of saline vehicle into RPa had no effect (Fig. 2) on any of the leptin-evoked increases in the parameters measured in these experiments.
To establish the specificity of the effects of 8-OH-DPAT to its activation of 5-HT1A receptors in RPa, the selective, 5-HT1A receptor antagonist WAY-100635 was microinjected into RPa 17 ± 1 min after the 8-OH-DPAT microinjection in five rats. Microinjection of WAY-100635 into RPa produced a prompt reversal (Fig. 2) of the 8-OH-DPAT-evoked inhibition of BAT thermogenesis and reinstated the leptin-evoked activation of BAT SNA (maximum increase, +1,628% of the untreated control before leptin) and the accompanying increases in BAT temperature (peak increase, +2.8°C), in expired CO2 (maximum rise, +1.6%), and in HR (peak tachycardia, +129 beats/min). Microinjection of WAY-100635 into RPa also increased MAP (peak, +17 mmHg) and core temperature (peak, +0.7°C). The mean peak values of the BAT thermogenic and cardiovascular parameters after local microinjection of WAY-100635 into RPa in rats treated sequentially with intravenous leptin and microinjection of 8-OH-DPAT into RPa are presented in Table 1. After leptin and 8-OH-DPAT administration, microinjection of WAY-100635 into RPa evoked a mean peak increase in BAT SNA of +1,156 ± 320% (P < 0.01) of the untreated control SNA before leptin, which produced a mean peak increase in BAT temperature of +2.2 ± 0.3°C (P < 0.005). These were accompanied by a mean peak increase in expired CO2 of +1.5 ± 0.2% (P < 0.002), a mean peak tachycardia of +99 ± 21 beats/min (P < 0.01), and a mean pressor response of +22 ± 7 mmHg (P < 0.05). The peak levels of BAT thermogenic and cardiovascular variables after WAY-100635 application to RPa neurons in leptin- and 8-OH-DPAT-treated rats were not different from those initially evoked by the leptin administration.
These data provide the first demonstration that neurons in the rostral ventromedial medulla, including the RPa, play a critical role in the stimulation of the BAT thermogenic and HR responses to intravenous administration of leptin in the rat. This conclusion was reached using the specific 5-HT1A receptor agonist 8-OH-DPAT to hyperpolarize local neurons in RPa (3, 4), potentially reducing their responsiveness to activation of leptin-sensitive pathways arising from the arcuate and caudal dorsomedial nuclei of the hypothalamus (11, 12, 16). The specific 5-HT1A receptor antagonist WAY-100635 reversed the BAT sympathoinhibitory effect of 8-OH-DPAT, as well as the falls in BAT temperature, expired CO2, and HR. The ability of a microinjection of 8-OH-DPAT into RPa to inhibit BAT SNA extends to thermoregulation the initial suggestion (19) that the RPa is one of the central sites of 5-HT1A receptors whose activation is responsible for the changes in autonomic function that occur with the clinical use of 5-HT1A receptor agonists in the treatment of depression and anxiety. Similarly, the absence of an available 5-HT1A receptor-mediated inhibitory input to RPa neurons may contribute to the exaggerated hyperthermic and HR responses to stress in 5-HT1A receptor knockout mice (37).
The finding that microinjection of 8-OH-DPAT into RPa resulted in a dramatic fall in leptin-stimulated levels of BAT SNA, BAT temperature, and expired CO2 (providing an index of acute changes in oxidative metabolism, likely reflecting that in BAT and heart) indicates the existence of 5-HT1A receptors on neurons in the RPa that influence the sympathetically driven thermogenic component of the increase in energy expenditure produced by leptin. The rostral ventromedial medulla, including the RPa, is hypothesized to be the site of sympathetic premotor neurons controlling lipid metabolism and thermogenesis in BAT (30, 32). This conclusion is based on 1) the demonstration of direct projections from neurons in RPa to the thoracic intermediolateral nucleus containing sympathetic preganglionic neurons (1, 23), 2) the identification of RPa as one of the earliest sites of retrogradely infected neurons after pseudorabies virus injections into BAT (2, 9, 34), and 3) the large increases in BAT SNA and thermogenesis produced uniquely by activation of medullary neurons in RPa, either with local microinjection of the GABAA receptor antagonist bicuculline (32) or of the glutamate receptor agonists N-methyl-d-aspartic acid or kainic acid (25). Additionally, interruption of the activity of neurons in RPa eliminates the stimulation of BAT SNA (31) and BAT thermogenesis (33) during the acute febrile response produced by central administration of PGE2 in anesthetized rats and elicits a marked reduction in core temperature in awake rats maintained at room temperature(44). Thus it seems likely that the reversal of the leptin-stimulated increase in BAT thermogenesis by microinjection of 8-OH-DPAT into RPa results from an inhibition of the discharge of BAT sympathetic premotor neurons located there. The most direct explanation for our results is that leptin activates hypothalamic neurons, which through pathways yet to be elucidated activate BAT sympathetic premotor neurons in the RPa, which in turn stimulate BAT thermogenesis. Alternatively, a certain level of tonic activity in BAT sympathetic premotor neurons in the RPa may be necessary to facilitate the excitation of BAT sympathetic preganglionic neurons by other descending pathways that are activated by leptin, such as the cocaine- and amphetamine-regulated transcript-containing neurons in the retrochiasmatic area and lateral arcuate nucleus (14).
While the data presented here do not indicate whether 8-OH-DPAT microinjected into RPa binds to 5-HT1A receptors located on BAT sympathetic premotor neurons or on an antecedent population of local excitatory interneurons, they do suggest that the well-described hypothermic effect of 5-HT1A receptor agonist administration (22, 28) could occur, at least in part, through a reduction in sympathetically regulated heat production effected by inhibition of the raphe sympathetic premotor neurons controlling the sympathetic activation of thermogenic tissues. Although Berner et al. (5) used a similar experimental design to show that injections of 8-OH-DPAT into the raphe magnus inhibited the increases in oxygen consumption and shivering normally elicited by cooling the preoptic/anterior hypothalamus (5), the large injection volumes used in their study would not distinguish 8-OH-DPAT-mediated effects on neurons in raphe magnus from those on neurons in neighboring RPa. The demonstration that 8-OH-DPAT inhibits the normal, cold-evoked increase in cutaneous sympathetic vasoconstrictor tone (35) suggests that an increase in heat loss also contributes to 5-HT1A receptor-evoked hypothermia. Interestingly, sympathetic premotor neurons regulating cutaneous blood flow are also postulated to be in the same RPa region of the ventromedial medulla (6, 7, 39), providing a potential substrate for the cutaneous heat loss contribution to 5-HT1A receptor-mediated hypothermia that would parallel that revealed here for a reduction in thermogenesis.
An anatomic substrate for the effects on BAT SNA and thermogenesis seen with microinjection of 8-OH-DPAT into RPa is found in immunocytochemical studies localizing 5-HT1A receptor binding sites on neurons in the RPa and the parapyramidal regions of the ventromedial medulla (20, 43) and in particular on raphespinal neurons projecting to the intermediolateral cell column, including a significant number of 5-HT-containing neurons in these regions (18). These results coupled with the demonstration in dorsal raphe, that 5-HT1A receptor activation hyperpolarizes both 5-HT-immunopositive and non-5-HT neurons (21), preclude any conclusion on the specific role of 5-HT-containing neurons in RPa in the regulation of BAT thermogenesis to be drawn from the 8-OH-DPAT-induced reduction in BAT SNA found in the present study. That 5-HT neurons in RPa might contribute to activation of BAT SNA is supported by the finding that some of the neurons in RPa are infected at short survival times after injection of the retrograde tracer pseudorabies virus into interscapular BAT were tryptophan hydroxylase immunoreactive (9), that serotonergic neurons in the RPa of freely moving cats increase their discharge during exposure to a low ambient temperature (26), and that cold exposure increases 5-HT synthesis, metabolism, and utilization in the rat thoracic spinal cord (36). Electron microscopic examination of labeled 5-HT1A receptors in the dorsal raphe indicated an exclusive somatodendritic location (40) suggestive of a role in mediating serotonergic effects on neuronal firing rather than transmitter release. Whether a similar localization of 5-HT1A receptors occurs in medullary RPa and parapyramidal neurons remains to be determined. Regarding a role for RPa neurons in mediating the increased energy expenditure stimulated by leptin, anatomic experiments using induction of c-fos to identify neural pathways activated by leptin administration (13, 15) did not reveal activation of neurons in the RPa region of the medulla. Although the reason for this discrepancy is unclear, it may be that the appropriate RPa neurons do not express c-fos (however, see Refs. 8, 32), that the number or distribution of RPa neurons expressing c-fos in response to leptin was not remarkable, or that activity in RPa neurons projecting to BAT sympathetic preganglionic neurons is only necessary in a permissive role to observe an increase in BAT SNA after leptin administration.
Microinjection of 8-OH-DPAT into RPa reversed the increase in HR resulting from the intravenous administration of leptin. This result indicates that the activity of neurons in RPa region is necessary not only for the leptin-evoked increase in BAT thermogenesis but also for the stimulation of cardiac sympathetic outflow mediating the accompanying tachycardia. Coupled with the demonstrations that activation of RPa neurons elicits a sympathetically mediated tachycardia (10), that neuronal activity in RPa is required for the tachycardic component of the response to central administration of PGE2 (31), and that the air-jet stress-induced increase in HR and that after disinhibition of neurons in the dorsomedial hypothalamus are mediated, at least in part, by neurons in the RPa (41, 45), the current finding is consistent with the proposal that RPa contains a population of cardiac sympathetic premotor neurons (10) that do not contribute to resting cardiac sympathetic tone or resting HR (44) but rather mediate the increases in HR that comprise an important component of the cardiovascular support for the responses to a variety of thermoregulatory, metabolic, and stress-related challenges.
In conclusion, the present results indicate the importance of activation of neurons in the rostral ventromedial medulla, including RPa, in the leptin-evoked increase in BAT SNA, BAT thermogenesis, and HR. These data provide further evidence supporting a model in which BAT sympathetic premotor neurons in RPa are the final common medullospinal pathway mediating the excitation of BAT sympathetic preganglionic neurons regulating metabolic activity, energy consumption, and thermogenesis in BAT. This study also suggests a significant role for 5-HT1A receptors in the RPa region in mediating the hypothermic effects in several species, including rat and human, of 5-HT1A receptor agonist treatment. Elucidating the local circuitry and sources of inputs to thermogenic neurons in the RPa region will further our understanding of the function of this brain region critical to control of sympathetically regulated effectors in thermoregulation and energy expenditure.
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-20378 and DK-57838.
I thank Dr. J. B. Young for insightful discussions during the course of these experiments and for performing the plasma catecholamine determinations.
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