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, 33 (5), 2017-28

α2 Adrenergic Receptor-Mediated Inhibition of Thermogenesis

Affiliations

α2 Adrenergic Receptor-Mediated Inhibition of Thermogenesis

Christopher J Madden et al. J Neurosci.

Abstract

α2 adrenergic receptor (α2-AR) agonists have been used as antihypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since α2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat brown adipose tissue (BAT) and shivering thermogenesis by α2-AR agonists. Nanoinjection of the α2-AR agonist clonidine (1.2 nmol) into the rostral raphe pallidus area (rRPa) inhibited BAT sympathetic nerve activity (SNA) and BAT thermogenesis. Subsequent nanoinjection of the α2-AR antagonist idazoxan (6 nmol) into the rRPa reversed the clonidine-evoked inhibition of BAT SNA and BAT thermogenesis. Systemic administration of the α2-AR agonists dexmedetomidine (25 μg/kg, i.v.) and clonidine (100 μg/kg, i.v.) inhibited shivering EMGs, BAT SNA, and BAT thermogenesis, effects that were reversed by nanoinjection of idazoxan (6 nmol) into the rRPa. Dexmedetomidine (100 μg/kg, i.p.) prevented and reversed lipopolysaccharide-evoked (10 μg/kg, i.p.) thermogenesis in free-behaving rats. Cholera toxin subunit b retrograde tracing from rRPa and pseudorabies virus transynaptic retrograde tracing from BAT combined with immunohistochemistry for catecholaminergic biosynthetic enzymes revealed the ventrolateral medulla as the source of catecholaminergic input to the rRPa and demonstrated that these catecholaminergic neurons are synaptically connected to BAT. Photostimulation of ventrolateral medulla neurons expressing the PRSx8-ChR2-mCherry lentiviral vector inhibited BAT SNA via activation of α2-ARs in the rRPa. These results indicate a potent inhibition of BAT and shivering thermogenesis by α2-AR activation in the rRPa, and suggest a therapeutic potential of α2-AR agonists for reducing potentially lethal elevations in body temperature during excessive fever.

Figures

Figure 1.
Figure 1.
Effect of activation of α2-ARs in the rRPa on BAT thermogenesis evoked by PGE2 or cooling. A, C, The stimulation of BAT SNA evoked by nanoinjection of PGE2 into the medial preoptic area (A) or cooling (C) was reversed by nanoinjection of the α2-AR agonist clonidine into the rRPa. Subsequent nanoinjection of the α2-AR antagonist idazoxan into the rRPa restored the PGE2-evoked (A) or cooling-evoked (C) BAT SNA, whereas nanoinjection of saline failed to reverse the effect of clonidine. Changes in TBAT, expired CO2, and HR paralleled those in BAT SNA. However, due to the short time period between injections, the thermogenic increase in TCORE and the thermal inertia of the BAT, TBAT, remained significantly elevated following nanoinjection of clonidine. A, D, Insets, Diagrams of anatomical drawings depict the locations of the centers of the nanoinjection sites. 3V, Third ventricle; ac, anterior commissure; OX, optic chiasm; LPO, lateral preoptic area; py, pyramidal tract. B, The means ± SEM (n = 4) of the time courses of the physiological variable (each point is a 30 s average of the variable value) for the PGE2-evoked responses are shown. *p < 0.05 (significant increases compared with the basal value before PGE2 administration); #p < 0.05 (compared with the peak PGE2-evoked value just before clonidine administration). D, Means ± SEM of the peak BAT SNA during cooling, the nadir within 10 min of nanoinjection of clonidine into the rRPa, and the peak within 10 min of subsequent injection of idazoxan (n = 4) or saline into the rRPa (n = 4). *p < 0.05 (compared with cooling evoked peak); #p < 0.05 (compared with clonidine-evoked nadir).
Figure 2.
Figure 2.
Effect of systemic administration of an α2-AR agonist and subsequent blockade of α2-ARs specifically in the the rRPa on BAT thermogenesis evoked by PGE2. A, Nanoinjection of PGE2 into the MPA increased BAT SNA, TBAT, expired CO2, mean arterial pressure (MAP), and HR. Subsequent intravenous administration of clonidine reversed the increases in BAT SNA, TBAT, expired CO2, and HR, whereas MAP was increased further. Nanoinjection of the α2-AR antagonist idazoxan into rRPa restored the PGE2-evoked BAT SNA, TBAT, expired CO2, and HR. Inset, Diagrams of anatomical drawings depict the locations of the centers of the nanoinjection sites. 3V, Third ventricle; ac, anterior commissure; LPO, lateral preoptic area; OX, optic chiasm; py, pyramidal tract. B, Means ± SEM (n = 6) of the time courses of the physiological variables (each point is a 30 s average of the variable value). *p < 0.05 (significant increases compared with the basal value before PGE2 administration or cooling); #p < 0.05 (compared with the peak PGE2- or cooling-evoked value just before clonidine administration).
Figure 3.
Figure 3.
Effect of systemic administration of an α2-AR agonist and subsequent blockade of α2-ARs specifically in the rRPa on shivering thermogenesis evoked by PGE2. A, Nanoinjection of PGE2 into the MPA increased shivering EMG activity in the nuchal, gastrocnemius, and masseter muscles and increased TCORE. Subsequent intravenous injection of dexmedetomidine (DEX) completely inhibited all EMG activity and decreased TCORE. Nanoinjection of the α2-AR antagonist idazoxan into the rRPa reversed the DEX-induced inhibition of shivering EMG activity in all recorded muscles. Inset, Diagrams of anatomical drawings depict the locations of the centers of the nanoinjection sites. ac, Anterior commissure; LPO, lateral preoptic area; OX, optic chiasm; py, pyramidal tract. B, Means ± SEM (n = 6) of the time courses of the physiological variables (each point is a 30 s average of the variable value). *p < 0.05 (significant increases compared with the basal value before PGE2 administration); #p < 0.05 (compared with the peak PGE2-evoked value just before DEX administration).
Figure 4.
Figure 4.
The caudal C1 area of the VLM provides the principal source of catecholaminergic input to the rRPa. A, Photomicrograph of DβH-ir highly varicose fibers in the rRPa. B, Representative example of a CTb injection site in the rRPa. C, Diagram drawing of the four CTb injection sites, each encompassing the entirety of the rRPa. Numbers represent the approximate distance caudal to bregma (Paxinos and Watson, 2007). D, Composite photomicrograph of a brainstem section representative of those in which CTb labeling was found in DβH-containing neurons in the caudal C1 region of the VLM; the white box depicts the area that is presented at higher magnification in E–G. CTb-ir neurons (red), and DβH-ir neurons (green) in the VLM and pons are illustrated in E, F, K, and L. G, Double-labeled (CTb-ir, DβH-ir) neurons (orange, indicated by yellow arrowheads) in the VLM. H, Counts (mean ± SEM, n = 4) of double-labeled neurons in the VLM with respect to the distance from bregma. I, Total number of CTb-ir neurons (mean ± SEM, n = 4) in the VLM. J, Composite photomicrograph of a pontine section through the region containing the A7 catecholaminergic cell population. The white box depicts the area presented at higher magnification in K–M. Note that there are no double labeled neurons in the pons (M). Scale bars: A, 250 μm; B, 500 μm; G (for E–G), M (for K–M), 200 μm.
Figure 5.
Figure 5.
Neurons in the medulla and pons are synaptically connected to BAT. A–F, Photomicrographs are taken from rats that were killed 90 h (A–C, E) or 98 h (D, F) after injection of PRV into BAT. A, PRV-infected neurons (red) and DβH-ir fibers (green) in the rRPa (approximate boundaries indicated by the dashed white line), note the density of varicose DβH-ir fibers in close apposition to the PRV-ir neurons in the rRPa. py, Pyramidal tract. Photomicrographs illustrating PRV-ir (red), DβH-ir (green), and double-labeled (yellow) neurons in the VLM, ∼12.0 mm caudal to bregma (B) and 13.5 mm caudal to bregma (C). Note the double-labeled (yellow) neurons in the rostral VLM (B) but not the caudal VLM (C) at 90 h post-inoculation. D, Photomicrograph illustrating PRV-ir (green), TH-ir (red), and double-labeled (yellow) neurons in the VLM ∼13.3 mm caudal to bregma. Note that caudal VLM neurons become infected at long postinoculation times (98 h). E, A7 neurons of the rostral pons are infected at early postinoculation times, as illustrated by the double-labeled (DβH-ir, PRV-ir, yellow) neurons. F, At later postinoculation times, in the region of the pons containing the caudal aspect of the A7 population (DβH-ir, green neurons) there are many PRV-ir (red) neurons that are not DβH-ir. Scale bars: A, E, 100 μm; B, C, D, 50 μm; F, 200 μm.
Figure 6.
Figure 6.
A, B, Counts (mean ± SEM) of PRV-only (white circles) and PRV-ir, DβH-ir (black squares) neurons throughout the VLM (from 11 to 15 mm caudal to bregma) at intermediate (A; n = 2) and late infection times (B; n = 3). Note the progression of infection: at the intermediate infection time, very few catecholaminergic neurons are infected with PRV in the caudal VLM (∼12.75–13.5 mm caudal to bregma), whereas at the late infection time, many catecholaminergic neurons of this region begin to become infected with PRV.
Figure 7.
Figure 7.
Photostimulation of VLM neurons inhibits BAT SNA via activation of α2-adrenergic receptors in the rRPa. A–C, Photomicrographs of the same area of the VLM, illustrating neurons expressing DβH (green), PRSx8-ChR2-mCherry lentivirus (red), and both DβH and lentivirus (yellow). D, Photomicrograph of the rRPa (approximate borders are denoted by the white dotted line) containing highly varicose fibers expressing the lentivirus (red). E, G, BAT SNA and TBAT during photostimulation of the VLM at several stimulus frequencies before (E) and after nanoinjection of idazoxan (G) into the rRPa. G, Inset, Diagram depicts the centers of the injection sites. F, H, The group data (mean ± SEM; n = 4) of the time course of changes in BAT SNA and TBAT evoked by photostimulation (blue shaded region) at 5 Hz (black circles), 10 Hz (red triangles), 20 Hz (green squares), and 40 Hz (yellow diamonds) stimulation frequencies before (F) and after nanoinjection of idazoxan (H) in the rRPa. I, J, The photostimulation frequency response curve of the percentage of inhibition of BAT SNA (I) and the maximal change in TBAT (J) before (filled circles) and after nanoinjection of idazoxan (open circles) in rRPa. The asterisk indicates two-way repeated measures ANOVA: drug effect, p < 0.05; frequency effect, p < 0.001; and no significant interaction effect, p = 0.114. py, Pyramidal tract. Scale bars: 100 μm.
Figure 8.
Figure 8.
Systemic administration of the α2-adrenergic receptor agonist dexmedetomidine (100 μg/kg) prevents and reverses LPS (10 μg/kg)-evoked thermogenesis. Symbols represent the group means ± SEM of TBAT just before administration of LPS, at the time of LPS administration (time 0), and at 30 min intervals following injection of LPS. A, The change in TBAT following intraperitoneal injection of LPS preceded by an intraperitoneal injection of saline (open circles; n = 5) or dexmedetomidine (filled circles; n = 4). The asterisk indicates two-way repeated measures ANOVA: drug effect, p < 0.001; time effect, p < 0.001; and interaction effect, p < 0.001. B, The increase in TBAT following an intraperitoneal injection of LPS is reversed 1.5 h later by an intraperitoneal injection of dexmedetomidine (filled circles; n = 5) but not saline (open circles; n = 4). The asterisk indicates two-way repeated measures ANOVA: drug effect, p < 0.005; time effect, p < 0.001; interaction effect, p < 0.001.

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