Energetic responses to cold temperatures in rats lacking forebrain-caudal brain
Katherine M. Nautiyal,1Megan Dailey,2Nilton Brito,3Marcia N. d. A. Brito,3Ruth B. Harris,4
Timothy J. Bartness,4and Harvey J. Grill1
1Graduate Groups of Psychology and Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania;2Department
of Biology and Center for Behavioral Neuroscience, Georgia State University, Atlanta;4Department of Foods and Nutrition,
University of Georgia, Athens, Georgia; and3Department of Morphophysiological Sciences, State University of Maringa ´,
Maringa ´, Brazil
Submitted 28 April 2008; accepted in final form 10 July 2008
Nautiyal KM, Dailey M, Brito N, Brito MN, Harris RB, Bart-
ness TJ, Grill HJ. Energetic responses to cold temperatures in rats
lacking forebrain-caudal brain stem connections. Am J Physiol Regul
Integr Comp Physiol 295: R789–R798, 2008. First published July 16,
2008; doi:10.1152/ajpregu.90394.2008.—Hypothalamic neurons are
regarded as essential for integrating thermal afferent information from
skin and core and issuing commands to autonomic and behavioral
effectors that maintain core temperature (Tc) during cold exposure and
for the control of energy expenditure more generally. Caudal brain
stem neurons are necessary elements of the hypothalamic effector
pathway and also are directly driven by skin and brain cooling. To
assess whether caudal brain stem processing of thermal afferent
signals is sufficient to drive endemic effectors for thermogenesis,
heart rate (HR), Tc, and activity responses of chronic decerebrate
(CD) and control rats adapted to 23°C were compared during cold
exposure (4, 8, or 12°C) for 6 h. Other CDs and controls were exposed
to 4 or 23°C for 2 h, and tissues were processed for norepinephrine
turnover (NETO), a neurochemical measure of sympathetic drive.
Controls maintained Tc for all temperatures. CDs maintained Tc for
the 8 and 12°C exposures, but Tc declined 2°C during the 4°C
exposure. Cold exposure elevated HR in CDs and controls alike.
Tachycardia magnitude correlated with decreases in environmental
temperature for controls, but not CDs. Cold increased NETO in brown
adipose tissue, heart, and some white adipose tissue pads in CDs and
controls compared with their respective room temperature controls.
These data demonstrate that, in neural isolation from the hypothala-
mus, cold exposure drives caudal brain stem neuronal activity and
engages local effectors that trigger sympathetic energetic and cardiac
responses that are comparable in many, but not in all, respects to those
seen in neurologically intact rats.
energy balance; thermoregulation; anterior hypothalamus; heart rate;
body temperature; sympathetic drive; norepinephrine turnover; white
adipose tissue; brown adipose tissue
THERE IS BROAD SUPPORT FOR localizing the neural control of
thermoregulation (see, e.g., Refs. 7, 8, 13, 38, 53, 58) and
energy expenditure to the hypothalamus (see, e.g., 20, 47, 57).
Specifically, a variety of data support the view that neurons of
the medial preoptic area (POa) respond to and integrate thermal
afferent signals arising in skin, core, and brain and issue
efferent commands to downstream autonomic and behavioral
effectors that maintain core temperature (Tc) in response to
environmental challenges (8, 13, 38, 47, 58). The dorsomedial
hypothalamus (DMH) also is implicated in thermoregulatory
control (18, 35). POa and DMH neurons project to the nucleus
raphe pallidus (RPa) in the caudal brain stem. RPa neurons are
a critical element in the hypothalamic efferent pathway that
triggers sympathetic responses, including interscapular brown
adipose tissue (IBAT) thermogenesis, increased heart rate
(HR) and blood pressure, and tail vasoconstriction (18, 35, 38,
42). RPa-mediated sympathetic responses contribute to the
maintenance of Tcwhen rodents are exposed to cold environ-
ments. When RPa neurons are inactivated, cold-induced regu-
latory responses are blocked (38, 42).
A variety of data suggest that, in addition to their contribu-
tion to efferent control, neurons of the caudal brain stem
(midbrain and hindbrain) receive skin thermal afferent infor-
mation. For example, neurons in the midbrain reticular forma-
tion respond to the cooling of the skin (40). Similarly, the
neurophysiological response of neurons of the medullary raphe
(raphe magnus and RPa) are highly sensitive to mild cooling of
the skin (46; see also Refs. 32 and 41). Some of these neurons
also respond to alterations in Tc (46). Skin cooling-driven
neurophysiological responses also can be recorded from the
medullary raphe of acutely decerebrated, anesthetized rats (17,
54), indicating that the caudal brain stem responses are not
secondary to forebrain processing. Skin cooling also increases
the neurophysiological activity of lateral parabrachial neurons
(lPBN) (34, 39). Perhaps most interesting are findings that
spinal projections from thermally responsive medullary raphe
neurons contribute to brown adipose tissue (BAT) activation
and other thermoregulatory defenses against environmental
cold challenges (46). The thermal sensitivity of these caudal
brain stem neurons and their participation in sympathetic
outflow to the periphery suggest that they may be part of a
“cold-defense” pathway (46).
Although the anterior hypothalamus is commonly described
as the thermoregulatory center, there is precedent in the liter-
ature for the idea that neurons within more caudal levels of the
neuraxis also play an integrative role in thermoregulatory
control. These “caudal neurons” are activated by thermal
afferents and appear to engage circuits that trigger sympathetic
and behavioral responses, thereby participating in some degree
of feedforward or feedback regulation of Tc. This view, that the
thermoregulatory control system is neurally distributed with
contributions arising from both hypothalamic and caudal, ex-
trahypothalamic sites, is emphasized in seminal papers by
Address for reprint requests and other correspondence: H. J. Grill, Graduate
Groups of Psychology and Neuroscience, Univ. of Pennsylvania, 3720 Walnut
St., Philadelphia, PA 19104 (e-mail: email@example.com).
The costs of publication of this article were defrayed in part by the payment
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Am J Physiol Regul Integr Comp Physiol 295: R789–R798, 2008.
First published July 16, 2008; doi:10.1152/ajpregu.90394.2008.
0363-6119/08 $8.00 Copyright © 2008 the American Physiological Societyhttp://www.ajpregu.org R789
Lipton (31), Chambers et al. (14), and Amini-Sereshki (1, 2)
and in a synthetic review by Satinoff (50) published in the
1970s but has apparently disappeared from discussions of
thermoregulatory control. Among the relevant findings, cool-
ing the skin does not elicit shivering in spinal animals. How-
ever, with a transection higher than the medulla and lower
pons, the shivering response to cooling of the extremities is
restored. This suggests a role for the caudal brain stem in
thermoregulatory control. Perhaps the lack of continued dis-
cussion of the findings from these reports is due to their
conclusion that, when compared with the thermoregulatory
competence achieved by hypothalamic circuits in the intact
brain, the degree of regulatory control achieved by more caudal
levels of the neuraxis is “incomplete” and “fragmentary.” It
was asserted, for example, that the initiation of thermoregula-
tory responses in these transected neurological preparations
requires more extreme thermal challenges than in intact prep-
arations (50). These hypotheses are evaluated here through
studies that examine the thermoregulatory competence of cold-
exposed, freely moving, chronically maintained decerebrate
rats (i.e., complete severing of the forebrain from the caudal
brain stem at the mesodiencephalic junction). This was accom-
plished by measuring Tc, sympathetic (HR), and behavioral
responses (locomotor activity) in awake, chronic supracollicu-
lar decerebrate rats and identically maintained neurologically
intact control rats exposed to 4°C and to intermediate cold
environmental temperatures (8 and 12°C). In addition, norepi-
nephrine turnover (NETO), a neurochemical measure of sym-
pathetic drive, was measured in the heart, IBAT, and several
white adipose tissue (WAT) depots. In these experiments, we
tested the hypothesis that, in the absence of hypothalamic
neural processing and hypothalamic-caudal brain stem com-
munication, caudal brain stem neurons responsive to thermal
afferent signals will engage local neurons that activate sympa-
thetic outflows and trigger energetic responses that result in a
level of energetic and thermoregulatory control that is compa-
rable in many, but perhaps not all, respects to that seen in
neurologically intact controls.
Maintenance and surgery. Male Sprague-Dawley rats (Charles
River Laboratories) weighing 275–325 g on arrival were housed
individually in plastic bins in a room maintained at 23 ? 2°C and a
12:12-h light-dark cycle (lights on 8:00 a.m. to 8:00 p.m.). All animals
were maintained in accordance with the guidelines of the University
of Pennsylvania Institutional Animal Care and Use Committee. Pel-
leted food (Purina Rodent Chow 5001) and water were available ad
libitum initially. All rats were then habituated to four, daily 9-ml
gavage feedings of powered rodent diet suspended in water (L1001;
Research Diets, New Brunswick, NJ). This feeding regime provided
rats with 79 kcal/day and ample hydration; chow and water were no
longer available. All procedures used were approved by the University
of Pennsylvania animal care and use committee.
Control sham surgery or unilateral brain transection at the supra-
collicular level was performed with a blunt L-shaped surgical spatula
(22) under ketamine (90 mg/kg), xylazine (2.7 mg/kg), and aceproma-
zine (0.64 mg/kg) anesthesia and Meloxicam analgesia (3.0 mg/kg).
After hemitransection (1 wk), full decerebration was completed, and
control rats were anesthetized and subject to a second sham surgery.
At the time of the second hemitransection, rats in the telemetry
experiment were implanted with an ER-4000 transponder (Mini-
Mitter, Bend, OR). The transponder core was positioned in the
peritoneal cavity for activity and Tcmeasurement. The two attached
wire leads were positioned subcutaneously in the upper right and
lower left quadrants of the rat’s chest and secured for HR measure-
ment. A minimum of 2 wk was allowed for recovery before the start
of thermal exposure experiments.
Procedure for transponder experiments. Naive chronic decerebrate
(CD; n ? 10) and pair-fed intact (n ? 5) rats were exposed to three
thermal conditions of 4, 8, or 12°C in a counterbalanced order; in each
case, rats also were evaluated under room temperature (23°C) condi-
tions for a period preceding cold exposure. Four days intervened
between each of the three test conditions. On the day of each
condition, rats received two gavage feedings (at 1.5 and 4 h after
lights-on) and were then transferred to an inactive chromatography
refrigerator with open doors in a room maintained at 23°C. Rats were
placed individually in plastic cages positioned on receivers on the
shelves of the refrigerator unit. No bedding was provided to preclude
nesting. For all conditions, rats remained in this environment for 2.5 h
(at 23°C); this time is referred to as the precold baseline period. Two
hours into the precold baseline period, rats were gavage fed a third
meal. The 6-h period of cold thermal exposure commenced 30 min
later. The doors were closed, and the refrigerator was activated to
achieve one of the three targeted (4, 8, or 12°C) temperatures. Target
ambient temperatures were achieved within 20 min. Following the 6-h
exposure period, the doors were opened, and the refrigerator was shut
off; temperature in the unit returned to 23°C within 20 min. Recording
continued in the postcold period. During the entire period, rats were
positioned in the refrigerator unit, and HR, Tc, and activity were
measured telemetrically every 30 s, 5 min, and 5 min, respectively.
Experimenters entered the room periodically to monitor posture,
position, and behavior of the rats.
Following completion of all experimental conditions, CD rats were
anesthetized with ketamine, xylazine, and acepromazine (as above)
and then perfused transcardially with heparinized saline followed by
10% formalin. Brains were removed from the crania, washed in saline,
and then submerged in 10% formalin. Brains were cyroprotected in a
20% sucrose solution before being embedded in albumin gelatin and
cut with a freezing microtome in 50-?m sagittal sections. Sections
stained with cresyl violet were examined to verify completeness of the
Procedure for NETO experiments. Additional naive CD (n ? 18)
and pair-fed intact (n ? 18) rats were used for NETO measurements
using the ?-methyl-p-tyrosine (?-MPT) method. ?-MPT is a compet-
itive inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in
catecholamine biosynthesis. This allows the estimation of NETO by
the rate of norepinephrine (NE) disappearance from the sympathetic
nerve terminals in the tissues of interest. The ?-MPT methyl ester
hydrochloride (Sigma Aldrich, St. Louis, MO) was prepared by first
adding ?0.5 ml of glacial acetic acid (1 ?l/mg ?-MPT) and then
diluting to the final concentration with 0.15 M NaCl. Basal levels of
NETO were determined for one-third of the animals in the control and
CD groups (n ? 6 each) that were untreated and killed (time 0), and
tissues were harvested for subsequent NE content measures. The
remaining rats (n ? 12/group) were injected intraperitoneally with
?-MPT (250 mg ?-MPT/kg; 25 mg/ml) between 0800 and 1000.
One-half of these rats was immediately placed in the cold (4°C) unit,
and the other one-half was placed in a room temperature (23°C)
thermal environment. Later (2 h), rats were decapitated. Inguinal
WAT (IWAT), retroperitoneal WAT (RWAT), epididymal WAT
(EWAT), as well as IBAT and heart were rapidly removed, weighed,
frozen in liquid nitrogen, and then stored at ?80°C until assayed for
NE content to determine NETO as described below. NETO was
calculated on a per pad basis rather than per gram or per milligram
protein because total NETO per pad reflects the physiological impact
of the cold stimulus on the sympathetic drive to the tissue. This is the
procedure and data expression we have used previously (9, 51, 60).
The decline in NE content is linear using this procedure (9), a
requirement for accurate NETO estimation (16).
ENERGETIC RESPONSES TO COLD IN CHRONIC DECEREBRATE RATS
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11. Brodie BB, Costa E, Dlabac A, Neff NH, Smookler HH. Application of
steady state kinetics to the estimation of synthesis rate and turnover time
of tissue catecholamines. J Pharmacol Exp Ther 154: 493–498, 1966.
12. Cannon B, Nedergaard J. Brown adipose tissue: function and physio-
logical significance. Physiol Rev 84: 277–359, 2004.
13. Cano G, Passerin AM, Schiltz JC, Card JP, Morrison SF, Sved AF.
Anatomical substrates for the central control of sympathetic outflow to
interscapular adipose tissue during cold exposure. J Comp Neurol 460:
14. Chambers WW, Seigel MS, Liu JC, Liu CN. Thermoregulatory re-
sponses of decerebrate and spinal cats. Exp Neurol 42: 282–299, 1974.
15. Collins S, Daniel KW, Rohlfs EM. Depressed expression of adipocyte
beta-adrenergic receptors is a common feature of congenital and diet-
induced obesity in rodents. Int J Obes Relat Metab Disord 23: 669–677,
16. Cooper JR, Bloom SR, Roth RH. The Biochemical Basis of Neurophar-
macology. New York, NY: Oxford Univ Press, 1982.
17. Dickenson AH. Specific responses of rat raphe neurones to skin temper-
ature. J Physiol 273: 277–293, 1977.
18. Dimicco JA, Zaretsky DV. The dorsomedial hypothalamus: a new player
in thermoregulation. Am J Physiol Regul Integr Comp Physiol 292:
19. Dodt C, Lonnroth P, Fehm HL, Elam M. The subcutaneous lipolytic
response to regional neural stimulation is reduced in obese women.
Diabetes 49: 1875–1879, 2000.
20. Elmquist JK. Hypothalamic pathways underlying the endocrine, auto-
nomic, and behavioral effects of leptin. Physiol Behav 74: 703–708, 2001.
21. Garofalo MA, Kettelhut IC, Roselino JE, Migliorini RH. Effect of
acute cold exposure on norepinephrine turnover rates in rat white adipose
tissue. J Auton Nerv Syst 60: 206–208, 1996.
22. Grill HJ, Norgren R. The taste reactivity test. II. Mimetic responses to
gustatory stimuli in chronic thalamic and chronic decerebrate rats. Brain
Res 143: 281–297, 1978.
23. Harris RB, Bartness TJ, Grill HJ. Leptin responsiveness in chronically
decerebrate rats. Endocrinology 148: 4623–4633, 2007.
24. Harris RB, Kelso EW, Flatt WP, Bartness TJ, Grill HJ. Energy
expenditure and body composition of chronically maintained decerebrate
rats in the fed and fasted condition. Endocrinology 147: 1365–1376, 2006.
25. Harris RB, Martin RJ. Changes in lipogenesis and lipolysis associated
with recovery from reversible obesity in mature female rats. Proc Soc Exp
Biol Med 191: 82–89, 1989.
26. Hermann DM, Luppi PH, Peyron C, Hinckel P, Jouvet M. Afferent
projections to the rat nuclei raphe magnus, raphe pallidus and reticularis
gigantocellularis pars alpha demonstrated by iontophoretic application of
choleratoxin (subunit b). J Chem Neuroanat 13: 1–21, 1997.
27. Jocken JW, Goossens GH, van Hees AM, Frayn KN, van Baak M,
Stegen J, Pakbiers MT, Saris WH, Blaak EE. Effect of beta-adrenergic
stimulation on whole-body and abdominal subcutaneous adipose tissue
lipolysis in lean and obese men. Diabetologia 51: 320–327, 2008.
28. Kobayashi A, Osaka T. Involvement of the parabrachial nucleus in
thermogenesis induced by environmental cooling in the rat. Pflugers Arch
446: 760–765, 2003.
29. Leibel RL, Forse RA, Hirsch J. Effects of rapid glucose infusion on in
vivo and in vitro free fatty acid re-esterification by adipose tissue of fasted
obese subjects. Int J Obes 13: 661–671, 1989.
30. Leibel RL, Hirsch J, Berry EM, Gruen RK. Alterations in adipocyte
free fatty acid re-esterification associated with obesity and weight reduc-
tion in man. Am J Clin Nutr 42: 198–206, 1985.
31. Lipton JM. Thermosensitivity of medulla oblongata in control of body
temperature. Am J Physiol 224: 890–897, 1973.
32. Martin-Cora FJ, Fornal CA, Metzler CW, Jacobs BL. Single-unit
responses of serotonergic medullary and pontine raphe neurons to envi-
ronmental cooling in freely moving cats. Neuroscience 98: 301–309, 2000.
33. Mefford IN. Application of high performance liquid chromatography with
electrochemical detection to neurochemical analysis: measurement of
catecholamines, serotonin and metabolites in rat brain. J Neurosci Meth-
ods 3: 207–224, 1981.
34. Menendez L, Bester H, Besson JM, Bernard JF. Parabrachial area:
electrophysiological evidence for an involvement in cold nociception.
J Neurophysiol 75: 2099–2116, 1996.
35. Morrison SF. Central pathways controlling brown adipose tissue thermo-
genesis. News Physiol Sci 19: 67–74, 2004.
36. Morrison SF. Differential regulation of sympathetic outflows to vasocon-
strictor and thermoregulatory effectors. Ann NY Acad Sci 940: 286–298,
37. Nagashima K, Nakai S, Tanaka M, Kanosue K. Neuronal circuitries
involved in thermoregulation. Auton Neurosci 85: 18–25, 2000.
38. Nakamura K, Morrison SF. Central efferent pathways mediating skin
cooling-evoked sympathetic thermogenesis in brown adipose tissue. Am J
Physiol Regul Integr Comp Physiol 292: R127–R136, 2007.
39. Nakamura K, Morrison SF. A thermosensory pathway that controls
body temperature. Nat Neurosci 11: 62–71, 2008.
40. Nakayama T, Hardy JD. Unit responses in the rabbit’s brain stem to
changes in brain and cutaneous temperature. J Appl Physiol 27: 848–857,
41. Nason MW Jr, Mason P. Medullary raphe neurons facilitate brown
adipose tissue activation. J Neurosci 26: 1190–1198, 2006.
42. Ootsuka Y, Blessing WW, McAllen RM. Inhibition of rostral medullary
raphe neurons prevents cold-induced activity in sympathetic nerves to rat
tail and rabbit ear arteries. Neurosci Lett 357: 58–62, 2004.
43. Ootsuka Y, Heidbreder CA, Hagan JJ, Blessing WW. Dopamine D2
receptor stimulation inhibits cold-initiated thermogenesis in brown adi-
pose tissue in conscious rats. Neuroscience 147: 127–135, 2007.
44. Ootsuka Y, McAllen RM. Comparison between two rat sympathetic
pathways activated in cold defense. Am J Physiol Regul Integr Comp
Physiol 291: R589–R595, 2006.
45. Osaka T. Thermogenesis elicited by skin cooling in anaesthetized rats:
lack of contribution of the cerebral cortex. J Physiol 555: 503–513, 2004.
46. Rathner JA, Owens NC, McAllen RM. Cold-activated raphe-spinal
neurons in rats. J Physiol 535: 841–854, 2001.
47. Richard D. Energy expenditure: a critical determinant of energy balance
with key hypothalamic controls. Minerva Endocrinol 32: 173–183, 2007.
48. Saarela S, Hissa R. Metabolism, thermogenesis and daily rhythm of body
temperature in the wood lemming, Myopus schisticolor. J Comp Physiol
[B] 163: 546–555, 1993.
49. Saito M, Minokoshi Y, Shimazu T. Metabolic and sympathetic nerve
activities of brown adipose tissue in tube-fed rats. Am J Physiol Endocri-
nol Metab 257: E374–E378, 1989.
50. Satinoff E. Neural organization and evolution of thermal regulation in
mammals. Science 201: 16–22, 1978.
51. Shi H, Bowers RR, Bartness TJ. Norepinephrine turnover in brown and
white adipose tissue after partial lipectomy. Physiol Behav 81: 535–542,
52. Skibicka KP, Grill HJ. Energetic responses are triggered by caudal
brainstem melanocortin receptor stimulation and mediated by local sym-
pathetic effector circuits. Endocrinology 149: 3605–3616, 2008.
53. Tanaka M, McAllen RM. A subsidiary fever center in the medullary
raphe? Am J Physiol Regul Integr Comp Physiol 289: R1592–R1598,
54. Taylor DC. The effects of nucleus raphe magnus lesions on an ascending
thermal pathway in the rat. J Physiol 326: 309–318, 1982.
55. Uno T, Roth J, Shibata M. Influence of the hypothalamus on the
midbrain tonic inhibitory mechanism on metabolic heat production in rats.
Brain Res Bull 61: 129–138, 2003.
56. Voss-Andreae A, Murphy JG, Ellacott KL, Stuart RC, Nillni EA,
Cone RD, Fan W. Role of the central melanocortin circuitry in adaptive
thermogenesis of brown adipose tissue. Endocrinology 148: 1550–1560,
57. Wolfgang MJ, Lane MD. The role of hypothalamic malonyl-CoA in
energy homeostasis. J Biol Chem 281: 37265–37269, 2006.
58. Yoshida K, Konishi M, Nagashima K, Saper CB, Kanosue K. Fos
activation in hypothalamic neurons during cold or warm exposure: pro-
jections to periaqueductal gray matter. Neuroscience 133: 1039–1046,
59. Young JB, Landsberg L. Suppression of sympathetic nervous system
during fasting. Science 196: 1473–1475, 1977.
60. Youngstrom TG, Bartness TJ. Catecholaminergic innervation of white
adipose tissue in Siberian hamsters. Am J Physiol Regul Integr Comp
Physiol 268: R744–R751, 1995.
ENERGETIC RESPONSES TO COLD IN CHRONIC DECEREBRATE RATS
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