Effects of preoptic microinjections of α-MSH on fever and normal temperature control in rabbits

Brain Research

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Vol. 18, pp. 473-417. @Pergamon Journals Ltd., 1987. Printed in the U.S.A.

0361~9230187 $3.00 + .@O

Effects of Preoptic Microinje~tions of a-MSH on Fever and Normal Temperature Control in Rabbits J. D. FENG,

T. DA0

AND J. M. LIFTON

Physiology Department, University of Texas Health Science Center at Dallas 5323 Harry Hines Boulevard, Dallas, TX 75235 Received

29 January

1987

FENG, J. D., T. DA0 AND J. M. LIPTON. Effects of preopfic ~;cr~~ffjecf~~nsof BASE unfever and ~~r~~~ temperuture control in rabbits. BRAIN RES BULL BK.4)473-477, 1987.--a-MSH within the septal region of the brain has been implicated in fever control; this peptide and ACTH (l-24), which contains the a-MSH amino acid sequence, reduce fever when given intracerebroventricularly (ICV) or peripherally. These peptides also cause hypothermia when given in doses larger than those required to reduce fever. Both peptides occur naturally within the preoptic PO region of the brain, the CNS locus of primary temperature control. a-MSH (350 ng) injected bilaterally into the PO region via chronic cannulas

reduced fever caused in six rabbits by IV injection of IL-l (interleukin 1, endogenous or leukocyte pyrogen) but had no effect in afebrile animals. A larger dose (1.5 pg) not only reduced fever but caused hypothermia in 12 rabbits. In separate experiments PO injections of ACTB (l-24) (1 pg) reduced normal temperature. In the same six rabbits (Y-MSH(1 pg) caused slightly smaller hypothermia. a-MSH (1.5 Fg) also had no effect in 8 afebrile rabbits when injected into the septum. The primary conclusion is that a-MSH receptors within the PO region can contribute to both the antipyretic and hypothetic actions that are observed after ICV and peripheral administration of the peptide. CNS control of fever

Primary temperature

control

Neuropeptides

Surgical Procedures

CONSIDERABLE evidence suggests that a-MSH, the l-13 amino acid sequence of ACTH, participates in central nervous system mediation of temperature control 14, 9, 14, 17, 221. Increases in concentration of the peptide within the septal region during fever [19] and defervescence induced by local injections [81 suggest that the septum is one of the central sites where cu-MSH exerts its antipyretic effect. Aithough there is no previous evidence that ar-MSH acts directly within the preoptic PO region, the primary thermoregulatory control, the peptide occurs in high concentrations within this region [18]. Negative relations between circadian changes in preoptic a-MSH concentration and body temperature 121support the possibility that an action of the peptide within this region contributes to the antipyretic and/or hypothermic actions that are observed after intracerebroventribular and peripheral administration. To test this possibility we measured the effects on core temperature of PO microinjections of the peptide in afebrile and febrile rabbits.

Each rabbit was pretreated with ketamine hydrocNoride (Vetalar, Parke-Davis; 40 mgtkg, IM) and promazine (Acepromazine, Ayerst; 4 mglkg, IM), and placed in a modified Kopf rabbit stereotaxic instrument [5,16] equipped for gas anesthesia. Anesthesia was induced and maintained by inhalation) methox~uo~e (Metofane, ~tman-M~~, Inc.) and a N,O-0, mixture. Under aseptic conditions, four stainless steel 23 ga guide cannulae mounted in a plastic pedestal were implanted into the preoptic and septal regions (PO: 2-4 mm anterior to bregma 0.8-1.0 mm lateral to the midline, 12 mm below the dura; septum: 2-3 mm anterior to bregma 1 mm from the midline, 8 mm below the dura). Stainless steel screws and dental acrylic were used to attach the pedestal to the calvarium. The outer ends of the cannulae were protected with a plastic cap screwed onto the threaded pedestal. A postoperative period of at least 7 days elapsed prior to the first experiment. During this period penicillin G (150,000 U, IM, Crysticillin, Squibb Laboratories) was given to each rabbit daily for five days.

METHOD

Animals

Solutions and fnjections

The experiments were performed on twenty-one male New Zealand white rabbits weighing 34 kg. The rabbits were maintained on a 12 hr ligh~d~k cycle in an environmental temperature of 22 t 1°C. They were allowed food and water ad lib except during the period of experimentation.

CX-MSHand the structurally related peptide ACTH (l-24) (Sigma Chemical Co.) were dissolved in non-pyrogenic, isotonic saline (Abbott Laborato~es). The dosage differed with the aims of experiment: 1.5 pg a-MSH was injected into both PO and septal regions to determine the hypothermic

473

474

FENG,

effect of this peptide; 1.0 pg of a-MSH and of ACTH was injected into the PO region to compare the hypothermic effects of these two peptides. A non-hypothermic dose (0.35 pg) and 1.5 @g of (Y-MSH were injected into the PO region of febrile animals to examine the antipyretic action of this peptide. Bilateral injections were made through 30 ga stainless steel cannulae connected to two 10 ~1 syringes via polyethylene tubing (Intramedic PE 20). The syringes were driven in parallel by the same microdrive (Stoelting Inc.). The syringes, tubing and cannulae were washed with ethanol and dried with acetone and air 30 min prior to injection. All glassware and metal instruments used in preparation of the peptide solutions were heated to 200°C for 1 hr. Immediately before injections stylets were removed from indwelling cannulae, placed in an oven for decontamination, and injection cannulae were inserted bilaterally to a depth of 0.5-3.0 mm beyond the guide cannulae. One microliter of tr-MSH. ACTH (1-24). or of non-pyrogenic saline was injected bilaterally over 30 sec. The injection cannulae remained in place for at least 30 sec. At the end of the experiment heat-treated stylets were reinserted and the plastic cap was replaced. IL-I prepared from rabbit leucocytes incubated with Su1morlcllrr typhow endotoxin (Difco. No. 0901). using a method reported previously [S], was injected IV in a dose (60 FVkg) selected to produce a 0.6-l .O”C rise in temperature.

The rabbits were restrained in conventional holders and placed in an environmental chamber at an ambient temperature of 225 1°C. A thermistor probe (YeUow Springs International, No. 701) was inserted approximately 100 mm into the rectum and taped to the tail. Temperature measurements were made automatically at 10 min intervals with a MING-1 1 computer connected to a digital temperature recorder (United Systems, Inc.). In afebrile rabbits central injections of a-MSH, ACTH or saline were made after 1 hr baseline temperature had been established. In tests of the antipyretic action of (x-MSH. interleukin 1 (IL- I, leukocytic pyrogen) was injected into a marginal ear vein after a 1 hour baseline period and PO microinjections were given 30 min later during the chill phase of the febrile response. All injections of pyrogen were separated by at least 72 hr. In each series of experiments a cross-over design was used so that each animal served as its own control. Upon completion of the experiments, microinjection sites were verified histologically after microinjections of dye and the results were analyzed using the Wilcoxon matched pairs signed-ranks test. The fever index, the area under the temperature curve in “C/hr, was calculated for each rabbit. In pilot experiments it was clear that injections into the PO region caused late developing fever, beginning about 90 min post injection, no matter whether (r-MSH, ACTH or saline was injected (Fig. 1). It was difficult to completely avoid the fever but the febrile response was delayed and/or minimized through improvement of injection techniques, thus preventing the phenomenon from masking the real or full effect of cr-MSH within the preoptic region in the main experiments. It was generally possible to prevent the occurrence of a late rise in temperature until approximately 150 min after the preoptic injection. RESULTS Histolog> Location

of the intracerebral

injection

sites was deter-

DA0

AND

....O IL-I (IV) + (1 - MSH (PO) o-01.5pg Q-MSH (PO) 0-o Saline

LIPI’ON

l

1

I

0

1

. ;

i’

I

I

J

2

3

4

Time (Hours) FIG. 1. Examples of late-developing fever caused by PO injections of saline and (w-MSH in a single rabbit. PO injections at 0 time for acute saline and (u-MSH injections, 30 min after IL-l injection (0 point) when the rabbit was febrile.

mined from serial coronal sections of frozen brain tissue projected on figures from the atlas of Sawyer et al. [20] (Fig. 2). The injection sites in the eight animals used in the septal experiments were all within the medial septal region. The PO cannula placements were bilateral in 20 rabbits and unilateral in a single animal. Tests in Afehrilc

Rubbits

Both a-MSH and the related peptide ACTH (1 pglpl) caused hypothermia of approximately 05°C when microinjetted into the PO region of all six rabbits tested (Fig. 3). The fall in temperature began within 20 min and reached nadir about 90 min after infusion of either peptide. The temperature returned to baseline levels at about 150 min after infusion of a-MSH, and about 190 min after injection of ACTH. A small dose of (Y-MSH (0.35 pg) had no effect on temperature. A larger dose (1.5 &pl) caused hypothermia when injected into the preoptic region of 12 rabbits but had no effect on thermoregulation when injected into the septal region of eight animals (Fig. 4). Saline microinjections into either site had no effect on core temperature (Fig. 3). Tests in Febrile Rabbits

To test for antagonistic effects of a-MSH on fever, saline or 1.5 pg of the peptide, a dose that caused hypothermia in experiments described above was microinjected bilaterally into the PO region of the same rabbits 30 min after an injection of IL-l. This dose of (u-MSH markedly reduced fever (pcO.01, Fig. 5). cr-MSH injected into the PO region of six animals in a dose of 0.35 pg 30 min after an IV injection of IL-1 also reduced fever (p
EFFECTS OF a-MSH ON TEMPERATURE

475

CONTROL

10 -

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I I I I I I I I1 8642024

I,

1~~1~11111111 8642024

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FIG. 2. PO and septal sites of injection of peptide and saline in all 21 rabbits. Sites projected drawings from the stereotaxic atlas of Sawyer et al. 1201.

v 5

G ii5

.-.1.5pg 1.5bg

a-MSH (PO) N=12 a- MSH (Septum) N:8

o-o

+0.25

on

0.0

E g

-0.5

.-C

%

ii 5

Hours After

Injection

o-0 o-o A-A A-A

EJ

1

2

3

4

Hours After Injection

FIG. 3. Effects on rectal temperature of bilateral PO injections of a-MSH and ACTH (l-24). Scores are means%SEM.

6

0

FIG. 4. Comparison of the effects on afebrile temperature of PO and septal injections of 1.5 pg (Y-MSH. Scores are means?SEM.

(IV) + Saline (PO) N=6 (IV1 + Saline (PO) N=12 IL-1 (IV) + 0.35pg a-MSH N=6 IL-I(IV) + 1.5pg cl-MSH (PO) N=12

Saline IL-I

I

I

I

I

I

0

1

2

3

4

Time

(Hours)

FIG. 5. Antipyretic effects of non-hypothermic (0.35 pg) and hypothermic (1.5 pg) doses of cu-MSH injected into the PO region in rabbits made febrile by IV injection of IL-l. IL-l or saline injected IV at first arrow, cu-MSH or saline injected PO at second arrow.

476

FENG. DA0 AND LIPTON DISCUSSION

M~croinjection of cu-MSH into the preoptic region, a region with dense distribution of fibers containing this peptide [I, 3, 7, 12, 13, 231 caused hypothermia. The present experiments are the first to show that a-MSH can exert a direct action on the primary temperature control to reduce normal body temperature. These findings and increases in local cu-MSH concentration when body temperature tends to be low [2] raise the possibility that the peptide may have a role in PO mediation of thermoregulation. The discovery that a dose of (u-MSH which caused hypothermia after PO injection had no effect on afebrile rabbits when injected into septum may indicate that the PO region is a more important central site of action of this peptide in the mediation of hypothermia caused by large doses of centrally or peripherally administered a-MSH. From comparisons with the effect of LU-MSH it appears that ACTH is a more potent hypothermic agent, an idea that is consistent with findings of previous experiments [91. However, our results contrast with those of Thomhill and Saunders who reported that preoptic anterior hypothalamic administration of ACTH evoked a slight hype~he~ic response [2 I J. This discrepancy may be due to the difference in species used. to differences in the sites injected or to differences in the doses used. Thornhill and Saunders [21] injected 10 and 25 pg of ACTH bilaterally in the lateral PO region in their experiments on rats. The finding that preoptic administration of wMSH reduced IL-1 fever is consistent with previous results [4,5]

1. Abrams, G. M., G. Nilaver, D. Hoffman, E. A. Zirnrne~~,

M. Fein, D. T. Krieger and A. S. Liotta. Immuno~ytochemical distribution of corticotropin (ACTH) in monkey brain. Neurology 30: 1106-1110, 1980. 2. Abrams, R. and H. T. Hammel. Cyclic variations in hypothalamic temperature in unanesthetized rats. Am J Physiol 258: 689-702, 1965. 3. Atkins, E., F. Allison, Jr., M. R. Smith and W. B. Wood, Jr.

4. 5. 6. 7.

Studies on the antipyretic action of cortisone in pyrogeninduced fever. J Exp Med 101: 353366, 1955. &mea, A., C. Oliver and J. C. Porter. SubceIlular localization of a-melanocyte stimulating hormone in the rat hypothalamus. J Neurochem 29: 619-624, 1977. Crawford, I. L., J. I. Kennedy and J. M. Lipton. A simple “planilabe” for rapid establishment of the stereotaxic horizontal zero plane in rabbits. Bruin Res Bull 2: 397-398, 1977. Eisenman, J. S. and D. C. Jackson. Thermal response patterns of septal and preoptic neurons in cats. .Erp Nevrr,l 19: 33-45, 1967. Eskay, R. L., P. Giraud, C. Oliver and M. J. Brownstein. Distribution of cu-melanocyte-stimulating hormone in the rat brain: Evidence that a-MSH containing cells in the arcuate region send projections to extrahypothalamic areas. Brain Res 178: 55-67,

1979. 8. Glyn-Ba~inger,

J. R., G. L. Bemardini and J. M. Lipton. CX-MSHinjected into the septal region reduces fever in rabbits. Peprides 4: 199-203, 1983. 9. Glyn, J. R. and J. M. Lipton. Hypothermic and antipyretic effects of centrahv administered ACTH (l-24) and a-melanotropin. Peptides 2: 177-187, 1981. 10. Hardy, J. D., R. F. Hellon and K. Sutherland. Temperature sensitive neurons in the dog’s hy~th~amus. J Phys~ol 175: 242-253, 1964.

which indicate that this peptide is a potent naturally oc~rring antipyretic neuropeptide. The present results also support the idea that the septum is not the only central site where wMSH can exert an antipyretic effect. In view of the close anatomical and functional connections between the preoptic and septal regions 16,7], it may be that the neurons within these two regions contribute in common to the antipyretic actions that are observed after ICV and peripheral administration of the peptide. Because of the importance of the PO neurons to thermoregulation [6, 10, I I, 24, 251 it is not inappropriate to assume that stimulation or damage of local tissue caused by PO microinjections was responsible for the late non-specific elevations in temperature. Such an explanation may account for the similarity in the time at which the late fever caused by injection of different agents began, and the finding that late fever could be postponed by allowing a longer interval between tests or by improving manual aspects of the injection technique. This phenomenon may not be as detrimental in tests of pyrogenic agents. However. for tests of antipyretic or hypothermic agents it must be controlled, ruled out, otherwise the real or full effect of the agent tested may be masked.

ACKNOWLEDGEMENT

This research was supported by Grant NS-10046 from the National Institute of Neurological and Communicative Disorders and Stroke.

11. Hellon, R. F. Thermal stimulation of hy~thal~ic

neurons in un~esthetized rabbits. J Physinl 193: 318-395, 1967. 12. Hench, P. S., E. C. Kendall, C. H. Slocumb and H. F. Polley. The effect of a hormone of the adrenal cortex (17-hydroxy- 1ldehydrocorticosterone: compound E) and of pituitary adrenocorticotropic hormone on rheumatoid arthritis. Prac Staff Meet Mayo Clin 24: 181-197, 1949. 13. Kass, E. H. and M. Finland. Effect of ACTH on induced fever. N Engl J Med 243: 693-695, 1950, 14. Lipton, J. M. and J. R. Glyn. Central ~ministration of peptides alters thermoregulation in the rabbit. Peptides 1: 15-18, 1980. 15. Lipton, J. M., J. R. Glyn and J. A. Zimmer. ACTH and a-melanotropin in central temperature control. Fed Proc 40: 2760-2764, 1981. 16. Lipton, J. M. and W. E. Romans. Modification of rabbit head holder to increase speed and accuracy of stereotaxic surgery. Brain Res Bull 1: 159-160, 1976. 17. O’Donohue, T. L. and D. M. Dorsa. The opiomelanotropinergic neuronal and endocrine systems. Peptides 3: 353-395, 1982. 18. O’Donohue, T. L., R. L. Miller, R. C. Pendleton and D. M. Jacobowitz. A diurnal rhythm of immunoreactive oc-melanocytestimulating hormone in discrete regions of the rat brain. Neuroendocrinology 129: 281-287, 1979. 19. Samson, W. K., J. M. Lipton, J. A. Zimmer and J. R. Glyn. The effect of fever on central a-MSH concentration in the rabbit. Peptides 2: 419-423, 1981. 20. Sawyer, C. H., J. W. Everett and J. D. Green. The rabbit diencephalon in stereotaxic coordinates. J Camp Neural 101:

801-824, 1954. 21. Thomhill, J. A. and W. Saunders. Thermoregulation (core. surface and metabolic) response of unrestrained rats to repeated PO/AH injections of ~-endo~hin or adrenoco~icotropin. Peptides 5: 713-719, 1984.

EFFECTS OF a-MSH ON TEMPERATURE

CONTROL

22. Warberg, J., C. Oliver, R. L. Eskay, C. R. Parker, Jr., A. Barnea and J. C. Porter. Release of cr-MSH from a synaptosome-enriched fraction prepared from rat hypothalamic tissue. In: Melanocyte Stimulating Hormdne, edited by F. J. H. Tilders, D. F. Swaab and T. J. B. Van Wimersma Greidanus. New York: S. Karger, 1977, pp. 167-169. 23. Watson, S. J., C. W. Richard, III and J. D. Barchas. Adrenocorticotropin in rat brain: immunocytochemical localization in cells and axons. Science 200: 1180-1182, 1978.

477

24. Wit, A. and S. C. Wang. Temperature-sensitive neurons in preoptic anterior hypothalamic region: Effects of increasing ambient temperature. Am J Physiol 215: 1151-1159, 1968. 25. Wit, A. and S. C. Wang. Temperature-sensitive neurons in preoptic anterior hypothalamic region: Actions of pyrogen and acetylsalicylate. Am .I Physiol 215: lb%-1169, 1968.