Regulatory Peptides, 2 (1981) 43-52 43 Elsevier/North-Holland Biomedical Press

THE REGIONAL DISTRIBUTION OF )'3-MELANOTROPIN-LIKE PEPTIDES IN BOVINE BRAIN lS CORRELATED WITH ADRENOCORTICOTROPIN IMMUNOREACTIVITY BUT NOT WITH /3-ENDORPHIN

TAMOTSU SI4IBASAKI a , . NICHOLAS LING a ROGER GUILLEMIN a, MICHAEL SILVER t~,** and FLOYD BLOOM b

a Laboratories fi:~r Neuroendocrmology, ~:,d b The A. V. Davis Center for Behavioral Neurobiology, 77:e Salk htstitute for BioloFicaI Studies, La Jolla, CA 9203 7, U.S.A.

Accepted for publication 23 January 1981

SUMMARY

Immunoreact ive (lR)-),3-mf;lanotropin (MSH), -adrenocor t icot ropi r (ACTH) and -/3-endorphin in v~ ious areas o f bovine brain were measured with their respective radioimmunoassays (RIA). The concentrat ions of IR-. 3"3-MSH were almost the same as those o f IR-ACTH in most areas. Further- more, in all brain regions, the concentrat ions of both peptides were lower than those o f IR~-endorphin . The hghes t concentrat ion of IR-7. rMSH was found in hypothalamus, followed by thalamus, midbrain and striatum. Gel permeat ion chromatographic studies ~howed that the main 73-MSH-Iike pep- tide in the hypothalamus, str iatum and midbrain was a small form, whose molecular weight is about 4500. The,,;e brain 73-MSH-like peptides were also found to be glycosylated.

3,-melanocyte-st~mulating hormone; pro-opiometanocort in; ~ycopep t ide ; radioimmunoassay; hypothalamus; pi tui tary

* Postdoctoral fellow from the Department of Medicine, Tokyo Women's Medical Col- lege, Tokyo, Japan. ** Present address: Department of Anatomy, Cohtmbi~ University, College of Fhysicians & Surgeons, New.York, NY. Address all correspondence to: Dr. N.C. Ling, The Salk Institute; P.O. Box 85800, San Diego, CA 92138, U.S.A,

0 t 6 %0115]81 ]0000-0000/$02 q0 © Elsevier/Nor th-Holland Biomedical Press

44

tNTRODUCTION

With the development of RIAs for /3-1ipotropin (/3-LPH), /3-endorphin, adrenocorticotropin (AC'I'H) and a-melanotropin (a-MSH), the distribution of these peptides in the brain of several species has been studied [1 -7 ] . In 1977, Mains et al. [8] reported that ACTH and/3-LPH were derived from a c o m m o n precursor protein and that this prohormone could be processed differet~tly in various tissues to yield /3-LPH and ACTH as well as their related peptides. Recently, the complete amino acid sequence of this precur- sor protein was characterized and a new fragment, 7-MSH, which shares a common amino acid sequence H i s -Phe -Arg -Trp with a-MSH and 13-MSH w~s discovered in the NH~-terminal region of the precursor [9]. To ascertain whether this 3,-MSH fragment was also processed into secretory products we l~ave developed three different RIAs [10,11] by raising antibodies against three synthetic y-MSH-like peptides, ~,t-MSH, 3,2-MSH and ys-MSH [ 12]. The amino acid sequences of these three peptides, as well as tkose of c~- and B-MSII. are presented in Fig. 1. Using these three specific antisera we have reported that at least two 3,~-MSH-like peptides with molecular weights of 8800 and 4500 respectively [13] and two 3q-MSH-like pept!,des with mole- cular weights of 5600 and 1500 respectively and no 72-MSH-like peptides [ l t ] exist in bovine pituitary.

To further extend our study we report here the detection of 3,~-MSH-like peptides in different regions of the bovine brain and the comparison of its

r.~I~H Acetyl - Ser-Tyr-Ser-Met-Gl u-Hi s-Phe-Arg~Trp-Gly-Lys-Pro-Val-NH,

B~H Asp-Ser-Gly-P ro- Tyr- Lys- ~.~t,.G1u- ~ ~ s-Phe.Arg.Trp.G ly . Ser.P ro_P ro. Lys_Asp.OH

v,~.** TyY -Val-~t-G1y-Hi s-Phe-Arg-Trp-Asp-Arg-Phe-NH z

y~-M.SH Tyr-Val-~t~Gly-His-Phe-Arg~Trp-Asp-Arg-Phe-Gly.OH

y~.~H Tyr-Va1-~bt-G1y-His-Phe-;~rg-Trp.Asp-Arg-Phe-Gly.Arg.Arg.Asn.Gly.Fer.Ser.

Ser-Ser-G1y-Va|-G1y-G]y-Ala-Ala-G1n-OH

Fig. I. Amino acid sequences of'bovine ~-MSH, ~-MSH and synthetic ? i ' , 72-and ys-MSH derivedfrompro-opiomelanocortin of Nakanish~ et al. [9].

45

distribution with ACTH and 3-~mdorphir,,. We also present the :preliminary characterization of the detected brain T3-MSHs.

MATERIALS AND METHODS

Peptide exn~,ction Adult male bovine brains and pituitaries were obtained at a local slaughter-

house. After opening of the skull the brain was immediately dissected grossly to obtain the selected areas and the tissues were frozen on solid COs. Two hours after the dissection, the frozen tissues were homogenized by Polytron (Brinkmann) ~br 10 s in 7 volumes of cold 1 M ac~tic acid containilng 20 mM HC1 and 0.01% phenylmethylsulfonyl fluoride. The homogenates were cen- trifuged at 2500 X g at 4°C for 30 min and the supernatants were defatted witil an equal volume of diethyl ether twice, then lyophilized. The lyophilized mazerials were each reconstituted in 2.0 ml cold water and immed:iately used for RiAs of 3,~-MSH, 3,3-MSH, ACTH and 3-endorphi'a. Small aliquots of the solul:ions were used for protein concentration determination with the Bio- Rad protein assay kit (Bio-Rad).

Gel permeation chromatography The reconstituted extracts of hypothalamus, striatum and midbrain corre-

sponding to 612 rag, 783 mg and 642 mg of the respective wet ti:~;sues were applied to a 0.7 X 48 cm Sephadex G-75 (Pharmacia) column equilibrated in 1 M acetic acid and eluted with the same solvent at 1.4 ml/h at 4°C. Frac- tions of 0.6 ml were collected, lyophilized and then reconstituted in RIA buffer for RIA. The column was calibrated with ferritin, ovine 3..LPH, hu- man ACTH, synthetic 73-MSH and phenol red.

AffiniO: chromatography on concanavalh~ A-coupled Sepharose 413 A 1-ml siliconized glass syringe was packed with 0.4 ml cmlcanavalin

A-coupled Sepharose 4B (Pharmacia). The column was equilibrated with 10 ml concanv~An A (con A) buffer consisting of 0.01 M Tris-HC1, 0.7 mM MgCI~, 0.1% bovine serum albumin, 1.0 M NaC1 and 0.1% Triton X-!00 at pH 7.4. Lyo£!lilized extracts of hypothalamus (361 mg wet weight) and mid- brain (2500 mg wet weight) were reconstituted in 1.0 mt con A buffer, then applied to the column. After washing the colunm with 4.5 ml con A buffer, 5 ml 0.2 M ~-methyl-D-mannopyranoside (c~-MM) in con A buffer was used to elute the adsorbed material. The column was further washed with ~.5 ml 1 M ¢~-MM and 1.0 ml 0.i M acetic acid. 1-ml fractions were collected and lyophilized fo~ RIA of ~/3-MSH. A control run of the experiment was per- formed with iodinated follicle-stimulating honnone (FSH).

46

RIA /or peptMes ~'rMSH° and 73-MSH-like peptide~ were measured with the antisera RB

282 and RB 294, respectivel3,, as previously described [ 10,11 ]. On a weight basis RB 282 showed 0.06% and 13.02% cross-reactivity with 3,,,-MSH and y3-MSH respectively, but no sb;ni~icant cross-reactivity with a-MSH,/3-MSH, ~en,~orphin or ACTH l 11 ]. The cross-reactivity of a-, ~-, 3't-, 72-MSH, ACTH ~md B-endorphin with RB 294, on a weight basis, was <0.01% at 50% dis- placement of t!~le tracer [ I 0]. RIA for t~-endorphin was performed with anti- ~crrum RB tO0 and that for ACTH was done with antiserum SI B2 (generous gift of Dr. D. Clrt'a) as previously reported [ 14].

RI~';ULTS

Table t shows the conce~tratior, of IR-f rMSH, -"/s-MSH, -ACTH and -~- endorphin in the various brain areas. The highest concentration o f these four peptides was found in hypothal~mus. High concentr,~.tions o f IR-"/s-MSH, -~(~-endorpbin a)~d -ACTH were also present in thalamus, midbrain, striatum ar~O amygdala. Significant quantities of these three peptides also existed in c,,'rcbral cortex, medulla, cerebellum, spinal cord, pons, pineal body and hippocampus. Vurthermore, there is a direct corcelation between the quanti- tative distribution of the three peptides in the areas of the brain examined.

IR-,,/rMSH, ,on the contrary, was observed only in the hypothalamus. The anterior lobe of the pituitary contained roughly equimolar amounts of IR- ~¢3-MSH, 43-t'ndorphin and -ACTH, with a lesser amount of I R ~ - M S H . How-

~OOr 5)nndetd curve of Yi-MSH

eo j- "" " \ 1 "\,

z: 60L

o[ t01 I02 104 105 }06 107 I08 TI-MSH (pg) ~ Protein Equivelent

ck "o

\

Fig. 2. Standard curve of 7rMSH RIA and dilution curves of pituitary and brain extracts o~ the displacement of the T1-MSH tracer. A------A intermediate pituitary extract; ~ . . . . . . . ~, anterior pituitary extract; ~---------~, hypothalamus e~;tract

"FA

BL

E I

Dis

trib

utio

ri o

f fR

--?3

-MS

tt, -

3q-M

SII

, -~

ead

orp

hin

a~

d -A

Cq'

tt i

n bo

vine

hra

isi

Reg

ion

No.

,~¢

|P 7

,~-M

Stl

1R-~

/,-M

S!!

iR-~

,.cnd

~iph

~m

iR-A

Url

t an

imal

s (n

gtm

g p

rote

itt)

(n

g/m

g p

rote

L, t

) (n

gim

g p

rote

in)

(ng/

ilri

g p,

otei

n}

Cer

ebra

l co

rtex

F

ront

al

3 O

c;ci

pita

J 3

Hyp

otha

tam

a~

3 H

~pp

ocam

pus

3 A

my

~al

a 3

S ,ri

~t~s

m

3 'T

ha~:

im~t

s 4

Mid

brzt

L'~

4 Pi

.~ea

i b

od

y

2 i'o

r~s

3 M

ed~.

llJa

3 C

ete~

llu

m

3 S~

,,~aI

co

rd

3

Pitu

Rar

~¢

A~*

terJ

ot

3 ln

term

edJa

*.e

2

* M

ean

* S

.E.g

L

"* N

ot d

etec

taM

e,

A7

± 9.

29

rod.

**

4.19

± 0

.55

1.33

± 0

.37

1.22

2 0

.t0

ro

d.

4.79

.t

0.}9

1.

28 k

0.2

6 5.

7".)

k 9.

86

0.23

± 0

.]4

9.

00 L

1.4

4 5,

51 ±

0,3

| 0.

12 ~

0X

)5

rod,

2A

l ,.*

- 6.6

2

0.5

4 ±

0,0

6 i

,25

* 0.

22

n.d.

3.

09 ±

0.4

4

t .56

± 0

.40

15

~0 ±

0A

S

t~/I.

6.

93 ~

OA

0

1.44

± 0

,37

2.27

± 0

.38

n.

d.

5.7

I ±

0.4

8

I.(.

J ±

0.22

2,

15 ±

0.2

3 n.

d.

5.50

± 0

.31

] °77

:t

O~O

g -,~

0.,

)l

OA

3 ,~:

0.0

5 n.

d:

0=21

*

¢ 0,

25 ±

0,0

2 0

.40

± 0

.05

r<d+

2.

g2 ±

(L5

2 0.

73 ±

0.J

1

0.55

z 0

.06

r~xl

: 4

,82

± 6

.93

0.6J

± 0

.02

0,5k

: :t

0.23

r~

,d.

3.06

± 0

°44

0,78

± 0

,05

0.56

, ±

0.t

9

n+d+

3.

75 ~

0.3

3 0.

74 :

t 0,0

2

t f3

X

|03

"£0

,02

O

.0Ig

XlO

:~±

6,0

02

0

.82

X~

,0~

20

.fH

~

.02

XH

):~

±0

.20

2.

24 X

t0~

.~ 0

,70

0

A9

O X

102

± 6

Xjt

g

2.25

X t

0~

2 f

).70

0.

78 X

H):~

~ 0

57

4g

L 0 ~ _ _ _ _ & _ _

i0 + I02 IO + 104 t05 106 107 108

?3~MSH (pg) p(J Protein Equivalent

F+g. 3+ StarJdar: + cu;v~ of T3-MSH RIA and dilution curves of pituitary and brain extracts o~ the disptacet~-e~t of the 73-MSH tracer. A-+ -% intermediate pituitary extract; , . . . . . . -<. anterior pituitary extract; * ~ - - . ~ , hypothalamus extract; • . . . . . -o, mid- i+rain e×tract; © - - - - - o , striatum extract; • =, frontal cortex extract.

ever, a high concentrat ion o f IR-%-MSH was detected in the intermediate lobe o f the pituiltary. Figs. 2 and 3 show the di lut ion curves o f the respective tissue extracts on the inhibit ion o f the binding o f the %-MSH and 7a-MSH tracers by their ~espective antisera, demonstrat ing their parallelism wi th the standard curves o f 7t-MSH and %-MSH respectively.

The Sephadex G-75 gel permeat ion chrom~itography profile o f !R+~a=MSH h u m the hypothalamus , striatt, m and m i d h m i n extracts is s h o w n in Fig. 4. The main IR~r3-MSH peak in these brain ext:cacts e luted at KD = 0 .54 which

A H f 90+hotOm~s

i~OF

+ /i ~ 5 [ , .

z ~ 0

+ i') ~'.J 2 4 6 8

Kd

8 SfrLa]tum b c C Mldbram b c

Q ,'3- L PH

b ACTH

c )'3- MSH

I 0 0 2 4 6 8 i 0 0 2 4 6 8 I0 K¢: Kd

Fig. 4. Sepbadex G-75 gel permeation chromatography of tR-~'3-MSH from hypothala- mus (A), striatum (B) and midbrain (C) extracts.

4o

cpm pQ ~Q t

O?M ~011t

1 1 i X~O I J 1 ,' I l l 1 ; i

000

,!,,,,!!1,... mk t 3 5 7 9 tl 13 3 5 7 9 tl 13 I 3 5 ~' 9 |1 t5

Fig. 5, Affinity chromatography of I~SI-FSH (a), hypothalamus extract (b) and midbrain extract (c) on concantwatin A-coupled Sephar~,~se 4B,

is the same position where ACTIt elutes. In the hypothalamic extract, a small IR-yrMStt peak, the molecular size of which is smaller than ~-LPIt, was also observed at KD = 0,36.

Fig, 5 shows tlae results from affinity chromatography of the hypothata- mus and midbrain extracts on concanavalin A-coupled Sepharose 4B. 1R-%- MSH in hypothalamus and midbrain had a similar etution profile to that of iodinated FSIL Forty to fifty percent of the total IR-y3-MSH applied to the column was retarded on the colmnn and eluted with a-,methyl-O.-manno- pyranoside. These results indicate that brain ~'~-MSIi-like peptides are also glycosylated as p{tuitary 7a-MStt-tike peptides.

D I S C U S S I O N

Tile data from Table I indicate that significant amounts of IR-?,vMSH are widely distributed in the brain, especially in hypothalamus, thalamus, mid- brain and striatm:,, and that the distribution pattern of IR-3,3-MStt is similar to that of IR-~-endorphin and IR-ACTH, Although the concentrations of IR-'Ta-MSH in the various brain areas are almost the same as these of IR- ACTtt, the concentrations of IR~-endorphin are generally higher than those of the other two peptides except in the pineal body, where all three are very

50

low. Krieger et al. [2] fi~und that hypothalanms, hippocampus, oll~actory bulb, amygdala and striatum extracts of the bovine brain contained high concentrations c,f IR-fl-endorphin compared with other brain areas. We have also detected high concentrations of IR43-endorphin in the hypothalamus, amygdala and striatum extracts. However, our values are lower than Krie- ger's, even though the/3-endorphin antiserum used in our study reads equi- molarly both /3-endorphin and ~3-LPH, and thus the concentration of 1R-/3- endorphin comprises the total amount of IR-/3-endorphin and -~3-LPH. In Krieger's study [2] the ¢I-LPH concentrations in various brain areas were generally higher than the ACTH concentrations on a molar basis. This obser- vation is in agreement with our findings that the concentrations of IR-/3- endorphin are higher than those of IR-ACTH and IR-T3-MSH in various brain areas.

lR-7:-MSH was detected only in the hypothalamic extract. When we tried to characterize the molecular size of this hypothalamic IR~cMSH by pas- sing the same extract through a Sephadex G-75 gel permeation column, no significant %-MSIt peak was found in the eluate (data not shown). This might be due to the low concentration of IR-%-MSIt present in the hypo- thalamus (see Table I).

According to the gel permeation chromatograpbic analyses, the molecular size of the predominant brain %-MSH4ike peptide is about 4500. On the contrary, in the anterior pituitary, gland the molecular size of the predomi- nant 73-MSH-like peptide is around 8800. Furthermore, in the anterior pituitary of many species ACTH and/3-LPtt are the predominant peptides, whereas smaller peptides such as ~-MSH and/3-endorphin, which are derived from ACTtt and ~-LPH respectively, are the main peptides in the interme- diate pituitary a,; well as in the brain [5,15,16]. The results from our present study show that this trend is also carried over to the 2r3-MSH-like peptides, since the big form of y3-MSH-like peptide _s dominant in the anterior pitu. itary and the small form of 3,3-MStt-like peptide is the main peptide in the pituitary as well as in the brain. Furthermore, like the pituitary, this small y3-MSH-like peptide in the brain was also found to be glycosylated.

Using the antisera against 73-MSH and /3-endorphin, Bloom et al. [171 discovered significant differences in immunohistological reactivity patten, s between 73-MSH and ~-endorphin in the rat brain. Cells stained with the /3-endorphin antiserum are larger, more numerous and more broadly dis- tributed in the hypothalamus than those showing 3,a-MSH staining, whereas more nerve-terminal fibers are stained by the %-MSH antiserum than by th.e ~q~ndorphin antiserum.

The values de'termined by RIA for the regional distribution of IR-y3-MSH in bovine brain correspond fairly closely, in general, to the regional distribu-

5t

tion of 3,.rMSH immtmorea,:tive fibers and celt bodies detected by immuno- cytochemistry in the rat brain [171. In both species inmmnoreacti,dty is greatest in tl~e hypothalamus, and quite pronounced in the thalamus and midbr~ in. However, in bovine regional dissections significant %-MSH immu- noreactivity was also detected in regions which, in the rat, show negligible reactivity, such as the striamm and cerebral cortex. However. it sho~ald be noted that RtA values tbr other products o f the pro-opiomelanocortin pre- cursor molecules were also relatively large in theae same regions o f the bo- vine brain, suggest:ing that significant species variations in the distribm:ion of these peptides may exist between rat and bovine brain, In the recent study by Dupont et al. [61, no values wine reported lkx" the cerebral cortex and striatum in the bovine bmim

In a preliminary study, Itenriksen et al. [18] observed that rats which had been intraventricularly or intracistemally iRiected with synthetic %-MSH showed transient behavioral hyperactivity, cortical desynch:onization a~d llypothermia,

Titus, the immu~ohistochemical localization and detection by EIA of %-MSH-tike peptides in various brain areas, and the observation of electro- graphic, behavioral a,,~d thermoregulatory effects following injection of synthetic %~-MSH, i:ndicate that native %-MStt-like peptides could play a neuroregulator3, role which is different from that of ,G-endorphin in the brain,

AC KNOW t,,I-DG E M E NTS

We art? grateful to Dr. D~N. Orth for the ger, erous gift of ACTlt antiserum S1 B2 as well as to B. Gayer for secretarial help. The research was supi~orted by N1 AM DD (AM- 1381 1-05) and the William R;mdolph tlearst Found:~ tion.

REFERENCES

i Oliver, C, and Porter, J.C., Distribution and chazact~rization of a-melanocyte-stimu- lating hormone in the rat brain, Endoczinology, 102 (1978) 697-705.

2 Krieger, D.T., Liolta, A~, Suda, T., Palkovits, M. mad ~rownstein, M.L. Presence of immunoassayable/3-1ipoproteia~ in b,~vine brain anti spinal cord: Lack of concccdance with ACrH concentrations, Biochem, Biophys. Res, Commun., 76 (1977)930-936.

3 Gramsch, C., H6Ut, V., Md~raein, P., Past, A. and Herz, A.. Regional distribt:tion of methionine,enkephalin-and beta-endorphir~qike ~mmunoreactivity in human brain and pituitary, Brain Res,, 171 (1979) 261--270.

4 Matsukura, S., Yoshhni, H., Sueoka, S., Kataoka, K.. Ono, T. and Ohtmshi, ,4., The regional distril~ution of immulmreactivc ~-endorphin in the monkey bra!~L Brain Res., 159 (I978) 228~ 233.

52

5 Gramsch, C., Kleber, G., H611t, V., Pail, A., Mehraein, P. and Herz, A., Pro-opiocortin fragments in human and rat brain:/~-endorphin and c~-MSH are the predominant pep- tides, Brain Res., 192 (1980) 109-119.

6 Dupont, A., Lepine, J., Langelier, P., Merand, Y., Rouleau, D., Vaudry, I!., Gros, C. and Barden, N., Differential distribution of ~-endorphin and enkephalin in rat anJ bovine brain, Regui. Peptides, 1 (1980) 43-52.

70gawa, N., Panerai, A.E., Lee, S., Forsbach, G., Havlicek, V. and Friesen, H.G., ~-Endorphin concentration in the brain of intact and hypophysectomized rats. Life Sci., 25 (1979) 317-326.

8 Mains, R.E., Eipper, B.A. and Ling, N., Common precursor to corticotrop~ns and endorphins, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3014-3018.

9 Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y., Cohen, S.N. and Numa, S., Nucleotide sequence of cloned eDNA for bovine corticotropin~-tipopro- tein precursor, Nature, 278 (1979) 423-427.

10 Shibasaki, T.:. Ling, N. and Guillemin, R., A radioimmunoassay for 7-melanocyte stimulating hormone, Life Sci., 26 (1980) 1781-1785.

11 Shibasaki, T., Ling, N. and Guillemin, R., A radioimnmnoassay for ~'l-metanotmpin and evidence that the smallest pituitary 7-melanotropin is amidated at the COOH- terminus, Biochem. Biophys. Res. Commun., 96 (1980) 1393--1399.

12 Ling, N., Ying, S., Minick, S. and Guiilemin, R., Synthesis and biological activity of four "[-melanotropin peptides derived from the cryptic region of the adrenocortico- tropin/~-lipop:~-otein precursor, Life Sci., 25 (1979) 1773-1780.

13 Shibasaki, T., Ling, N. and Gufllemin, R., PituitaD, immunoreactive T-melanotropins are glycosylated oligopeptides, Nature, 285 (1980) 416-417.

la Shibasaki, T., Deftos, L. and GuiUemin, R., Immunoreactive-/3-endorphin, -adre~o- corticotropin and -calcitonin in extracts of anaptastie or differentiated (rat) medul- lary thyroid carcincma, Biochem. Biophys. Res. Commun., 90 (I 979) 1266-1273.

15 Eipper, B.A. and Mains, R.E., Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituitary, J. Supramol. Struct., 8 (t978) 247-262.

' 6 Liotta. A.S., Suda, T. and Krieger, D.T.,/3-Lipotropin is the major opioid-like peptide o7 human pituitary and rat pars distalis; Lack of significant ~-endorphin, Proc. Natl, Acad. Sci. U.S.A., 75 (1978) 2950-2954.

i7 Bloom, F.E., Battenberg, E.L.F., Shibasaki, T., Benoit, R., Ling, N. and Guillemin, R., Localization of 7-melanocyte stimulating ~ormone (7-MSH) imnmnoreactivity in rat brain and pituitary, ReguI. Peptides, 1 (1980) 105- 222.

I8 ftenriksen, S.21., Benabid, A.L., Madamba, S,, Bloom, F.E and Ling, N., T3-Melano- cyte stimulating hormone (T3-MSH): Electrographic, thermoregulatory and beha~5oral effects, Soc. Neurosci. Abstr., 6 ( 1980"1 681.