Brain Research. 430 (1987) 3_3-3_8 323 Elsevier

BRE 13138

Pro-opiomelanocortin-derived peptides (ACTH/fl-endorphin/ a-MSH) in brainstem baroreceptor areas of the rat

• i "~ 1.2 Mikl6s Paikovlts "-', I~va Mezey and Robert L. Eskay 3 1Laboratory of (.'eli Biology. National Institute of Mental Heahh. Bethesda. MD 20892 t U. S. A.I. 2First Department of Anatomy.

Semmelweis University Medical School, Budapest t Hungaryt and ~Neurochemistry Section, Laboratory of Clinical Studies. National Institute on Alcohol Abuse and Alcoholism. Bethesda. MD 20892 (U. S. A. I

(Accepted 2 June 1987)

Key words: Barorcceptor area: Adrenocorticotropic hormone ( ACTH ): fl-Endorphin: a-Melanocyte-stimulating hormone (a-MSH): Brainstem lesion: Hypothalamus: Nucleus of the solitary tract

Relatively high concentrations of adrenocorticotropic hormone (ACTH), fl-endorphin and a-melanocyte-stimulating hormone Ca- MSH) were determined by radioimmunoassay in the nucleus of the solitary, tract (NTS) of rats. Dense networks of immunoreactive fi- bers for these peptides were most prominent in the commissural part of the nucleus, where immunostained perikarya (8-15 per sec- tion) were also seen in colchicine-treated rats. Moderate peptide levels and moderately dense immunoreactive networks of these pep- tides were found in the lateral reticular nucleus (including the A I and As-C i catecholaminergic cell groups) and the nucleus ambiguus. Ten different types of surgical lesions or transections were performed in the hypothalamus and the lower brainstem to determine the origin of ACTH, fl-endo~phin and a-MSH in the brait~stem baroreceptor centers. Except the commissural part of the NTS, the barore- ceptor areas receive ACTH, fl-endorphin and ~t-MSH innervations from both the hypothalamic arcuate cells and local neurons in the NTS. Fibers in the commissural part of the NTS seem to be of local origin. Hypothalamic fibers to the rostrai part of the NTS and the vasomotor As-C I cell groups descend in both a medial (through the periaqueductal central gray) and a lateral (ventrolateral tegmen- tal fibers) pathway, whereas fibers to the caudal lateral reticular nucleus (A, cell group) and the nucleus ambiguus may run only in the lateral pathway. The descending fibers may decussate somewhere in the caudal hypothalamus-rostral midb~ain, but caudal to that level they run and terminate ipsilaterally. Fibers from tla~e ACTH-, fl-endorphin- and a-MSH-containing cells in the NTS form a bundle arching between the NTS and the ventrolateral medulla and partially (40-55%) innervate the vasomotor and the vasodepressor areas, as well as the nucleus ambiguus.

INTRODUCTION

Baroreceptor areas in the lower brainstem that are

involved in the central regulation of blood pressure

consist of the: (1) primary baroreceptor center (nu-

cleus of the solitary tract); (2) vasodepressor area

(the caudal part of the lateral reticular nucleus, in-

cluding the A l noradrenergic cell grzup); (3) vaso-

motor area (rostral lateral reticular rmcleus, includ-

ing the A 5 and Cl catecholaminergic cell groups); and

(4) vasomotor preganglionic project ing neurons in

the dorsal vagal and ambiguus nuclei (for review see

ref. 19). All of these medul lary baroreceptor areas,

especially the nucleus of the solitary tract (NTS), are

rich in neuropeptides, including pro-opiomelanocor-

tin (POMC)-re la ted peptides (for review see ref. 12,

18,19).

Dense neuronal networks which immunostained

for the POMC precursor m or its derivatives such as

the adrenocorticotropic hormone ( A C T H ) 2a'24. fl-en-

dorphin ~' and a-melanocyte-st imulat ing hormone (a-

MSH) 15-31, have been demonstra ted in the nucleus of

the solitary tract (NTS), whereas moderately dense

POMC-positive staining has been observed in the

other medullary baroreceptor areas. Similarly, rela-

tively high concentrations of immunoassavable

Corresponde:we: M. Palkovits. First Department of Anatomy. Semmelweis University Medical School. 1450 Budapest, Tiizolt6 utca 58. Hungary.

0006-8993i.'~7,'$~ ~3.51}© ! 987 Elsevier Science Publishers B.V. ( Biomedical Division )

324

ACTH 2°, fl-endorphin 2° and a-MSH 5'15 have been repe~ted in the NTS and lesser levels in the other medullary baroreceptor areas. In addition, ACTHg.25-fl-endorphin m'25-, and a-MSH31-contain - ing cell bodies have been demonstrated in the NTS. It has been suggested that these local NTS cells might innervate a part of the medulla oblongata 23 but ~he origin of POMC-positive fibers and networks in the medullary baroreceptor areas has not been investi- gated systematically. On the basis of immunohisto- chemistry, descending ACTH 24-, fl-endorphin 6- and a-MSHlS-containing fibers, which arise from hypo- thalamic nuclei, have been reported recently. How- ever, the participation of these descending fibers in the innervation of medullary nuclei requires more specific analyses.

In the present study, the detailed innervation pat- terns of the baroreceptor areas by ACTH-, fl-endor- phin- and a-MSH-containing neurons were mapped with immunohistochemistry. In addition, a number of surgical interventions were performed in the rat brainstem to delineate more precisely the probable origins of ACTH-, fl-endorphin- and a-MSH-con- taining fibers through the combined use of immuno- histochemistry and radioimmunoassay.

MATERIALS AND METHODS

Male Sprague-Dawley (Osborne-Mendel) rats weighing 200 + 10 g were used. Animals were kept under standard laboratory conditions: 12 h of light and 12 h of dark (lights on at 06.00 h), dry rat chow and tap water were given ad libitum.

Experimental surgery Each animal was anesthetized with ether and the

head was fixed in a Kopf stereotaxic device. Ten dif- ferent types of surgical interventions with appro- priate sham operations (n = 6 per group) were per- formed (Fig. 1) as follows. (1) Complete hypotha- lamic deafferentation by means of a Hal~isz-knife 8.

The knife with a rotatory radius of 1.2 mm was low- ered with an anterior position 3.2 mm caudal to the level of bregma, and the medial-basal hypothalamus was cut around (Fig. 1A). (2) Caudal hypothalamic lesion. A stainless-steel electrode (outer diameter 0.2 mm) with an uninsulated bare tip of 1.0 mm was used for making electrolyt:~ lesions. After a midline penetration into the diencephalon (4.0 mm caudal to bregma, vertical coordinate: 6.0 mm below the skull), 10 V current was applied for 6 s through a radiofrequency lesion generator. The posterior hy° pothalamic nucleus and the central area of the dien- cephalic-mesencephalic border were lesioned at the rostral tip of the periaqueductal central gray (Fig. 1B). (3) Unilateral transection of the brainstem at the junction of the di- and mesencephalon with a 2- mm wide glass knife 2t, 4.5 mm caudal to bregma (Fig. 1C). (4) Bilateral transection of the mesence° phalic central gray matter. An 'arrowhead'-shaped 2- mm wide glass knife was lowered 5.8 mm down, and 6.3 mm caudal to bregma in the midline (Fig. 1D). The rostral part of the central gray and a part of the superior colliculus and the retrosplenial cortex were transected bilaterally. (5) A cut was made identical to that of No. 4, but only one side of the central gray was transected with a 1.0-mm wide glass knife. (6) Bilateral transection of the lateral ventral tegmental area. A glass knife 1.5 mm wide was lowered to the ventral surface of the midbrain 1.0 mm lateral to the midline and 6.3 mm caudal to bregma (Fig 1E). (7) A cut was made identical to that of No. 6, but only one side of the midbrain was transected. (8) Pontine hemisections with a 2.0-2.2 mm wide glass knife. From the midline, one side of the pons was com- pletely transected 8.0 mm caudal to 0regma (Fig. 1F). (9) Rostral medullary hemisections. The rostral portion (11.0 mm caudal to bregma) of the medulla oblongata was transected with a 2.5-mm wide glass knife from the midline to the lateral edge of the brainstem (Fig. 1G). (10) Unilateral parasagittal knife cut in the medulla oblongata. An area between

Fig. 1. Coronal sections of rat brains with the location of various types of lesions and transections. Drawings are taken from different levels: A 2.7, B 3.9, C 4.5, D and E 6.3. F 8.1. G 11.1 and H 13.8 mm caudal to the level of the bregma 17. Types of the lesions: A, deaf- ferentation of the medial-basal hypothalamus; B, caudal diencephalic lesion: C, hemisection at the diencephalic-mesencephalic junction: D. transection of the periaqueductal central gray matter; E, bilateral transection of the ventrolateral midbrain tegmentum: F, pontine hemisection: G, medullary hemisection; H, knife cut between the nucleus of the solitary tract and the ventrolateral medul- la.

c~

-1-

~

-I"1

i \

I'M

326

the nucleus of the solitary tract and the lateral ret- icular nucleus was transected with a 1.5-mm wide glass knife. The oblique cut (the top of the knife was medially oriented) was performed 1.0 mm lateral to the midline starting 0.5 mm rostral and ending 1.0 mm caudal to the obex (14.0 mm caudal to bregma). Fiber connections between the dorsomedial and ven- trolateral medulla were completely transected (Fig. 1H).

Animals were sacrificed by decapitation (for radio- immunoassay) or by perfusion under Nembutal anes- thesia (for immunohistochemistry) 8-14 days (Table III) after surgery. The exact location of surgical cuts was verified histologically on 10-~m thick sections from the lesioned brain areas.

Immunohistochemistry Intact, colchicine-treated (100/~g/10/zl colchicine

was injected into the lateral ventricle 48 h prior to perfusion) or operated rats were anesthetized with Nembutal and animals were perfused through the as- cending aorta with an ice-cold fixative solution con- taining 4% paraformaldehyde and 0.2% picric acid in 0.167 M phosphate buffer (pH 7.3). The lower brain- stems were dissected out and placed in the same solu- tion overnight at 4 °C. Next, brainstems were washed several times in phosphate buffer and 40-gm thick coronal sections were cut with a vibratome. Ad- jacent sections from each brain were collected in 3 separate wells, and incubated in the presence of either a 1:1000 dilution of ACTH, fl-endorphin or a- MSH antiserum overnight at 4 °C. Next, the sections were rinsed in phosphate buffer, and incubated in biotinilated anti-rabbit IgG (1:400) followed by bioti- nilated avidin-peroxidase complex (1:200) for 1 h each at room temperature. The peroxidase was de- veloped by diaminobenzidine tetrahydrochloride, as a substrate. The sections were placed on gelatin- coated slides, dried, dehydrated and mounted with a plastic mounting medium. Preabsorption of each an- tibody with either 10/~g of ACTHl_24, fl-endorphin or a-MSH eliminated the appropriate peptide-specif- ic staining. Reagents were purchased from Vector Labs, CA, unless otherccise indicated. The ACTH antiserum was obtained from S.J. Watson (Ann Ar- bor, MI) and has I:een reported to detect the mid (17-24) portion of the ACTH molecule 3°. The a- MSH antibody recognized the C-terminal portion of

the molecule, and its specificity has been previously reported 15. The antiserum to fl-endorphin was raised in rabbits against human fl-endorphin (Peninsula Labs). This antibody is midportion-directed and rec- ognizes the POMC precursor, the fl-endorphin (acet- ylated and deacetylated) and fl-lipotropin ~ - LPH)I_31.1_2714.

Radioimmunoassay After decapitation, rat brains (Groups 3 and 10)

were quickly removed and frozen on histological spe- cimen holders with dry ice. The medulla oblongata was cut into 300-gm thick coronal sections and the NTS (rostral and commissural parts separately), the A 1 and As-C 1 catecholaminergic cell groups, and the nucleus ambiguus were obtained by the punch tech- nique 16 according to their microdissection coordi- nates reported previous!y 17. Tissue punches were ho- mogenized in 300/~1 of chilled 0.1 N HCI by sonica- tion and 30-/d aliquots of each homogenate were re- moved for protein determination 13. Each sample was centrifuged and the supernatant fluid was lyophyi- ized. Residues were resuspended in assay buffer and the concentration of ACTH, fl-endorphin and a- MSH was determined by radioimmunoassay. Syn- thetic peptides were obtained from Peninsula Labs. The ACTH antiserum appears to recognize some portion of the sequence of amino acids contained within ACTH ll_x6, but does not recognize a-MSH 28. The fl-endorphin antiserum recognizes a midportion sequence of amino acids contained within fl-endor- phin17_27 and therefore it recognizes fl-LPH and the pro-opiomelanocortin precursor molecule 1.2. The a- MSH antibody recognizes the C-terminal portion of the molecule and therefore has no cross-reactivity with ACTH, fl-endorphin or fl-LPH 2,5.28.

Levels of each peptide were calculated and ex- pressed as ng/mg protein. Data from operated vs sham-operated rats were analyzed by the Student's t- test (two-tailed), and P-values less than 0.05 were re- garded as significant.

RESULTS

Intact and colchicine-treated rats Moderately high levels of a-MSH, fl-endorphin

and ACTH were found in the NTS by radioimmu- noassay (Tables I and II). Within this nucleus, the

327

T A B L E I

Concentrations of ACTH, fl-endorphin and (t-MSH tng/mg protein) in brainstem baroreceptor nuclei 8 days qfter hemi~ection b('t~teen the di- and mesetwephalon

Values are the means + S .E .M. Number s in parentheses are the num~cr of animals.

Sham operated Hem(section

Ipsilateral ('ontrah~teral

A C T H

Rostral NTS 11.23 + 0.02 (6) 0.12 _+ 0.03" ( 6 1 - 4 8 % 0.23 + 0.06 16) Commissura l NTS 11.24 + 0.04 (61 A.~-C t cell g roups 0.11 + 0.03 (5) A t cell g roup 0.13 + 0.02 (6) Nucleus ambiguus 0.07 + 0.01 (6)

f l -Endorphin Rostral NTS 11.3(I + 0.05 (6) Commissura l NTS 0.40 + 0.04 (6) A s - C t cell g roups 0.26 + 0.02 (6) A t cell g roup 0.26 + 0.02 (6) Nucleus ambigut.ls 0.22 + 0.03 (61

a-MSH Rostral NTS 11.50 + 0.09 (6) Commissura l NTS 0.78 + 0.18 (5) A.<-C t cell g roups 0.40 + 0.10 (4) A t cell g roup I).34 + 0.10 (7)

*P < 0.01.

()c~

11.18 + 0,(!3 (, ' ,)-22c~ 0.22 _+ ().(14 (6) - 4% U.~5 ± 0.02" ( 5 ) - 5 5 Q 0.10 _+ 0.05 (5) - 9 Q 0.05 +_ 0.01 * ( 6 ) - 6 2 c ; - 0.08 _+ 0.03 (5) - 3 8 % 0.04 + 11.02 ( 6 ) - 4 3 % 0.05 + 11.112 (41 - 2 9 %

0.08 _+ (1.02" (6) - 7 3 % 0.34 + 11.114 16) + 15% 0.32 + 0.06 1 6 ) - 2 0 Q 0.53 + 0.07 (5) + 3 2 % 0.16 + 1!.02" (6) - 3 8 Q 0.18 + 0.04 161 - 31%

0.__ z 0.02 161 - 15Q 0.07 + 0.04" ( 5 ) - 7 3 Q "v~ I).07 + 11.112" (6) -68c~ 0.26 + 0.02 161 + ISQ

0.07 + 0.03* (41-86c~ I).42 = t,. ;.4 (5) - 10% 0 . 5 7 + 0 . 1 0 ( 4 1 - 2 7 % 0.56 _*_ 0.12 (41 -28c~ 0.04 + 0.01" ( 6 1 - 9 0 Q !1.41 I + 11.114 (4) I)Q 11.05 + 0.03* ( 5 ) - 7 6 % I).27 + 0.04 (4) -21c/-

highest levels were found in the caudal (commissu- ral) subdivision. Concentrations of these peptides were lower in the A~ and A s - C 1 cell groups and in the nucleus ambiguus with peptide values reaching only about one-half of those observed in the NTS (Tables I and II).

The NTS can be easily recognized in lower brain- stem sections on the basis of the high density of ACTH-, fl-endorphin- and a-MSH-positive fibers. The a-MSH-stained neuronal network was partic- ularly impressive (Fig. 2). The density of the NTS im- munoreactive network increased continuously from

T A B L E II

Concentrations of A CTH and fl-endorphin (ng/mg proteb~J in brabrstem baroreceptor nuclei 8 days after a surgwal cut between tl, e NTS and the ventrolateral medulla

Values are the means + S .E .M. N u m b e r s in parentheses are the n u m b e r of animals.

Sham operated Transection

A C T H Rostra l NTS Commissura i NTS

A ~ - C t cell g roups A t cell g roup Nucleus ambiguus

f l -Endorphin Rostral NTS Commissura l NTS

A s - C t cell g roups A I cell g roup Nucleus ambiguus

11.23 + I).112 (6) 11.24 + (`).04 (6) 0.11 + 11.03 (5) 0.13 + 0.0216) I).117 + 0.01 161

0.30 + 0.05 161 0.4t . ) + 0.04 ( 6 )

0.26 + 0.02 (6) 11.26 + 0.02 (6) 0.22 + 0.03 (6)

Ipsilateral Contralateral

0.24 + 0.13 (5) + 4% 0.22 + 11.115 151 - 4% 0.20 + 0.04 (6) - 17% 11.24 + 11.114161 0% 0.05 + 0.01" 15) - 5 5 Q 0.09 + 0.02161 - ISQ 0.06 + 0.112" (5) - 5 4 Q 0.12 + 11.112 141 - 8% 0.114 + 0.01" (6) -43c/- 11.117 + 11.113 (4) ()Q

0.30 + 0.02 16) 0 % 0.31 + 11.113 (61 + 3cc

0.38 + 0.04 (6) - 5% 0.47 + 0.03161 + 17% 0.13 + 0.04* (5) -50% (.).26 + ( ).( 13 (51 (tci 0.15 + 0.03" (5) - 4 2 % 0.21 + 0.02 (5) - 1 9 % 0.12 +_ 0.02* (5) - 4 5 % /I.311 + 11.115 151 + 3 h %

* P < 0 . 0 1 .

328

Fig. 2. a-MSH-containing fibers and network in the NTS, especially in its commissural part. Dark-field photographs of coronal sec- tions, a and b. rostral; c and d, commissurai part of the NTS, sections are taken from levels 13.5.13.8.14.6 and 15.2 mm caudal to the bregma, respectively. 1. lateral part of the rostral NTS; m, medial part of the rostral NTS; A. area postrema; (3. fasciculus aod nucleus gracilis; H. motor hypoglossal nucleus; P. nucleus prepositus hypoglossi; T. solitary tract. The fourth ventricle is indicated with stars and the cemral canal with arrows. × 70.

rostral (Fig. 2a) to caudal (Fig. 2b,c) with the most

dense network appearing in the commissura! part of

the NTS (Fig. 2d). Several fibers cross over in the

NTS, mainly below the area postrema. Long fibers

run laterally from the NTS and car~ be divided into

two groups. The majority of these fibers appear to

run ventrolaterally with a few fibers running hori-

zontally (Figs. 2a, 4b and 7b). In the commissural

part of the NTS, 8-15 cell bodies per section immu-

nostained for ACTH, fl-endorphin and a-MSH (Fig.

3). The immunopositive cells did not respect the bor-

der of the NTS and were scattered laterally and ven-

trolaterally along the arc of fibers between the NTS

and the ventrolateral medulla. These long arching fi-

bers (Figs. 4a,b and 7a,b) can be recognized both ros-

tral and caudal to the level of the obex in a rostrocau-

dal segment of the medulla between 12 and 15 mm

caudal to bregma. Fine a-MSH-immunoposit ive neu-

ronal networks were seen in the lateral reticular nu-

cleus, mainly in its lateral portion (Fig. 4d) and in the

nucleus ambiguus (Fig. 4c). The demonstration of

several immunopositive cross-sectioned fibers indi-

r

o

-

°

H . r

0

. ~ , . •

I. , "

9

• :?, : ..... : :~%~'a ?

:"

N

329

e

b

, / p

Fig. 3. a-MSH-containing cell bodies in the commissural part of the bITS in colchicine-treated rats. a, 14.7 mm; b, 15.3 mm caudal to the bregma. G, fasciculus gracilis; H, motor hypoglossal nucleus. The central canal is indicated by an arrow, x 130.

cates the existence of a presumably descending spinal bundle close to the ventral surface of the ventrolater- al medulla (Fig. 4c,d).

ACTH- and fl-endorphin-immunostained fibers and networks were found in similar topographical positions but were less densely packed in all of the above medullary nuclei and areas.

POMC innervation of brainstem baroreceptor centers by hypothalamic neurons

Brainstem hemisection at the diencephalic-mes- encephalic border (Group 3) resulted in significant

depletions in the concentration of all 3 investigated peptides in the rostral part of the NTS, the A u and A 5 - C 1 catecholaminergic cell groups, as well as in the nucleus ambiguus, ipsilateral to the lesion (Table I). No significant alterations of peptide levels were measured in the commissural part of the NTS (Table

I). A substantial number of ACTH, fl-endorphin and

a-MSH fibers in the medullary baroreceptor areas arise from the hypothalamus. After a total deaf- ferentation of the medial-basal hypothalamus (Group l), almost all of the immunoreactive fibers

330

Fig. 4. ct-MSH-containing fibers and network in the caudal medulla oblongata. Dark-field photographs of coronal sections, a, 13.7 ram; b, 14.8 mm; c, 14.0 mm; and d, 14.4 mm caudal to the bregma, a and b: immunoreactive fibers arching between the NTS (T) and the lateral reticular nucleus (Al). c: a-MSH-containing neuronal network in the nucleus ambiguus (A). d: a-MSH-containing network in the lateral reticular nucleus and immunoreactive fibers in the descending spinal bundle (x). D, pyramidal decussation; IO, inferior olive; V, nucleus of the spinal trige:ninal tract, a and b, x31; c and d, x70.

disappeared from the rcstral part of the NTS and di-

minished significantly in the lateral reticular nucleus

(both in A l and A s - C I cell groups) and in the nucleus

ambiguus. Peptide levels did not change in the com-

missural part of the NTS (Table III). The observed

hypothalamic fibers may arise exclusively from the

TA

BL

E

III

Eff

ects

of

vari

ous

brai

n le

sion

s an

d tr

anse

ctio

ns o

n A

CT

H,

~-en

dorp

hin

and

a-M

SH i

mm

unor

eact

ivit

y in

bra

inst

em n

ucle

i in

voh,

ed i

n ba

rore

cept

or m

echa

nism

s

* *

*, D

isap

pea

ran

ce;

**,

stro

ng

dep

leti

on

; *,

dim

inu

tio

n o

f th

e im

mu

no

re a

ctiv

ity

; -,

w

ith

ou

t ch

ang

es;

Ipsi

., i

psi

late

ral;

Co

ntr

a.,

co

ntr

alat

eral

: L

at.,

lat

eral

: u

nil

., u

nil

ater

al:

bil

.,

bil

ater

al;

Me

d.,

med

ial;

I M

H,

imm

un

oh

isto

ch

em

istr

y;

RIA

, ra

dio

imm

un

oa

ssa

y.

Gro

up T

ype

of le

sion

Se

ctio

n of

P

ost-

]'o

r N

TS

NT

S A

~-C

t N

ucle

us

A t g

roup

Fi

g. 1

op

erat

ive

(ros

tral

) (c

omm

issu

ral)

gr

oups

am

bigu

us

days

lp

si.

Con

tra.

Ip

si.

Con

tra.

lp

si.

Com

m.

lpsi

. C

omra

. Ip

si.

Con

~ra.

lp

si.

Con

tra.

Spbl

al b

undl

e

1 H

yp

oth

ala

mic

isl

and

A

11

IM

H

***

***

2 H

yp

oth

ala

mic

lesi

on

s B

11

IM

H

**

**

3 H

yp

oth

ala

mu

s-

mid

bra

in c

ut

C

8 R

IA

**

- 4

Lat

. m

idb

rain

un

il.

E

14

IMH

-

- 5

Lat

. m

idb

rain

bil

. E

14

IM

H

**

**

6 M

ed.

mid

bra

in u

nil

. D

14

IM

H

- -

7 M

ed.

mid

bra

in b

il.

D

14

IMH

*

* 8

Po

nti

ne

hem

isec

tio

n

F 12

IM

H

***

- o

Med

ull

ary

hem

isec

tio

n

G

12

IMH

**

* -

10

Cu

t b

etw

een

N

TS

an

d A

t

H

8 IM

H-R

IA

- -

332

arcuate nucleus, where ACTH, fl-endorphin and a- MSH immunostained substances accumulated in cells retrogradely (Fig. 5), 3 days after medullary hemisection (Group 9).

Descending immunoreactive POMC fibers appear to run in two major pathways. One is in the periaque- ductal central gray matter and the other one is i a the ventrolateral tegmentum. Most of the long des- cending fibers seem to be present in the ventrolateral pathway, since bilateral transection of this area (Group 6) resulted in a significant loss of immuno-

reactive fibers in the rostra~ NTS, the A~ cell group, the nucleus ambiguus and in the descending spinal bundle (Table III). Caudal to the midbrain, these fi- bers may not cross over, since after unilateral lesion of the ventrolaterai tegmentum (Group 7) immuno- reactive fibers diminished only ipsilateral to the le- sion (Table III). The rostral part of the NTS was an exception in this group, since the unilateral cut caused no visible change in the peptide-positive fiber densities of this area. Furthermore, the rostral part of the NT$ may receive hypothalamic fibers through

°.•

b

?V " r i . " ~ . •

" 2 . '~.:"~','~.' "~'':: . ' , ~ ' . :

0

~ o .

V

% ."

%.

. - ¢

°

, " & . - . / 4

+

P IM

Fig. 5. ACTH-immunostained cells in the arcuate nucleus 3 days after medullary hemisection (without colchicine treatment). Coronal sections: a, 3.0 ram; b, 3.9 mm caudal to the bregma level. Due to the retrograde accumulation of the immunoreactive material, cells are visible mainly ipsilateral (*) to the transection, but scattered cells are also on the other side. IM, inframammillary recess of the third ventricle; M, median eminence; P, dorsal premammillary nucleus; V, third ventricle, x 130.

the periaqueductal central gray, since the number of immunoreactive fibers were reduced in the rostral NTS after bilateral transection of the periaqueductal gray matter (Group 7). A substantial number of fi- bers should also run to the vasomotor area (As-Cn

333

cell groups) through that dorsomedial pathway since both uni-(Group 6) and bilateral (Group 7) periaque- ductal gray transections were effective in decreasing the immunoreactivity in the vasomotor area (Table m).

- r

dP

o o •

. j

• °

,%

A 1

c d

. . , .

- , o

dl, --, .

" ~ ' l ,

0, , I

% ' , ~ t J2. ". .

o

• • ' , b " " , ."

. . . . . ... " .. ' .~"

. . . ~ . . , . . . •

4"" . - a, ~ " •

° - / ~ •

A 1 • . ~ t

I

- " r

. .

- 2

,

, - " . ; , ¢ • .

" . : . J r ' . , . " '

~ g ' o . ."

Fig. 6. a-MSH-immunoreactive fibers in the rostral part of the nucleus of the solitarv tract (a and b) and in the ventrolateral medulla oblongata (c and d). Coronal sections (a and b dark-field photographs) 13.8 mm (a and b) and 14.2 mm (c and d) caudal to bregma. The number of a -MSH fibers are diminished in the NTS (a), and almost completely disappeared (c) from the lateral reticular nucleus (A~), nucleus ambiguus (A) and the descending spinal bundle (*) in medullary hemisected rats ipsilateral to the lesion in comparison to those on the unoperated side (b and d). H. motor hypoglossal nucleus: V~, nucleus of the spinal trigeminal tract• a and b. x 13(1: c and d,

x llll.

334

G

Fig. 7. a-MSH-immunoreactive fibers in the caudal medulla oblongata 8 days after a knife cut between the NTS and the ventrolateral medulla, a: the location of the knife cut is indicated on a coronal section 14.0 mm caudal to the bregma level r . b -e : coronal sections, dark-field photographs, b, a-MSH fibers constitute an arc between the NTS and the ventrolateral nledulla (intact rat): c. tt-MSH fibers medial to the cut (in the direction of the NTS). Scattered fibers (x) are still present ventrolaterai to the cut (arrows). The number of tt- MSH fibers diminished in the later al reticular nucleus (A~) and remained unchanged in the descending spinal bundle (arrow) Ipsilatcr- al to the cut (c) in comparison to those in intact rats (d) I), pyramidal decussation: H, motor hypoglossal nucleus: N. nucleus of the soli- tarvtract, b, ×31:c , x 130: d and e, x l l0 .

Pontine (Group 8) and medullary (Group 9) hemi- sections clearly demonstrated that ACTH, fl-endor- phin and a-MSH fibers descend to the baroreceptor centers from higher brain regions and terminate in the medulla ipsilaterally (Table III). A substantial number of fibers disappeared from the rostral part of the NTS (Fig. 6a), the lateral reticular nucleus (Fig. 6c) and the nucleus ambiguus after hemisection (Fig. 6c) but not from the commissural part of the NTS. The long descending POMC-peptide-containing im- munoreactive fibers may run further down and inner- vate the spinal cord, since immunoreactive fibers ip- silateral to the lesions in the descending spinal bundle disappeared almost completely after pontine or med- ullary hemisections (Fig. 6c).

POMC innervation of medullao' baroreceptor areas by neurons in the NTS

A small group of immunopositive ACTH, fl-endor- phin and a-MSH cells is present in the commissural part of the NTS (Fig. 3). A dense bundle of fibers arc between this area and the ventrolaterai medulla (Fig. 7b). It appears that most of the fibers in this bundle arise from the NTS and are ventrolaterally directed, since after transecting this bundle (Group 10) fibers seemed to remain intact on the medial side of the

knife cut (in the direction of the NTS) but diminished substantially lateral to the transection (Fig. 7c). Fi- bers diminished in numbers but did not disappear to-

tally indicating that fibers in the arching bundle are also directed from the ventrolateral medulla to the NTS. The significance of the contribution of these fi- bers to the overall levels of ACTH and/3-endorphin

present in the NTS seems to be minor, since the con- centration of these two peptides in the NTS (both in its rostral and commissural parts) did not change 8

days after a transection of the bundle between the NTS and the ventrolateral medulla (Table II). On the other (lateral) side of the knife cut, ACTI- and fl-en-

dorphin levels in the A 1, As-CI cell groups and nu- cleus ambiguus depleted significantly (Table I!).

However, since about 45-55% of these peptides re- mained after such transections, it would appear that the baroreceptor areas may also receive POMC-in-

nervation from elsewhere. As has been demon-

strated by the other lesions (Groups 1-9), the re- maining portion of the POMC peptides may arise from the hypothalamus. In agreement with the radio-

-, .,.g

immunoassay data, ct-MSH-positive fibers were di- minished but did not entirely disappear in the lateral reticular nucleus (Fig. 7e) and the nucleus ambiguus, ipsilaterai to the transection between the NTS and the ventrolateral medulla (Fig. 7a,c). The number of a-MSH-immunopositive fibers in the descending spi- nal bundle diminished only slightly after the medul- lary knife cut (Fig. 7e), which suggests that a-MSH cells in the NTS, in addition to hypothalamic cells, may also project to the spinal cord.

DISCUSSION

POMC-derived peptides in the brainstem barore- ceptor areas have been strongly implicated as flaying a role in the central control of blood pressure (for re- view see refs. 11, 13). The baroreceptor centers are relatively rich in POMC peptides as compared to their concentrations in neighboring ~-~reas in the lower brainstem 2°. Concentrations of ACTH, fl-endorphin and a-MSH in the commissural part of the NTS are almost as high as their hypo',halamic levels ~4. Among the POMC peptides, a-M3H levels in the barorecep- tor areas are generally higher, while ACTH levels are generally lower ,'.nan those of fl-endorphin. Gel filtration studies o~ acid extracts of the dorsal caudal medulla 3 reveal::d that fl-endorphin-sized and a- MSH-sized mzterial represent the major and that fl- LPH and ACTH represent the minor POMC end products in the caudal medulla of the rat.

A relatively high density of immunoreactive ACTH z3"24, fl-endorphin 6 and a-MSH 15"31 fibers has

been reported in the baroreceptor areas, and our re- sults are consistent with these earlier studies. Not

only fibers, but ACTH -'25. fl-endorphin -l i..'.~ and ct- MSH31-positive neuronal perikarya have been re- ported to be present in the commissural part of the NTS. In agreement with these studies, we found 8-15 ACTH. fl-endorphin and tt-MSH cells per sec- tion in the commis~ural part of the NTS with ion,,= ax- ons arching between the NTS and the ventrolateral medulla. A less extensive distribution of POMC-pcp- tide fibers were seen in the lateral reticular nucleus and the nucleus ambiguus, but these nuclei contained

more dense fibers than neighboring areas. lnnervation of the brainstem baroreceptor areas

by POMC-derived peptides arrives from two groups

of cells: one is located in the hypothalamic nucleus

336

arcuatus and the other one in the nucleus of the soli- tary trac ". Previous reports, based on chemical 4'22'23 or surgical 5 lesions of the arcuate nucleus or tran- section of possible descending fibers from the hypo- thalamus 2°'22, indicated that hypothalamic A C T H 2°'23-. fl-endorphin 2°- and a-MSH4"5-contain - ing cells project to the lower brainstem. In the pres- ent study, the surgical separation of the arcuate nu- cleus from the lower brainstem through a complete deafferentation of the medial basal hypothalamus (Group 1) resulted in depletions of POMC peptides in baroreceptor areas as significant as that observed after brainstem hemisections, indicating that ACTH-. fl-endorphin- and aoMSHocontaining descending fi- bers arise from the arcuate nucleus. Indeed, 3 days after medullary hemisection, ACTH-, fl-endorphin- and a-MSH-immunostained substances accumulated retrogradely only in the arcuate nucleus and nowhere else in the forebrain. In addition, after brainstem hemisections, enhanced a-MSH-immunostained cells were not seen in the dorsolateral hypothalamus where a second, but non-POMC-related a-MSH cell population is prc:,ent 7'29. This dorsolateral hypotha- lamic group of cells may send projections to the spi- nal cord 1~'2°, but probably not to the medullary baro- receptor areas.

Descending POMC-peptide-containing fibers run in two major pathways. One pathway descends through the periaqueductal central gray and further down in the dorsomedial tegmentum, while the sec- ond pathway runs in the ventrolateral tegmentum along the entire lower brainstem 6'1°A5,24,26. Fibers ap-

pear to leave the hypothalamus mainly through the medial posterior hypothalamus. Lesioning the me- dial-posterior hypothalamus (Group 2) reduced ACTH, fl-endorphin and a-MSH immunoreactivity in the baroreceptor areas similar to that seen with hy- pothalamic deafferentation. A portion of the de- scending POMC-peptidergic fibers may decussate at the level of tt'e posterior hypothalamus 24. Brainstcm transections caudal to this level 2° or medullary hemi- sections resulted in a retrograde accumulation of ACTH, fl-endorphin and a-MSH in the arcuate nu- cleus, not only ipsi- but also contralateral to the tran- section. Previous immun°hist°chemical data 1°.23.24 demonstrated that descending POMC pathways are interconnected at several levels in the brainstem. A summary of our radioimmunoassay and immunohis-

tochemical findings, after various types of brainstem lesions, indicates that descending ACTH, fl-endor- phin and a-MSH fibers may use both avenues, as out- lined, on their way through the midbrain and pons to the baroreceptor cer~ters. Fibers reaching the rostral part of the lateral reticular nucleus (A5-C t cell groups) and the rostral part of the NTS run mainly through the periaqueductai central gray. These fi- bers join the ventral tegmental fibers more caudally and most probably at the level of the midbrain-pon- tine junction, where POMC-immunoreactive fibers arc between the two descending pathways 6J°'23"24.

Descending fibers to the caudal part of the lateral ret- icular nucleus (A 1 cell group) and the nucleus ambig- uus run mainly through the ventrolateral tegmental tract. Lesioning of the periaqueductal central gray did not influence ACTH, fl-endorphin or a-MSH im- munoreactivity in these nuclei. The density of immu- noreactive fibers in the commissural part of the NTS remained intact, after the hypothalamic, midbrain or pontine operations indicating that its POMC innerva- tion of the commissural part of the NTS is most prob- ably of local origin.

Immunohistochemical and radioimmunoassay data further indicate that the POMC innervation of the lower brainstem may not arise entirely from the hypothalamus, since 30-50% of the ACTH 2°, fl-en- dorphin 2° and a-MSH 4"5 remained in the lower brain- stem after hypothalamic lesioning. It would appear, that the portion of POMC-derived peptides which re- main in the brainstem may be of medullary origin, in the present study, systemic immunohistochemical analysis and radioimmunoassay measurements in combination with numerous types of lesions, lend qualitative and quantitative support to this hypothe- sis. ACTH-, fl-endorphin- and a-MSH-containing cell bodies in the NTS may innervate not only the commissural part of the nucleus but may also give rise to fibers innervating the vasomotor (As-C 1 cell groups) and vasodepressor (A l cell group) areas and to the nucleus ambiguus 25. In the coronal plane, these fibers are observed arching between the NTS and the ventrolateral medulla. Transection of these arching fibers, substantially reduces the number of POMC-peptide-containing fibers in medullary areas lateral to the cut. In addition, long immunopositive fibers were seen extending from the ventrolateral medulla to the cut. These dorsomedially oriented fi-

337

bers m a y be long to the vent ro la tera l tegmenta l

P O M C pa thway which innervates the lateral ret-

icular and ambiguus nuclei and some of their f ibers

may reach the NTS from a ventroh-teral direct ion as

a minor c o m p o n e n t of the arc be tween the NTS and

the vent ro la tera i medul la . The POMC-pep t ide -con-

taining cells in the NTS appear not to project to the

higher b ra ins tem regions 23"3t, but p robab ly send fi-

bers to the spinal cord, as has been suggested pre-

viously 27. A significant reduct ion in the n u m b e r of

P O M C - i m m u n o s t a i n e d fibers, af ter b ra ins tem hemi-

sections and knife cuts be tween the NTS and the ven-

trolateral medul la , indicates that hypotha lanf ic and

NTS POMC-pep t ide -con ta in ing f ibers both partici-

pate in the descending spinal bundle .

ACKNOWLEDGEMENT

The authors wish to thank Chris t ina G. Palkovits

and Marjor ie K. W a r d e n for their assistance with im-

munohis tochemis t ry .

REFERENCES

1 Beaulieu, M., Goldman, M.E., Miyazaki, K., Frey, E.A., Eskay, R.L., Kebabian, J.W. and Cote, T.E., Bromocrip- tine-induced changes in the biochemistry physiology, and histology of the intermediate lobe of the rat pituitary gland, Endocrinology, 114 (1984) 1871-1884.

2 Dave, J.R., Rubinstein. N. and Eskay, R.L., Evidence that fl-endorphin binds to specific receptors in rat peripheral tis- sues and stimulates the adenylate cyclase-adenosine 3'.5'- monophosphate system. Endocrinology, 117 (1985) 1389-1396.

3 Dores, R.M., Jain, M. and AkiL H., Characterization of the forms of fl-endorphin and a-MSH in the caudal medulla of the rat and guinea pig, Brain Research, 377 (1986) 251-260.

4 Eskay, R.L., Brownstein, M.J. and Long, R.T., a-Melano- cyte-stimulating hormone: reduction in adult rat brain after monosodium glutamate treatment of neonates, Science, 205 (1979) 827-829.

5 Eskay, R.L., Giraud, P., Oliver, C. and Brownstein, M.J., a-Melanocyte-stimulating hormone in the rat brain, evi- dence that a-MSH containing cells in the arcuate region send projections to extrahypothalamic sites, Brain Re- search, ! 78 ~ ~ :~ 79) 55-67.

6 Finley, J.C.W., LindstrOm, P. and Petrusz, P., Immuno- cytochemicai localization of fl-endorphin-containing neu- rons in the rat brain, Neuroendocrinology, 33 (1981) 28-42.

7 Guy, L., Leclerc, R., Vaudry, H. and Pelletier, G., Identi- fication of a second category of a-melanocyte-stimulating hormone (a-.MSH) neurons in the rat hypothalamus, Brain Research, 199 (1980) 135-146.

8 Halfisz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all neuron pathways to the hypophysiotropic area, Endocrinology, 77 (1965) 553-562.

9 Joseph, S.A., Pilcher, W.H. and Bennett-Clarke, C., lm- munocytochem~cal localization of ACTH perikarya in nu- cleus tractus solitarius: evidence for a second opiocortin neuronal system, Neurosci. Leu., 38 (1983) 221-225.

10 Khachaturian, H., Lewis, M.E., Tsou, K. and Watson, S.J., fl-Endorphin, a-MSH, ACTH, and related peptides. In A. Bj6rklund and T. HOkfelt (Eds.), Handbook of Chemical Neuroanatomv. Vol. 4, GABA and Neuropep- tides in the CNS, Par: !. Elsevier, Amsterdam, 1985, pp. 916-9T~ ~ . , .

11 K6hler, C., Haglund, L. and Swanson, L.W., A diffuse a-

MSH-immunoreactive projection to the hippocampus and spi.n.a! cord from Z, ndividual neurons in the lateral hypotha- lamic area and zona incerta, J. Comp. Neurol.,. 223 (1984) 501-51,4.

12 Leslie, R.A., Neuroactive substances in the dorsal vagal complex of the medulla oblongata: nucleus of the tractus solitarius, area postrema, and dorsal motor nucleus of the vagus, Neurochem. Int.. 7 (1985) 191-211.

13 Lowry, O., Rosebrough, N., Farr, A. alid Randall, R., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.

14 Mezey, E., Kiss, J.Z., Mueller, G.P., Eskay, R.L., O'Do- nohue, T.L. and Palkovits, M., Distribution of the pro opiomelanocortin derived peptides, adrenocorticotropin, a-melanocyte-stimulating hormone and fl-endorphin (ACTH, a-MSH, fl-END)in the rat hypothalamus, Brain Research, 328 (1985) 341-347.

15 O'Donohue, T.L., Miller, R.L. and Jacobowitz, D.M., Identification, characterization and stereotaxic mapping of intraneuronal u-melanocyte-stimulating hormone-like im- munoreactive peptides in discrete regions of the rat brain, Brain Research, 176 (1979) 101-123.

16 Palkovits, M., Isolated removal of hypo:halamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450.

17 Palkovits. M.. Guide and Map for the Isolated Removal of h;dividual Cell Groups from the Rat Brain, (Hungarian text), Akad6miai Kiad6, Budapest, 1981}.

18 Palkovits, M., Distribution of neuroactive substances in the dorsal vagal complex of the medulla oblongata, Neuro- chem. hat., 7 (1985) 213-219.

19 Palkovits, M., Neuronal circuits in central baroreceptor mechanism. In I.H. Parvez. H. Saito, T. Nagatsu, and S. Parvez (Eds.), Progress in Hypertension, Vo;. 1, Neuro- transmitters as Modulators of Blood Pressure, V.N.U. In- ternational Science Press, Utrecht, in press.

2(~ Palkovits, M. and Eskav, R.L., Distribution and possible origin of fl-endorphin and ACTH in discrete brainstem nu- clei of rats, Neuropeptides. 9 (1987) 123-137.

21 Palkovits, M.. Tapia-Arancibia, L., Kordon, C. and Epel- baum. J., Somatostatin connections between the hypothal- amus and the iimbic system of the rat, Brain Research. 250 (198_) _,3-,_8.

22 Pelletier, G., Leclerc, R., Saavedra, J.M., Brownstcin, M.J., Vaudrv, H., Ferland, L. and Labrie, F., Distribution of fl-lipotropin (fl-LPH), adrcnocorticotropin (ACTH) and a-melanocvte-stimulating hormone (a-MSH) in the rat brain. I. ()rio.qin of the extrah.vpothalamic fibers, Brain Re-

338

search, 192 (1980) 433-440. 23 Pilcher, W.H. and Joseph, S.A., Differential sensitivity of

hypothalamic and medullary opiocortin and tyrosine hy- droxylase neurons to the neurotoxic effects of monosodium glutamate (MSG), Peptides, 7 (1986) 783-789.

24 Romagnano, M.A. and Joseph, S.A., lmmunocytochem- ical localization of ACTHI_39 in the brainstem of the rat, Brain Research, 276 (1983) 1-16.

25 Schwartzberg, D.J. and Nakane, P.K., ACTH-related pep- tide containing neurons within the medulla oblongata of the rat, Brain Research, 276 (1983) 351-356.

2,5 Shiosaka, S., Kawai, Y., Shibasaki, T. and Tohyama, M., The descending a-MSHergic (a-melanocyte-stimulating hormone-ergic) projections from the zona incerta and later- al hypothalamic area to the inferior colliculus and spinal cord in the rat. Brain Research, 338 (1985) 371-375.

27 Tsou, K., Khachaturian, H., Akil, H. and Watson, S.J., Immunocytochemical localization of pro-opiomelanocor- tin-derived peptides in the adult rat spinal cord, Brain Re-

search, 378 (1986) 28-35. 28 Usategui, R., Oliver, C., Vaudry, H., Lombardi, G., Ro-

zenberg, I. and Mourre, A.M., Immunoreactive a-MSH and ACTH levels in rat plasma and pituitary, Endocrinolo- gy, 98 (1976) 189-196.

29 Watson, S.J. and Akil, H.. a-MSH in rat. brain: occurrence within and outside of /~-endorphin neurons, Brain Re- search, 182 (1980) 217-223.

30 Watson, S.J. and Akil, H., Opioid peptides and related sub- stances: immunocytochemistry. In J.B. Martin, J. Reichlin and K.L. Bick (Eds.), Neurosecretion and Brain Peptides, Advanc. Biochem. Psychopharmacol., Vol. 28, Raven, New York. 1981, pp. 78-88.

31 Yamazoe, M., Shiosaka, S., Yagura, A., Kawai, Y., Shiba- saki, T., Ling, N. and Tohyama, M., The distribution of a- melanocyte stimulating hormone (a-MSH) in the central nervous system of the rat: an immunohistochemical study. II. Lower brain stem, Peptides, 5 (1984) 721-727.