IMMUNOHISTOCHEMICAL LOCALIZATION OF HYPOTHALAMIC

864

THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY

Copyright #{174}1976 by The Histochemical Society, Inc.

Vol. 24, No. 7, pp. 864-871, 1976

Printed in U.S.A.

IMMUNOHISTOCHEMICAL LOCALIZATION OF HYPOTHALAMIC

HORMONES

GEORGES PELLETIER, RACHEL LECLERC AND DONALD DUBE�

Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier de l’Uniuersit#{235}Laval, Quebec Gi V 4G2, Canada

Using an immunoperoxidase technique at the light and electron microscope levels, thelocalization of somatostatin and luteinizing hormone-releasing hormone (LH-RH) has beenperformed in the brain, as well as the pancreas and stomach. The following generalizationsmay be drawn on the localization of LH-RH and somatostatin. (a) LH-RH and somatostatinare contained in axons and nerve endings of the median eminence and organum vasculosumof the lamina terminalis. (b) LH-RH and somatostatin are present in different nerve endings.(c) In the subcomn�issural organ, subfornical organ and area postrema, the two neurohor-mones are present in the cytoplasm of ependymal and subependymal cells. (d) In the

endocrine pancreas and gastric mucosa, somatostatin is localized within the secretorygranules of specific endocrine cells.

The activity of the anterior pituitary gland is

controlled by neurohormones produced by the

hypothalamus. During the last few years, two

hypothalamic releasing hormones and one re-

lease-inhibiting hormone have been character-

ized and synthesized (3, 4, 7, 8, 22, 30). While it

is well known that hypothalamic regulatory

hormones are concentrated in the median cmi-

nence (19), it has been recently reported that

thyrotropin-releasing hormone and somato-

statin are present in some extrahypothalamic

areas (5, 6). Moreover, a direct inhibitory effect

of somatostatin in the secretion of insulin (15),

glucagon (15) and gastnin (13) has also been

reported, thus suggesting that this neurohor-

mone could be produced in the pancreas and

gastrointestinal tract. By measuring contents of

very small pieces oftissue obtained by microdis-

section (5, 6) or purified nerve endings (33), it is

not possible to determine which cells and gran-

ules contain the hormone in the nervous tissue.

Only use of immunohistochemical techniques at

both light and electron microscope levels can

achieve a precise identification of the cellular

elements responsible for the production of the

hypothalamic hormones. In order to gain a

better understanding of the mechanisms of

packaging and secretion of luteinizing hormone-

releasing hormone (LH-RH) and somatostatin

in the brain and target tissues, detailed im-

munohistochemical studies were performed on

the localization of these peptides.

IMMUNOHISTOCHEMICAL METHODS

The development by Sternberger (32) of a sensitive

immunoperoxidase technique involving the use of a

penoxidase-antiperoxidase (PAP) complex has pen-mitted the localization of neurohormones. With thistechnique, which is 100-1000 times more sensitive

than immunofluorescence, it has been possible todemonstrate small polypeptides such as vasopnessin(20), which has only eight amino acids. At the lightmicroscope level, the localization studies were per-formed in thick (7-�.cm) and semithin (2-�.cm) sections.The antisera against LH-RH and somatostatin were

supplied by Dr. A. Arimura (New Orleans) and wereproduced in our laboratory (by J. C#{244}t#{233}).The anti-serum to vasopressin was supplied by Dr. J. Roth(Bethesda, Md.), whereas the antiserum to oxytocinwas prepared in our laboratory (by Dr. A. Belanger).

The PAP was a generous gift from Dr. L. A. Stem-berger (Edgewood Arsenal, Md.). All of the im-munohistochemical reactions were carefully con-

trolled by immunoabsorption of the antiserum withthe synthetic hormone. Detection of different hor-

mones in consecutive sections was also used as a

control of specificity.

LOCALIZATION OF LH-RH

Luteinizing hormone-releasing hormone (LH-

RH), a decapeptide, has been studied by many

groups who used either fluorescence or peroxi-

dase labeling methods. In many species,

LH-RH was consistently observed in the exter-

nal zone of the median eminence (1, 2, 17, 18,

23, 31). The reaction was generally more con-

centrated in the lateral portions of the median

eminence (Fig. 1A) and appeared along the

entire sweep of the external zone, although more

concentrated in the caudal median eminence.

By immunoelectron microscopic studies, we

have clearly established that LH-RH is present

exclusively in nerve endings of the external zone

by guest on October 5, 2016jhc.sagepub.comDownloaded from

http://jhc.sagepub.com/

“.4 �__r�3.�#{149}.

‘-‘I

- .._�. �

4

--a- %5,4� -

+‘�, ,-.

1�

�1’ -

4,4

-; �#{149}�-4�-:,,

-tf-

-f

.4#{149}s

-- -‘I, � #{149}#{234}��#{149}#{149}-�-

-

:‘� � � - .4y -- -

-, -- -

-t

‘4

.�

4-S #{149}#{149}

NE *#{149}-#{149}#{216}l

-1 -.

i

‘I.

�:

- �‘4_ -

- � �

1��,

-‘- ��.e’’ ‘�c-i�

V � - � ..4S. A..&

q:..

- -- � 0

-- . -

‘,‘- � ...� ;‘-�

. #{149}-i�’� .-�(‘. - -. --- -;#. ,�

I � �4__

� � h�’�’ � � � ‘i4v� � � �

41’- --, -�j� - - - _“f- -

I � �

#{149} -.

- -

4-

FIG. 1. A, immunohistochemical localization of LH-RH in a coronal section of the caudal portion of themedian eminence. The reaction product (arrow) is concentrated in the dorsal region of the external zone. x 190.B, localization of somatostatin in a section adjacent to that shown in A. The reaction (arrows) is present in theventral and lateral portion of the external zone, whereas the dorsal region is relatively free of reaction. x 190.

FIG. 2. Electron micrograph showing a section of the external zone of the median eminence. A nerve ending

containing granules positive for LH-RH (arrow) is present. Other nerve endings (NE) are negative. x32,000.

865



866 PELLETIER, LECLERC, DUBE

of the median eminence (24). As evidenced in

Figure 2, the reaction is localized in the secre-

tory granules of the positive nerve endings. The

diameter of the LH-RH-containing granules

ranges from 75 to 95 nm. Absolutely no reaction

was observed in the tanycytes of the median

eminence.

Most investigators have been unable to show

immunostained cell bodies in the hypothalamus

(1, 17, 18, 31). In guinea pigs previously injected

with colchicine, Barry et al. (2), using immuno-

fluorescence, observed a few LH-RH positive

cell bodies scattered in a large area extending

from the preoptic area to the caudal part of the

tuber. Zimmerman et al. (36) described the

presence of LH-RH in the penikaryon of a few

neurons of the arcuate nucleus in the mouse.

However, the picture seems to indicate that

most of the staining is really around the neurons

instead of being inside the perikarya. Using

serial paraffin sections, we have been unable to

find any immunostained cell bodies in the rat

brain (23).

At the light microscope level, LH-RH was

localized around the organum vasculosum of

the lamina tenminalis (OVLT) of the mouse

(36), guinea pig (9) and rat (Fig. 3A). The

reaction was usually very strong and located in

close proximity to the capillaries of the onganum

vasculosum. With the electron microscope,

LH-RH was cleanly observed in the secretory

granules of a few nerve endings located in

contact with the basement membrane of the

fenestrated capillaries. These observations sug-

gest that LH-RH is secreted from this organ into

the general circulation. As in the median emi-

nence, all of the secretory granules (of a diame-

ter of 70-100 nm) were labeled in a positive axon

(23).

The other peniventnicular organs were also

studied for LH-RH detection. It was found that

the subfonnical organ, the subcommissural

organ (Fig. 4A) and the area postrema showed a

positive reaction. A common pattern for the

distribution of LH-RH was observed in these

organs: a strong reaction was localized in the

ependymal and subependymal layers and also

at the vicinity of blood vessels. At the ultra-

structural level, a diffuse reaction was observed

in the cytoplasm of the ependymal and subep-

endymal cells (Fig. 5). This reaction was not

clearly associated with any onganelles. Some

cell processes were also found to be labeled

mainly at the proximity of the capillaries.

LOCALIZATION OF SOMATOSTATIN IN THE BRAIN

By immunopenoxidase, somatostatin, the

most recently characterized hypothalamic hor-

mone, has been localized in the external zone of

the median eminence of both rat and guinea pig

(9, 16, 25, 26). The immunostaining was gener-

ally more intense in the lateral parts of the

median eminence, although the somatostatin

fibers are generally medial to LH-RH fibers

(Fig. 1B). Using an immunofluonescence tech-

nique, Hockfelt et a!. (14) have localized soma-

tostatin in the external zone of the median

eminence and the ventnomedial nucleus of the

guinea pig brain. With the aid of ultrastructural

localization, we have localized somatostatin

within secretory granules in about 30% of the

nerve endings in the external zone (25). These

granules were slightly larger than those contain-

ing LH-RH (90-110 nm versus 75-95 nm).

These results cleanly indicate that somatostatin

and LH-RH fibers, although belonging to the

tuberoinfundibular system, are separate sub-

systems.

Using serial paraffin, it was demonstrated

that somatostatin was located in the OVLT, the

subcommissural organ, the subfornical organ

and the pineal gland (10, 23, 26). In the OVLT,

immunostaining was found close to the capil-

laries, whereas ependymal cells remained un-

stained. As demonstrated on serial sections, the

localization of somatostatin was completely

different from that of LH-RH (Fig. 3B). With

high resolution immunohistochemistry, soma-

tostatin was detected in the secretory granules

(of a diameter of 90-120 nm) of many nerve

endings. In the subfornical organ, subcommis-

sunal organ and area postrema, the reaction was

FIG. 3. A, coronal section through the organum vasculosum of the lamina terminalis. LH-RH (arrows) islocalized in proximity to the capillaries (C) of the organum vasculosum. x440. B, detection of somatostatin of asection adjacent to that shown in A. The distribution of reaction product (arrows) dlose to the capillaries (C) isdifferent from that observed for LH-RH. x 440.

FIG. 4. Localization of LH-RH (A) and somatostatin (B) in two consecutive sections of the subcommissuralorgan. The distribution of the two neurohormones, which are mainly concentrated in the ependymal layers(arrows), is very similar. x540.



LOCALIZATION OF HYPOTHALAMIC HORMONES867

�-4� ‘4 1

� I,-� - --

� �; i� ‘Ik

4, 4 j� �.-r -�

;4 �

C � � .�

-� -I

� ‘.

-�-�-‘4- --

R �..

- 4’ ‘#_‘ *.e .‘ ,4,- -- .�.

L’�.

- ..

4��t

� � -

.�,‘-

#{149}:�‘� �

S. - __-�.S.� -

-r - - � � - - -�

._..-4�- ..-� .JI�



- -�r

----.4----’- -:

- - - .L,�--y--;--..

�4�4�4

I-’ -. - - - -

- �- --

- I

- .4.-. -

-�,:

�1

-.-,-,

-‘. -�t-.1�

I

1�

I

:-‘.---�‘

�d ‘4.’. �S. -,

-- �e�- � -. -�-:

� - - ----‘�- -, � -. --.

-- - - - - - -

I,- - - ��1”

- �:4. ;� �

� �, Y

--)�:�, �1:�1 - ,--

- ‘�- $�-:I �

4, -,- - ,--

p-v�s. � -

-1

�‘1��’-

‘-4

-, Si!

--

-- EC

I.

FIG. 5. Electron micrograph showing the localization of LH-RH in the subcommissural organ. A diffusepositive reaction is observed in the cytoplasm of many ependymal cells (E). V, third ventricle. x 16,000.

Fic. 6. Immunohistochemical localization of somatostatin in the gastric mucosa. Accumulation of PAPmolecules can be detected over the secretory granules (arrows) and the cytoplasm of an endocrine cell. Anotherendocrine cell (EC) contains granules which are completely negative. x 17,000.

868



LOCALIZATION OF HYPOTHALAMIC HORMONES 869

similar to that observed for LH-RH (Fig. 4B).

The accumulation of PAP molecules was pres-

ent over the cytoplasm of ependymal and sub-

ependymal cells. In the pineal gland, a diffuse

reaction was found in the cytoplasm of many

cells surrounding the capillaries.

In the OVLT, the presence of somatostatin in

nerve endings suggests that this organ, besides

its possible role in the control of gonadotropins

secretion, may also play a role in the control of

growth hormone secretion. In the other peniven-

triculan organs, the presence of LH-RH and

somatostatin in the same cells, which are not

secreting neurons, may simply represent an

uptake of the neurohonmone.

LOCALIZATION OF SOMATOSTATIN IN PANCREAS AND

STOMACH

In the pancreas of many species, a cell type

present in Langerhans islets has been shown to

contain somatostatin (10, 12, 27, 29). Absolutely

no immunostaining was found in the exocnine

cells or nerve endings. In the rat pancreas, the

positive reaction was mainly observed in the

secretory granules of a few cells located at the

periphery of the Langerhans islets (12, 27). The

diameter of the somatostatin granules was

about 175-210 nm. The a and � cells were

always unstained by the antisomatostatin se-

rum. This somatostatin cell seems to come-

spond to the D cell type (23, 29). These results

suggest that somatostatin, which inhibits the

release of both insulin and glucagon by the

endocrine pancreas, may have some physiologic

importance in the regulation of the secretion of

these two hormones.

Somatostatin cells were also found in the

gastrointestinal tract (23, 29). They are particu-

larly abundant in the antral mucosa in the

neighborhood of gastnin-secreting cells. These

positive cells were generally located in the basal

region of the villi. At the ultrastructural level,

the accumulation of PAP molecules was local-

ized over the secretory granules (150-250 nm in

diameter) with some degree of diffusion in the

cytoplasm of a cell type which has not been

found in contact with the pyloric humen (Fig.

6). On the basis of histochemical and ultra-

structural characteristics, the somatostatin cell

appears to correspond very well to the intestinal

D cell (23, 29).

NEUROPHYSIN IN THE EXTERNAL ZONE OF THE

MEDIAN EMINENCE

Neurophysin and vasopnessin have rarely

been observed in the external zone of the rat

median eminence (20, 34). Since at the ultra-

structural level they are localized in granules

(150-200 nm in diameter) similar to those

observed in axons ofthe internal zone (20), they

are thought to belong to the hypothalamo-

neurohypophyseal system. Very recently, an

increase in the content of neumophysin in the ex-

tennal zone of the rat median eminence after

adrenalectomy has been noted (34, 35). Since

the origin of this neurophysin, possibly as-

sociated with corticotropin-neleasing hormone,

was not known, an ultrastructural study was

performed to identify cleanly the structures con-

taming neurophysin and vasopressin in the

adrenalectomized rat. It was found that both

neumophysin and vasopnessin are present in

small granules (about 80-100 nm in diameter)

in the external zone of the adrenalectomized

animals (Fig. 7A and B). These positive nerve

endings are very different from the axons of

the internal zone of the median eminence,

which are also positive for neurophysin and

vasopressin and contain granules about 150 nm

in diameter (28). Thus it appears that there are

two neurophysin-vasopressin systems. The first

one is a constituent of the hypothalamo-

neurohypophyseal tract, whereas the second

one probably originates from a parvicellulan

system and is dramatically influenced by

adrenalectomy. The origin and role of this

second neurophysin-vasopressin system remain

to be established.

SUMMARY AND CONCLUSION

With the help of a sensitive immunohisto-

chemical technique, many important conclu-

sions have recently been obtained in the field of

neu.rosecnetion. LH-RH and somatostatin have

been found within secretory granules of nerve

endings located near the portal capillaries in the

median eminence. These findings confirm the

earlier theory that regulatory hormones are

produced by neurons and stoned in axon endings

before being released into the capillaries of the

pituitary portal plexus. In the OVLT, LH-RH

and somatostatin have also been localized in

nerve endings near the fenestrated capillaries of



870 PELLETIER, LECLERC, DUB�

1�

- �--

;5

i�-�--$ �-- -- S

-.---.‘ I

I-,

#{149}‘*�-

.‘e

#{149}:- S

--�--‘4

S

3--

-.5

4

.4,.

�-#{149}:��

�.

‘�;,..#{149}

�

#{149}.

�p-

-. -,- #{149}e_#{149}�4_ -�

1

Ftc. 7. Sections of the external zone of the median eminence of adrenalectomized rats treated for

neurophysin (A) and vasopressin (B) detection. Both neurophysin and vasopressin have been localized in nerveendings (arrows) containing granules of about 80-100 nm. x 17,500.

�-. -$!_S -

5-

54 ‘ -

� -.� :

the onganum vasculosum. These findings, simi-

lam to that observed in the median eminence,

suggest that OVLT could be involved in the

secretion of these hypothalamic hormones. Fur-

then studies are needed to evaluate the impor-

tance of this organ.

In the endocrine pancreas and the gastroin-

testinal mucosa, somatostatin has been found

in specific secretory cells. These somatostatin-

producing cells have always been found in close

proximity to endocrine cells (pancreatic a and ficells and G cells) of which they inhibit the

secretion. The wide distribution of somatostatin

cells in the gastrointestinal tract suggest that

they can influence the secretion of other hor-

mones.

Finally, we have shown that neurophysin and

vasopressin are also constituent of a parvicellu-

lan system. Since this system seems to be

related with adrenocorticotrophic secretion, it is

possible that a corticotropin-releasing hormone,

immunologically related to vasopressin, had

been detected by immunocytochemistry.

LITERATURE CITED

1. Baker, BL, Dermody WC, Reel JR: Distribution

of gonadotropin-releasing hormone in the rat

brain as observed with immunocytochemistry.

Endocrinology 97:125, 19752. Barry J, Dubois MP, Poulain P: LRF producing

cells in the mammalian hypothalamus. Z Zell-forsch Mikrosk Anat 146:351, 1973

3. B#{216}ler J, Enzman F,- Folkers K, Bowers CY,Schally AV: The identity of chemical and hormo-nal properties of the thyrotropin-releasing hor-mone and pyroglutamyl-histidyl-proline amide.Biochem Biophys Res Commun 37:705, 1969

4. Brazeau P, Vale W, Burgus R, Ling V, ButcherM, Rivier J, Guillemin R: Hypothalamic polypep-tide that inhibits the secretion of immunoreactivepituitary growth hormone. Science 179:77, 1973

5. Brownstein M, Arimura A, Sato H, Schally AV,Kizer JS: The regional distribution of somato-statin in the rat brain. Endocrinology 96:1456,1975

6. Brownstein M, Palkovits M, Saanedra JM, Bas-sin RM, Utiger RD: Thyrotropin-releasing hor-mone in specific nuclei of the brain. Science185:267, 1974

7. Burgus R, Butcher M, Ling N, Monahan M,

Rivier J, Blackwell R, Vale W, Guillemin R:



LOCALIZATION OF HYPOTHALAMIC HORMONES 871

Structure mol#{233}culaine du facteur hypothalamique(LRF) d’onigine ovine contn#{244}lant la s#{233}cr#{233}tiondel’honmone goandotrope hypophysaire de lut#{233}ini-sation. C R Acad Sci [Paris] 273:1611, 1971

8. Burgus R, Dunn TF, Desiderio D, Guillemin R:Structure mol#{233}culaire du facteur hypothalamiqueTRF d’origine ovine: mise en evidence parspectnom#{233}trie de masse de la sequence PCA-His-ProNH2. C R Acad Sci [Paris] 269:1870, 1969

9. Dub#{233}D, Leclerc R, Pelletier G: Immunohisto-chemical detection of growth hormone-release

inhibiting hormone (somatostatin) in the guinea-

pig brain. Cell Tissue Res 161:385, 197510. Dubois PM: Immunoreactive somatostatin is

present in discrete cells in the endocrine pan-creas. Proc Natl Acad Sci USA 72:1340, 1975

11. Fonssman WG, Orci L, Pictel R, Renold AE,Rouiller C: The endocrine cells in the epitheliumof the gastrointestinal mucosa of the rat. Anelectron microscope study. J Cell Biol 40:692,1969

12. Goldsmith PC, Rose JC, Arimura A, Ganong WF:Ultrastructural localization of somatostatin in

pancreatic islets of the rat. Endocrinology97:1061, 1975

13. Hayes JR, Johnson PG, Koerker P, Williams RH:Inhibition of gastnin release by somatostatin invitro. Endocrinology 96: 1374, 1975

14. Hokfelt T, Efendic S. Johansson D, Luft R,Animura A: Immunohistochemical localization ofsomatostatin (growth hormone release-inhibitingfactor) in the guinea pig brain. Brain Res 80:165,

1974

15. Johnson DG, Ensinck JW, Koerker P, Palmer J,

Goodner CJ: Inhibition of glucagon and insulinsecretion by somatostatin in the rat pancreasperfused in situ. Endocrinology 96:370, 1975

16. King JC, Gerall AA, Fishback JB, Elkind KE,Arimura A: Growth hormone-release inhibitinghormone (GH-RIH) pathway of the rat hypothal-amus revealed by the unlabeled antibody peroxi-

dase-antiperoxidase method. Cell Tissue Res160:423, 1975

17. King JC, Parsons JA, Erlandsen SL, WilliamsTH : Luteinizing hormone-releasing hormone(LH-RH) pathway of the rat hypothalamus re-vealed by the unlabeled antibody peroxidase-antiperoxidase method. Cell Tissue Res 153:211,

1974

18. Kordon C, Kerdelhu#{233} B, Pattou E, Jutisz M:

Immunocytochemical localization of LH-RH inaxons and nerve terminals of the rat medianeminence. Proc Soc Exp Biol Med 147:122, 1974

19. Krulich L, Quijada M, Illner P, McCann SM: Thedistribution of hypothalamic hypophysiotropic

factors in the hypothalamus of the rat. Int UnionPhysiol Sci 9:326, 1971

20. Leclerc R, Pelletier G: Electron microscope im-munohistochemical localization of vasopressin inthe hypothalamus and neunohypophysis of thenormal and Brattleboro rat. Am J Anat 140:583,1974

21. Luft R, Efendic 5, H#{246}kfeltT, Johansson 0,Arimura A: Immunohistochemical evidence forthe localization of somatostatin-like im-

munoreactivity in a cell population of the pan-cneatic islets. Med Biol 52:428, 1974

22. Matsuo H, Baba Y, Nair RMG, Arimura A,Schally AV: Structure of the porcine LH- andFSH-releasing hormone. I. The proposed amino

acid sequence. Biochem Biophys Res Commun43:1334, 1971

23. Pelletier G: Immunohistochemical localization ofhypothalamic hormones at the electron micro-

scope level, Hypothalamus and Endocrine Func-tions. Edited by F Labnie, J Meites, G Pelletier.Plenum Press Co., New York, in press

24. Pelletier G, Labnie F, Arimura A, Schally AV:Electron microscope immunohistochemical local-ization of luteinizing hormone-releasing hormonein the rat median eminence. Endocrinology95:314, 1974

25. Pelletier G, Labrie F, Arimura A, Schally AV:Electron microscopic immunohistochemical lo-calization of growth hormone-release inhibitinghormone (somatostatin) in the rat median emi-nence. Am J Anat 140:445, 1974

26. Pelletier G, Leclerc R, Dub#{233}D, Labnie F, PuvianiB� Arimura A, Schally AV: Localization of growthhormone-release inhibiting hormone (somato-

statin) in the rat brain. Am J Anat 142:397, 1975

27. Pelletier G, Leclerc R, Animura A, Schally AV:

Immunohistochemical localization of somato-

statin in the rat pancreas. J Histochem Cytochem23:699, 1975

28. Pelletier G, Leclerc R, Labrie F, Puviani R:Electron microscopic immunohistochemical lo-calization of neurophysin in the rat hypothalamusand pituitary. Mol Cell Endocrinol 95:314, 1974

29. Polak JM, Pearse AGE, Grimelius L, Bloom SR.Animura A: Growth hormone-release-inhibitinghormone in gastrointestinal and pancreatic D-cells. Lancet 1:1220, 1975

30. Schally AV, Dupont A, Arimura A, Redding TW,Lenthicum GL: Isolation of porcine GH-releaseinhibiting hormone (GH-RIH): The existence of 3forms of GH-RIH. Fed Proc 34:584, 1975

31. S#{233}t#{225}l#{243}G, Vigh 5, Schally AV, Animura A, Flerk#{243}B: LH-RH containing neural elements in the rathypothalamus. Endocrinology 96: 135, 1975

32. Sternberger LA: Immunocytochemistry. PrenticeHall Inc., Englewood Cliffs, N.J., 1974

33. Taber CA, Karovolas HJ: Subcellular localizationof LH releasing activity in the rat hypothalamus.Endocrinology 96:446, 1975

34. Vandesande F, De Mey J, Dienicks K: Identifica-tion of neurophysin producing cells. I. The originof the neurophysin-like substance-containingnerve fibers of the external zone of the medianeminence. Cell Tissue Res 151:187, 1974

35. Watkins WE, Schwabedal P, Bock R: Im-munohistochemical demonstration of a CRF-associated neurophysin in the external zone of therat median eminence. Cell Tissue Res 152:411,1974

36. Zimmerman EA, Hsu KC, Ferin M, KozlowskiGP: Localization of gonadotropin-releasing hor-mone (Gn-RH) in the hypothalamus of the mouseby immunopenoxidase dose technique. Endocri-nology 95:1, 1974



Documents

IMMUNOHISTOCHEMICAL LOCALIZATION OF HYPOTHALAMIC