Hypothalamic thyrotropin-releasing hormone (TRH)-containing neurons involved in the hypothalamic-hypophysial-thyroid axis. Light microscopic immunohistochemistry

Brain Research, 345 (1985) 205-218 Elsevier

205

BRE 11060

Research Reports

Hypothalamic Thyrotropin-Releasing Hormone (TRH)-Containing Neurons Involved in the Hypothalamic-Hypophysial-Thyroid Axis. Light Microscopic Immunohistochemistry TOHRU NISHIYAMA l, HITOSHI KAWANOL YOSHIHIRO TSURUO 1, MASAHIKO MAEGAWA I, SETSUJI HISANO I . T O H R U ADACHI 1. SHIGEO DAIKOKU 1and MITSUO SUZUKI 2

JDepartment of Anatomy, School of Medicine, The University of Tokushima, Tokushima 770 and 2Department of Physiology. Institute of Endocrinology, Gunma University, Maebashi 371 (Japan) (Accepted January 8th, 1985)

Key words: hypothalamus - - immunohistochemistry - - thyrotropin-releasing hormone (TRH) - - rat

The localization of neurons containing immunoreactive thyrotropin-releasing hormone (TRH) was examined in the hypothalamus of intact, propylthiouracil (PTU)-treated, and colchicine-treated adult rats. In intact animals, immunoreactive TRH neurons were occasionally found in the paraventricular and dorsomedial nuclei I and in the anterior and lateral hypothalamic areas. In PTU-treated animals, the cellular appearance of the hypothalamus with the exception of the paraventricular nucleus was almost similar to that of intact animals. In the paraventricular nucleus, only the cells localized in the periventricular and medial parvocellular subdivisions significantly increased in number and became hypertrophic in comparison with intact animals. The distribution of immunoreactive fibers in the hypothalamus was almost equal among the 3 animal groups with the exception of that in the median eminence, in which the fibers werc most densely concentrated in intact animals, and most sparse in PTU-treated rats. The fibers projecting into the median eminence were distinguished into the periventricular and lateral pathways, which are derived from the neurons in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus, respectively. Thus, among immunoreactive TRH neurons in the hypothalamus, only those in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus may be involved in the hypothalamic-hypophysiabthyroid axis,

INTRODUCTION

t h a l a m u s or electric s t i m u l a t i o n of certain h y p o t h a lamic nuclei h a v e b e e n p e r f o r m e d by m a n y r e s e a r c h -

Although immunoreactive TRH-containing

neu-

ers 25. In rats b e a r i n g the d e a f f e r e n t e d island o f the

rons are localized in m a n y r e g i o n s of the h y p o t h a l a -

m e d i o b a s a l h y p o t h a l a m u s , B r o w n s t e i n et al. 7 h a v e

mus ~,~,el,~6, a c c u r a t e l o c a l i z a t i o n of the n e u r o n s in-

d e m o n s t r a t e d that the c o n t e n t of T R H in the island

v o l v e d in h y p o p h y s i o t r o p h i c

f u n c t i o n has not yet

was 76% l o w e r t h a n in the tissue f r o m intact c o n t r o l

b e e n d e t e r m i n e d . I n d e e d , the n e u r o n s are f o u n d in

rats, a l t h o u g h t h e r e was no r e d u c t i o n in the h o r m o -

the s u p r a c h i a s m a t i c - p r e o p t i c n u c l e u s , p a r v o c e l l u l a r

nal levels in r e g i o n s o u t s i d e of the island. T h e s e find-

subdivisions of the p a r a v e n t r i c u l a r n u c l e u s , p e r i f o r -

ings suggest that a m a j o r part of the T R H i n c l u d e d in

nical area, d o r s o m e d i a l n u c l e u s , and lateral hypo-

the h y p o t h a l a m u s m a y be s y n t h e s i z e d o u t s i d e of the

t h a l a m u s , and their axons a p p e a r to be p r o j e c t e d

island. In a similar e x p e r i m e n t , H e f c o et al.=2 h a v e

widely t h r o u g h o u t the h y p o t h a l a m u s , and, p r e s u m ably, e v e n in e x t r a h y p o t h a l a m i c brain regions. T o

p r o p o s e d that m o s t if not all of the T R H necessary for n o r m a l T S H s e c r e t i o n u n d e r resting c o n d i t i o n s o r

d e t e r m i n e the localization of T R H i n v o l v e d in the re-

during a h y p o t h y r o i d state is p r o d u c e d in the island

lease of h y p o p h y s i a l t h y r o i d - s t i m u l a t i n g h o r m o n e ( T S H ) , various lesions, d e a f f e r e n t a t i o n s of the hypo-

and r e l e a s e d into the h y p o p h y s i a l portal vessels at the m e d i a n e m i n e n c e . T h e r e f o r e , we suggest that al-

(½rrespondence: S. Daikoku, Department of Anatomy, School of Medicine, The University of Tokushima 770, Japan. 0006-8993/85/$03,30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

though neurons containing T R H distributed widen throughout the hypothalamus are involved in the hypophysiotrophic function as well as in neurotrophic functions, most of T R H which is concerned with emergent or unusual demand of TSH may be synthesized in certain areas, outside of the basal medial hypothalamus. The purpose of the present study is to determine immunohistochemically, the localization of T R H neurons responding to a thyroid hormone deficiency in the rat hypothalamus. MATERIALS AND METHODS Adult male Sprague-Dawley rats, weighing 180-290 g, were used. The animals consisted of the following 3 groups: 13 intact rats; 14 propylthiouracil-treated rats which were administered per os with a suspension of the drug (50 mg in 2 ml distilled water/ rat) once a day for 9 days and killed 24 h after the last treatment; and 12 colchicine-treated animals which were treated with an intraventricular administration of colchicine (50/~g in 5/tl saline) and killed 24 h after the treatment. The details of the treatments have been described in previous papers ls,26. The animals were killed by cervical dislocation without anesthesia. The hypothalamus was removed from the brain with hypophysis and fixed overnight in a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 °C. The thyroid gland was also taken and fixed in the same fixative. Tissue taken for the hypothalamic fragment was bounded anteriorly by the anterior margin of the optic chiasm, posteriorly by the anterior margin of the mammillary nucleus, laterally by the hypothalamic sulcus, and dorsally by a plane which was parallel to the ventral surface of the brain passing through the dorsal margin of the anterior commissure. The hypothalamus was dehydrated with an ascending alcohol series, embedded in paraffin, and cut serially at 5 p m thicknesses in the frontal plane. Every 20th section was mounted on glass slides and immunostained for histological examination. To verify the effect of PTU, thyroid glands were also examined histologically. The glands fixed were weighed, and made in paraffin sections 5 #m thick for light microscopic observation. The tissue sections were stained with hematoxylin and eosin, and the height of the follicular epithelium was determined

with a micrometer under x4110 magnification. l h c data obtained were analyzed statistically. For immunostaining of T R H , the double-bridge peroxidase-antiperoxidase (PAP) method by Vacca et al? 5 was employed. The antiserum was produced against T R H conjugated with thyrogl0bulin in rabbit. and the specificity of the serum was determined > . During staining, the serum was diluted 1:1000 with 0.l M phosphate buffer containing rat liver extract. The staining specificity of the serum was determined by preabsorbing the antibody with synthetic TRH26. No specific immunoreaction was obtained when the sections were stained with the preabsorbed antiserum. Determination of the number of immunoreactive cell bodies in tissue sections was performed only in the animals in which the tissue preparation was suitably performed. The immunoreactive cell bodies with immunonegative nucleus were counted in every 20th section cut in 5/*m thicknesses and passing through the paraventricular nucleus, dorsomedial nucleus, perifornical area and anterior hypothalamic nucleus. The total number of the cells was determined in each nucleus, and the data were treated statistically to examine significant differences among the animal groups. In the paraventricular nucleus, the cells were further estimated in each subdivision33, since the cells appeared in groups in the nucleus. The determination of the number of T R H neurons in the PTUtreated animals was especially critical in the present study. Therefore, the examination of the thyroid gland was performed in conjunction with the hypothalamus in all animals. If the PTU treatment did not result in hypertrophy of the thyroid gland, the animals were omitted. In the present study, there was one such animal, and the numerical data on the hypothalamic T R H neurons were rejected. In addition, one of the intact animals having an abnormally hypertrophied thyroid gland was also omitted from the statistical analysis. Student's t-test and the AspinWelch method were employed for statistical analyses of the data on the number of T R H neurons and cell height of the thyroid follicular epithelium. RESULTS

Hypothalamus General view (Fig. 1). TRH-like immunoreactivity

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Fig. 1. Schematic illustrations showing the localizations of immunoreactive TRH-containing cell bodies (large dots) and fibers (small dots) in the frontal sections of the hypothalami of PTU-treated (PTU), intact and colchicine-treated (Col) rats The sections (A-F), which are arranged from top to bottom, were obtained at distances 2300, 2100, 1400, 1100, 300 and 0/xm from the rostral border of the infundibulum, respectively One large dot approximately corresponds with 3 cell bodies• The populations of the cell bodies are d i s tinctive in the 3 animal groups but those of the fibers arc almost similar

208 is evident in neuronal fibers and cell bodies in many regions of the hypothalamus. The fibers extend from the rostral part of the p r e o p t i c nucleus to the mammillary complex, and are c o n c e n t r a t e d in certain nuclear areas of the hypothalamus, forming dense networks. In the m e d i a n eminence, the fibers a p p e a r as b e a d e d strings a r r a n g e d in a palisade structure to the external surface of the m e d i a n eminence. The staining density and architecture of the fiber networks seem to be somewhat variable in animals but do not differ among the 3 animal groups. I m m u n o r e a c t i v e cell bodies, however, are distinctive in population and stainability among the 3 animal groups. Distribution of immunoreactive TRH fibers. The most dense concentration of immunoreactive T R H fibers is found t h r o u g h o u t the rostral-caudal extent of the external layer of the m e d i a n eminence, juxtaposed to the portal capillaries, where T R H m a y be discharged from the fiber terminals to the capillaries. These secretory fibers are b e a d e d strings which are distributed from the lateral portion of the m e d i a n eminence to its midregion, where immunoreactivity was most heavily concentrated. The a p p e a r a n c e of the fibers varies considerably among the 3 animal groups. The accumulation and varicosity of the fibers

Fig. 3. Immunoreactive TRH fibers (dots) distributed m the peripheral zone and at the center of the hypophysial stalk (StL V, third ventricle, x 175. are most p r o m i n e n t in intact animals and least prominent in P T U - t r e a t e d animals (Fig. 2). The fibers were also found as dots in the internal layer of the

Fig. 2. Median eminence of intact (a), colchicine-treated (b) and Fl'U-treated (c) rats. Beaded immunoreactive TRH fibers are concentrated in the external layer surrounding the capillaries (cap) of the portal vessels. The fibers accumulate most densely in a, and most scarcely in c. Ep, ependym, x 560.

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b Fig. a Anterior p o m o n of the hypothalamus Rectangular area in a ( x 56) is shown with a larger magnification (x250) in b. Immunoreactive T R H fibers appear to traverse into the stria terminalis (arrows in b). Arrowheads show immunoreactive cell bodies. Frontal section.

median eminence. They may extend posteriorly to the neural lobe of the pituitary. In fact, the fibers were followed into the hypophysial stalk (Fig. 3). Immunoreactive fibers are also condensed in the organum vasculosum laminae terminalis, where long beaded strings of immunoreactive fibers are present, running dorsoventrally. This seems to be derived from the neurons containing TRH in the medial preoptic nucleus. The fibers accumulate under the anterior commissure (Fig. 4). They seem to be derived from the cells localized in the anterodorsal portion of the paraventricular nucleus and ascend anterodorsally to the anterior commissure. The fibers then turn laterally and enter into the stria terminalis and disperse. Their further pathway, however, can not be determined presently. Fiber networks are not evident in this area. In the paraventricular nucleus (Fig. 5), the fibers

accumulate densely in the parvocellular portion but loosely in the magnocellular region. In the periventricular region, many fibers traverse dorsoventrally as long beaded strings. These represent the periventricular fiber pathway to the median eminence. From the lateral portion of the paraventricular nucleus, thc fibers traverse laterally, turn ventrally surrounding the fornix, then further turn medially ahmg the dorsal margin of the optic tracts to enter the median eminence laterally. Along this fiber pathway, various numbers of immunoreactive cells are observed in both colchicine-treated and PTU-treated animals. In the dorsomedial nucleus (Fig. 6), the fibers accumulate densely and extend into the lateral hypothalamic area. They appear to surround immunonegative cell bodies, and occasionally immunoreactive cell bodies, if present, suggesting the presence of synaptic contacts (Fig. 7). In the lateral hypothalamic

210

Fig. 5. Frontal section of the hypothalamus showing the paraventricular nucleus with immunoreactive TRH neurons and the lateral (l) and periventricular (m) pathways (arrows) derived from the nucleus, Note that the lateral fiber pathway is accompanied by a numbcr of immunoreactive cell bodies (arrowheads). V, third ventricle, x56. Fig. 6. Frontal section of the hypothalamus. The dorsomedial nucleus shows a dense accumulation of immunoreactivc fibers which fu~ther extend laterally (arrows) to surround the fornix. A long arrow shows the periventricular immunoreactive fiber pathway. V. third ventricle. × 56. area, the fibers appear around the fornix and turn ventrally and further, posteromedially. The continuing pathway of these fibers is uncertain at present, but they seem to pass into the premammillary regions, sending some branches to the arcuate nucleus. In the perifornical area, the fibers seem to conjoin with the fibers derived from the lateral portion of the paraventricular nucleus. A moderate accumulation of immunoreactive fibers is found in the dorsal and ventral premammillary region and in the arcuate nucleus, where the fibers appear as dots.

Distribution of immunoreactive TRH-containing perikarya. In intact animals, the perikarya are found only occasionally in the paraventricular nucleus, dorsomedial nucleus, anterior and lateral hypothalamic areas, but in colchicine-treated animals, they appear abundantly in many nuclear regions. The cells found in intact animals are small and stained lightly or moderately. In colchicine-treated animals, many perikarya are localized in the ventral portion of the medial preoptic

nucleus, diagonal band of Broca. anterior, medial and periventricular parvocellular subdividions of the paraventricular nucleus and dorsomedial nucleus. and some in the lateral hypothalamic area. perifornical area, arcuate nucleus and premammiilary regions. This distribution pattern of the perikarya is similar to the findings of Lechan and Jackson 2~ using vibratome thick sections. In the lateral hypothalamic area, the perikarya are found along the fiber bundle. which are probably derived from the medial parvocellular subdivision of the paraventricular nucleus. It remains to be elucidated whether the fibers and celt bodies correspond with the classical neurosecretory fibers and cell bodies. In the parvocellular subdivisions of the paraventricular nucleus, periventricular area and dorsomedial nucleus, most of the immunoreactive neurons are small, densely stained and bipolar, whereas the cells in the premammillary area. the diagonal band of Broca, lateral hypothalamic area and anterior hypothalamic area are large, lightly stained and multipolar (Fig. 8). The most dense accumulation of the perika-

21l ventricular parvocellular subdivisions. In the lateral and dorsal parvocellular subdivisions, the cells arc less numerous. Only a few cells, if any, are encountered in the magnocellular portion of the paraventricular nucleus (Fig. 9). In PTU-treated animals, the appearance of the cells in the hypothalamic areas is the same as in intact animals with the exception of the cells in the paraventricular nucleus, where they are more n u m e r o u s than in intact animals although less n u m e r o u s than in colchic±he-treated animals. In the paraventricular nucleus, the cell bodies are more hypertrophic than the cells which appeared in colchicine-treated and in intact animals, and the immunostainability is less dense (Fig. 10). They are localized in the medial and periventricular parvocellular portions of the nucleus. Quantitative analysis of T R H cell n u m b e r s indicates (Tables I and II) that T R H cells are significantly more n u m e r o u s in colchicine-treated animals in all nuclear regions examined in comparison with intact controls. P T U treatment also resulted in a significant increase in T R H cell n u m b e r of the paraventricular nucleus in comparison with intact animals, although less than in colchicine-treated animals. The n u m b e r of T R H - c o n t a i n i n g n e u r o n s is further examined quantitatively in each parvocellular subdivision of the paraventricular nucleus (Table I1). Colchicine administration induced a significant increase

Fig. 7. Dorsomedial nucleus of the hypothalamus. Beaded immunoreactive fibernets (arrowheads) surround an immunonegative cell body (a) and an immunoreactive cell body (b). a and b are from animals treated with colchicine and PTU, respectively. ×900.

in cell n u m b e r in all subdivisions of the nucleus in comparison with intact and P T U - t r e a t e d rats. However, in PTU-treated rats, the T R H cells increased significantly in n u m b e r only in the periventricular subdivision and medial parvocellular subdivision of the nucleus in comparison with untreated animals.

rya is found in the paraventricular nucleus, where they are confined to the medial, anterior and per±-

TABLE I Effects of propylthiouracil ( PTU) and colchicine (Col) on numbers of hypothalarnic irnmunoreactive TRH cells

Supplemental data obtained from a PTU-treated animal (ineffect) and an untreated animal (abnorm) are rejected from the statistical evaluation due to an abnormal appearance of their thyroids (Table Ill). Animals

Hypothalamic nuclei PVN

DMH

PFA

A HA

Intact PTU Col

106.4 ± 9.98 193.3 ± 18.14" 457.9 + 2(/.25"

53.0 _+8.48 57.9 _+9.(/9 257.1 _+21.79~

29.8 ± 3.30 24.9 _+3.46 109.5 _+ 15.15"

23.8 ± 2.56 28.7 _+4.90 68.2 _+7.06*

±neffcot abnorm

29 172

43 88

21 -

30 6(I

* P < (I.001 vs intact control.

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Fig. 8. Various types of immunoreactive T R H neurons found in the medial parvocellular subdivision of paraventricular nucleus (PVN) (at, lateral hypothalamic area (b, c), premammillary area (d), and diagonal band of Broca (e). The cells in a and b are oval and bipolar, and the others are spherical and seem to be multipolar. Note that the immunoreactive granules exist even in the processes (a and b) of colchicine-treated rats. x 1050.

T A B L E II

Effects of propylthiouracil (PTU) and colehicine (Col) on numbers of immunoreactive TRH containing cells in the subdivisions o]'the paraventricular nucleus Animals

Intact PTU Col

Paraventricular subdivisions mp

pv

ap

dp

lp

59.1 + 6.1 118.8 + 14.3" 209.0 + 15.7"*

1(1.1 +_ 3.3 24.7 + 313" 98.4 + 7.3**

26.6 + 4.6 34.4 + 7.4 107.2 _+ 6.5**

3.8 + 1.0 2.9 + 1.1 17.3 + 1.8"*

2.1 + 0.5 1.6 _+ 0.5 9.7 +_ 1.8 **

* P < 0.01 vs intact control. ** P < 0.001 vs intact control.

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Fig. 9. Schematic representations of immunoreactwe TRH-containing neurons (dots) localized in the subdivisions (Swanson and Kuypers 33) of the paraventricular nucleus in frontal sections of the hypothalami of PTU-treated (PTU), intact and colchicine-treatcd (Col) rats. The sections are arranged rostrocaudally from top to bottom (A-D). One dot corresponds to one cell.

Thyroid gland For morphological criteria of the function of the thyroid gland, we determined the weight of the thyroid gland and the height of the follicular epithelial cells of the gland (Table III). As shown in Table III, the thyroid weight and the height of follicular epithelium increase significantly in PTU-treated animals in

comparison with untreated and cholchicine-treated animals. Colchicine treatment does not affect the weight of the gland or the height of the epithelium. The results clearly indicate that PTU treatment acts as an effective goitrogen, although the serum content of thyroid hormones was not determined.

214

Fig. 10. Paraventricular nuclei of intact (a, b), colchicine-treated (c, d) and PTU-treated (e, f) rats. Rectangular areas in a. c and u ~rc shown in b, d and f, respectively, under greater magnification (x600). Note that the immunoreactive cells arc accumulated in the medial parvocellular subdivision of the nucleus. In colchicine-treated animals, the cells appear in the greatest abundance, and the immunostainability of the perikarya is strongest. However. in e and f, the cells are hypertrophic but weakly immunoreactive. V, third ventricle. x 175.

DISCUSSION The present study is the first to show immunohistochemically that although T R H - c o n t a i n i n g cells exist in many nuclear regions in the hypothalamus, only those localized in the medial and periventricular parvocellular subdivisions of the paraventricular nucleus respond to P T U treatment with a numerical increase,

hypertrophy and diminished stainability of the perikarya. This finding suggests that this population of neurons is involved in the negative feedback mechanism of the hypothalamic-hypophysial-thyroid axis. Due to difficulties with fixation, the immunohistochemical determination of T R H was delayed in comparison with other hypophysiotrophic peptides which were isolated after T R H 2,4,9,24. Lechan and Jackson 21

215 T A B L E III

Effect of propylthiouracil and colchicine on the thyroid gland Supplemental data (ineffect and abnorm) are rejected from the statistical evaluation.

Animals

Body weight (g)

Thyroid gland

Numbers

At the beginning of treatment

At sacrifice

Weight (rag)

Intact

10

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2 1 . 6 _+ 1.46

PTU Colchicine

11 9

235.0 + 5.3 243.8 _+7.3

228.5 _+6.0 222.2 _+6.7

46.8 _+ 1.52" 20.6 + 0.64

18.36 + I).81" 4.49 _+0.19

255 -

255 245

41.8 29.2

5.26 19.67

Treatments

ineffect abnorm

1 1

Height of follicular epithelium (l,m) 4.65 _+ I).35

* P < 0.001 vs intact control.

d e m o n s t r a t e d well-preserved T R H - c o n t a i n i n g structures in the h y p o t h a l a m u s after applying acrolein as a fixative. Acrolein has been introduced recently into immunohistochemistry as a fixative useful in preserving the peptides 19. Previously, we also found out that acrolein fixes T R H well in the brain tissues 10. However, acrolein is a highly toxic tear gas which causes much difficulty during tissue p r e p a r a t i o n . In the present study, therefore, we employed a mixture of glutaraldehyde and p a r a f o r m a l d e h y d e . The present study indicates that in the hypothalamus, the cells and fibers containing i m m u n o r e a c t i v e T R H are distributed in the same regions as described by Lechan and Jackson 21. A l t h o u g h the populations of the cells and fibers seem to be less p r o m i n e n t than those in their findings as j u d g e d by p h o t o g r a p h s , accurate comparison may be difficult since their tissue sections were 60 ~m in thickness and ours are 5 ~tm in thickness. As shown in many other n e u r o p e p t i d e s synthesized in the hypothalamusl3.17.18, 34, intraventricular administration of colchicine was effective in demonstrating T R H - c o n t a i n i n g cell bodies in the hypothalamus 14,16,21. This was also the case in the present study. As shown in Tables 1 and II, T R H - c o n t a i n i n g cell bodies were much m o r e n u m e r o u s in colchicinetreated animals in comparison with u n t r e a t e d animals in many nuclear regions of the hypothalamus. In the P T U - t r e a t e d animals, the cells were n u m e r o u s only in the periventricular and medial parvocellular subdivisions of the p a r a v e n t r i c u l a r nucleus with a few in other regions as shown in u n t r e a t e d animals. In P T U - t r e a t e d animals, it was also recognized that the

thyroid gland b e c a m e hypertrophic and the follicular epithelium was high in comparison with intact and colchicine-treated animals. Of interest is that such an increase in T R H cell n u m b e r in the parvocellular subdivisions of the paraventricular nucleus was not o b t a i n e d in an animal in which no significant thyroid h y p e r t r o p h y occurred after the PTU treatment. Since P T U disturbs iodine incorporation of thyroid follicular cells, these findings suggest that there is a reverse relationship between the a m o u n t of circular thyroxine and the numerical increase in T R H - c o n taining neurons in the parvocellular divisions in the paraventricular nucleus. Therefore, it seems reasonable to say that the T R H neurons in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus may be involved in the negative f e e d b a c k mechanism in the h y p o t h a l a m i c - h y p o physial-thyroid axis. In a r e t r o g r a d e transport e x p e r i m e n t using wheat germ agglutinin as a m a r k e r , Lechan et al. 22 d e m o n strated that the anterior, periventricular and medial parvocellular subdivisions of the p a r a v e n t r i c u l a r nucleus project the axons to the median eminence. Similarly, using True Blue as a tracer, Sawchenko and Swanson 32 have found that the periventricular and medial parvocellular subdivisions of the paraventricular nucleus project axons to the median eminence. D e a f f e r e n t a t i o n of the medial-basal h y p o t h a l a m u s suggests that much of the T R H that is normally present in the tissue may be synthesized by neurons outside of this region. These findings would be consistent with the physiological studies by Martin and

216 Reichlin 23 which showed that the greatest TSH response to hypothalamic electrical stimulation occurred when electrodes were placed bilaterally in the paraventricular nucleus, and by Aizawa and Greeri showing that a prompt reduction in plasma TSH occurs after lesions which were confined to the paraventricular nucleus. Earlier work by H6kfelt et al.~4 implicated the midline hypothalamic regions between the paraventricular nucleus and the anterior lip of the infundibulum as the thyrotropic area. Further experiments have been performed on the origins of immunoreactive T R H fibers in the median eminence. Using a combination of anterolateral, bilateral and frontal cut of the hypothalamus, Palkovits et al. 2s proposed that the T R H fibers pass through a small portion localized in the lateral retrochiasmatic area from the outside of the medial-basal hypothalamus to the median eminence. As shown here, among T R H cells existing outside of the medial-basal hypothalamus, only the cells existing in the medial and periventricular parvocellular subdivisions were reactive to PTU treatment. Although Palkovits et al.2~ have refused the existence of the periventricular pathway of TRH-containing fibers since they could not obtain a statistically significant reduction of T R H contents after coronal transection of the anterior hypothalamus, their data show a remarkable reduction of the contents. We showed a number of T R H fibers running dorsomediaily in the periventricular area of the third ventricle. They appeared to be derived from the neurons in the periventricular parvocellular division of the paraventricular nucleus, which was affected by the PTU treatment. Thus, it is likely that there are two pathways of TRH-containing fibers from the paraventricular nucleus to the median eminence: the lateral and medial or periventricular fiber pathways derived from the medial and periventricular parvocellular subdivisions of the paraventricular nucleus, respectively. To confirm this point a study combined with TRH-immunohistochemistry and retrograde axonal transport of wheat germ agglutinin as a marker is in progress in our department. A complete deafferentation of the basal medial hypothalamus 7 and lesion of the paraventricular nucleus 8 indicated a partial remainder of T R H in the basal medial hypothalamic island. Using various sized hypothalamic deafferentations, Hefco et al. t2 have suggested that the T R H necessary for normal TSH secretion under resting conditions or during the hypo-

thyroid state may be produced in the median era> nence-arcuate nuclear area, and released into the h~,o pophysial portal vessels. Studying the topograph 3, ol immunoassayable T R H in the hypothalamus, Krulich et al. e0 have assumed that the dorsomedial and preoptic areas are sites of T R H production, from which the peptide is conveyed presumably by long axons to the median eminence where ir is stored in axon terminals. Additionally, they have proposed that the median eminence-arcuate region may be a production site of TRH. However, as we have shown, the T R H cells in the dorsomedial nucleus, preoptic area and arcuate nucleus did not respond to PTU treatment, rendering their assumption incompatible with our findings. The wide distribution of immunoreactive T R H throughout the hypothalamus suggests that much of the hypothalamic T R H has a neurotrophic function other than regulation of the anterior pituitary The finding that immunoreactive fibers appear to surround immunonegative and immunolabeled cell bodies in the dorsomedial nucleus suggests the existence of synaptic connections between them. In a retrograde transport experiment, Lechan et al. 22 could not recognize wheat germ agglutinin administrated to the median eminence in the dorsomedial nucleus. T R H is known to have both inhibitory 3.3x and excitatory 3 effects on neurons in the hypothalamus, cerebral cortex and cerebellum, and further, to have an important role in the thermoregulation 5.11.30. The T R H cells in the anterior parvocellular subdivision of the paraventricular nucleus did not respond to PTU treatment. This is in sharp contrast to the penventricular and medial parvocellular subdivisions of the same nucleus. Since the fibers presumably derived from T R H cells in the anterior parvocellular subdivision seem to conjoin in the stria terminalis, they may be related to a neurotrophic function of T R H in the amygdala. The amygdala has been shown to have a remarkable amount of T R H 7. and has much more T R H receptor than the hypothalamus z7.2~ ACKNOWLEDGEMENTS The authors wish to thank Mr. I. Shimada for his excellent technique of preparation of photographs. This work was supported by Grant-in-Aid for Scientific Research (59370001) from the Ministry of Education, Science and Culture, Japan.

217 LIST OF ABBREVIATIONS AHA Arc DMH LHA PFA POA PVN VMH ap ca ch

anterior hypothalamic area arcuate nucleus dorsomedial hypothalamic nucleus lateral hypothalamic area perifornical area preoptic area paraventricular nucleus ventromedial hypothalamic nucleus anterior parvocellular subdivision of PVN anterior commissure optic chiasma

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