Cell and Tissue Research

Cell Tissue Res. 201,377-408 (1979)

9 by Springer-Verlag 1979

Cellular Organization of the Lateral and Postinfundibular Regions of the Median Eminence in the Rat* E.M. Rodriguez, C.B. Gonz/tlez, and L. Delannoy** Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile

Summary. The structural organization of the rostral, lateral and postinfundibular regions of the median eminence (ME) of 5-day cyclic diestrous rats was studied with light and electron microscopic methods. The ependymal cells lining (i) the floor of the infundibular recess (IR) at rostral levels, (ii) the lateral extensions of the IR, and (iii) the floor of the premammillary recess appear to represent the same type of tanycyte ependyma (fl 1 tanycytes). In the entire width of the rostral and postinfundibular palisade regions, as well as in the lateral palisade region of the preinfundibular ME, the processes of the fl 1 tanycytes form a continuous cuff. This cuff separates the nerve endings from the blood vessels and the pars tuberalis. At this level, synaptoid contacts between neurosecretory axons and the ependymal cuff can be observed. The ultrastructural characteristics of the fl 1 tanycytes are described and their ependymal endings tentatively classified into three types. In the lateral regions of the ME, the Golgi study revealed the presence of two fiber systems: (i) one possessing a latero-medial trajectory and distributed in the subependymal region; (ii) the other formed by a loose longitudinal tract originating from neurons of the arcuate nucleus. Some functional implications of the cellular organization of the rat M E are discussed. Key words: Median eminence - Tanycytes - Nerve tracts - Regional organization - Rat.

The ultrastructure of the rat median eminence (ME) has been studied repeatedly applying conventional electron microscopical techniques. Most of these investigations have been published between 1960 and 1970 (for references, see Send offprint requests to: Dr. Esteban M. Rodriguez, Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile * Supported by Grants from PLAMIRH (92.171.2.77) and from the Direcci6n de Investigaciones, Universidad Austral (S-77-28) ** The authors wish to thank Miss Rosario Andrade, Mrs. Elizabeth Santibhfiez and Mr. Armando Bilbao for their assistance

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Kobayashi and Matsui, 1969; Kobayashi et al., 1970; Knigge and Scott, 1970). It appears, however, that in all these studies regional differences within the rat ME were not considered. Furthermore, these papers were mainly concerned with the cellular organization of the medial region of the ME. Evidence obtained from immunocytochemical and catecholamine-fluorescence studies clearly indicate a regional organization of the rat ME. There is agreement with respect to the distribution within the rat ME of the luteinizing hormonereleasing hormone (LH-RH)-containing nerve fibers. This tract and the endings of its axons are mainly concentrated in the lateral regions of the ME and in the medial region of the "post-infundibular ME" (Baker et al., 1974, 1975; King et al., 1974; S6t/tl6 et al., 1975; Baker and Dermody, 1976; Barry and Dubois, 1976; King and Gerall, 1976; Kobayashi et al., 1978; Krisch, 1978). A similar distribution of the LH-RH-containing axons has been found in the mouse ME (Gross, 1976). In other mammalian species the location of the L H - R H tract at the level of the ME is slightly different from that in the rat and mouse (Barry et al., 1973; Barry and Dubois, 1976; Silverman, 1976). On the other hand, axons containing the growth hormone-release inhibiting hormone appear to be mainly distributed in the medial region of the rat ME (H6kfelt et al., 1975; King et al., 1975). In the rat ME the aminergic fibers also follow a regional pattern of distribution. Thus, the subependymal layer appears to be mainly innervated by noradrenergic fibers, whereas in the lateral palisade region the dopaminergic fibers are the predominant aminergic elements. In the medial palisade region both noradrenergic and dopaminergic fibers appear as a mixed population (L6fstr6m, 1977; L6fstr6m et al., 1976a, b). To our knowledge neither the ultrastructural organization of the postinfundibular ME nor that of the lateral region of the rat ME have been studied in detail. A reference to the latter region was, however, given in the legend to Fig. 12 Of the paper by Scott and Knigge (1970) and in a review by Knigge et al., (1976). All the evidence available indicates that the rat ME is zonated not only in the ventro-dorsal direction but also in the latero-medial and rostro-caudal directions. The lack of information with respect to the ultrastructural organization of some regions of the rat ME led us to perform a systematic light and electron microscopic study of the entire ME in the rat. In the present paper the results obtained from the study of the lateral and post-infundibular regions of the ME will be presented. Since the different hormonal states of the estrous cycle can probably induce ultrastructural changes in these regions, the study was performed in diestrous 5-day cyclic rats.

Materials and Methods

Thirty-eight5-day cyclicrats (body weight approximately200 g) derivedfrom a Holtzman stock were used. The rats werekilledby decapitationwithout anesthesia, at diestrus and between10a.m. to 12a.m. Electron Microscopy

The brain was quicklyremoved,the hypothalamusdissectedout and immersedin a threefoldaldehyde mixturebufferedto pH 7.4 with phosphate(Rodriguez,1969).After 30rain the blockof tissuewas placed

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under a dissecting microscope to separate the medial basal hypothalamus (MBH). Taking into consideration the length of the median eminence (ME) and stalk (about 2 mm), the MBH was divided into two halves which were regarded as the rostral ME (RME) and the caudal ME (CME) (Fig. 1.A). The blocks of tissue were then immersed again in the same fixative for 1.5 h. The tissues were further processed as previously described (Rodriguez, 1969). In an additional group of four rats, the animals were anesthetized with ether and a cannula was stereotaxically placed into the third ventricle. Then, 30~tl of the threefold aldehyde mixture were perfused at a rate of 1 lal/min. At the end of the 30rain perfusion period the animal was still alive although a decrease in the respiratory frequency was observed. The brain was removed and processed as above. In a third group of four diestrous rats the brains were dissected out and the MBH fixed in glutaraldehyde-forrnaldehyde fixative (Karnovsky, 1965) buffered to pH 7.4 with 0.2 M monosodicdisodic phosphate. The tissues were then processed with a ZIO procedure (K-P.7.4-V.7.4-ZIO-4~ h) as described previously (Rodriguez and Gimenez, 1979). This procedure reveals the Golgi complexes and the endoplasmic reticulum. The CME was orientated in order to obtain frontal sections starting from the cephalic end. Sections 1 Ixm thick were stained with toluidine blue-borax or mounted whithout staining to be studied under the phase contrast microscope. Parallel ultrathin sections of the entire surface of the block were mounted on 200 mesh grids and stained with uranyl acetate and lead citrate. The thick section was studied under the light microscope and a semischematic drawing was prepared. As shown in Fig. 1 B, the arcuate nucleusmedian eminence region was divided into several areas. The ultrathin section was first studied using low magnifications ( ~ 500 x ) in order to recognize the different areas represented in the drawing. When pictures were obtained, the number of the picture was registered in the corresponding area of the drawing. The first sections obtained from the cephalic end of the CME were regarded as region I of the caudal median eminence (CI). When the study of this region was completed, 100 subsequent sections about I lam thick were available. The last one of this series was regarded as region II of the CME (CII) (Fig. 1 C). Parallel ultrathin sections were studied as described for region I. The same procedure was repeated to obtain and study regions III, IV, V and VI (Fig. 1 D, E, F and G). In the first CME studied, 10 pictures from each area of each region were taken, making a total of 600 pictures. In the other CMEs studied the same procedure was applied but only a few pictures were obtained from each region. In total, about 2000 pictures were available.

Light Microscopy Lipids. To study the distribution of lipid inclusions, the entire MBH was fixed in acetic acid-formaline fixative for 4 h. It was then immersed in Benoit's fixative (see Gabe, 1968) for 4 h, dehydrated in a graded series of ethanol and embedded in paraffin. Frontal serial sections obtained from the entire MBH were stained either with alkaline toluidine blue or with orange G.

GolgiStudies. Ten hypothalami were processed with the Golgi method according to the modification of Valverde (1970). Since the nerve profiles of the core of the tissue blocks (periventricular area) were poorly impregnated, a new group of five hypothalami was prepared. In the latter the ventricular cavity was exposed by removing one side of the hypothalamus so that the ventricular wall was bathed with the fixative and the silver salt. This procedure rendered a completely different Golgi picture of the hypothalamus. The blocks of tissue were embedded in a soft plastic medium (Pfischel et al., 1973). Serial frontal and longitudinal sections about 50 ~tm thick were obtained by using razor blades. In every case and for comparative and descriptive purposes, the serial sections obtained from the entire MBH were grouped into two sets: one corresponded to the CME, the other to RME. The sections from each set were, in turn, classified into different regions as shown in Fig. 1A. Results

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Fig. 1. A Longitudinal section through the medial basal hypothalamus of the rat. MB mammillary body. x 56. The wavy line indicates the approximate site where the ME was divided into a rostral (R) and a caudal (C) half. C1 to C6 represent the caudal levels shown schematically in Figs. l b - l g . R 1 - R 4 represent the rostral levels. B-G Schematic representation of the 1 pan thick sections obtained at levels C1 to CVL The areas framed by broken lines were studied under the electron microscope. D dorsal ependyma, V ventral ependyma, A angular ependyma; LE latero-external, L I latero-internal, T transition, M E medio-external, M I medio-internal; M middle regions, 117 infundibular recess. P M premammillary recess

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infundibular recess. From level RIV to level CII the infundibular recess presented the typical triangular shape characterized by the two lateral recesses. At these levels, the lipid droplets showed a consistent pattern of distribution. At the lateral recesses the lipid inclusions were mainly found in the perikaryon of the ependymal cells (Fig. 2). On the other hand, in the medial third of the floor of the infundibular recess the lipid droplets were principally present in the basal processes of the ependymal cells and rare in the perikaryon. Furthermore, at these levels lipid droplets filled most of the cytoplasm of glial cells located in the medial palisade region (Figs. 2, 3). 9Levels CIII to CVI included the stalk and the basal region of the caudal hypothalamus that has been called "postinfundibular median eminence" (PI ME) by Gross (1976). At these levels the lipid inclusions were mostly found in the perikaryon of the ependymal cells lining the floor of the premammillary recess. At levels CVII and CVIII (Fig. 1A) lipid inclusions in the ependyma were no longer found.

Golgi Studies. The Golgi method revealed both ependymal tanycytes and nerve profiles.

Tanycytes. In animals in which the hypothalamus was processed without exposing the ventricle, the Golgi-positive tanycytes had a uniform appearance and distribution. At levels RI and RII the cell bodies of these cells were located in the floor of the infundibular recess and their basal processes reached the medial palisade region. At level RIII the stained cell bodies were found in the lateral regions of the ventricular floor and their processes projected toward the lateral palisade region. From level RIV to level CII the Golgi-positive tanycytes were observed in two different locations. A few tanycytes were present in the medial third of the ME. The basal process of these cells was characterized by a rather smooth surface, straight trajectory and a distal ramification formed by several thin, long and parallel branches (Fig. 3). According to the nomenclature proposed by Akmayev (1976, 1977), these cells will be called f12 tanycytes. The perikaryon of these tanycytes was generally located in the ependymal layer, although it could also be found in deep subependymal layers. In the second location the stained tanycytes had their perikarya in the dorsal wall of the lateral recesses of the infundibular recess. Their basal processes followed an arch-like trajectory and presented three distinct segments (Fig. 3). The segment proximal to the cell body was thicker than the other segments and displayed spine-like formations (Fig. 4). The second segment was the longest and had a rather uniform diameter (Figs. 3, 4). Distally, the ependymal process divided into a few, short branches which contacted the external limiting membrane (Fig. 3). These ependymal cells may correspond to the fll tanycytes of Akmayev's nomenclature. At levels CIII and CIV the stained tanycytes were numerous. Their cell bodies were mainly located in the lateral walls of the premammillary recess and were rare in the floor of the latter. Their basal processes formed a rather compact tract that, after an archiform trajectory, ended at the level of the tuberoinfundibular sulcus. At levels CV and CVI the Golgi-positive tanycytes were concentrated in the

Fig. 2. Frontal section at level CI. In the ependyma the lipid inclusions are concentrated in the cells lining the lateral recess (LR) of the infundibular recess (IR) (broken line). The lateral palisade region (LPR) lacks ceils with lipid inclusions. Benoit method, x 150 Fig.3. Frontal section at level CI. Golgi method, unexposed ventricle, fll and /32 tanycytes are observed. The lateral palisade region (LPR) lacks cells with lipid inclusions, in contrast to the medial palisade region which appears filled that lipids (white arrow). IR infundibular recess, PT pars tuberalis. • 170 Fig. 4./31 tanycyte showing the proximal segment (between arrowheads) with spine-like formations and the smooth-surfaced distal segment (DS). Golgi method, unexposed ventricle. • 200

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floor of the premammillary recess and their basal processes traversed the whole width of the postinfundibular ME. No stained tanycytes were found at levels CVII and CVIII. In the hypothalami, where the ventricular cavity had been exposed before fixation, all the tanycytes lining the ventro-lateral walls of the third ventricle appeared stained (Fig. 5). In contrast, only a small proportion of the tanycytes of the median eminence (fi2) were impregnated. The study of these specimens, where the basal process of virtually all the tanycytes could be traced, made it possible to determine the distribution of these processes within the different hypothalamic areas as well as their relationships with neural and vascular elements. If the nomenclature proposed by Akmayev is considered from the anatomical point of view only, it can be said that most of the basal processes of the fl 1 tanycytes, after following a latero-ventral trajectory, end in the vicinity of the tuberoinfundibular sulcus (Figs. 5, 6). The same arrangement was observed for the e2 tanycytes lying more ventrally. Only few fl 1 tanycytes end on capillaries located in the region of the arcuate nucleus, and none was observed to end on a neuron. On the other hand, bundles formed by several processes of e2 tanycytes were seen to end either on a single capillary vessel or on a single neuron of the ventro-medial nucleus (Fig. 6). It is not within the scope of this paper to analyze further the distribution and interrelationships of the tanycyte processes.

Nerve Profiles. From level RII to RIV the Golgi method revealed the presence of a bundle of fine, beaded nerve fibers in the lateral regions of the ME. The tract could be traced laterally as far as the region of the ventro-medial nucleus, where it became intermingled with a dense network of cell processes (Figs. 7-9). Only very few fibers of this tract were traced throughout their entire length and found to originate in neurons located in the ventral portion of the ventromedial nucleus (Fig. 7). The tract ran latero-medially and extended into the subependymal region, especially the layer underlying the lateral recesses of the infundibular recess (Figs. 7-9). This resulted in the formation of a dense network of nerve fibers located immediately beneath the ependymal lining (Fig. 8). F r o m the lateral subependymal networks a few fibers were found to descend and to end in the palisade region of the lateral ME (Fig. 8). Longitudinal sections at the level of the lateral recess of the infundibular recess showed a network of nerve fibers in the ependymal region that corresponded to the network found in the frontal sections. In addition, these lateral longitudinal sections revealed a loose nerve tract running along the palisade region of the lateral ME. From each axon of this tract several collaterals branched off at right angles, ran ventrally and ended in the vicinity of the portal capillaries (Fig. 11). Some fibers of this tract were traced back and found to originate in neurons of the arcuate nucleus (Figs. 10, 11). Although the present paper is mainly concerned with the lateral region of the ME, the fiber distribution in the medial region will be briefly presented for comparative purposes. Longitudinal sections of the medial region of the ME showed two longitudinal nerve tracts. One was located in the subependymal region, and the other ran along the border between the hilar and the palisade region. Axons of the two tracts sent collaterals, at right angles, toward the palisade layer, where

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Fig. 5. Frontal section at level RIV. Golgi method, exposed ventricle. Virtually all tanycytes o f the lateral wall of the infundibular recess (IR) are stained. Neurons of the ventromedial nucleus (VMN) are also visualized. M E median eminence, TIS tuberoinfundibular sulcus, x 85 Fig. 6. Higher magnification of a region shown in Fig. 5. The white bracket indicates a bundle of ependymal processes ending on a blood capillary (C). The black bracket indicates a bundle ofependymal processes ending at the level of the tuberoinfundibular sulcus. The black arrows point to a neuron of the V M N and its dendritic process directed toward the region of the arcuate nucleus. L R lateral recess, P T pars tuberalis, x 170. Insert: Ependymal endings contacting a capillary, x 300

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Fig. 7. Frontal section at level R II. Golgi method, unexposed ventricle. A latero-medial Golgi-positive bundle of nerve fibers is observed. The black arrows indicate nerve fibers in the medial palisade region. The white arrow points to a neuron, the axon of which enters the latero-medial tract. AN arcuate nucleus, L P R lateral palisade region, P T pars tuberalis, x 200 Fig. 8. Frontal section at level RIII. Golgi method, unexposed ventricle. The latero-medial tract forms a subependymal network. Descending fibers (arrow) project toward the lateral palisade region (LPR). P T pars tuberalis. • 200

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Fig. 9. Frontal section at level RIV. Golgi method, unexposed ventricle. The latero-medial tract is prominent and is distributed in the medial subependymal region. The lateral palisade region (LPR) appears clearly delimited and devoid of nerve fibers and lipid inclusions. MPR medial palisade region, IR infundibular recess. PT pars tuberalis, x 200 they became intermingled. These descending collaterals were also clearly seen in frontal sections, whereas the axons from which they originated appeared as faint dots (Figs. 7, 8). In frontal sections of hypothalami impregnated after exposing the ventricle, several Golgi-positive neurons of the arcuate nucleus were observed. According to the dendritic pattern, at least two types of neurons were distinguished. One of them was characterized by a pyramidal cell body and by several long and beaded dendrites. M a n y of the latter ran medially and extended into the area underlying the ependymal lining, where they became intermingled with the basal processes of tanycytes. The same arrangement was observed for the dendrites of some neurons of the ventromedial nucleus (Fig. 6).

Electron Microscopy

Since the tanycytes lining the lateral recesses of the infundibular recess (Akmayev's fl I tanycytes) project their basal processes toward the lateral region of the ME, they will be regarded as a component of this region. Ultrastructural differences between the tanycytes lining the lateral recesses ventrally and those located at the dorsal circumference were consistently found. The former will be regarded as f l l V tanycytes and the latter as fl 1 D tanycytes. In addition, since the ependymal cells lining the bottom of the premammillary recess (CIII, CIV, CV and CVI levels) were identical to those of the dorsal wall of the lateral recesses of the infundibular recess (RIII to CII levels), they will also be regarded as fll D tanycytes.

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Fig. 10. Paramedian sagittal section of the medial basal hypothalamus. Golgi method, unexposed ventricle. The body (S) and the axon (A) of a n arcuate neuron are present. PR lateral palisade region, IR infundibular recess, EP ependymal processes, x 200. lnsert: High magnification o f the same neuron shown in Fig. 10. S neuronal body, D dendrite, .4 axon. x 450 Fig. 11. Longitudinal section at the level of the lateral recess (LR) o f the infundibular recess. Golgi method, unexposed ventricle. Descending axons (arrowheads) extend into the lateral palisade region (LPR). • 260. lnsert: High magnification o f an axon shown in Fig. 11. Collaterals at right angles are observed, x 520

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fllD Tanycytes. These cells possess elongated nuclei lying at various levels and giving the tissue a stratified appearance. Therefore, the amount of apical cytoplasm varies from one cell to another. In most cases the apical cytoplasm appeared as an elongated apical cell process with an irregular outline. Its lateral surface presented spine-like protrusions. The most distinct features of the apical cytoplasm were a highly developed smooth endoplasmic reticulum, abundant polysomes and numerous dense and elongated mitochondria (Fig. 12). Lipid droplets, filaments, Golgi complexes, multivesicular bodies, coated vesicles, smooth-surfaced vesicles with and without an electron dense content were also distributed throughout the apical cytoplasm (Fig. 12). In the material processed with the K-P.7.4-V.7.4-ZIO4 ~ h method, the lumen of the smooth endoplasmic reticulum appeared partially or completely filled with a material of high electron density. In these specimens the high development of this organelle became convincingly clear. It was also possible to observe the intimate spatial relationship between the smooth endoplasmic reticulum and the lipid droplets (Fig. 13). Sections 0.5 ~tm thick were observed under the electron microscope using 100 kV accelerating voltage and revealed that the smooth endoplasmic reticulum forms a dense network around each lipid droplet. The lateral protrusions of the apical cytoplasm were mainly occupied by polysomes and lipid inclusions (Fig. 12). At the ventricular surface three types of protrusions were distinguished (Fig. 12): (1) Scarce and short microvilli were mainly found near the cell junctions. (2) Small and numerous bulbous protrusions about 350 nm in diameter appeared connected to the apical cytoplasm by a thin stalk. They displayed a flocculent material of low electron density and a membrane-bound structure containing dense particles and clear vesicles (Fig. 12, insert). (3) Large and scarce bulbous protrusions were connected to the cytoplasm by a thick stalk. They contained polysomes, a flocculent material and occasionally and isolated smooth surfaced cisterna, the lumen of which was stained by the ZIO procedure. Among these surface projections there were a few nerve terminals filled with clear and dense cored vesicles (Fig. 12). Laterally, and near the free surface, the cells interdigitated. With the method used only one type of cell junction was observed, namely, zonula adhaerens. Typical tight junctions were not found. The perikaryon of the fl 1 D tanycytes appeared as a dilated portion of the cell where the nucleus was excentrically located (Fig. 15). The SER was poorly developed as compared with that of the apical cytoplasm, whereas microtubules were numerous and regularly arranged. The proximal segment of the basal process was characterized by an irregular outline due to the presence of several irregular spine-like protrusions. At this level two distinct cytoplasmic regions were distinguished. The core of the basal process was occupied by a few filaments and Golgi complexes, several cisternae of the SER and by numerous microtubules and mitochondria (Figs. 14, 15, 19). The latter were elongated and had a very dense matrix. However, their most peculiar feature was the ability of the external limiting membrane to form long tubular formations of a rather uniform diameter (about 80nm), so that the external mitochondrial compartment was considerably enlarged. These mitochondrion-born tubules displayed a distinct structure and were readily distinguishable from all the other tubular formations of the cytoplasm (Figs. 14, 19); therefore, it was not necessary to

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Fig. 12. Apical cytoplasm of/31 D tanycytes. The electron micrograph was taken in the region indicated in the top-left insert. S spine-like protrusions, G Golgi complex, arrowheads smooth endoplasmic reticulum, SP small ventricular protrusions, MV microvilli, N E nerve ending. Between tanycytes large nerve profiles (NP) and cross sections of axons (arrow) x 17,000. Bottom insert: Small ventricular protrusions showing the inner structure of the membrane-bound bodies. • 50,000

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Fig. 13./~1 V tanycyte showing the intimate relationship between the smooth endoplasmic reticulum (stained by the ZIO method) and the lipid droplets. IR infundibular recess. 0.5 ~rn thick section studied at 100kV. K-P.7.4-V.7.4-ZIOprocedure, x 50,000

view their connection to mitochondria in order to characterize them. These mitochondrial tubules were also found, although only occasionally, in the perikaryon, apical cytoplasm and the distal segment of the basal process. Sometimes the external mitochondrial compartment was seen in continuity with the lumen of the SER. Polysomes were virtually absent from the axial cytoplasm. On the other hand, the inner structure of the spine-like protrusions differed completely from that of the axial cytoplasm. They all contained a large number of polysomes (Figs. 14, 19). In addition, they contained lipid droplets, a few mitochondria and SER. Smooth-surfaced cisternae, concentrically arranged, and similar to those found in some ependymal endings were more rarely observed. The distal segment of the basal process of the/~ 1 D tanycytes presented a rather uniform diameter. Spine-like protrusions were not seen at this level. The cytoplasm was mainly occupied by mitochondria, microtubules and cisternae of the SER. Polysomes, filaments and vesicles were less numerous than in the other segments of the cell. In the ZIO-processed material the SER of the basal processes appeared as a loose network distributed throughout the cytoplasm (Fig. 18). This technique made it possible to visualize clearly the SER cisternae connected to the external mitochondrial membrane. The mitochondrion-born tubules were not stained by this procedure. The ependymal endings are described in detail in the text.

Interrelationships Between [31D Tanycytes and Adjacent Structures Between the proximal segments of these tanycytes numerous nerve profiles were observed. They belonged to three types: Type I profiles were small (about 150 nm in diameter), formed bundles and contained microtubules (Fig. 12). Only once was one of them seen to be the post-synaptic component of a typical synaptic structure.

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Fig. 14. A Electron micrograph obtained from the region indicated in the insert. T E tanycyte ependyma, N P large nerve profiles. The framed area is shown in Fig. 14B. x 12,000 B High magnification of the area indicated in Fig. 14A. Mitochondrion-connected tubules are indicated by the arrow and the stars. Arrowheads point to microtubules. S E R smooth endoplasmic reticulum, x 37,000

The second type o f nerve profile was represented by pale structures ranging in size between 1 and 3 p.m in diameter. They were distributed t h r o u g h o u t the ependymal layer o f the dorsal wall in the lateral recesses o f the infundibular recess, but were especially a b u n d a n t at the level o f the nuclear layer (Figs. 14, 15). M a n y o f them were seen in the intercellular space close to the junctional complexes. They all contained a fine flocculent material, and some o f them displayed a m i t o c h o n d r i o n , smooth-surfaced vacuoles and cisternae (Fig. 17). F o r m a t i o n s with an identical inner structure displayed a variable n u m b e r o f dense core vesicles, but m o s t frequently they contained only one or two ot these vesicles (Fig. 12). Occasionally

Fig. 15. Proximal segment of the basal process of a/~ 1 tanycyte surrounded by large nerve profiles (NP). R ribosomes, G Golgi complex. Arrows indicate synapses. • 18,000 Fig. 16. High magnification of the synapse framed in Fig. 15. • 60,000 Fig. 17. Large nerve profile (NP) connected to a thin fiber. The arrow points to a microtubule. • 20,000

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Fig. 18. Processes of fl 1 V tanycytes. K-P.7.4-V.7.4-ZIO procedure. The smooth endoplasmic reticulum is stained, x 18,000

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Fig. 19. Proximal segment of the basal process of a fl 1 tanycyte (for location, see insert) displaying two spine-like protrusions (bent arrows). The process is filled with microtubules, smooth endoplasmic reticulum (SER), mitochondria, mitochondrion-connected tubules (stars and large arrow) and a Golgi complex (G). Two axo-dendritic synapses can be observed ($1, $2). In the dendrites (D) the small arrows point to coated vesicles. • 28,000

these structures were seen to be connected by a thin profile, thus appearing as dilatations o f small fibers (Fig. 17). The thin segment contained microtubules which disappeared abruptly at the dilatations (Fig. 17). The entire structure resembled beaded nerve fibers, but with very few organelles and inclusions in the dilated regions. They established side-by-side contacts with the f l l D tanycytes, but synaptoid contacts were never observed. Frequently, they were found to be the post-synaptic c o m p o n e n t o f typical synapses (Figs. 15, 16). Type I I I profiles were typical dendrites. They contained n u m e r o u s microtubules, mitochondria, SER and coated vesicles (Fig. 19). W h e n sectioned

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longitudinally, they appeared beaded. They received synaptic contacts of two types. In one type, the axon terminal contained only small clear vesicles, and in the other type the presynaptic cytoplasm contained clear and dense-cored vesicles (Fig. 19). In their trajectory toward the lateral palisade region, the basal processes of the f l l D tanycytes established "en passant" contacts with blood capillaries. These capillaries were characterized by their lack of a perivascular space. The perivascular basal lamina possessed several processes penetrating the surrounding tissue. This membrane was completely covered by processes and endings that resembled the ependymal endings found in the lateral palisade region of the ME. Golgi preparations also showed that some ependymal processes ended on capillaries of the arcuate nucleus. However, the possibility that many or all of the endings contacting these capillaries were, in actuallity, the endings of glial cell processes, cannot be discarded.

fil V Tanycytes. Their main differences from the f l l D tanycytes were: (i) The small ventricular protrusions lacked the membrane-bound dense structures. (ii) The spine-like protrusions were seen in a very short segment of the basal process. (iii) Lipid inclusions, although also concentrated in the perikaryon, were much more numerous than in the fi 1 D tanycytes. (iv) The basal processes of these tanycytes projected toward the transitional zone between the lateral and medial palisade regions of the ME. In addition, the large and pale nerve profiles and the dendrites found between the fl 1 D tanycytes were very rarely observed between the fl 1V tanycytes.

Ependymal Endings. At level CI and CII, the latero-external, latero-internal, laterodorsal and transition regions, as represented in Fig. 1 B and 1 C, will be regarded as the lateral palisade region (LPR). The medio-external, medio-internal and middle regions will be regarded as the medial palisade region (MPR). From levels CIII to CVI, the medial, transition, latero-internal and latero-external areas will be considered as the palisade region of the postinfundibular ME (PIPR) (Fig. 1 D-1 G). The ultrastructure of the ependymal endings found in both the LPR and the PIPR varied considerably. These endings have tentatively been classified into three types: Type L The most distinct feature of these endings was the presence of numerous dense and elongated mitochondria, filaments, microtubules and SER. Isolated and long smooth-surfaced cisternae of unknown nature (SSC) were also a consistent finding. They differed from the RER in that they did not have ribosomes, and from the SER in that they had a rather uniform width and in their inability to connect to each other to form a reticulum (Fig. 22). Type II. These endings were characterized by the presence of a large number of short tubular formations with an electron dense content. They virtually lacked microtubules, filaments, SER and glycogen particles (Figs. 20, 24).

Type III. The endings of this type contained several SSC. Most frequently, the latter were concentrically arranged. In these onion-like structures, the cisternae remained

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Fig. 20. Processes (large arrows) and endings (EE) of fll tanycytes (for location, see insert on the top left). The nerve endings establish synaptoid contacts with the preterminal portion of the ependymal processes (bent arrows). E L M external limiting membrane, PT pars tuberalis, x 16,000. Bottom insert: High magnification of a synaptoid contact. At the "presynaptic" side (NE) the small vesicles appear embedded in a dense material. The white arrow points to a dense projection. The "intersynaptic" space appears widened and filled with an electron dense material. At the ependymal side (EP) no special arrangement is observed, x 70,000

separated from each other by thin cytoplasmic sheaths containing glycogen r-particles. These r-particles were seen t h r o u g h o u t the ending. These endings also displayed granules o f different sizes filled with a filamentous material. Filaments and microtubules were virtually absent from these endings (Fig. 23). All types o f ependymal endings displayed hemidesmosome-like structures at their surface contacting the external limiting m e m b r a n e o f the brain. Also, they all contained coated vesicles opening t o w a r d the limiting membrane. Some ependymal endings clearly appeared as transitional stages between the three types described. A t levels CI and C I I the type I I I ependymal endings were almost exclusively f o u n d in the latero-external and latero-internal regions. Type II endings were more n u m e r o u s in the latero-internal and transition regions. The n u m b e r o f t y p e I

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Fig. 21. Ependymal cuff separating the nerve endings (stars) from the external limiting membrane. Type I and II ependymal endings are observed (EL Ell). C capillary, PT secretory cell of the pars tuberalis, x 10,000. The insert indicates the area from which the electron micrograph was obtained

endings increased progressively from the LPR toward the MPR. In the postinfundibular ME (levels CIII to CVI) types I and II were the most abundant elements. They were distributed in the medial, transition and latero-internal regions. E p e n d y m a l Barrier. In the LPR (levels CI and CII) the ependymal endings formed a continuous cuff that separated the nerve terminals from the external limiting membrane. In turn, the latter separated the ependymal cuff from the blood capillaries and the pars tuberalis. In the transition region, the ependymal cuff was "perforated" by a few nerve fibers which then made direct contact with the limiting membrane. At the middle region, and rather abruptly, the ependymal cuff disappeared and the external limiting membrane was principally contacted by nerve endings.

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Fig. 22. Type I ependymal endings (for location, see insert). S S C smooth-surfaced cisternae, F filaments, T microtubule, N E nerve ending, E L M external limiting membrane. The short arrow points to a hemidesmosome-like structure, x 27,000

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Fig. 23. TypeIII ependymal ending containing smooth-surfaced cisternae (SSC), granules (GR) and glycogen/~-particles(GY). Mitochondria and filamentsare lacking. The arrow indicates a coated vesicle opening toward the external limitingmembrane (ELM). GC glial cell process, x 20,000. Insert: location of the region shown in the electron micrograph

In the postinfundibular ME, the ependymal cuff was present in the entire palisade region (Fig. 21). Only in the medial region the ependymal barrier appeared occasionally interrupted by nerve endings. In the LPR (levels CI and CII), where the ependymal cuff was continuous, the nerve fibers were seen to end on the preterminal portion of the ependymal process and to establish synaptoid contacts (Fig. 20). The latter were characterized by an accumulation of small clear vesicles and neurosecretory granules embedded in an electron dense matrix. At the synaptoid contact the intercellular space became wider and was filled with a fine electron dense material. No special structure was observed in the ependymal cytoplasm facing the synaptoid contact. The preterminal portion of the basal process of/~ 1 tanycytes was the only site of these cells that received synaptoid contacts. Although nerve and ependymal endings were closely related spatially, no synaptoid contacts were observed in the PIPR. Only a few sections of the rostral half of the M E were studied electron microscopically. It was observed that the LPR at levels R I I I and R I V resembled that

Fig. 24. A Schematic representation of the perikaryon of a/~1 D tanycyte. S P and L P small and large ventricular protrusions, M V microvilli, E R and S E R rough and smooth endoplasmic reticulum, G Golgi complex, L lipid droplets, R ribosomes, F filaments, Tmicrotubules, SP small nerve fibers, D dendrite. B Proximal segment of the basal process of a/~ 1 D tanycyte. Stars indicate spine-like structures. C Median eminence at level CII showing the location of/~1D tanycytes and the preferential distribution of the three types of ependymal endings (I, I~, lII). The area with small triangles shows the location of the continuous ependymal cuff. L R lateral recess of the infundibular recess, L M E lateral region of the ME, P T pars tuberalis. D, E and F Types of ependymal endings. F filaments, T microtubules, S S C smoothsurfaced cisternae, GR granules, G Y glycogen, arrowhead coated vesicle; short arrow hemidesmosome, bent arrow mitochondrial tubule, star electron dense tubules, N E synaptoid contact

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Fig. 25. Schematicrepresentationof the rat medianeminenceshowingthe presenceof a continuouslayer of ependymal endings in the rostral (RME), lateral (LPR)and postinfundibular(PIME)regions. At these levels the axons do not contact the blood vessels but rather the ependymalcuff. In the medial palisade region (MPR)and the stalk (S) the ependymalcells do not form a continuous layer, and the axons establish direct contact with blood vessels

of level CI, whereas the cellular organization of the palisade region at levels RI and RII resembled the PIPR. A systematic study of the rostral half of the M E is in progress.

Discussion

Types ofEpendymal Tanycytes. In the rat hypothalamus tanycytes are mainly found in the lateroventral wall of the third ventricle (Bleier, 1971; Millhouse, 1971, 1972, 1975). There is evidence indicating that these tanycytes do not constitute a homogeneous cell population. Thus, enzyme-histochemical studies have shown that the tanycytes lining the lateral walls of the infundibular recess differ from those of the median eminence (Bock and Goslar, 1969; Goslar and Bock, 1970). Similarly, a light and electron microscopic analysis of the distribution of adenosine triphosphatase clearly indicated that this enzyme is confined to the tanycytes facing the ventromedial nucleus (VMN) (Firth and Bock, 1976). Combined histochemical and experimental studies have led Akmayev and coworkers to classify the hypothalamic tanycytes into four types (Akmayev et al., 1973; Akmayev and Fidelina, 1976). These authors recognized e I tanycytes as the elements facing the VMN, e2 lying close to the arcuate nucleus (AN), /~1 located in the lateral extensions of the infundibular recess, and /~2 lining the floor of the ventricle (ME proper). Cytometric studies performed by Oksche et al. (1974) showed that, in the mouse, the tanycytes in juxtaposition with the A N and those lining the median eminence react in a different manner after ovariectomy. The different types of tanycytes also become distinguishable when studying the distribution of lipid droplets. As shown

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in the present paper, in the fl 1 tanycytes (following Akmayev's nomenclature) the lipid inclusions are confined to the perikaryon, whereas in the f12 tanycytes they are mostly found in the basal processes, a tanycytes virtually lack lipid droplets. Akmayev and Popov (1977) have described the fine structure of the a and fl tanycytes. These authors did not make a distinction between fi 1 and f12 tanycytes despite the fact that they had previously demonstrated histochemical and experimental differences between these two types of fl tanycytes (Akmayev et al., 1973; Akmayev and Fidelina, 1976). It is our impression that most of the ultrastructural characteristics ascribed by these authors to the fl tanycytes actually correspond to the fl 2 tanycytes. Thus, for example, the presence of lipid inclusions both in the ependymal processes and in the ependymal endings, the existence in the latter of what Akmayev and Popov (1977) have called type IV vesicles, and the numerous synaptoid contacts found along the basal process are all distinct features of the f12 tanycytes (Rodriguez and Gonz/dez, unpublished observations). These and other features differentiate the latter cell type from the fl 1 tanycytes. According to Akmayev and Popov (1977), the small globular protrusions containing a multivesicular body, and described in the present paper as membrane-bound structures containing dense particles, are one of the characteristic features of a tanycytes. Our results clearly indicate that this structural feature is shared by the fl 1 tanycytes lining the dorsal wall of the lateral recesses of the infundibular recess (fl 1 D tanycytes) but not by those lining the ventral wall (fl IV tanycytes).

fl 1 Tanycytes. Since the basal processes of fl 1 tanycytes end in the lateral palisade region of the ME (LPR), their fine structural organization appears as a relevant matter of discussion. The most prominent features of the perikaryon of these cells are the large number of lipid inclusions, the high development of the smooth endoplasmic reticulum (SER) and the intimate spatial relationship between the former and the latter. These characteristics are shared by cells engaged either in steroid or in lipid synthesis, or in lipid metabolism. Enzymes involved in lipid metabolism have been histochemically demonstrated in these tanycytes (Bock and Goslar, 1969; Akmayev et al., 1973, 1976). Both the large ventricular globular protrusions and the spine-like protrusions display the same characteristics, namely, they lack cell organelles and are filled with polysomes. These cytoplasmic regions thus appear as local areas specialized in protein synthesis. In the same manner as the number of dendritic spines is taken as an indicator of the synaptic pattern of a neuron, the extension and number of the spine-like structures of tanycytes could be indicative of the polysome population. The basal process of the fl 1 tanycytes presents two distinct features, the network of SER and the large number of elongated mitochondria orientated paraxially (Brawer, 1972). The ability of these mitochondria to form and/or connect to tubular structures appears as an intriguing feature that requires further investigation. The central region of the basal process appears as a "channel" clearly distinguishable from the peripheral region, especially the spine-like structures. Based on the distribution of organelles, it might be postulated that the "channel" is involved in a transport function, whilst the spine-like formations could be engaged in protein synthesis. The transport capacity of the ME-tanycytes has repeatedly been discussed (Wagner and Pilgrim, 1974; Knigge et al., 1976; Rodriguez, 1976).

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Another important feature of the basal processes of fl 1 tanycytes is that, with the exception of their preterminal portion, they lack synaptoid contacts. The cellular organization of the fll tanycytes raises several questions. What is the functional correlate for the high development of the SER and the lipid inclusions? Why are lipid droplets concentrated in the perikaryon and absent from the basal processes, in contrast to the f12 tanycytes, the lipid inclusions of which are mainly found in their processes? What is the functional meaning of the spine-like protrusions ? Why do fl 1 tanycytes, as compared with fl 2 tanycytes, virtually lack "innervation" ?

Ependymal Endings. The presence of transitional forms between the three types of ependymal endings described in the present report suggests that actually there is only one type of ependymal terminal. The different appearance of these endings probably reflects either different functional states or differences in the function they perform. Since type III endings are only present in the lateral and postinfundibular regions of the ME, it seems obvious that they are related to events occurring in these regions. The same can be said with respect to type I endings and the medial palisade region. That the basal processes of the arcuate ependyma end in the lateral region of the ME had been advanced by Brawer (1972). Brawer also established that these ependymal endings contain concentric ring structures regarded as SER. The functional significance of several components of the ependymal endings, e.g., the smooth-surfaced cisternae, the granules filled with a filamentous material (type III endings), and the short tubular formations containing an electron dense material (type II endings), remains to be elucidated. Ependymal processes of hypothalamic tanycytes have been reported to end or to establish specialized contacts with the blood vessels of several hypothalamic nuclei (Bleier, 1971, 1972; Millhouse, 1975). Our own observations from the Golgi material indicate that the processes of a large number of~2 and fl 1 D tanycytes end on capillaries supplying the AN region. The ultrastructural study of these vessels reveals that they are completely surrounded by endings identical to type I and II ependymal endings. The vessels of the AN communicate with the portal capillaries of the ME (Duvernoy, 1972; Ambach et al., 1976). Furthermore, Krisch et al. (1978) have shown that the perivascular spaces in the region of the AN communicate with those of the ME. These authors have postulated that the presence of tight junctions between the perivascular ependymal endings of the AN prevents the diffusion of substances from the subendothelial space into the surrounding neuropil. We have not seen these junctions, but it appears that they are rudimentary and appreciable only in freeze-etch replicas (Brightman et al., 1975; Krisch et al., 1978). Bleier (1971, 1972) has reported "intricate relationships between the ependymal processes and the neurons of various hypothalamic cell groups". On the other hand, Millhouse (1975) stated that "there has not been any indication in the rat brain of unique structural arrangements between tail processes and neurons other than possible side-by-side juxtaposition'. We agree with Millhouse (1975) with respect to the arcuate ependyma, however, el tanycytes as judged from Golgi preparations, appear to end on neurons of the VMN.

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Ependymal Barrier. In the lateral region of the ME and in the entire palisade region of the postinfundibular ME, the ependymal endings form a continuous cuff. From the Golgi material and the study of a few ultrathin sections of the rostral ME, it seems that in this region there is also an uninterrupted ependymal cover. This means that only the medial region of the "preinfundibular" ME lacks an ependymal barrier. Here, a large surface of the external limiting membrane is directly contacted by nerve endings (Fig. 25). Where the ependymal barrier is present (rostral, lateral and postinfundibular regions), it separates the nerve endings from the blood vessels and cells of the pars tuberalis. As judged from the distribution of lipid and the ultrastructural characteristics, all the processes forming the ependymal barrier originate from/31 tanycytes. Knigge et al. (1976) has already called attention to the fact that in the rostral and lateral regions of the ME the tanycyte endings occupy most of the abluminal basal lamina. An important question to be answered is whether this ependymal cuff actually behaves as a barrier. In this respect it should be interesting to investigate in freeze-etch replicas the probable existence of tight junctions between these ependymal endings (see Krisch et al., 1978). Different types of nerve endings establish typical synaptoid contacts, resembling those described by Gtildner and Wolff (1973), with the ependymal cuff. This finding and the fact that these endings never contact the limiting membrane could be taken as indirect evidence for a transependymal transport of the substances released from the nerve endings. There is a remarkable topographical correlation between the distribution of the ependymal cuff (/~ 1 tanycyte endings) and that of the LH-RH tract. In the rat, such a tract is found to be spread throughout the entire palisade region of the rostral and postinfundibular ME and concentrated in the LPR of the "preinfundibular" ME (Baker et al., 1975; S6tfil6 et al., 1975; Baker and Dermody, 1976; Krisch, 1978). Therefore, in all probability, the LH-RH-containing axons do not contact the limiting membrane but the ependymal cuff. This seems evident in Fig. 9 of Baker and Dermody (1976). The cellular organization of these regions of the rat ME supports the possibility of a functional relationship between the LH-RH tract and the/~ 1 tanycytes. By using a different methodological approach, Akmayev and Fidelina (1976) have advanced a similar hypothesis. These authors have suggested that/~ 1 tanycytes are involved in the mechanism of sexual differentiation of the hypothalamus. Taking into account the observations of Wittkowski and Scheuer (1974), it seems possible that the ependymal cuff is not a permanent structure and that under certain functional conditions it can undergo a reorganization allowing the nerve endings to establish direct contact with the limiting membrane. However, seven and also thirty days after ovariectomy the ependymal cuff of the rat LPR remains an efficient anatomical barrier (Rodriguez, Gonzfilez and Bilbao, unpublished). Neuronal Connections in the Lateral Region of the ME. According to Bj6rklund et al. (1973, 1974), "the fibres of the reticulo-infundibular noradrenergic system ascend in the medial forebrain bundle up to the level of the rostral ME where they turn sharply medially along the ventral surface to enter the rostral ME from the lateral side. The fibres terminate bilaterally in the internal and subependymal layers". There is complete correlation between this description and the distribution in the rostral ME (RII, RIII and RIV levels) of the bundle of Golgi-positive fibers

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entering the ME lateroventrally (see Results). Considering the distribution within the ME and the direction of its fibers, the Golgi-positive lateral tract (GLT) corresponds neither to the L H - R H tract nor to the supraoptico-hypophysial tract. It seems highly probable that the G L T and the ascending noradrenergic (NA) tract are one and the same fiber system. In the M B H the afferent NA fibers innervate the V M N (Fuxe et al., 1974), the A N (Bj6rklund et al., 1974) and the subependymal layer of the ME (Bj6rklund et al., 1970, 1973, 1974; Cuello et al., 1973; Fuxe et al., 1974; L6fstr6m et al., 1976 ). The G L T emerges from the ventral portion of the VMN, but due to the dense nerve network it was not possible to see whether this tract innervates certain groups of neurons. A few axons of the G L T are distributed in the rostroventral portion of the AN. Most of the axons of this tract end in the lateral subependymal region o f the ME, where they form a dense network. The distribution of this tract in the medial region of the ME will not be discussed in the present context. Only a few axons of the G L T reach the LPR. Immunohistofluorescence studies suggest that a small proportion of NA fibers end in the LPR (Fuxe et al., 1974). Ultrastructural (Ajika and H6kfelt, 1973) and fluorescence histochemical studies (L6fstr6m et al., 1976) have shown that in the LPR the monoaminergic terminals are mainly dopaminergic. In the present report a loose longitudinal tract running along the latero-external region of the ME and originating in neurons of the AN has been described. It seems possible that this AN-LPR tract is the Golgi correlate of the AN-LPR dopaminergic system. The lack of evidence for the existence of LH-RH-secreting neurons in the rat A N would favor this possibility. Since in the LPR not a single nerve fiber was seen to establish direct contact with the external limiting membrane (see ependymal barrier), both DA and NA axons must end on the preterminal and terminal regions of the fl 1 tanycyte processes. Although different types of nerve endings were seen in the present conventional ultrastructural study of the LPR, they were not classified. Electron microscopic histochemistry is undoubtedly the appropriate tool to characterize the different types of nerve endings contacting the ependymal cuff. The neuro-ependymal arrangement of the rat LPR resembles that in the neural lobe of reptiles (Rodriguez and La Pointe, 1969). The functional meaning of such an arrangement is enterily unknown and it certainly poses a challenging question. Only on two occasions axons of the G L T were found to originate in neurons located in the ventral region of the VMN. At present we are not able to decide whether the contribution of the VMN to the innervation of the ME is a consistent feature. There are contradictory statements with respect to the existence in the ME of axons originating in the VMN. Szent~gothai (1964) and Millhouse (1973), among others, were not able to find V M N neurons innervating the ME. On the other hand, fibers originating from nerve cells of the V M N and entering the ME have been described by several authors (Dellmann, 1962; Christ, 1966; and others). Neurons of the ventral portion of the V M N send long dendritic processes toward the neuropil underlying the cell bodies offl 1 tanycytes. Some neurons o f the A N also project their dendrites toward this region. Typical beaded dendrites and beaded profiles (probably dendrites) were revealed by the ultrastructural study of this region. Both types of beaded profiles received numerous synaptic contacts. One probable source of these axon endings is the GLT, which is distributed in this region

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(see above). After laterodorsal deafferentation Cuello et al. (1973) f o u n d d e g e n e r a t i n g a x o d e n d r i t i c synapses in the neuropil o f the A N a n d suggested that the d e g e n e r a t i n g fibers b e l o n g to the afferent noradrenergic tract. The n e u r o p i l u n d e r l y i n g the lateral recesses of the i n f u n d i b u l a r recess thus appears as a synaptic region integrating nerve c o m p o n e n t s of at least three different sources, namely, (i) the V M N , (ii) the A N , a n d (iii) the r e t i c u l o - i n f u n d i b u l a r N A system. I n this respect it is interesting to recall the findings o f C h a t e a u et al. (1976) suggesting that the V M N is involved in the m e c h a n i s m s controlling the d u r a t i o n of the estrous cycle in the rat, a n d the results o f L 6 f s t r 6 m (1977) d e m o n s t r a t i n g differences in the N A t u r n o v e r in the s u b e p e n d y m a l layer between 4- a n d 5-day cyclic rats.

References Ajika, K., H6kfelt, T.: Ultrastructural identification of catecholamine neurons in the hypothalamic periventricular - arcuate nucleus - median eminencecomplex with special reference to quantitative aspects. Brain Res. 57, 97-117 (1973) Akmayev, I.G., Fidelina, O.V.: Morphological aspects of the hypothalamic-hypophyseal system. VI. The tanycytes: Their relation to the sexual differentiation of the hypothalamus. An enzymehistochemical study. Cell Tissue Res. 173, 407416 (1976) Akmayev, I.G., Fidelina, O.V., Kabolova, Z.A., Popov, A.P., Schnitkova, T.A.: Morphological aspects of the hypothalamic-hypophyseal system. IV. Median basal hypothalamus. An experimental morphological study. Z. Zellforsch. 137, 493-512 (1973) Akmayev, I.G., Popov, A.P.: Morphological aspects of the hypothalamic-hypophysealsystem.VII. The tanycytes: Their relation to the hypophyseal adrenocorticotropic function. An ultrastructural study. Cell Tissue Res. 180, 263-282 (1977) Ambach, G., Palkovits, M., Szentfigothai, J.: Blood supply of the rat hypothalamus. IV. Retrochiasmatic area, median eminencearcuate nucleus.Acta Morphol. Acad. Sci. Hung. 24, 93-119 (1976) Baker, B.L., Dermody, W.C.: Effect of hypophysectomy on immunocytochemicallydemonstrated gonadotropin releasing hormone in the rat brain. Endocrinology 98, 1116-1122 (1976) Baker, B.L., Dermody, W.C., Reel, J.R.: Localization of luteinizinghormone-releasinghormone in the mammalian hypothalamus (1). Am. J. Anat. 139, 129-134 (1974) Baker, B.L., Dermody, W.C., Reel, J.R.: Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocytochemistry. Endocrinology 97, 125-135 (1975) Barry, J., Dubois, M.P.: Immunoreactive neurosecretory pathways in mammals. Acta anat. (Basel)94, 427-503 (1976) Barry, J., Dubois, M.P., Poulain, P.: LRF producing cells of the mammalian hypothalamus. Z. Zellforsch. 146, 351 366 (1973) Bj6rklund, A., Falck, B., Hromek, F., Owman, Ch., West, K~.: Identification and terminal distribution of the tubero-hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectrofluorimetric techniques. Brain Res. 17, 1-23 (1970) Bj6rklund, A., Falck, B., Nobin, A., Stenevi, U.: Organization of the dopamine and noradrenaline innervations of the median eminence-pituitary region in the rat. In: Neurosecretion. The final neuroendocrine pathway (F. Knowles, L. Vollrath, eds.), pp. 209-222. Berlin-Heidelberg-New York: Springer 1974 Bj6rklund, A., Moore, R.Y., Nobin, A., Stenevi, U.: The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res. 51, 171-191 (1973) Bleier, R.: The relations of ependyma to neurons and capillaries in the hypothalamus: A Golgi-Cox study. J. Comp. Neurol. 142, 439464 (1971) Bleier, R.: Structural relationship of ependymal cells and their processes within the hypothalamus. In: Brain-Endocrine Interaction I. Median Eminence: Structure and Function (K.M. Knigge, D.E. Scott, A. Weindl, eds.), pp. 306-318. Basel: S. Karger 1972

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Accepted June 9, 1979