Zeitschrift fiir Zellforschung 81,264--282 (1967)

Uhrastructure of the Hypothalamic Neurosecretory System of the Dog*

DAVID ZAMBRANO * * and EDUARDO DE I:~OBERTIS

Instituto de Anatomia General y Embriologia, Faeultad de Medicina, Universidad de Buenos Aires, Argentina

Received November 29, 1966

Summary. The hypothalamic neurosecretory system of normal dogs was studied by light and electron microscopy after perfusion-fixation. In the supraoptie nucleus most neurons are loaded with elementary neurosecretory granules having a content of low electron density. Neurons with less neurosecretory material and signs of enhanced synthetic activity, as recog- nized by the changes in the endoplasmic reticulum, were also observed.

The vesiculated neurons of JEWELL were studied under the electron microscope and various stages of development were described. I t was postulated that they originate by a localized process of cytoplasmic cytolysis which ends in the formation of a large aqueous intracellular cavity limited by a plasma membrane. The possible significance of these vesiculated neurones is discussed. Some few myelinated neurosecretory axons are found in the supraoptic nucleus.

The neurons of the paraventricular nucleus are smaller and contain less neuroseeretory material. This is abundant and very pale in the axons. The median eminence consists of an inner zone, mainly occupied by the neurosecretory axons of the hypothalamic-neurohypo- physial tracts, and an outer zone in which some neurosecretory axons end on the capillary of the portal system. This outer zone contains numerous axons with the axoplasm rich in neuro- filaments and some containing granulated and non-granulated synaptic vesicles. Some neurons with granulated vesicles were observed in this region. The adrenergic nature of these neurons and axons is postulated.

The infundibular process of the neurohypophysis shows small axons with discrete amounts of elementary granules and vesicles of synaptic type at the endings. Some enlarged axons having, in addition, large polymorphic bodies are observed and related to the Herring bodies.

The size and morphology of the granules are analyzed along the entire hypothalamic- neurohypophysial system. The changes in diameter and electron density are related to the maturation of the granules and the possible significance of such evolution.

Among the hypothalamic neurosecretory systems of mammals , tha t of the dog displays certain special features which are of considerable interest. One of them is represented by the large accumula t ion of neurosecretory mater ial along the entire system: i.e. the supraoptic and paravent r icular nuclei, the median eminence, the hypotha lamic-neurohypophys ia l t ract and the neurohypophysis (BARGMANN, 1949 a and b). Another morphological characteristic is the occurrence of vesiculated neurons shown by SCHARRER (1933) and SCHARRER and SCttARRER (1940), OLIVEIRA E SILVA (1937 and 1939) and extensively studied by JEWELL (1953). Such vesicu- lated neurons have also been observed in the fox bu t not in other species. F rom the biochemical viewpoint, in the dog there is lack of correlation of the vasopressin/ oxytocin ratio between the hypotha lamus and the neurohypophysis (VoGT, 1953; LEDERIS, 1962).

* Supported by grants from the Consejo Nacional de Investigaciones Cientifieas y T6cnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-66).

** From the Departamento de Anatomla, Fisiologia y Farmacologia Experimental, Facultad de Farmicia y Bioquimica, Universidad de Buenos Aires, Argentina.

Neurosecretion in the Dog 265

The aim of the present paper is t ha t of s tudying under the electron microscope the n i t ras t ruc tu ra l characteristics of this neurosecretory system. Par t icular a t ten- t ion will be devoted to the s t ructure of the vesiculated and non-vesicula ted neu- rons, and the significance of such vesicles will be clarified. Emphasis will be made on the differences between the neurons of the supraoptic and paravent r icu la r nuclei and on the s t ructure of the median eminence and in fund ibu la r process of the neurohypophysis . The special changes t ha t the neurosecretory e lementary granules undergo along the neurosecretory system will also be considered and compared with those previously described in this labora tory for amphib ians (GERSCHENFELD et al., 1960) and rodents (ZAMBRANO and DE ROBE~TIS, 1966).

Material and Methods Only male individuals, weighing between 4 and 7 kgs were used in this study. This was

done to avoid the cyclic changes, observed in female animals, probably due to the effect of the female hormones on the water metabolism during the sexual cycle (PAsQVALI~O and RA- GONESE, 1958; RAGONES~, 1958). In order to have reproducible results prior to fixation the animals were kept for 1O days under standard conditions of feeding and with water ad libitum. Ten animals were studied under the electron microscope and two under the light microscope.

After intraperitoneal anesthesia with sodium pentobarbital (50 mg/kg) the abdominal aorta was cannulated according to the technique of GONZALEZ-AGUILAR and DE ROBERTIS (1963). Several fixing solutions and methods were tried to overcome the difficulties arising from the large size of the animals. The best results were obtained with a similar solution as that of the above authors but containing 2 % formaldehyde and 30/0 glutaraldehyde instead of formaldehyde alone. After cannulation it was necessary to wash the vascular system of the animal with the solution of GO~IZ~L]~z-AGuILAR and DE ROBERTIS (1963) for 5 or 6 min, instead of the 3 0 4 0 see needed in the rat. After 1 hour of continuous flow of the fixative the brain was removed, and slices of the hypothalamus and neurohypophysis, 2 mm thick, were kept for 24 hours in the same fixing solution at 4~ under slow mechanical agitation. I~o differences were found between animals injected with washing and fixing solutions kept cold or at room temperature but the addition of heparin improved the preservation. The slices were dissected in order to obtain the following portions: the supraoptie nucleus, divided into its anterior and posterior divisions, the paraventricular nucleus, the median eminence and the neurohypophysis (Fig. 1). The blocks, 1 mm thick, were washed 24 hours in phosphate buffer 0.1 M with sucrose at 10% and after further trimming "refixed" for 2 hours in 1.5% osmium in phosphate buffer and treated with uranyl acetate. After embedding in Epon 812 the sections were stained with lead citrate according to REYNOLDS (1963) or VENABLE and COGGESHALL (1965) and observed in a Siemens Elmiskop I. For light microscopy the animals were also perfused, but the washing solution was 0.9% sodium chloride and the fixing solution Bouin's fluid. The hypothalamie system was cut in frontal sections, which were stained with chrome-alum haematoxylin- phloxine according to GOMORI (1941).

Observations

Supraoptic Nucleus According to JEWELL (1953), the supraoptic nucleus of the dog m a y be divided

in to two regions: anter ior and posterior (Figs. 1, 2). Wi th the l ight microscope two kinds of neurosecretory neurons may be recognized: the non-vesicula ted and the vesiculated neurons (Fig. 3). The former show the common feature of other neurosecretory neurons, i.e. the presence of Gomori-posit ive mater ia l in the cyto- plasm. However, in the dog the special feature is the very large a m o u n t of such mater ia l present in the per ikaryon and also in the surrounding axons (Fig. 3). The vesiculated neurons are much larger because of the presence of the vesicles which

266 D. ZAmBRANO and E. DE ROB~RTIS:

may have a diameter of 40 to 1O0 ~. These vesicles appear as if they were empty, without visible material inside. In some favorable sections the vesicles appear completely surrounded by neuronal cytoplasm, which is also Gomori positive (Fig. 3). Sometimes the vesicle appears to be formed by two adjacent neurones.

The electron microscopy study shows that the supraoptic nucleus is richly vascularized. Some arterioles innervated with adrenergic nerve endings having granulated and non-granulated synaptic vesicles (DE ROBERTIS and P~LL~GRI~O

~n

Fig. 1. Diagram of a sagittal view of the hypothalamic-neurohypophyseal system of the dog. Insets show the main anatomical regions studied under the light and electron microscopes. The presence of vesiculated neurons in the anterior and posterior divisions of the supraoptic nucleus is indicated. Legends for all figures: As astroglia; ASO anterior division of the supra- optic nucleus; Ax axon; Ce capillary endothelium; Cl capillary lumen; Co collagen; E synaptic ending; er endoplasmic reticulum; G golgi complex; g/ gliofilaments; gp glial process; Hb Herring body; I P infundibular process; Is infundibular stem; ly lysosome; M B mammillary body; N nucleus; Nu nucleolus; n/neurofilaments; ng neurosecretery granule; nt neurotubule; OC optic chiasma; P pituicyte; Pd pars distalis; Ppn pars paranuclearis; PSO posterior division of the supraoptic nucleus; Pt pars tuberalis; P V paraventricular nucleus; SO supraoptic

nucleus; V vesicle; Vm vesicular membrane; Ze zona externa; Zi zona interna

DE IRALDI, 1961) may be observed. Most frequently the neurons are situated near a capillary (Fig. 4). Both the anterior and posterior divisions of the supraoptic nucleus are very close to the subarachnoidal space only separated by a septum of glial processes.

With electron microscopy the two kinds of neurons are easily recognized. The non-vesiculated is by far the most common type (Figs. 4, 5). The nucleus is oval or indented, with a well developed nucleolus. This is sometimes attached to the inner surface of the nuclear envelope. Some dense chromatin material may be found in between the nucleolus and the nuclear envelope. The cytoplasm shows an inner or perinuclear zone which is poor or devoid of the components of the endoplasmic reticulnm (i.e. cisternae with ribosomes) and contains numerous mitochondria and Golgi complexes. Two types of granules may be recognized in this region. Some are larger and with the limiting membrane in close contact with

Neurosecretion in the Dog 267

Figs. 2 and 3. Photomicrographs of various anatomical regions of the hypothalamic-neurohypo- physeal system of the dog. Gomori's stain. Fig. 2 Low power view of the supraoptie and para- ventricular nuclei. Some vesiculated neurons are observed in the supraoptic and only one in the paraventricular, x 35. Fig. 3 High power view of the supraoptie neurons; five of which are

vesiculated. Most neurons are non-vesiculated. X 350

a dense cent ra l core. T h e y range be tween 0.5 and 0.8 ~ and b y morpho logy t h e y are s imilar to the lysosomes of o ther cells.

The granules more closely in con tac t wi th the Golgi complexes and having a dense core are p r o b a b l y smal l lysosomes developing f rom Golgi membranes (Fig. 4). The t r u l y neurosecre to ry granule has a ve ry conspicuous l imi t ing m e m b r a n e a n d an e x t r e m e l y l ight core. They v a r y in d i ame te r be tween 1900 and 2600 A wi th a

mean of 2260 A (Fig. 6).

18 Z. Zellforsch., Bd. 81

Fig. 4. Electronmicrograph of a neuron of the supraoptic nucleus containing small amounts of neurosecretory granules. The perinuclear zone is rich in Golgi complexes, mitoehondria and lysosomes. The endoplasmic reticulum is abundan t and with dilated cisternae, a sign of active synthesis. Observe the close relationship of the neuron with a capillary and the thin

Fig. 5. Eleetronmicrograph of a neuron of the supraoptic nucleus loaded with elementary neuroseeretory granules. The endoplasmie reticulum is confined to small areas and the cisternae

are closed. This perikaryon is in storage stage. • 8,000

layer of axons and nerve endings situated in between. With arrows are indicated some granu- lated vesicles of adrenergic type within the endings. In the inset, a detail of the dilated cisternae

of the endoplasmic reticulum may be observed. • 17,550. Inset • 40,000

18.

270 D. ZAMBRANO and E. DE ROBERTIS:

o6

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Fig. 6 a and b. Histograms of the size of neurosecretory granules in various regions of the hypothalamic-neurohypophyseal system of the dog (see description in the text)

Similar elementary granules of low electron density may be observed in the axons of the supraoptic nucleus and median eminence, but as it will be described later, they undergo a condensation upon reaching the neurohypophysis. In Fig. 5a non-vesiculated neurone is observed in which the cytoplasm is filled with masses of elementary granules which reduce the cytoplasmic organoids - - particularly the endoplasmic reticulum - - to small islands.

The cisternae of the endoplasmic reticulum may show different degrees of flattening or dilatation among the various neurons. This may indicate various stages of activity similar to those previously described in the supraoptic of the rat (ZAM~A~O and DE ROBERTIS, 1966). When the cisternae are dilated, as in the rat, they show a filamentous material inside (Fig. 4, inset). The neurons which are loaded with elementary granules have in general flattened cisternae.

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Neurosecretion in the Dog

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N e u r o h y p o p h y s i s

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In the neuropile surrounding the neurons numerous glial processes containing gliofilaments, fine axons and numerous nerve endings with synaptic vesicles may be observed (Fig. 4). The axons contain very conspicuous neurotubules and the endings show some granulated vesicles of 700--850 A similar to those described by PELLEGRINO DE IRALDI et al. (1963) in the anterior hypothalamus, in addition to the more common non-granulated vesicles.

The vesiculated neurons described by JEWELL (1953) are mainly found in the posterior division of the supraoptic nucleus (Fig. 1). The observation of such vesi- cles with the electronmicroscope is made difficult by the fact tha t most neurons are non-vesiculated and by the enormous size of these structures. Only the smaller vesicles, or tangential sections to them, may be contained within the field of a low power electronmicrograph. In general only partial views of the vesicle with the surrounding rim of cytoplasm may be obtained (Figs. 7 and 8).

272 D. ZAMBRANO and E. DE ROBERTIS."

Fig. 7. Partial view of a vesiculated neuron near a capillary. The vesicle contains some macro- molecular material but it is limited from the rim of cytoplasm by a rather discontinuous plasma membrane. The cytoplasm is loaded with neurosecretory granules. Axons, glial processes and

some collagen are observed in the vicinity of the capillary. • 30,000

The e lect ronmicroscope clear ly demons t ra t e s t h a t the vesicle is conta ined with- in the cy top la sm of the neuron. The smal ler vesicles such as t h a t i l lus t ra ted in Fig. 7 conta in a macromolecn la r ma te r i a l suggesting the presence of some pro te in prec ip i ta te . I t is in teres t ing t h a t in such cases the cy top lasm is only pa r t i a l ly covered b y a p l a sma membrane and t h a t a t some points the vesicular f luid seems to be in d i rec t con tac t wi th the cy top lasmic m a t r i x (Fig. 7). I n the larger vesicles, toge the r wi th the lack of macromolecu la r ma te r i a l inside, there is a definite l imit ing m e m b r a n e covering the cy top lasm (Fig. 8). The band of cy top lasm a round the vesicle conta ins some mi toehondr ia , endoplasmic re t ieu lum and neuro tubnles b u t in general is f i l led wi th densely packed e l emen ta ry granules (Figs. 7 and 8). As i l lus t r a t ed in Fig. 7 the vesicles are s i tua ted near a capi l la ry and only separa ted b y a few microns of neuropi le conta ining th in axons and numerous glial processes. I n Fig. 8 such processes, filled wi th gl iofi laments, appea r to form a k ind of sup- por t ing shell a round the vesicle.

Neurosecretion in the Dog 273

Fig. 8. Partial view of a vesiculated neuron with completely clear content. There is a more complete limiting membrane along the rim of cytoplasm loaded with elementary granules. An astrocyte with many processes forms a glial shell around the neuron. •

Two types of neurosecretory axons may be recognized in the proximity of the supraoptic neurons. The most common is unmyelinated and filled with numerous elementary granules the size of which show a small increase in size with respect to the granules in the cytoplasm (Fig. 6). The other type, less frequent, is represented by some poorly myelinated fibers which contain elementary granules. In one of such axons cut longitudinally a Ranvier node was observed.

Paraventricular Nucleus

Paraventricular neurons share many of the features already described for the supraoptic nucleus (Fig. 9). They are smaller and contain less neurosecretory granules in the perikaryon. The elementary granules are somewhat smaller with a mean diameter of 2040 A (Fig. 6). Around the neurons, large axons filled with neurosccretory granules are observed (Fig. 9). Most of them are unmyelinated but some myelinated ones are also found. The size of the granules is larger than in the perikaryon, the mean diameter being 2590 A. In spite of the fact that some vesieu- lated neurons may be observed at the light microscope level, they are too scarce and this has made impossible to recognize them with the electron microscope. The vascularization of this nucleus is also similar to that of the supraoptic nucleus.

Median Eminence

According to KNOCHE (1953), HAGEN (1957) and ROTHBALL]~R and Srzo~YNA (1960) the median eminence of the dog may be divided into an inner zone, which lies beneath the ependyma of the infundibular recess and an outer zone which is immediately below (Fig. 1). The inner zone is occupied by the axons of the supra- optic and paraventricular tracts which end in the infundibular process of the

274 D . ZAMBRANO and E. DE ~OBERTIS :

Fig. 9. Electronmicrograph of a neuron from the paraventricular nucleus of the dog containing few neurosecretory granules. ~ear the perikaryon there are large axons filled with neurosecre- tion, some nerve endings with synaptic vesicles and glial processes. With an arrow some

granulated adrenergic vesicles are indicated, x 21,000

l~eurosecretion in the Dog 275

Fig. lOa--d . Variat ion of the elementary neurosecretory granules in different regions of the hypothalamic neurohypophyseal system, a in the perikaryon of a supraoptie neuron; b in a

supraoptie axon; e in an axon of the inner zone of the median eminence; d in the infundibular process. X 60,000

276 D. Z~BRANO and E. D~ ROBERTS:

Fig. 11. Electronmicrograph of the infundibular process of neurohypophysis (see description in the text). The large axon in the centre corresponds to a Herring body. The axons contain dense and smaller neurosecretory granules and some vesicles of synaptic type. Neurotubules are

abundant . • 39,000

Neurosecretion in the Dog 277

neurohypophysis. There are unmyelinated nerve fibers and a few myelinated ones containing elementary granules (Fig. 10 c). These granules have slightly increased in size as compared with those in the hypothalamic nuclei and the core is denser (Fig. 6).

The outer zone consists of a mass of t ightly packed unmyelinated axons of which only few contain neurosecretory material. These axons are irregularly shaped and contain abundant neurofilaments and only few neurotubules. Some of these axons contain typical synaptic vesicles and others have the granulated type of vesicles with a very dense core and a light zone below the membrane. In the outer zone, besides unmyelinated axons it is possible to observe neurons which probably are of adrenergic nature. Within the cytoplasm of these neurons there are several granulated vesicles of 800--900 A, identical to those observed in the axons of the neuropile and similar to those previously described by P~r~L~G~INO D~, IRALDI et al. (1963) in the anterior hypothalamus and interpreted as containing cateeholamines. This adrenergic neuron contains Golgi complexes, lysosomes and a rather fibrillar matr ix rich in neurofilaments.

Neurohypophysis

In the dog, the infundibular process of the neurohypophysis shows certain features which differ from those from other species (Fig. I1). Compared to the ra t the axons are rather small and contain few neurosecretory granules. In the vicinity of the contact with the capillaries small vesicles similar to the synaptic ones appear. In such axons the neurotubules are also very conspicuous (Fig. 11). The elementary neurosecretory granules show considerable differences with those observed either in neurons or axons in the hypothalamic nuclei. They are considerably denser and smaller. The mean diameter is of 1090 A which represents a large reduction in volume (Fig. 6).

The electron micrograph of Fig. 11 shows most of the described features and in addition that the neurosecretory axons are in intimate contact with long processes of pitnicytes which are filled with filamentous material. Another special characteristic of the dog neurohypophysis is the presence of greatly enlarged axons which contain abundant neurotubules and some elementary granules. In these, the bulkier component is represented by round or rather irregular dense bodies of 0.3---0.5 ~t with a definite limiting membrane. The content of such bodies is rather polymorphic. In some there is a dark amorphous mass, in others the prevalent structure is a multi laminar organization with irregular masses included.

Discussion The Ultrastructure o/ Vesiculated Neurons

The electron microscope observations presented here throw definite light on the localization and nature of the large "vesicles" found in the hypothalamic neurosecretory nuclei of the dog and suggest a possible mechanism of origin for such vesicles.

JEWELL (1953) found tha t only about 0.1 to 0.2% of the neurosecretory neu- rons are vesiculated. Of these 76 % are located in the posterior division of the supra- optic nucleus, 18% in the anterior division and only 6% in the paraventricular

278 ~), ZAMBRANO a n d E. DE ROBERTIS:

nucleus. The exact relationship of these large vesicles with the neurosecretory neurons is now determined by our electron microscope observations. Some sug- gestions tha t the vesicles could represent dilated or ruptured capillaries produced by the perfusion at pressure (GI~EEN, 1952) are completely disproven. The vesicles, although close to capillaries, are always surrounded by a rim of neurosecretory cytoplasm which is covered by a sheath formed by glial and neuronal processes. An idea of the degree of development of the vesicle may also be obtained by electronmicroscopy. The finding of a macromolecular precipitate within the smaller vesicles, together with the lack of a definite limiting membrane, suggest that they are probably in an early stage of formation. The mechanism could be that of a localized intracytoplasmic cytolysis with fluid accumulation. In the larger vesicles now limited from the cytoplasm by a continuous plasma membrane, the precipitate is no longer present and the cavity looks empty. The finding of intermediary stages with various amounts of macromolecular content and partial limiting membranes suggest tha t there is a progressive evolution with many intermediary stages. The interpretation may be advanced that the vesiculated neurons originate from the non-vesiculated by a local zone of cytoplasmic cytolysis and that the resulting cavity is finally separated by a plasma membrane.

These findings and interpretations may have some bearing on the physiological function which has been attr ibuted to these structures. Because of the close re- lationship of the vesicles with capillaries VERN]~Y (1947) and JEWELL and VERN]~Y (1957) suggested tha t the vesiculated neurons could represent the osmoreceptors. This hypothesis was based on the assumption that the osmolarity of the blood could influence the composition of the vesicular fluid and this in time could activate or inhibit "intraneuronally" the neurosecretory neuron. This hypothesis is a t present untenable. In other species such as the cat - in which there are no vesicu- lated neurons - many cells of the supraoptic nucleus change their rate of fh~ng by osmotic and ionic changes in the blood (KoizvMi et al., 1964; ISH~KAWA et al., 1966). Furthermore, even in the dog the number of vesiculated neurons is too small to explain only by them the osmoreceptor properties of the supraoptic nucleus.

State o/Activity o/the Neurosecretory Neurons In recent years, due to the use of specific staining techniques and especially

to the ultrastructural studies with the electron microscope considerable informa- tion has been gathered on the activity of neurosecretory neurons (see DE ROB]~RTIS, 1964; BARGMA~N, 1966). In the normal supraoptic nucleus of the rat both with Gomori staining (ZAMBRA~O and MORDOH, 1966) and electron microscopy (ZAM- BRANO and DE ROB]~RTIS, 1966) variations in the amount of neurosecretion and in the structure of the various cytoplasmic organoids were observed from one neuron to another. Such variations, which were also enhanced by water deprivation of surcharge, led to postulate a cycle of activity in the neurosecretory neurons and to the concept tha t in normal conditions the degree of activity may also oscillate in individual neurons.

The observations reported here suggest tha t such differences of activity may be also present in the supraoptic nucleus of the dog. Most neurons are in a storage stage, with the cytoplasm loaded with elementary granules and the cisternae of the endoplasmic reticulum flattened. Fewer neurons, while containing less granules,

~qeurosecretion in the Dog 279

have the cisternae dilated and filled with a filamentous material. Such characteristics correspond to an active stage of secretion (ZAMBRA~O and DE ROBERTIS, 1966).

According to their ultrastructural characteristics in the paraventricular nucleus most neurons would be actively secreting while the product is accumulated in the axons.

Evolution o/ the Neurosecretory Granules GE~SCHENFELD et al., (1960), in the toad, demonstrated tha t the neurosecretory

granules increase in size along the preoptic-neurohypophysial t ract and postulated that their axonic flow is accompanied by a progressive protein synthesis or accu- mulation. Similar observations were made in the ra t by ZAMERA~O and DE RO- BERTIS (1966) and KOEAYASHI et al., 0966). The dog has some special features in the evolution of the elementary granules which are apparently at variance with the above mechanism. Fig. 10 illustrates the wide variation in the size and density of the granules observed along the entire neurosecretory system. The actual measure- ments are recorded in Fig. 6. Both in the cytoplasm of the supraoptic neurons and in the axons neuroseeretion appears as round vacuoles with a limiting membrane and a very diluted content of low electron density (Fig. 10a, b). In the median eminence, while the size is about the same, there is an increase in electron density which varies from one granule to another (Fig. 10c). The most striking change is observed in the neurohypophysis where the granule has suffered a great reduction in size, which is accompanied by a considerable increase in electron density (Fig. 10d).

These structural variations may reflect differences in the properties of the granule. SACHS and TABAKATAKE (1964) and TABAKATAKE and SACHS (1964) have observed that in vitro the median eminence is able to incorporate labeled cisteine into vasopressin, indicating that at this level there is still synthesis of the hormone, while the neurohypophysis is unable to incorporate the amino acid precursors. These authors have explained the increase in vasopressin act ivi ty along the t ract as due to the breakage into active polypeptide groups given off by a high molecular weight precursor synthetized in the perikaryon. I t is possible to postulate tha t the large and less dense granule found in the perikarya and axons of the hypothalamie nuclei and the in medium eminence are still able of synthesis or protein accumula- tion, while those smaller and denser of the infundibular process have lost such property and have a purely storage function. This interpretation may explain the apparent discrepancy with the neurosecretory system of the toad (GERSCHE~FEL]) et al., 1960) and ra t (ZAMBRA~O and DE ROBERTIS, 1966).

Ultrastructure o/the Median Eminence

The median eminence is not only a site of passage for the hypothalamic neurohypophyseal tracts but an important effector neurosecretory organ in which products from the hypothalamic neurons are released into the portal system. The anatomical division into an inner zone, rich in passing neurosecretory axons (ROTHB~LER and SKORYNA, 1960), and an outer zone, in relation with the capil- lary plexus of the portal vessels, is here corroborated with the electron microscope. BARGMANN (1966), distinguishes two types of vesicular (i.e. granular) elements in the median eminence. Type I is larger and corresponds to the true neuro- secretory elementary granule, which is more frequent in the axons of the inner

280 D. ZAMBRANO and E. DE ROBERTIS:

zone. Type I I is made of smaller vesicles with a very dense core and a light rim below the membrane which are similar to the granulated vesicles, previously described by PELLEGRINO et al., (1963) in the anterior hypothalamus. Such granulated vesicles were isolated by cell fractionation from the anterior hypo- thalamus and their high content of noradrenaline was demonstrated by DE ROBERTIS et al., (1965). These vesicles were previously observed by KOBAYASHI et al., (1966) in the median eminence of the rat and as shown here, they are promi- nent in axons of the outer zone of the median eminence of the dog.

Using the fluorescence technique FuxE (1964) and FuxE and HOKFELT (1964) have found a sy s t em- - assumed to be dopaminergic-- originating from the nucleus arcuatus of the hypothalamus and ending in the median eminence. Studies are now being carried out in laboratory to study the neurons of such a nucleus under the electron microscope. The finding of a neurone present in the outer zone of the medium eminence and containing the adrenergic type of vesicle is of great interest because it may be related to the numerous axons and endings containing such adrenergic type of vesicles. The outer zone of the median eminence appears to be a very complex effector structure in which adrenergie (and/or dopaminergic) and typical neurosecretory axons end on the capillaries of the portal system.

Ultrastructure o/the Neurohypophysis In the infundibular process of the dog the size of the axons and the amount

of neurosecretion is smaller than in other species such as: the rat (PALAY, 1957; ZAMB~ANO and D~, ROBERTIS, 1966), the opossum (BODIAN, 1963), the monkey (BoDIAN, 1966) and man (LEDERIS, 1965), this in contrast with the considerable amounts of elementary granules loading the hypothalamic nuclei. We have already pointed out the differences in growth and maturation of the granules which are at variance with the other species studied in this laboratory. I t can be assumed that in the dog full maturation of the granule is produced in the last portion of the neurosecretory tract, with a considerable reduction in size accompanied by a great increase in molecular packing. As shown by SACHS and TAKABATAKE (1964) the neurohypophysis of the dog is unable to incorporate labeled cysteine or tyro- sine into vasopressin as it occurred in the median eminence. This is definite indi- cation that at this level the dense mature granules have lost the capacity of synthesis or accumulation of polypeptides.

The findings of large axons containing polymorphic bodies of 0.3--0.5 ~ in diameter, in addition to neurosecretory granules, may be interpreted as correspon- ding to the so-called Herring bodies. Such granular components are Gomori positive and cannot be differentiated from neurosecretion at the level of the light micro- scope. BODIAN (1963, 1966) has observed similar structures in the opossum and monkey.

References B~OMAN~, W.: 13ber die neurosekretorische Verkniipfung von Hypothalamus und Neuro-

hypophyse. Z. Zellforsch. 34, 610--634 (1949a). - - Ober die neurosekretorische Verkniipfung von Hypothalamus und Hypophyse. Klin Wschr.

27, 617--622 (1949b). - - Neurosecretion. Int. Rev. Cytol. 19, 183--202 (1966).

Neurosecretion in the Dog 281

BODIAN, D.: Cytological aspects of neurosecretion in opossum neurohypophysis. Bull. Johns Hop. Hosp. 118, 57--93 (1963).

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Dr. DAVID ZAMBRANO Instituto de Anatomia General y Embriologla, Facultad de Medicina Paraguay 2155, Buenos Aires, Argentina