Cell and Tissue Research
Cell Tiss. Res. 191, 405419 (1978)
9 by Springer-Verlag 1978
Parenchymal Fine Structure of the Subfornical Organ in the Japanese Quail, Coturnix coturnix japonica Kazuhiko Tsuneki, Yoshio Takei, and Hideshi Kobayashi Misaki Marine BiologicalStation, Universityof Tokyo, Misaki, Kanagawa-ken, Japan
Summary. The parenchyma of the subfornical organ (SFO) of the Japanese quail was studied by light and electron microscopy. The SFO consists of ependymal, intermediate, and basal (perimeningeal) layers. In the intermediate layer, neurons, glial cells, and their processes are found. Axons containing dense core granules approximately 80 nm in diameter are numerous, some of which make synaptic contact with the neuronal perikarya or dendrites. Synaptic vesicles in some axons contain a dense dot in the interior after treatment with 5-hydroxydopamine. The activity of the SFO, which is probably concerned with elicitation of drinking by angiotensin II, may be regulated at least partly by afferent monoaminergic axons. Capillaries with a non-fenestrated endothelium are occasionally found in the parenchyma. The basal layer is occupied by glial processes abutting on the digitating layer of perivascular connective tissue of meningeal vessels. The endothelium of these vessels is occasionally fenestrated. Trypan blue injected systemically accumulated in the SFO, but not in the deeper areas of the brain. The absence of a blood-brain barrier is suggested in the SFO. Key words: Subfornical organ - J a p a n e s e quail, Coturnix coturnix j a p o n i c a Monoaminergic innervation - Electron microscopy.
Introduction The subfornical organ (SFO), consisting of parenchyma and specialized ependyma, is located on the rostral wall of the third ventricle just below the interventricular foramen (Akert, 1969). Several functions of the S FO have been proposed relating to gonadal activity (rat, Stumpf, 1970; mouse, George and Penrose, 1975), osmorecepSend offprint requests to: YoshioTakei, Misaki MarineBiologicalStation, Universityof Tokyo, Misaki,
Kanagawa-ken, 238-02Japan Acknowledgements. The authors wouldlike to expresstheirgratitudeto Dr. Ebert A. Ashbyfor reading the manuscript. This work was supported by grants from the Ministry of Educationof Japan and from the Ford Foundation
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tion (frog, Dierickx, 1963; rat, Sarrat, 1968; mouse, George, 1973, 1974), and reception o f cholinomimetics for induction o f drinking (rat, Simpson and Routtenberg, 1974). Recently, the SFO has been shown to play an essential role in drinking induced by angiotensin II (AII) in the rat (Simpson and Routtenberg, 1973, 1975; Abdelaal et al., 1974) and the Japanese quail (Takei, 1977), based on the following observations: (1) Direct application o f A l l to the SFO induces copious shortlatency drinking, (2) electrolytic lesioning o f the SFO reduces the a m o u n t o f water intake induced by intravenous A I I , and (3) SFO lesioning attenuates water intake induced by A l l injected into the preoptic area (POA), another area showing high sensitivity to All. Furthermore, in the Japanese quail, an afferent monoaminergic p a t h w a y connects the preoptic area (POA) with the SFO (Takei et al., 1979). These nerve fibers p r o b a b l y transfer the stimulus generated by A I I at the P O A to the SFO, since transection o f these fibers eliminates the dipsogenic action o f preoptically applied A l l (Takei et al., 1979). F o r these reasons, it is o f particular interest to investigate the ultrastructure of the SFO in order to determine the character o f nerve cells, afferent synaptic terminals and specialized perivascular structures. Ultrastructural studies o f the SFO o f several m a m m a l i a n species have previously been reported (Andres, 1965; Rohr, 1966a, b; Rudert et al., 1966, 1968; Pfenninger et al., 1967; Akert et al., 1967; Dempsey, 1968; Schinko et al., 1972; L e o n h a r d t and Lindemann, 1973; Dellmann and Simpson, 1975, 1976). In avian species, however, morphological studies o f the SFO are few, even at the light microscopical level (Pines and Scheftel, 1929; Reichold, 1942; Wetzig and Palkovits, 1968; Takei, 1977). To the knowledge o f the authors no report has so far appeared at the ultrastructural level. The present study was undertaken to assess the functionally important structures o f the quail SFO as well as to elucidate general organization o f the organ. The surface ultrastructure o f the quail SFO was described in a previous paper (Takei et al., 1978).
Materials and Methods Male Japanese quail, Coturnix coturnix japonica Temminck et Schlegel, were obtained from a commercial source at the age of 28 days. They were kept individually in small wire cages under a short day light cycle(8L/16D). The birds were used at the age of more than 35 days for the following purposes: (1) For general light microscopical observation, the SFO of several birds were fixed in Bouin fluid. Serial paraffin sections were cut at 10 ~tmthickness and stained with either paraldehyde fuchsin or hematoxylin and eosin. (2) For study of the vascularization, several birds were perfused intracardially with India ink. The brains of the perfused birds were fixed in Bouin fluid and embedded in paraffin. Serial sections 25 ~tmthick were observed without counterstaining. (3) In order to clarify whether a blood-brain barrier is present in the SFO, daily injections of 0.7 ~o trypan blue in saline were made intravenously (i.v.) or intraperitoneally (i.p.) in a volume of 0.5 ml (i.v.) or 1 ml (i.p.) for one week in several birds. The brains of these birds were f'Lxedin Bouin fluid and serial paraffin sections of 25 ~tm thickness were examined without staining. (4) For electron microscopy, the SFO of eight birds were fixed by either immersion or systemicperfusion with modified Karnovsky fixative. The details of fixation and further procedures for electron microscopy were given in a previous paper (Takei et al., 1978). (5) Two birds were infused intracerebro-ventricularly with 5-hydroxydopamine (5-OHDA) dissolved in saline containing 0.1 ascorbic acid. Doses of 5-OHDA were 0.5 mg and 2 mg/100 g BW, respectively. Infusion continued for 30 min at a rate of 1.33 ~tl/min. The birds were decapitated 30 min after the termination of the infusion. The SFO of these birds were fixed by immersion in modified Karnovsky fixative and further processed for electron microscopy.
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Fig. 1. Transverse section through the body of the SFO in the Japanese quail. Dotted line represents the delineation of the organ. Note densely arranged ependymal cells. Cell bodies form loose clusters on both sides offissura longitudinalis cerebri (tic). The region facing the fissure is not occupied by cell bodies, but rather by membrana limitans gliae superficialis. The trapezoid shows a region in which most of the electron micrographs were taken. BL Basal layer, cp choroid plexus, E L ependymal layer, 1L intermediate layer, s sinus-like vessel, I I I third ventricle. Hematoxylin and eosin, x 260 Fig. 2. Sagittal section through the SFO of the quail injected with India ink intracardially. Rostroventral to the left. The SFO is limited by dotted line. In the center of the SFO, meningeal capillary net and large sinus partially filled with India ink are seen. The vascularization of the SFO parenchyma is rather poor. The meningeal vessels are also seen in the upper left corner, cp Choroid plexus, pc pallial commissure, I I I third ventricle. • 100 Fig. 3. Sagittal section through the SFO of the quail injected with trypan blue. Rostroventral to the left. The SFO is limited by dotted line. Note deposition of grains of trypan blue in the SFO. Such grains are not observed in the deeper region of the brain, cp Choroid plexus, pc pallial commissure, 1II third ventricle, x 100
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Results
Light Microscopical Observations The SFO of the Japanese quail consists of a body and a stalk. The body occupies the rostroventral portion and the stalk occupies the caudodorsal portion of the organ (for details, see Takei et al., 1978). In transverse section, the body of the organ is semicircular in outline (Fig. 1). The ventral region projects slightly into the ventricle. The dorsal region is continuous with the brain parenchyma and divided into two halves by thefissura longitudinalis cerebri. The dorsal and lateral boundary of the organ is indistinct. The parenchyma approximately 2 mm in depth from the ventricular surface represents roughly the limits of the organ (Fig. 1). Within this region, neurons constitute a loosely clustered arrangement (nucleus). Capillaries are occasionally seen in the parenchyma. Meningeal blood vessels are located on the wall of the fissure. The vessel located most ventrally in the fissure is sinus-like in appearance and proceeds longitudinally along the SFO including the stalk (Fig. 1). Some vessels on the wall of the fissure are also seen in Figure 2. The SFO of the birds injected daily with trypan blue was characterized by the deposition of blue granules (Fig. 3). They were distributed in the parenchymal tissue near blood vessels. The blue granules were also observed in the median eminence, the organum vasculosum of the lamina terminalis (OVLT) and the choroid plexus. No granules were found in the other brain areas including the paraventricular organ and the subcommissural organ. The pineal organ, neural lobe, and area postrema were not studied.
Electron Microscopical Observations The SFO of the Japanese quail can be roughly divided into three layers: ependymal, intermediate, and basal (perimeningeal) layers (Figs. 1, 4). Although the whole area of the SFO was studied, the region indicated by a trapezoid in Figure 1 was given particular attention.
Ependymal Layer. The fine structure of this layer was described in a previous paper (Takei et al., 1978). The nuclei of ependymal cells are arranged at different levels, possibly indicating a pseudostratified epithelium (Fig. 4). The cells located relatively distant from the ventricle may be classified as subependymal cells. Neurons are occasionally present in the subependymal region. In the stalk of the SFO, only one layer of ependymal cells is found. Intermediate and basal layers are absent. These ependymal cells directly contact the perivascular space of the underlying vessel.
Intermediate Layer. This layer is occupied by neurons, dendrites, axons, glial cell bodies, glial processes, ependymal processes, and capillaries (Figs. 4-13). The neurons are usually scattered. In some instances, however, two neurons are in direct contact (Fig. 6) or are separated only by a very thin glial process. The nucleus of the neurons is round or elliptical and large, containing a small amount of dense
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Fig. 4. Transverse section through a portion of the body of the SFO. The third ventricle and ependymal cells of the adjacent ventricular wall are seen in the upper left corner. BL Basal layer, ct connective tissue, EL ependymal layer, gl glial cell, IL intermediate layer, n neuron, t process of tanycyte, x 4400
chromatin (Figs. 5, 6). The nucleolus is usually prominent. Two types o f neurons m a y be present; the first type shows a small cytoplasmic area (Fig. 6), the other a more expansive cytoplasm (Figs. 4, 5). The cytoplasm contains mitochondria, neurotubules, lysosomes, irregularly shaped dense bodies, Golgi apparatus, tubular profiles o f rough surfaced endoplasmic reticulum, and polysomes. The last three organelles are especially well developed. Electron dense granules are only
Fig. 5. Part of Figure 4 (intermediate layer) seen at higher magnification. Note intermingled neuronal and glial processes in the center. Arrow points out the junctional complex between glial cells which contain m a n y microfilaments, gl Glial cells, n neuron, sr subependymal cell. x 11,100 Fig. 6. Two neurons with prominent nucleoli and scanty cytoplasm are in direct contact. Arrow indicates axo-dendritic synapse, x 11,100. The inset shows small axons with neurotubules and the perikaryon containing one granule (arrow). x 22,200
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Fig. 7. Three axon terminals making synaptic contacts(arrows) on a perikaryon. Two terminals contain both dense core granules and synaptic vesicles, one terminal has only synaptic vesicles. • 22,200 Fig. 8. Axons in the SFO of a quail treated with 2 mg 5-OHDA. Dense dots are seen in synaptic vesiclesin axons shown in the upper left and upper right corners. The dense granules in the axon shown in the upper right corner are approximately 120 nm in diameter; the granules in the other axons are about 80 nm in diameter. Arrows indicate axo-dendritic synapse. • 22,200
infrequently f o u n d in the perikarya o f some neurons (Fig. 6, inset). Their diameter measures 90 to 100nm. A l t h o u g h large neurons containing a b u n d a n t electron dense granules approximately 180 to 200 n m in diameter are occasionally found in the periphery o f the organ, they m a y represent peripherally scattered neurons o f the secretory paraventricular nucleus. Corresponding to this observation, the light microscopical preparations stained with paraldehyde fuchsin revealed several neurosecretory cells o f the paraventricular nucleus located a r o u n d the ventrolateral border o f the SFO. Neither fibers n o r cell bodies staining with paraldehyde fuchsin were f o u n d except for occasional, possibly specialized glial cells (microglia ?) in the vicinity o f meningeal vessels. Dendrites and axons are abundant. Small axons contain only neurotubules (Fig. 6). Large axons frequently have dense core granules approximately 70 to 90 nm in diameter in addition to synaptic vesicles and m i t o c h o n d r i a (Fig. 7). The axons containing larger dense core granules (110 to 120 n m in diameter) are rarely found. Some axons contain only synaptic vesicles. N o myelinated axons are observed. A x o n s containing either dense' core granules (approximately 80 n m in diameter) and synaptic vesicles, or only synaptic vesicles, frequently m a k e synaptic contact with dendrites (Figs. 6, 10) or neuronal perikarya (Fig. 7). As m a n y as five synapses can be seen on one neuronal perikaryon. These axo-dendritic and axosomatic synapses show typical synaptic characters such as the aggregation o f
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Fig. 9. Parenchymaof the SFO of the same bird as shownin Figure 8. Dots are seenin synapticvesicles of some axons (al), but not in others (a2). One "positive"axon is in synaptic contact (arrows) with a neuronal perikaryon, x 20,800
synaptic vesicles on the presynaptic membrane and thickening of both pre- and postsynaptic membranes. In the preparations treated with 2 mg 5-OHDA, synaptic vesicles in some axons with dense core granules (approximately 80 nm in diameter) contained a dense dot in the interior (Figs. 8, 9). This structure was found in every vesicle in some axons, but only in a small proportion of vesicles in others. Axons with dotted synaptic vesicles also made synaptic contacts with either dendrites or perikarya (Fig. 9). Axons containing dense core granules with a diameter of 120 nm possessed synaptic vesicles with a dense dot (Fig. 8). No synapses were formed in connection with these axons, which can perhaps be accounted for by the fact that possibly occurring synaptic contacts were overlooked due to the rarity of the axons. Axons in the SFO of the quail injected with 0.5 mg 5-OHDA showed fewer dotted synaptic vesicles. Glial cells are dispersed in the parenchyma. Their shape is variable; round, elliptical or even irregular (Figs. 5, 10, 11). Dense chromatin is usually more abundant in the nucleus of glial cells than neurons (Fig. 11). Small, dense bodies possibly identified as primary lysosomes are occasionally found in the cytoplasm of some glial cells (Figs. 10, 11). The density of the cytoplasm is extremely variable among glial cells. In some cases, two adjacent glial cells are joined by a long junctional complex (Fig. 5). These glial cells contain abundant microfilaments. Slender straight processes filled with microfilaments course the subependymal layer to the basal region of the parenchyma (Fig. 4). These are probably tanycytes. A single cilium was found in the parenchymal cells in several instances.
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Fig. 10. Glial cell (probably astrocyte) in the intermediate layer. Arrows point out axo-dendritic synapses, x 11,100 Fig. 11. Glial cell (probably microglia)in the intermediatelayer, x 11,100
Vacuoles of varying sizes are occasionally observed in the parenchymal cells. They are usually irregular in outline (Fig. 12). The large vacuoles exceed the average size of a cell. They are rimmed by a very thin cytoplasmic covering. Flocculent material is occasionally seen in the lumen. The perivascular space of parenchymal capillaries is thin (Fig. 13). Endothelial cells are not fenestrated. Pericytes are observed in the vicinity of some capillaries.
Basal (Perirneningeal) Layer. The basal glial processes abutting on the extraparenchymal connective tissue in a palisade fashion are usually voluminous and electron lucent (Fig. 14). They contain round or tubular vesicles of varying densities, relatively large vacuoles, and occasionally very large mitochondria filled with lamellae (Fig. 15, inset). Microfilaments are infrequently found in the processes. The slender dense processes and glial cell bodies themselves also sometimes contact the connective tissue (Fig. 15). The branches of the connective tissue sheet penetrate deep into the basal layer of the parenchyma (Fig. 14). In some sections, the branches of the connective tissue sheet are isolated apparently from the perivascular region. The extent of branching decreases toward the dorsal (distal to the ventricle) region of the SFO. The perivascular space of specimens fixed by immersion is relatively homogeneously dense, but the material fixed by systemic perfusion tends to show a basal lamina and many collagen fibrils more clearly (compare Fig. 14 with Fig. 15).
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Fig. 12. Intracytoplasmic vacuole surrounded by a very thin rim of cytoplasm; gl glial cell. x 17,800 Fig. 13. Capillary with non-fenestrated endothelium (en) in the intermediate layer. The lumen is occupied by an erythrocyte (er). Perivascular connective tissue surrounding the endothelium and pericyte (p) is thin. x 14,800
Fig. 14. Basal layer consisting of light glial processes (probably processes of astrocytes) abutting on the digitation of the perivascular connective tissue (ct). en Endothelium, er erythrocyte, f fibroblast. x 11,100. The inset shows fenestrated endothelium (arrow) and a fibroblast (f) in the perivascular space. • 27,900 Fig. 15. Basal layer of material fixed by vascular perfusion. The basal lamina and collagen fibrils in the perivascular space (pvs) are clearly seen. In this section, glial cells (gO directly contact the perivascular space as dark ( dp) and light processes ( lp). x 17,800. The inset shows light glial processes containing large mitochondria; pvs perivascular space, x 16,400
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The meninx covering the parenchyma consists of blood vessels, of which the most ventral one is sinus-like, collagen fibrils, ground substance of the connective tissue, and accompanying cells (Fig. 14). The pattern of intermingling of these elements is occasionally very complicated. The main cellular components appear to be fibroblasts. Plasma cells are also occasionally found. In some specimens, only perivascular connective tissue (collagen fibrils+ ground substance) separates the parenchyma from endothelial cells. Since the wall of vessels does not contain smooth muscle cells, the vessels may not be arterial, but rather venous or, more likely, capillary specializations draining this region of the brain. The thickness of the endothelial cells of the vessels is extremely variable. Fenestrations are occasionally found (Fig. 14, inset).
Discussion
The present study on the SFO of the Japanese quail shows that this organ has many characteristics in common with circumventricular organs, such as a thickened ependymal layer, rich vascular supply and the presence of tanycytes. However, the paucity of apparent neurosecretory elements is in contrast to the abundance of these elements in other circumventricular organs of the quail such as the median eminence (Ishii et al., 1975) and the OVLT (Mikami, 1976). The SFO of the quail differs from mammalian SFO in that its delineation as an organ is indistinct and the basal layer is contiguous with the meninx. This difference may be due to the absence of the fornix in birds. However, the similarity between the quail and mammalian SFO at the ultrastructural level is more striking. Scattered neurons and glial cells, neuronal processes with or without dense core granules, large vacuoles developed in cells, digitations of perivascular connective tissue, long junctional complexes between adjacent glial cells, and axo-dendritic or axo-somatic synapses have also been described in mammalian SFO (Andres, 1965; Rohr, 1966a, b; Rudert et al., 1966,1968; Pfenninger et al., 1967; Akert et al., 1967; Dempsey, 1968; Schinko et al., 1972; Leonhardt and Lindemann, 1973; Dellmann and Simpson, 1975). The quail SFO possesses a small number of poorly granulated perikarya, which may represent a transitional form between SFO apparently lacking a granulated perikaryon (dog, Andres, 1965; cat, Rohr, 1966 b; Pfenninger et al., 1967; rabbit, Rudert et al., 1968; mouse, Schinko et al., 1972) and SFO with perikarya containing some granules (approximately 120 nm) (rat and other mammals, Dellmann and Simpson, 1975). The presence of dense core granules in perikarya may imply secretory activity of neurons, but its significance is unclear in the SFO. The axons with neurosecretory granules larger than 150 nm (peptidergic neurosecretory axons ?) were absent in the present material. This type of axon is rare in the rat et al. (Dellmann and Simpson, 1975), and seems to be absent in the dog (Andres, 1965), and mouse (Schinko et al., 1972). Although Rohr (1966 b) and Pfenninger et al. (1967) reported neurosecretory axons in the cat SFO, their electron micrographs show rather irregular granules seemingly different from so-called elementary neurosecretory granules. The relation between SFO and the peptidergic neurosecretory nucleus was suggested in the frog (Rudert, 1965) and some other species (Legait and Legait, 1957, etc.), but such a relation is apparently negligible in the Japanese quail.
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In the quail SFO, non-fenestrated capillaries have been found in the parenchyma, whereas fenestrated capillary-like vessels were observed in the meninx. Non-fenestrated capillaries were found in the SFO of the dog (Andres, 1965) and rabbit (Rudert et al., 1966). In contrast, fenestrated capillaries were found in the cat (Rohr, 1966a) and mouse (Schinko et al., 1972). Consequently, both types of capillaries seem to be present in mammalian and avian SFO (see also Dellmann and Simpson, 1975). It has become increasingly clear that the SFO of the rat and the Japanese quail contain dipsogenic receptors for AII, since AII applied directly to the SFO induces immediate copious drinking (Simpson and Routtenberg, 1973; Kucharczyk et al., 1976; Takei, 1977). The most probable location of these receptors is the nerve cells in the SFO. Two types of nerve cells are distinguished in the quail SFO according to the relative abundance of cytoplasm. AII may act on the cells of one type or both types to induce drinking. However, nothing is yet known as to the efferent pathway from the SFO to the drinking center. Intravenously injected AII may gain access to nerve cells of the SFO, since the dipsogenic effect of AII is attenuated by prior lesioning of the SFO in the Japanese quail (Takei, 1977) as well as in the rat (Abdelaal et al., 1974; Simpson and Routtenberg, 1975). The occasional fenestrated endothelium in the meningeal vessels may represent a possible pathway for rapid exchange of materials between these vessels and the parenchyma. The well developed digitations of the perivascular connective tissue may increase the area of contact between the vascular system and the parenchyma. Furthermore, deposition of intravenously injected trypan blue in the SFO shows the absence of a blood-brain barrier, similar to the situation in the mammalian SFO and other circumventricular organs (see Koella and Sutin, 1967). These results support the concept that AII reaches parenchymal nerve cells of the SFO. In addition, some of glial processes abutting on the digitations of perivascular connective tissue contain extremely large mitochondria, which are sites of energy production for active transport of material. The intracerebro-ventricularly injected AII may act on neurons of the SFO through the ependymal layer. In the Japanese quail, neuronal perikarya are sometimes located in the subependymal region. These perikarya may be easily affected by AII penetrating from the ventricle. The functional significance of ependymal specialization has already been discussed in a previous paper (Takei et al., 1978). In the stalk region of the SFO, no neuronal perikarya are present and ependymal cells directly contact the perivascular space. This organization is similar to that of the choroid plexus. However, nothing definite is known on the function of the stalk. The present study revealed many axon terminals containing both dense core granules (approximately 80 nm in diameter) and synaptic vesicles, or synaptic vesicles only. Synaptic vesicles in some axon terminals containing dense core granules contained a dense dot in their interior after 5-OHDA treatment. Therefore, these axon terminals are considered monoaminergic (see Richards and Tranzer, 1970). The presence of monoaminergic terminals in the quail SFO is further supported by the histochemical finding that terminal-like varicosities showing monoamine-specific fluorescence are present (Takei et al., 1979). Since no fluorescent cell bodies are found in the SFO of the quail, the origin
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o f these m o n o a m i n e r g i c terminals must be outside the SFO. The P O A is one o f the possible sources o f afferent m o n o a m i n e r g i c fibers, since fluorescent perikarya are observed in the P O A (Takei, unpublished), and fluorescent fibers can be traced from the P O A to the SFO (Takei et al., 1979). These fluorescent fibers m a y transport dipsogenic information f r o m the P O A to the SFO, since transection o f these fibers eliminates drinking induced by preoptically applied A I I (Takei et al., 1979), and direct application o f noradrenalin or serotonin to the SFO induces drinking in the Japanese quail (Takei, unpublished). Some m o n o a m i n e r g i c axon terminals observed in the present study make synaptic contact with dendrites and perikarya, which is considered as ultrastructural evidence for m o n o a m i n e participation in the dipsogenic information transport to the SFO. The significance o f rare axons containing dense core granules with a diameter o f 1 2 0 n m is u n k n o w n . They also seem to be monoaminergic, since their synaptic vesicles showed a dense dot after 5 - O H D A treatment. M a n y axons did not show a dense dot in their synaptic vesicles after 5 - O H D A treatment, irrespective o f the presence of dense core granules. Some o f these axons might originate from perikarya within the SFO. It is also highly probable that afferent axons containing neurotransmitters other than m o n o a m i n e s are involved in the activity o f the SFO. Some functions o f the SFO in relation to gonadal activity, osmoreception and dipsogenic reception o f cholinergic drugs have been suggested in mammals. It has yet to be clarified whether these functions are present in the qail SFO and whether they are mediated t h r o u g h cholinergic or other mechanisms.
References
Abdelaal, A.E., Assaf, S.Y., Kucharczyk, J., Mogenson, G.I.: Effect of ablation of the subfornical organ on water intake elicited by systemicallyadministered angiotensin-II. Canad. J. Physiol. Pharmacol. 52, 1217-1220 (1974) Akert, K.: The mammalian subfornical organ. J. neuro-visc. Relat., Suppl. 9, 78-93 (1969) Akert, K., Pfenninger, K., Sandri, C.: The fine structure of synapses in the subfornical organ of the cat. Z. Zellforsch. 81, 537-556 (1967) Andres, K.H.: Der Feinbau des Subfornikalorgans vom Hund. Z. Zellforsch. 68, 445-473 (1965) Dellmann, H.-D., Simpson, J.B.: Comparative ultrastructure and function of the subfornical organ. In: Brain-endocrine interaction II (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Isbii, eds.), pp. 166--189. Basel: Karger 1975 Dellmann, H.-D., Simpson, J.B.: Regional differences in the morphology of the rat subfornical organ. Brain Res. 116, 389-400 (1976) Dempsey, E.W.: Fine-structure of the rat's intercolumnar tubercle and its adjacent ependyma and choroid plexus, with especial reference to the appearance of its sinusoidal vessels in experimental argyria. Exp. Neurol. 22, 568-589 (1968) Dierickx, K.: The subfornical organ, a specialized osmoreceptor. Naturwissenschaften 50, 163-164 (1963) George, J.M.: Localization in hypothalamus of increased incorporation of 3H cytidine into RNA in response to oral bypertonic saline. Endocrinology 92, 1550-1555 (1973) George, J.M.: Hypothalamic sites of incorporation of [3H]cytidine into RNA in response to oral hypertonic saline. Brain Res. 73, 184-187 (1974) George, J.M., Penrose, M.: Increased incorporation of [3H]uridine into RNA in the brain subfornical organ of ovariectomized mice. Brain Res. 97, 167-170 (1975)
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Ishii, S., Wada, M., Oota, Y.: Identification of neurosecretory granules carrying adenohypophysial hormone-releasing hormone. In: Brain-endocrine interaction II (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 70-79. Basel: Karger 1975 Koella, W.P., Sutin, J.: Extra-blood-brain-barrier brain structures. Int. Rev. Neurobiol. 10, 31-55 (1967) Kucharczyk, J., Assaf, S.Y., Mogenson, G.I.: Differential effects of brain lesions on thirst induced by the administration of angiotensin-II to the preoptic region, subfornical organ and anterior third ventricle. Brain Res. 108, 327-337 (1976) Legait, H., Legait, E.: Les vois extra-hypophysaires des noyaux neuros6crrtoires hypothalamiques chez les Batraciens et les Reptiles. Acta anat. (Basel) 30, 429-443 (1957) Leonhardt, H., Lindemann, B.: Surface morphology of the subfornical organ in the rabbit's brain. Z. Zellforsch. 146, 243-260 (1973) Mikami, S.: Ultrastructure of the organum vasculosum of the lamina terminalis of the Japanese quail, Coturnix coturnixjaponica. Cell Tiss. Res. 172, 227-243 (1976) Pfenninger, K., Akert, K., Sandri, C., Bruppacher, H.: Zum Feinbau des Subfornikalorgans der Katze. III. Nerven- und Gliazellen. Schweiz. Arch. Neurol. Neurochir. Psychiat. 100, 232-254 (1967) Pines, L., Scheftel, M.: Ist bei den niederen Vertebraten ein Homologon des subfornikalen Organes der S~iugetiere festzustellen? Anat. Anz. 67, 203-216 (1929) Reichold, S.: Untersuchungen fiber die Morphologie des subfornikalen und des subcommissuralen Organes bei S/iugetieren und Sauropsiden. Z. mikr.-anat. Forsch. 52, 455- 479 (1942) Richards, J.G., Tranzer, J.-P.: The ultrastructural localization of amine storage sites in the central nervous system with the aid of a specific marker, 5-hydroxydopamine. Brain Res. 17, 463-469 (1970) Rohr, V.U.: Zum Feinbau des Subfornikalorgans der Katze. I. Der Gef'~iBapparat. Z. Zellforsch. 73, 246-271 (1966a) Rohr, V.U. : Zum Feinbau des Subfornikalorgans der Katze. II. Neurosekretorische Aktivit/it. Z. Zellforsch. 75, 11-34 (1966b) Rudert, H.: Das Subfornikalorgan und seine Beziehungen zu dem neurosekretorischen System im Zwischenhirn des Frosches. Z. Zellforsch. 65, 790-804 (1965) Rudert, H., Schwink, A., Wetzstein, R.: Die Feinstruktur des Subfornikalorgans beim Kaninchen. I. Die Blutgef,iBe. Z. Zellforsch. 74, 252-270 (1966) Rudert, H., Schwink, A., Wetzstein, R.: Die Feinstruktur des Subfornikalorgans beim Kaninchen. II. Das neuronale und gliale Gewebe. Z. Zellforsch. 88, 145-179 (1968) Sarrat, R.: Enzymhistochemische Untersuchungen am Subfornikalorgan der Ratte. Experientia (Basel) 24, 1239-1240 (1968) Schinko, I., Rohrschneider, I., Wetzstein, R.: Elektronenmikroskopische Untersuchungen am Subfornikalorgan der Maus. Z. Zellforsch. 123, 277-294 (1972) Simpson, J.B., Routtenberg, A.: Subfornical organ: Site of drinking elicitation by angiotensin II. Science 181, 1172-1174 (1973) Simpson, J.B., Routtenberg, A.: Subfornical organ: Acetylcholine application elicits drinking. Brain Res. 79, 157-164 (1974) Simpson, J.B., Routtenberg, A.: Subfornical organ lesions reduce intravenous angiotensin-induced drinking. Brain Res. 88, 154-161 (1975) Stumpf, W.E.: Estrogen-neurons and estrogen-neuron systems in the periventricular brain. Amer. J. Anat. 129, 207-218 (1970) Takei, Y.: The role of the subfornical organ in drinking induced by angiotensin in the Japanese quail, Coturnix coturnix japonica. Cell Tiss. Res. 185, 175-181 (1977) Takei, Y., Tsuneki, K., Kobayashi, H.: Surface fine structure of the subfornical organ of the Japanese quail, Coturnix coturnixjaponica. Cell Tiss. Res. (in press, 1978) Takei, Y., Kobayashi, H., Yanagisawa, M., Bando, T.: Involvement of monoaminergic fibers in angiotensin-induced drinking in the Japanese quail, Coturnix coturnix japonica. (In preparation, 1979) Wetzig, H., Palkovits, M.: Die Entwicklung des Organon subfornicale bei der Sturmmrve Larus canus L. Z. mikr.-anat. Forsch. 79, 283-291 (1968) Accepted May 9, 1978
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Parenchymal Fine Structure of the Subfornical Organ in the Japanese Quail, Coturnix coturnix japonica Kazuhiko Tsuneki, Yoshio Takei, and Hideshi Kobayashi Misaki Marine BiologicalStation, Universityof Tokyo, Misaki, Kanagawa-ken, Japan
Summary. The parenchyma of the subfornical organ (SFO) of the Japanese quail was studied by light and electron microscopy. The SFO consists of ependymal, intermediate, and basal (perimeningeal) layers. In the intermediate layer, neurons, glial cells, and their processes are found. Axons containing dense core granules approximately 80 nm in diameter are numerous, some of which make synaptic contact with the neuronal perikarya or dendrites. Synaptic vesicles in some axons contain a dense dot in the interior after treatment with 5-hydroxydopamine. The activity of the SFO, which is probably concerned with elicitation of drinking by angiotensin II, may be regulated at least partly by afferent monoaminergic axons. Capillaries with a non-fenestrated endothelium are occasionally found in the parenchyma. The basal layer is occupied by glial processes abutting on the digitating layer of perivascular connective tissue of meningeal vessels. The endothelium of these vessels is occasionally fenestrated. Trypan blue injected systemically accumulated in the SFO, but not in the deeper areas of the brain. The absence of a blood-brain barrier is suggested in the SFO. Key words: Subfornical organ - J a p a n e s e quail, Coturnix coturnix j a p o n i c a Monoaminergic innervation - Electron microscopy.
Introduction The subfornical organ (SFO), consisting of parenchyma and specialized ependyma, is located on the rostral wall of the third ventricle just below the interventricular foramen (Akert, 1969). Several functions of the S FO have been proposed relating to gonadal activity (rat, Stumpf, 1970; mouse, George and Penrose, 1975), osmorecepSend offprint requests to: YoshioTakei, Misaki MarineBiologicalStation, Universityof Tokyo, Misaki,
Kanagawa-ken, 238-02Japan Acknowledgements. The authors wouldlike to expresstheirgratitudeto Dr. Ebert A. Ashbyfor reading the manuscript. This work was supported by grants from the Ministry of Educationof Japan and from the Ford Foundation
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tion (frog, Dierickx, 1963; rat, Sarrat, 1968; mouse, George, 1973, 1974), and reception o f cholinomimetics for induction o f drinking (rat, Simpson and Routtenberg, 1974). Recently, the SFO has been shown to play an essential role in drinking induced by angiotensin II (AII) in the rat (Simpson and Routtenberg, 1973, 1975; Abdelaal et al., 1974) and the Japanese quail (Takei, 1977), based on the following observations: (1) Direct application o f A l l to the SFO induces copious shortlatency drinking, (2) electrolytic lesioning o f the SFO reduces the a m o u n t o f water intake induced by intravenous A I I , and (3) SFO lesioning attenuates water intake induced by A l l injected into the preoptic area (POA), another area showing high sensitivity to All. Furthermore, in the Japanese quail, an afferent monoaminergic p a t h w a y connects the preoptic area (POA) with the SFO (Takei et al., 1979). These nerve fibers p r o b a b l y transfer the stimulus generated by A I I at the P O A to the SFO, since transection o f these fibers eliminates the dipsogenic action o f preoptically applied A l l (Takei et al., 1979). F o r these reasons, it is o f particular interest to investigate the ultrastructure of the SFO in order to determine the character o f nerve cells, afferent synaptic terminals and specialized perivascular structures. Ultrastructural studies o f the SFO o f several m a m m a l i a n species have previously been reported (Andres, 1965; Rohr, 1966a, b; Rudert et al., 1966, 1968; Pfenninger et al., 1967; Akert et al., 1967; Dempsey, 1968; Schinko et al., 1972; L e o n h a r d t and Lindemann, 1973; Dellmann and Simpson, 1975, 1976). In avian species, however, morphological studies o f the SFO are few, even at the light microscopical level (Pines and Scheftel, 1929; Reichold, 1942; Wetzig and Palkovits, 1968; Takei, 1977). To the knowledge o f the authors no report has so far appeared at the ultrastructural level. The present study was undertaken to assess the functionally important structures o f the quail SFO as well as to elucidate general organization o f the organ. The surface ultrastructure o f the quail SFO was described in a previous paper (Takei et al., 1978).
Materials and Methods Male Japanese quail, Coturnix coturnix japonica Temminck et Schlegel, were obtained from a commercial source at the age of 28 days. They were kept individually in small wire cages under a short day light cycle(8L/16D). The birds were used at the age of more than 35 days for the following purposes: (1) For general light microscopical observation, the SFO of several birds were fixed in Bouin fluid. Serial paraffin sections were cut at 10 ~tmthickness and stained with either paraldehyde fuchsin or hematoxylin and eosin. (2) For study of the vascularization, several birds were perfused intracardially with India ink. The brains of the perfused birds were fixed in Bouin fluid and embedded in paraffin. Serial sections 25 ~tmthick were observed without counterstaining. (3) In order to clarify whether a blood-brain barrier is present in the SFO, daily injections of 0.7 ~o trypan blue in saline were made intravenously (i.v.) or intraperitoneally (i.p.) in a volume of 0.5 ml (i.v.) or 1 ml (i.p.) for one week in several birds. The brains of these birds were f'Lxedin Bouin fluid and serial paraffin sections of 25 ~tm thickness were examined without staining. (4) For electron microscopy, the SFO of eight birds were fixed by either immersion or systemicperfusion with modified Karnovsky fixative. The details of fixation and further procedures for electron microscopy were given in a previous paper (Takei et al., 1978). (5) Two birds were infused intracerebro-ventricularly with 5-hydroxydopamine (5-OHDA) dissolved in saline containing 0.1 ascorbic acid. Doses of 5-OHDA were 0.5 mg and 2 mg/100 g BW, respectively. Infusion continued for 30 min at a rate of 1.33 ~tl/min. The birds were decapitated 30 min after the termination of the infusion. The SFO of these birds were fixed by immersion in modified Karnovsky fixative and further processed for electron microscopy.
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Fig. 1. Transverse section through the body of the SFO in the Japanese quail. Dotted line represents the delineation of the organ. Note densely arranged ependymal cells. Cell bodies form loose clusters on both sides offissura longitudinalis cerebri (tic). The region facing the fissure is not occupied by cell bodies, but rather by membrana limitans gliae superficialis. The trapezoid shows a region in which most of the electron micrographs were taken. BL Basal layer, cp choroid plexus, E L ependymal layer, 1L intermediate layer, s sinus-like vessel, I I I third ventricle. Hematoxylin and eosin, x 260 Fig. 2. Sagittal section through the SFO of the quail injected with India ink intracardially. Rostroventral to the left. The SFO is limited by dotted line. In the center of the SFO, meningeal capillary net and large sinus partially filled with India ink are seen. The vascularization of the SFO parenchyma is rather poor. The meningeal vessels are also seen in the upper left corner, cp Choroid plexus, pc pallial commissure, I I I third ventricle. • 100 Fig. 3. Sagittal section through the SFO of the quail injected with trypan blue. Rostroventral to the left. The SFO is limited by dotted line. Note deposition of grains of trypan blue in the SFO. Such grains are not observed in the deeper region of the brain, cp Choroid plexus, pc pallial commissure, 1II third ventricle, x 100
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Results
Light Microscopical Observations The SFO of the Japanese quail consists of a body and a stalk. The body occupies the rostroventral portion and the stalk occupies the caudodorsal portion of the organ (for details, see Takei et al., 1978). In transverse section, the body of the organ is semicircular in outline (Fig. 1). The ventral region projects slightly into the ventricle. The dorsal region is continuous with the brain parenchyma and divided into two halves by thefissura longitudinalis cerebri. The dorsal and lateral boundary of the organ is indistinct. The parenchyma approximately 2 mm in depth from the ventricular surface represents roughly the limits of the organ (Fig. 1). Within this region, neurons constitute a loosely clustered arrangement (nucleus). Capillaries are occasionally seen in the parenchyma. Meningeal blood vessels are located on the wall of the fissure. The vessel located most ventrally in the fissure is sinus-like in appearance and proceeds longitudinally along the SFO including the stalk (Fig. 1). Some vessels on the wall of the fissure are also seen in Figure 2. The SFO of the birds injected daily with trypan blue was characterized by the deposition of blue granules (Fig. 3). They were distributed in the parenchymal tissue near blood vessels. The blue granules were also observed in the median eminence, the organum vasculosum of the lamina terminalis (OVLT) and the choroid plexus. No granules were found in the other brain areas including the paraventricular organ and the subcommissural organ. The pineal organ, neural lobe, and area postrema were not studied.
Electron Microscopical Observations The SFO of the Japanese quail can be roughly divided into three layers: ependymal, intermediate, and basal (perimeningeal) layers (Figs. 1, 4). Although the whole area of the SFO was studied, the region indicated by a trapezoid in Figure 1 was given particular attention.
Ependymal Layer. The fine structure of this layer was described in a previous paper (Takei et al., 1978). The nuclei of ependymal cells are arranged at different levels, possibly indicating a pseudostratified epithelium (Fig. 4). The cells located relatively distant from the ventricle may be classified as subependymal cells. Neurons are occasionally present in the subependymal region. In the stalk of the SFO, only one layer of ependymal cells is found. Intermediate and basal layers are absent. These ependymal cells directly contact the perivascular space of the underlying vessel.
Intermediate Layer. This layer is occupied by neurons, dendrites, axons, glial cell bodies, glial processes, ependymal processes, and capillaries (Figs. 4-13). The neurons are usually scattered. In some instances, however, two neurons are in direct contact (Fig. 6) or are separated only by a very thin glial process. The nucleus of the neurons is round or elliptical and large, containing a small amount of dense
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Fig. 4. Transverse section through a portion of the body of the SFO. The third ventricle and ependymal cells of the adjacent ventricular wall are seen in the upper left corner. BL Basal layer, ct connective tissue, EL ependymal layer, gl glial cell, IL intermediate layer, n neuron, t process of tanycyte, x 4400
chromatin (Figs. 5, 6). The nucleolus is usually prominent. Two types o f neurons m a y be present; the first type shows a small cytoplasmic area (Fig. 6), the other a more expansive cytoplasm (Figs. 4, 5). The cytoplasm contains mitochondria, neurotubules, lysosomes, irregularly shaped dense bodies, Golgi apparatus, tubular profiles o f rough surfaced endoplasmic reticulum, and polysomes. The last three organelles are especially well developed. Electron dense granules are only
Fig. 5. Part of Figure 4 (intermediate layer) seen at higher magnification. Note intermingled neuronal and glial processes in the center. Arrow points out the junctional complex between glial cells which contain m a n y microfilaments, gl Glial cells, n neuron, sr subependymal cell. x 11,100 Fig. 6. Two neurons with prominent nucleoli and scanty cytoplasm are in direct contact. Arrow indicates axo-dendritic synapse, x 11,100. The inset shows small axons with neurotubules and the perikaryon containing one granule (arrow). x 22,200
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Fig. 7. Three axon terminals making synaptic contacts(arrows) on a perikaryon. Two terminals contain both dense core granules and synaptic vesicles, one terminal has only synaptic vesicles. • 22,200 Fig. 8. Axons in the SFO of a quail treated with 2 mg 5-OHDA. Dense dots are seen in synaptic vesiclesin axons shown in the upper left and upper right corners. The dense granules in the axon shown in the upper right corner are approximately 120 nm in diameter; the granules in the other axons are about 80 nm in diameter. Arrows indicate axo-dendritic synapse. • 22,200
infrequently f o u n d in the perikarya o f some neurons (Fig. 6, inset). Their diameter measures 90 to 100nm. A l t h o u g h large neurons containing a b u n d a n t electron dense granules approximately 180 to 200 n m in diameter are occasionally found in the periphery o f the organ, they m a y represent peripherally scattered neurons o f the secretory paraventricular nucleus. Corresponding to this observation, the light microscopical preparations stained with paraldehyde fuchsin revealed several neurosecretory cells o f the paraventricular nucleus located a r o u n d the ventrolateral border o f the SFO. Neither fibers n o r cell bodies staining with paraldehyde fuchsin were f o u n d except for occasional, possibly specialized glial cells (microglia ?) in the vicinity o f meningeal vessels. Dendrites and axons are abundant. Small axons contain only neurotubules (Fig. 6). Large axons frequently have dense core granules approximately 70 to 90 nm in diameter in addition to synaptic vesicles and m i t o c h o n d r i a (Fig. 7). The axons containing larger dense core granules (110 to 120 n m in diameter) are rarely found. Some axons contain only synaptic vesicles. N o myelinated axons are observed. A x o n s containing either dense' core granules (approximately 80 n m in diameter) and synaptic vesicles, or only synaptic vesicles, frequently m a k e synaptic contact with dendrites (Figs. 6, 10) or neuronal perikarya (Fig. 7). As m a n y as five synapses can be seen on one neuronal perikaryon. These axo-dendritic and axosomatic synapses show typical synaptic characters such as the aggregation o f
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Fig. 9. Parenchymaof the SFO of the same bird as shownin Figure 8. Dots are seenin synapticvesicles of some axons (al), but not in others (a2). One "positive"axon is in synaptic contact (arrows) with a neuronal perikaryon, x 20,800
synaptic vesicles on the presynaptic membrane and thickening of both pre- and postsynaptic membranes. In the preparations treated with 2 mg 5-OHDA, synaptic vesicles in some axons with dense core granules (approximately 80 nm in diameter) contained a dense dot in the interior (Figs. 8, 9). This structure was found in every vesicle in some axons, but only in a small proportion of vesicles in others. Axons with dotted synaptic vesicles also made synaptic contacts with either dendrites or perikarya (Fig. 9). Axons containing dense core granules with a diameter of 120 nm possessed synaptic vesicles with a dense dot (Fig. 8). No synapses were formed in connection with these axons, which can perhaps be accounted for by the fact that possibly occurring synaptic contacts were overlooked due to the rarity of the axons. Axons in the SFO of the quail injected with 0.5 mg 5-OHDA showed fewer dotted synaptic vesicles. Glial cells are dispersed in the parenchyma. Their shape is variable; round, elliptical or even irregular (Figs. 5, 10, 11). Dense chromatin is usually more abundant in the nucleus of glial cells than neurons (Fig. 11). Small, dense bodies possibly identified as primary lysosomes are occasionally found in the cytoplasm of some glial cells (Figs. 10, 11). The density of the cytoplasm is extremely variable among glial cells. In some cases, two adjacent glial cells are joined by a long junctional complex (Fig. 5). These glial cells contain abundant microfilaments. Slender straight processes filled with microfilaments course the subependymal layer to the basal region of the parenchyma (Fig. 4). These are probably tanycytes. A single cilium was found in the parenchymal cells in several instances.
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Fig. 10. Glial cell (probably astrocyte) in the intermediate layer. Arrows point out axo-dendritic synapses, x 11,100 Fig. 11. Glial cell (probably microglia)in the intermediatelayer, x 11,100
Vacuoles of varying sizes are occasionally observed in the parenchymal cells. They are usually irregular in outline (Fig. 12). The large vacuoles exceed the average size of a cell. They are rimmed by a very thin cytoplasmic covering. Flocculent material is occasionally seen in the lumen. The perivascular space of parenchymal capillaries is thin (Fig. 13). Endothelial cells are not fenestrated. Pericytes are observed in the vicinity of some capillaries.
Basal (Perirneningeal) Layer. The basal glial processes abutting on the extraparenchymal connective tissue in a palisade fashion are usually voluminous and electron lucent (Fig. 14). They contain round or tubular vesicles of varying densities, relatively large vacuoles, and occasionally very large mitochondria filled with lamellae (Fig. 15, inset). Microfilaments are infrequently found in the processes. The slender dense processes and glial cell bodies themselves also sometimes contact the connective tissue (Fig. 15). The branches of the connective tissue sheet penetrate deep into the basal layer of the parenchyma (Fig. 14). In some sections, the branches of the connective tissue sheet are isolated apparently from the perivascular region. The extent of branching decreases toward the dorsal (distal to the ventricle) region of the SFO. The perivascular space of specimens fixed by immersion is relatively homogeneously dense, but the material fixed by systemic perfusion tends to show a basal lamina and many collagen fibrils more clearly (compare Fig. 14 with Fig. 15).
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Fig. 12. Intracytoplasmic vacuole surrounded by a very thin rim of cytoplasm; gl glial cell. x 17,800 Fig. 13. Capillary with non-fenestrated endothelium (en) in the intermediate layer. The lumen is occupied by an erythrocyte (er). Perivascular connective tissue surrounding the endothelium and pericyte (p) is thin. x 14,800
Fig. 14. Basal layer consisting of light glial processes (probably processes of astrocytes) abutting on the digitation of the perivascular connective tissue (ct). en Endothelium, er erythrocyte, f fibroblast. x 11,100. The inset shows fenestrated endothelium (arrow) and a fibroblast (f) in the perivascular space. • 27,900 Fig. 15. Basal layer of material fixed by vascular perfusion. The basal lamina and collagen fibrils in the perivascular space (pvs) are clearly seen. In this section, glial cells (gO directly contact the perivascular space as dark ( dp) and light processes ( lp). x 17,800. The inset shows light glial processes containing large mitochondria; pvs perivascular space, x 16,400
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The meninx covering the parenchyma consists of blood vessels, of which the most ventral one is sinus-like, collagen fibrils, ground substance of the connective tissue, and accompanying cells (Fig. 14). The pattern of intermingling of these elements is occasionally very complicated. The main cellular components appear to be fibroblasts. Plasma cells are also occasionally found. In some specimens, only perivascular connective tissue (collagen fibrils+ ground substance) separates the parenchyma from endothelial cells. Since the wall of vessels does not contain smooth muscle cells, the vessels may not be arterial, but rather venous or, more likely, capillary specializations draining this region of the brain. The thickness of the endothelial cells of the vessels is extremely variable. Fenestrations are occasionally found (Fig. 14, inset).
Discussion
The present study on the SFO of the Japanese quail shows that this organ has many characteristics in common with circumventricular organs, such as a thickened ependymal layer, rich vascular supply and the presence of tanycytes. However, the paucity of apparent neurosecretory elements is in contrast to the abundance of these elements in other circumventricular organs of the quail such as the median eminence (Ishii et al., 1975) and the OVLT (Mikami, 1976). The SFO of the quail differs from mammalian SFO in that its delineation as an organ is indistinct and the basal layer is contiguous with the meninx. This difference may be due to the absence of the fornix in birds. However, the similarity between the quail and mammalian SFO at the ultrastructural level is more striking. Scattered neurons and glial cells, neuronal processes with or without dense core granules, large vacuoles developed in cells, digitations of perivascular connective tissue, long junctional complexes between adjacent glial cells, and axo-dendritic or axo-somatic synapses have also been described in mammalian SFO (Andres, 1965; Rohr, 1966a, b; Rudert et al., 1966,1968; Pfenninger et al., 1967; Akert et al., 1967; Dempsey, 1968; Schinko et al., 1972; Leonhardt and Lindemann, 1973; Dellmann and Simpson, 1975). The quail SFO possesses a small number of poorly granulated perikarya, which may represent a transitional form between SFO apparently lacking a granulated perikaryon (dog, Andres, 1965; cat, Rohr, 1966 b; Pfenninger et al., 1967; rabbit, Rudert et al., 1968; mouse, Schinko et al., 1972) and SFO with perikarya containing some granules (approximately 120 nm) (rat and other mammals, Dellmann and Simpson, 1975). The presence of dense core granules in perikarya may imply secretory activity of neurons, but its significance is unclear in the SFO. The axons with neurosecretory granules larger than 150 nm (peptidergic neurosecretory axons ?) were absent in the present material. This type of axon is rare in the rat et al. (Dellmann and Simpson, 1975), and seems to be absent in the dog (Andres, 1965), and mouse (Schinko et al., 1972). Although Rohr (1966 b) and Pfenninger et al. (1967) reported neurosecretory axons in the cat SFO, their electron micrographs show rather irregular granules seemingly different from so-called elementary neurosecretory granules. The relation between SFO and the peptidergic neurosecretory nucleus was suggested in the frog (Rudert, 1965) and some other species (Legait and Legait, 1957, etc.), but such a relation is apparently negligible in the Japanese quail.
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In the quail SFO, non-fenestrated capillaries have been found in the parenchyma, whereas fenestrated capillary-like vessels were observed in the meninx. Non-fenestrated capillaries were found in the SFO of the dog (Andres, 1965) and rabbit (Rudert et al., 1966). In contrast, fenestrated capillaries were found in the cat (Rohr, 1966a) and mouse (Schinko et al., 1972). Consequently, both types of capillaries seem to be present in mammalian and avian SFO (see also Dellmann and Simpson, 1975). It has become increasingly clear that the SFO of the rat and the Japanese quail contain dipsogenic receptors for AII, since AII applied directly to the SFO induces immediate copious drinking (Simpson and Routtenberg, 1973; Kucharczyk et al., 1976; Takei, 1977). The most probable location of these receptors is the nerve cells in the SFO. Two types of nerve cells are distinguished in the quail SFO according to the relative abundance of cytoplasm. AII may act on the cells of one type or both types to induce drinking. However, nothing is yet known as to the efferent pathway from the SFO to the drinking center. Intravenously injected AII may gain access to nerve cells of the SFO, since the dipsogenic effect of AII is attenuated by prior lesioning of the SFO in the Japanese quail (Takei, 1977) as well as in the rat (Abdelaal et al., 1974; Simpson and Routtenberg, 1975). The occasional fenestrated endothelium in the meningeal vessels may represent a possible pathway for rapid exchange of materials between these vessels and the parenchyma. The well developed digitations of the perivascular connective tissue may increase the area of contact between the vascular system and the parenchyma. Furthermore, deposition of intravenously injected trypan blue in the SFO shows the absence of a blood-brain barrier, similar to the situation in the mammalian SFO and other circumventricular organs (see Koella and Sutin, 1967). These results support the concept that AII reaches parenchymal nerve cells of the SFO. In addition, some of glial processes abutting on the digitations of perivascular connective tissue contain extremely large mitochondria, which are sites of energy production for active transport of material. The intracerebro-ventricularly injected AII may act on neurons of the SFO through the ependymal layer. In the Japanese quail, neuronal perikarya are sometimes located in the subependymal region. These perikarya may be easily affected by AII penetrating from the ventricle. The functional significance of ependymal specialization has already been discussed in a previous paper (Takei et al., 1978). In the stalk region of the SFO, no neuronal perikarya are present and ependymal cells directly contact the perivascular space. This organization is similar to that of the choroid plexus. However, nothing definite is known on the function of the stalk. The present study revealed many axon terminals containing both dense core granules (approximately 80 nm in diameter) and synaptic vesicles, or synaptic vesicles only. Synaptic vesicles in some axon terminals containing dense core granules contained a dense dot in their interior after 5-OHDA treatment. Therefore, these axon terminals are considered monoaminergic (see Richards and Tranzer, 1970). The presence of monoaminergic terminals in the quail SFO is further supported by the histochemical finding that terminal-like varicosities showing monoamine-specific fluorescence are present (Takei et al., 1979). Since no fluorescent cell bodies are found in the SFO of the quail, the origin
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o f these m o n o a m i n e r g i c terminals must be outside the SFO. The P O A is one o f the possible sources o f afferent m o n o a m i n e r g i c fibers, since fluorescent perikarya are observed in the P O A (Takei, unpublished), and fluorescent fibers can be traced from the P O A to the SFO (Takei et al., 1979). These fluorescent fibers m a y transport dipsogenic information f r o m the P O A to the SFO, since transection o f these fibers eliminates drinking induced by preoptically applied A I I (Takei et al., 1979), and direct application o f noradrenalin or serotonin to the SFO induces drinking in the Japanese quail (Takei, unpublished). Some m o n o a m i n e r g i c axon terminals observed in the present study make synaptic contact with dendrites and perikarya, which is considered as ultrastructural evidence for m o n o a m i n e participation in the dipsogenic information transport to the SFO. The significance o f rare axons containing dense core granules with a diameter o f 1 2 0 n m is u n k n o w n . They also seem to be monoaminergic, since their synaptic vesicles showed a dense dot after 5 - O H D A treatment. M a n y axons did not show a dense dot in their synaptic vesicles after 5 - O H D A treatment, irrespective o f the presence of dense core granules. Some o f these axons might originate from perikarya within the SFO. It is also highly probable that afferent axons containing neurotransmitters other than m o n o a m i n e s are involved in the activity o f the SFO. Some functions o f the SFO in relation to gonadal activity, osmoreception and dipsogenic reception o f cholinergic drugs have been suggested in mammals. It has yet to be clarified whether these functions are present in the qail SFO and whether they are mediated t h r o u g h cholinergic or other mechanisms.
References
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