521
J. Anat. (1979), 129, 3, pp. 521-530 With 10 figures Printed in Great Britain
A scanning electron microscope study of the pineal recess of the adult brush-tailed possum (Trichosurus vulpecula)* R. S. TULSI
Department of Anatomy, University of Adelaide, Adelaide 5001, South Australia
(Accepted 11 October 1978) INTRODUCTION
In the past decade, correlative studies, and especially those which include the use of the scanning electron microscope, have revealed much new information about the lining of the brain ventricles, particularly as regards the distribution of cilia, microvilli, intraventricular nerve processes and the heterogeneous population of supraependymal cells. Some investigators, including the present author, Samarasinghe & Delahunt (in New Zealand, personal communication), Ribas (1977) and Krapp (1978), are paying particular attention to the ventricular surface features of the pineal recess and the immediately adjacent regions. The pineal lies close to the ventricular cavity (in the possum at least) and in addition to secreting melatonin it contains serotonin, acetylcholine, noradrenaline and histamine. While it is widely believed that melatonin is released from the pineal into the bloodstream, direct release into the CSF has been suggested (Reiter, Lukaszyk, Vaughan & Blask, 1976), with the possibility of transport across the third ventricle to influence the hypothalamo-hypophysial system. As yet there is no positive evidence that melatonin is secreted directly into the CSF nor do we have information on the fine surface features of the pineal recess where such secretion might take place. A preliminary observation which stimulated the present study was the finding that in the brush-tailed possum Trichosurus vulpecula the pineal is a saccular pouch without a stalk and therefore is in direct contact with the CSF as in fishes and birds. In contrast, the pineal of eutherian mammals is often separated from the third ventricle by a stalk of some length. The major aim of the present investigation was to study the fine structural features of the pineal recess and to attempt to relate these to the movement of cerebrospinal fluid locally. MATERIALS AND METHODS
A total of 35 adult animals (14 male and 21 female) were used. They were caught in the vicinity of Adelaide throughout the year and used within 3 or 4 days of capture. While in captivity they were in enclosures in the open, and were not exposed to artificial light. The animals were anaesthetized with chloroform and perfused through the ascending aorta with 3 % glutaraldehyde (TAAB) and 2 % formaldehyde in cacodylate buffer (0-05 M, pH 7T2, 395 mOsm). This fixative was allowed to run for 20 minutes and then the cranial cavity was opened, incisions made in the meninges, * A preliminary report of this study was presented at the Fourteenth Annual Conference of the Anatomical Society of Australia and New Zealand held at the University of Queensland in May 1976.
0021-8782179/2828-6780 $02.00 ©1979 Anat. Soc. G.B. & I.
522
R.S. TULSI
overnight°C
and the entire skull with the brain in situ left at 4 in the same fixative. A block of tissue containing the region of the pineal recess was then carefully dissected out using fine instruments and a dissecting microscope. After further fixation for two hours the block was washed in three 10 minute changes of distilled water. After post-fixation in 2 % osmium tetroxide, and dehydration, the specimen was subjected to critical point drying (Denton DCP 1) using amyl acetate as the intermediate liquid and carbon dioxide as the transitional liquid. The specimen was immediately put on an aluminium stub and coated (Denton D 502) with 60/40 gold/palladium to a thickness of 15 to 20 nm. A Siemens Autoscan was used for kV. viewing the block, usually at 20 RESULTS
The pineal recess, as viewed with the SEM, shows three zones which are sufficiently consistent and clearly defined to warrant calling them central, paracentral and peripheral (C, P, Per in Figs. 1 and 2). Variability is greatest in the central zone but this does not seem to be associated with sex, age or season. In most specimens the central zone occupies one third to one half of the pineal recess but occasionally it is much larger (C in Fig. 1) while in other specimens it is very small or fragmented into several small colonies of cells. The central zone projects into the pineal recess, and when well developed it may be seen with the unaided eye. The main cells of the central zone are non-ciliated, non-microvillous, morphologically heterogeneous, and often project slightly into the ventricular cavity (C in Fig. 2). A close inspection of the surface of the central zone (at higher magnification than Fig. 2) reveals one of the following three appearances. First, the cells may be free of any flocculent covering with clefts or deep spaces between adjacent cells. These clefts open into a labyrinth of interconnecting spaces lying deeply between the cells of the central zone, an observation confirmed by transmission electron microscopy. Second, the cells over wide areas may be covered by a layer of material which obscures surface details and cell boundaries. In the third type of appearance the cells of the central zone are overlaid with small highly branched supraependymal cells. The processes of these latter cells vary greatly in appearance from short tapering projections to veil-like protrusions. The paracentral zone is chiefly characterised by a heterogeneous population of supraependymal cells lying on ependymal cells covered with microvilli (P in Figs. and H in Figs. 5 and 6). However, as one moves away towards the 1 and 2, Mzone ciliated cells make their appearance (Figs. 7-10). The variability of peripheral the supraependymal cells does not appear to be related to sex, age, or season. The lowest number of supraependymal cells counted on the block face was 14, but they could be so numerous and interwoven that counting was impossible. The most interesting cell type looks remarkably like a neuron (S in Figs. 3-5) and has several processes which form a 'network' with adjacent cell processes. Additionally, and an outstanding feature, is that the longer processes extend over several hundred microns, either reaching and disappearing into the peripheral ciliated zone, or else diving deeply between the ventricular lining cells, disappearing by passing deeply to the cells of the central zone, or occasionally ending in a bulbous terminal (N in Fig. 5). A more exhaustive study using a wide variety of methods is currently in progress to determine the details of the density and ramifications of these intraventricular neuron-like cells. are much less common than the The largest supraependymal cells
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J. Anat. (1979), 129, 3, pp. 521-530 With 10 figures Printed in Great Britain
A scanning electron microscope study of the pineal recess of the adult brush-tailed possum (Trichosurus vulpecula)* R. S. TULSI
Department of Anatomy, University of Adelaide, Adelaide 5001, South Australia
(Accepted 11 October 1978) INTRODUCTION
In the past decade, correlative studies, and especially those which include the use of the scanning electron microscope, have revealed much new information about the lining of the brain ventricles, particularly as regards the distribution of cilia, microvilli, intraventricular nerve processes and the heterogeneous population of supraependymal cells. Some investigators, including the present author, Samarasinghe & Delahunt (in New Zealand, personal communication), Ribas (1977) and Krapp (1978), are paying particular attention to the ventricular surface features of the pineal recess and the immediately adjacent regions. The pineal lies close to the ventricular cavity (in the possum at least) and in addition to secreting melatonin it contains serotonin, acetylcholine, noradrenaline and histamine. While it is widely believed that melatonin is released from the pineal into the bloodstream, direct release into the CSF has been suggested (Reiter, Lukaszyk, Vaughan & Blask, 1976), with the possibility of transport across the third ventricle to influence the hypothalamo-hypophysial system. As yet there is no positive evidence that melatonin is secreted directly into the CSF nor do we have information on the fine surface features of the pineal recess where such secretion might take place. A preliminary observation which stimulated the present study was the finding that in the brush-tailed possum Trichosurus vulpecula the pineal is a saccular pouch without a stalk and therefore is in direct contact with the CSF as in fishes and birds. In contrast, the pineal of eutherian mammals is often separated from the third ventricle by a stalk of some length. The major aim of the present investigation was to study the fine structural features of the pineal recess and to attempt to relate these to the movement of cerebrospinal fluid locally. MATERIALS AND METHODS
A total of 35 adult animals (14 male and 21 female) were used. They were caught in the vicinity of Adelaide throughout the year and used within 3 or 4 days of capture. While in captivity they were in enclosures in the open, and were not exposed to artificial light. The animals were anaesthetized with chloroform and perfused through the ascending aorta with 3 % glutaraldehyde (TAAB) and 2 % formaldehyde in cacodylate buffer (0-05 M, pH 7T2, 395 mOsm). This fixative was allowed to run for 20 minutes and then the cranial cavity was opened, incisions made in the meninges, * A preliminary report of this study was presented at the Fourteenth Annual Conference of the Anatomical Society of Australia and New Zealand held at the University of Queensland in May 1976.
0021-8782179/2828-6780 $02.00 ©1979 Anat. Soc. G.B. & I.
522
R.S. TULSI
overnight°C
and the entire skull with the brain in situ left at 4 in the same fixative. A block of tissue containing the region of the pineal recess was then carefully dissected out using fine instruments and a dissecting microscope. After further fixation for two hours the block was washed in three 10 minute changes of distilled water. After post-fixation in 2 % osmium tetroxide, and dehydration, the specimen was subjected to critical point drying (Denton DCP 1) using amyl acetate as the intermediate liquid and carbon dioxide as the transitional liquid. The specimen was immediately put on an aluminium stub and coated (Denton D 502) with 60/40 gold/palladium to a thickness of 15 to 20 nm. A Siemens Autoscan was used for kV. viewing the block, usually at 20 RESULTS
The pineal recess, as viewed with the SEM, shows three zones which are sufficiently consistent and clearly defined to warrant calling them central, paracentral and peripheral (C, P, Per in Figs. 1 and 2). Variability is greatest in the central zone but this does not seem to be associated with sex, age or season. In most specimens the central zone occupies one third to one half of the pineal recess but occasionally it is much larger (C in Fig. 1) while in other specimens it is very small or fragmented into several small colonies of cells. The central zone projects into the pineal recess, and when well developed it may be seen with the unaided eye. The main cells of the central zone are non-ciliated, non-microvillous, morphologically heterogeneous, and often project slightly into the ventricular cavity (C in Fig. 2). A close inspection of the surface of the central zone (at higher magnification than Fig. 2) reveals one of the following three appearances. First, the cells may be free of any flocculent covering with clefts or deep spaces between adjacent cells. These clefts open into a labyrinth of interconnecting spaces lying deeply between the cells of the central zone, an observation confirmed by transmission electron microscopy. Second, the cells over wide areas may be covered by a layer of material which obscures surface details and cell boundaries. In the third type of appearance the cells of the central zone are overlaid with small highly branched supraependymal cells. The processes of these latter cells vary greatly in appearance from short tapering projections to veil-like protrusions. The paracentral zone is chiefly characterised by a heterogeneous population of supraependymal cells lying on ependymal cells covered with microvilli (P in Figs. and H in Figs. 5 and 6). However, as one moves away towards the 1 and 2, Mzone ciliated cells make their appearance (Figs. 7-10). The variability of peripheral the supraependymal cells does not appear to be related to sex, age, or season. The lowest number of supraependymal cells counted on the block face was 14, but they could be so numerous and interwoven that counting was impossible. The most interesting cell type looks remarkably like a neuron (S in Figs. 3-5) and has several processes which form a 'network' with adjacent cell processes. Additionally, and an outstanding feature, is that the longer processes extend over several hundred microns, either reaching and disappearing into the peripheral ciliated zone, or else diving deeply between the ventricular lining cells, disappearing by passing deeply to the cells of the central zone, or occasionally ending in a bulbous terminal (N in Fig. 5). A more exhaustive study using a wide variety of methods is currently in progress to determine the details of the density and ramifications of these intraventricular neuron-like cells. are much less common than the The largest supraependymal cells
flocculent
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