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Seasonal variation in the secretory activity of the subcommissural organ (SCO) of reptiles
ELSEVIER
Neuroscience Letters 219 (1996) 9-12
HEUROSCIENC[ IETT[RS
Seasonal variation in the secretory activity of the subcommissural organ (SCO) of reptiles M a n u e l C i f u e n t e s , J u a n P d r e z , J e s f s M. G r o n d o n a , P e d r o F e r n 4 n d e z - L l e b r e z * Departamento de Biolog[a Animal, Facultad de Ciencias, Universidad de M(tlaga, 29071 M61aga, Spain Received 23 September 1996; revised version received 7 October 1996; accepted 7 October 1996
Abstract Seasonal variations in the secretory activity of the subcommissural organ (SCO) of snakes and turtles was studied by immunocytochemistry, lectins, and electron microscopy. In animals sacrificed in summer, immunoreactive material, mostly devoid of sialic acid, occupied the whole cytoplasm. Cells showed many distended cistemae of rough endoplasmic reticulum and secretory granules. In animals sacrificed in winter, patches of immunoreactive sialic acid-rich material occupied the apical cytoplasm. Cells lacked distended cistemae and the secretory granules formed clusters. These results suggest a decreased synthesis and release of secretory material in the SCO of lethargic reptiles. Keywords: Lethargy; Immunocytochemistry; Electron microscopy; Lectins; Natrix maura snake; Mauremys caspica turtle
The subcommissural organ (SCO) is an ependymal brain gland located in the roof of the third cerebral ventricle. It releases into the cerebrospinal fluid glycoproteins that polymerise and form the Reissner's fiber (RF), extending along the aqueduct, fourth ventricle and central canal of the spinal cord [12,16]. The secretory activity of the SCO undergoes seasonal variations in hibernating or lethargic species. The SCO of frogs sacrificed in winter (torpid state) contained more secretory material than in summer [11]. However, autoradiographic studies have shown a negative linear correlation between the amount of secretory material in the frog SCO and the growth rate of the RF [1]; thus, more material in the organ indicated less release. In the lizard, more secretory material was reported in summer than in winter [4]. Also in mammals, immunocytochemical studies revealed a drastic decrease of secretory material during hibernation [10]. Then, irrespective of the amount of secretory material present, it seems that the SCO is inhibited during lethargy. The aim of the present investigation was to investigate the seasonal variation in the secretory activity of the SCO
* Corresponding aulhor. Tel.: +34 5 2131858; fax: +34 5 2132000: e-mail: [email protected]
of reptiles by using methods that allow us to distinguish between mature and immature materials such as immunocytochemistry, lectins and electron microscopy. Snakes NatrLr maura and turtles Mauremys caspica were captured, under authorisation, in M41aga (Spain) in spring and kept in aquaria-terraria, at environmental temperature and photoperiod. Snakes were fed living frogs and turtles fresh meat ad libitum. In summer, snakes and turtles were active and regularly fed whereas in winter the animals, although having access to food, did not eat and entered in a torpid state. Six snakes and four turtles were sacrificed in late June when environmental temperature averaged 24°C. A similar number of specimens were sacrificed in mid January with temperature averaging 12°C. Handling, care and processing of the animals were carried out according to principles approved by the council of the American Physiological Society and national laws (B.O.E. 67, 1988, Spain). All animals were anaesthetised by intraperitoneal injection of tricaine (Sigma, St. Louis MO, USA) [6] and perfused transcardially with Bouin fluid, for light microscopy (snakes, 4 summer (s) + 4 winter(w); turtles, 4 s + 4 w) and Kamovsky's fixative [6], for electron microscopy (snakes, 2 s + 2 w). Dissected brains were put in fresh fixative for 48 h at room temperature (Bouin) or for 4 h
0304-394(I/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S 0 3 0 4 - 3 9 4 0 ( 9 6 ) I 3 I 61 -X
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M. Cifuentes et al. / Neuroscienee Letters 219 (1996) 9-12
M. Cifuentes et al. / Neuroscience Letters 219 (1996) 9-12
at 4°C (Karnovsky). For light microscopy, brains were embedded in paraffin (snakes, 2 s + 2 w; turtles, 2 s + 2 w) and butyl-methyl methacrylate (snakes, 2 s + 2 w; turtles, 2 s + 2 w) for thin and semithin sections, respectively. For electron microscopy, pieces containing the SCO (snakes, 2 s + 2 w) were postfixed in 1% osmium tetroxide for 2 h at 4°C, dehydrated and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate. A rabbit antiserum against the bovine Reissner's fiber (AFRU) [14] was used to selectively stain the secretory products of the subcommissural organ as described previously [6]. For lectin histochemistry, Concanavalin A (Con A) (affinity, mannose, glucose) and L i m a x f l a v u s agglutinin (LFA) (affinity, sialic acid) were used. Con A was labelled with horseradish peroxidase (HRP) and L F A was not labelled. Lectins were revealed as described previously [9]. Both in summer and winter, the SCO and R F of snakes and turtles showed immunoreactive material with a distinct pattern of distribution. In summer, secretory materials distributed evenly throughout the apical, perinuclear, and basal cytoplasm (Fig. 1(1,2)). In winter, the perinuclear and basal cytoplasm showed a weak immunoreactivity whereas the apical cytoplasm showed an uneven staining with prominent patches of strongly immunoreactive materials on a weakly immunoreactive background (Fig. 1(4,5)). Staining pattern of Con A was similar to that obtained with AFRU. However staining with L F A was quite different. In summer, L F A revealed scarce granules in the apical cytoplasm and elongated profiles near the nuclear zone. The glycocalix of the ependymal cells and the R F were strongly stained (Fig. 1(3)). In contrast, in winter, strongly stained patches of different sizes distributed through the perinuclear and apical cytoplasm, excepting the cell apexes, (Fig. 1(6)). The ultrastructure of the SCO of summer snakes have been described elsewhere [5]. Briefly, in the apical cytoplasm, granules of different shapes, sizes and electrondensity and cisternae of endoplasmic reticulum were the most prominent features (Fig. 1(7)). The perinuclear and basal cytoplasms were occupied by typical highly distended cisternae of rough endoplasmic reticulum RER filled with pale material (Fig. 1(9)). The SCO of winter
11
snakes showed an apical band (1 /~m thick) devoid of organules. Under this band, the apical cytoplasm was rich in clusters of granules of different sizes, shapes and electron density (Fig. 1(8)), The perinuclear and basal cytoplasm showed flat parallel cisternae of RER, Golgi complexes, granules, mitochondria, and glycogen particles but, in sharp contrast to the animals sacrificed in summer, no distended cisternae of RER were seen (Fig. 1(10)). The amount of secretory material in the SCO is the result of a balance between the rates of biosynthesis and release. More material could reflect either an increased synthesis or a decreased release; and vice-versa, less material could reflect either a decreased synthesis or an increased release. The SCO is a gland that synthesises and releases very large glycoproteins (molecular weight 2 0 0 - 6 0 0 kDa) [8,9]. The precursor forms are produced in typical distended cisternae of RER in the perinuclear cytoplasm. A part of the precursor glycoproteins is processed in the Golgi apparatus and released into the ventricle within 1 h after synthesis [15]. A second fraction is retained in the dilated RER cisternae and slowly processed and released over a period of 3 - 5 days [2,16]. In mammals, the bulk of secretion of the SCO is stored in the RER and only few mature secretory granules can be visualised in the apices of the secretory cells [ 16]. In non-mammalian vertebrates, both RER and the secretory granules may represent important storage sites [7,13]. In the present work, we used immunocytochemistry to identify secretory material of the SCO. The antiserum employed, A F R U , selectively recognises the secretory material in the SCO and the R F of all vertebrate species investigated [14] including the reptilian species used in the present investigation [6]. Using A F R U we were then able to reveal all forms of secretory material (precursor and mature) in the SCO of summer and winter specimens. In addition, we have used the lectin L F A , that selectively binds to sialic acid, to identify mature (post-Golgi) SCO secretory material containing sialic acid. Thus, by the combined use of A F R U and L F A in adjacent sections we had an idea of the amount of immature and mature secretory materials inside the SCO in a given situation. On the other hand, electron microscopy led the identification of the organules involved in the synthesis (RER) and those containing mature secretory materials (secretory granules).
Fig. 1. (1-6). Transverse sections through the subcommissural organ of snakes (1,4) and turtles (2,3,5,6) sacrificed in summer (1-3) and winter (4-6) immunostained with an antiserum against the bovine Reissner's fiber (1,2,4,5) (semithin methacrylate sections) and the lectin LFA (3,6) (paraffin sections). Summer animals show immunoreactive secretory materials homogeneously distributed throughout apical (a), perinuclear (p) and basal (b) cytoplasms whereas winter animals show patches in the apical cytoplasm. Note immunonegativeapical band in winter snakes (arrows in (4)). LFA stained the secretory materials and the glycocalix (arrowheads). Summer turtles displayed scarce LFA-positive granules (small arrow in (3)) whereas winter turtles showed many in the apical cytoplasm. Large arrows, Reissner's fiber; V, third ventricle. (7-10) Electron microscopic views of the apical (7,8) and perinuclear (9,10) cytoplasms of SCO cells of snakes sacrificed in summer (7,9) and winter (8,10). In the apical cytoplasm, summer snakes show different types of secretory granules (sg) whereas, in the winter snakes, secretory granules (sg) accumulate beneath an apical region devoid of organules (bar). In the perinuclear cytoplasm, summer snakes show many distended cisternae of RER that were absent in winter snakes. N, Nuclei of SCO cells. Scale bar, (1,2,4,5) x 850; (3,6) x 350; (7) × 19800; (8) x 23000; (9,10) x 22000.
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M. Ci[uentes et al. / Neuroscience Letters 219 (1996) 9-12
O u r results s h o w e d s e a s o n a l d i f f e r e n c e s in the r e p t i l i a n S C O . In s u m m e r , d i s t e n d e d c i s t e r n a e o f R E R a n d few mature (LFA-positive) secretory granules indicated an a c t i v e s y n t h e s i s a n d release. In winter, a b s e n c e o f distended RER and accumulation of mature secretory granules in the apical c y t o p l a s m s u g g e s t e d a n i m p a i r e d s y n t h e s i s a n d release. In c o n c l u s i o n , o u r results s u g g e s t t h a t in s n a k e s a n d turtles the S C O d e c r e a s e s its activity (synthesis and release) during winter, a season of lethargy for b o t h species. A m o n g the v e r t e b r a t e s p e c i e s studied, d i f f e r e n c e s e x i s t w i t h r e s p e c t to the a m o u n t o f s e c r e t o r y m a t e r i a l s i n s i d e S C O cells b e t w e e n a c t i v e a n d t o r p i d a n i m a l s . W h e r e a s t o r p i d p o i q u i l o t h e r m i c v e r t e b r a t e s s h o w e d , in g e n e r a l , s e c r e t o r y m a t e r i a l s in t h e S C O ([1,4,11] p r e s e n t results), h i b e r n a t i n g m a m m a l s s h o w e d v e r y little s e c r e t o r y m a t e r i als d e t e c t a b l e b y i m m u n o h i s t o c h e m i c a l p r o c e d u r e s [10]. In b o t h cases, h o w e v e r , i r r e s p e c t i v e o f the a m o u n t o f s e c r e t o r y m a t e r i a l p r e s e n t , the S C O h a s b e e n s h o w n to b e less a c t i v e d u r i n g torpor. I n h i b i t i o n o f the S C O activity was d e m o n s t r a t e d to b e a r e s p o n s e in a c t i v e frogs subm i t t e d to low t e m p e r a t u r e [1,3]. If arrest o f S C O activity is a m e r e r e s p o n s e to l o w t e m p e r a t u r e or if it is a true s e a s o n a l b i o r r i t h m c o n t r o l l e d b y central i n f l u e n c e s is not yet k n o w n . Supported by grants DGICYT PB93-0979 Madrid, and FIS 9 5 - 1 5 9 1 M a d r i d , Spain. ll] Diederen, J.H.B. and Vullings, H.G.B., Comparison of several parameters related to the secretory activity of the subcommissural organ in European green frogs, Cell Tissue Res., 212 (1980) 383394. [2] Diederen, J.H.B., Vullings, H.G.B. and Legerstee-Oostveen, G.G., Autoradiographic study of the production of secretory material by the subcommissural organ of frogs (Rana temporaria) after injection of several radioactive precursors, with special reference to the glycosylation and turnover rate of the secretory material, Cell Tissue Res., 248 (1987) 215-222. [3] Diederen, J.H.B. and Vullings, H.G.B., Dynamic aspects of the secretory process in the amphibian subcommissural organ. In A. Oksche, E.M. Rodrfguez and P. Fem~indez-Llebrez (Eds.), The Subcommissural Organ. An Ependymal Brain Gland, SpringerVerlag, Berlin, 1993, pp. 111-120. [4] D'Uva, V., Ciarcia, G. and Ciarletta, A., The subcommissural organ of the lizard Lacerta s. sicula Raf, Ultrastructure and secretory cycle, J. Submicrosc. Cytol., 8 (1976) 175-191.
[5] Femfindez-Llebrez, P., Pdrez-Figares, J.M., Becerra, J., P6rez, J. and Matin Gir6n, F., Morphological evidence for the presence of two cell types in the ependyma of the subcommissural organ of the snake Natrix maura, Cell Tissue Res., 238 (1984) 407-409. [6] Fern~indez-Llebrez, P., P6rez, J., Nadales, A.E., Pdrez-Ffgares, J.M. and Rodrfguez, E.M., Vascular and leptomeningeal projections of the subcommissural organ of reptiles, Lectirl histochemical, immunocytochemical and ultrastructural studies, Histochemistry, 87 (1987) 607-614. [7] Grondona, J.M., P6rez, J., Cifuentes, M., L6pez-Avalos, M.D., Nualart, F.J., Peruzzo, B., Fernfindez-LLebrez, P. and Rodrfguez, E.M., Analysis of the secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula), Mol. Brain Res., 26 (1994) 299 308. [8] L6pez-Avalos, M.D., Pdrez, J., P6rez-Ffgares, J.M., Peruzzo, B., Grondona, J.M. and Rodrfguez, E.M., Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms, Cell Tissue Res., 283 (1996) 75-84. [9] Nualart, F., Hein, S., Rodrfguez, E.M. and Oksche, A., Identification and partial characterization of the secretory glycoproteins of the bovine subcommissural organ-Reissner's fiber complex. Evidence for the existence of two precursor forms, Mol. Brain Res., 11 (1991) 227-238. [10] Ntirberger, F. and Pena Salazar, P.A., Effects of hibernation and hypotermia on the secretory activity of the subcommissural organ. In A. Oksche, E.M. Rodrfguez and P. Femfindez-Llebrez (Eds.), The Subcommissural Organ. An Ependymal Brain Gland, Springer-Verlag, Berlin, 1993, pp. 257-264. [11] Oksche, A., Histologische, histochemische und experimentelle Studien am Subkommissuralorgan von Anuren (mit Hinweisen auf den Epiphysenkomplex), Z. Zellforsch., 57 (1962) 240-326. [12] Oksche, A., Rodrfguez, E.M. and Fernfindez-Llebrcz, P., The Subcommissural Organ. An Ependymal Brain Gland, Springer-Verlag, Berlin, 1993, 333 p. [13] Peruzzo, B., P6rez, J., Fern~indez-Llebrez, P., P6rez-Ffgares, J.M., Rodrfguez, E.M. and Oksche, A., Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections, Histochemistry, 93 (1990) 269278. [14] Rodrfguez, E.M., Oksche, A., Hein, S., Rodriguez, S. and Yulis, R., Comparative immunocytochemical study of the subcommissural organ, Cell Tissue Res., 237 (1984) 427-441. [15] Rodriguez, E.M., Herrera, H., Peruzzo, B., Rodriguez, S., Hein, S. and Oksche, A., Light- and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: evidence for processing of secretory material, Cell Tissue Res., 243 (1986) 545-559. [16] Rodriguez, E.M., Oksche, A., Hein, S. and Yulis, C.R., Cell biology of the subcommissural organ, Int. Rev. Cytol., 135 (1992) 39121.
Neuroscience Letters 219 (1996) 9-12
HEUROSCIENC[ IETT[RS
Seasonal variation in the secretory activity of the subcommissural organ (SCO) of reptiles M a n u e l C i f u e n t e s , J u a n P d r e z , J e s f s M. G r o n d o n a , P e d r o F e r n 4 n d e z - L l e b r e z * Departamento de Biolog[a Animal, Facultad de Ciencias, Universidad de M(tlaga, 29071 M61aga, Spain Received 23 September 1996; revised version received 7 October 1996; accepted 7 October 1996
Abstract Seasonal variations in the secretory activity of the subcommissural organ (SCO) of snakes and turtles was studied by immunocytochemistry, lectins, and electron microscopy. In animals sacrificed in summer, immunoreactive material, mostly devoid of sialic acid, occupied the whole cytoplasm. Cells showed many distended cistemae of rough endoplasmic reticulum and secretory granules. In animals sacrificed in winter, patches of immunoreactive sialic acid-rich material occupied the apical cytoplasm. Cells lacked distended cistemae and the secretory granules formed clusters. These results suggest a decreased synthesis and release of secretory material in the SCO of lethargic reptiles. Keywords: Lethargy; Immunocytochemistry; Electron microscopy; Lectins; Natrix maura snake; Mauremys caspica turtle
The subcommissural organ (SCO) is an ependymal brain gland located in the roof of the third cerebral ventricle. It releases into the cerebrospinal fluid glycoproteins that polymerise and form the Reissner's fiber (RF), extending along the aqueduct, fourth ventricle and central canal of the spinal cord [12,16]. The secretory activity of the SCO undergoes seasonal variations in hibernating or lethargic species. The SCO of frogs sacrificed in winter (torpid state) contained more secretory material than in summer [11]. However, autoradiographic studies have shown a negative linear correlation between the amount of secretory material in the frog SCO and the growth rate of the RF [1]; thus, more material in the organ indicated less release. In the lizard, more secretory material was reported in summer than in winter [4]. Also in mammals, immunocytochemical studies revealed a drastic decrease of secretory material during hibernation [10]. Then, irrespective of the amount of secretory material present, it seems that the SCO is inhibited during lethargy. The aim of the present investigation was to investigate the seasonal variation in the secretory activity of the SCO
* Corresponding aulhor. Tel.: +34 5 2131858; fax: +34 5 2132000: e-mail: [email protected]
of reptiles by using methods that allow us to distinguish between mature and immature materials such as immunocytochemistry, lectins and electron microscopy. Snakes NatrLr maura and turtles Mauremys caspica were captured, under authorisation, in M41aga (Spain) in spring and kept in aquaria-terraria, at environmental temperature and photoperiod. Snakes were fed living frogs and turtles fresh meat ad libitum. In summer, snakes and turtles were active and regularly fed whereas in winter the animals, although having access to food, did not eat and entered in a torpid state. Six snakes and four turtles were sacrificed in late June when environmental temperature averaged 24°C. A similar number of specimens were sacrificed in mid January with temperature averaging 12°C. Handling, care and processing of the animals were carried out according to principles approved by the council of the American Physiological Society and national laws (B.O.E. 67, 1988, Spain). All animals were anaesthetised by intraperitoneal injection of tricaine (Sigma, St. Louis MO, USA) [6] and perfused transcardially with Bouin fluid, for light microscopy (snakes, 4 summer (s) + 4 winter(w); turtles, 4 s + 4 w) and Kamovsky's fixative [6], for electron microscopy (snakes, 2 s + 2 w). Dissected brains were put in fresh fixative for 48 h at room temperature (Bouin) or for 4 h
0304-394(I/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S 0 3 0 4 - 3 9 4 0 ( 9 6 ) I 3 I 61 -X
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M. Cifuentes et al. / Neuroscienee Letters 219 (1996) 9-12
M. Cifuentes et al. / Neuroscience Letters 219 (1996) 9-12
at 4°C (Karnovsky). For light microscopy, brains were embedded in paraffin (snakes, 2 s + 2 w; turtles, 2 s + 2 w) and butyl-methyl methacrylate (snakes, 2 s + 2 w; turtles, 2 s + 2 w) for thin and semithin sections, respectively. For electron microscopy, pieces containing the SCO (snakes, 2 s + 2 w) were postfixed in 1% osmium tetroxide for 2 h at 4°C, dehydrated and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate. A rabbit antiserum against the bovine Reissner's fiber (AFRU) [14] was used to selectively stain the secretory products of the subcommissural organ as described previously [6]. For lectin histochemistry, Concanavalin A (Con A) (affinity, mannose, glucose) and L i m a x f l a v u s agglutinin (LFA) (affinity, sialic acid) were used. Con A was labelled with horseradish peroxidase (HRP) and L F A was not labelled. Lectins were revealed as described previously [9]. Both in summer and winter, the SCO and R F of snakes and turtles showed immunoreactive material with a distinct pattern of distribution. In summer, secretory materials distributed evenly throughout the apical, perinuclear, and basal cytoplasm (Fig. 1(1,2)). In winter, the perinuclear and basal cytoplasm showed a weak immunoreactivity whereas the apical cytoplasm showed an uneven staining with prominent patches of strongly immunoreactive materials on a weakly immunoreactive background (Fig. 1(4,5)). Staining pattern of Con A was similar to that obtained with AFRU. However staining with L F A was quite different. In summer, L F A revealed scarce granules in the apical cytoplasm and elongated profiles near the nuclear zone. The glycocalix of the ependymal cells and the R F were strongly stained (Fig. 1(3)). In contrast, in winter, strongly stained patches of different sizes distributed through the perinuclear and apical cytoplasm, excepting the cell apexes, (Fig. 1(6)). The ultrastructure of the SCO of summer snakes have been described elsewhere [5]. Briefly, in the apical cytoplasm, granules of different shapes, sizes and electrondensity and cisternae of endoplasmic reticulum were the most prominent features (Fig. 1(7)). The perinuclear and basal cytoplasms were occupied by typical highly distended cisternae of rough endoplasmic reticulum RER filled with pale material (Fig. 1(9)). The SCO of winter
11
snakes showed an apical band (1 /~m thick) devoid of organules. Under this band, the apical cytoplasm was rich in clusters of granules of different sizes, shapes and electron density (Fig. 1(8)), The perinuclear and basal cytoplasm showed flat parallel cisternae of RER, Golgi complexes, granules, mitochondria, and glycogen particles but, in sharp contrast to the animals sacrificed in summer, no distended cisternae of RER were seen (Fig. 1(10)). The amount of secretory material in the SCO is the result of a balance between the rates of biosynthesis and release. More material could reflect either an increased synthesis or a decreased release; and vice-versa, less material could reflect either a decreased synthesis or an increased release. The SCO is a gland that synthesises and releases very large glycoproteins (molecular weight 2 0 0 - 6 0 0 kDa) [8,9]. The precursor forms are produced in typical distended cisternae of RER in the perinuclear cytoplasm. A part of the precursor glycoproteins is processed in the Golgi apparatus and released into the ventricle within 1 h after synthesis [15]. A second fraction is retained in the dilated RER cisternae and slowly processed and released over a period of 3 - 5 days [2,16]. In mammals, the bulk of secretion of the SCO is stored in the RER and only few mature secretory granules can be visualised in the apices of the secretory cells [ 16]. In non-mammalian vertebrates, both RER and the secretory granules may represent important storage sites [7,13]. In the present work, we used immunocytochemistry to identify secretory material of the SCO. The antiserum employed, A F R U , selectively recognises the secretory material in the SCO and the R F of all vertebrate species investigated [14] including the reptilian species used in the present investigation [6]. Using A F R U we were then able to reveal all forms of secretory material (precursor and mature) in the SCO of summer and winter specimens. In addition, we have used the lectin L F A , that selectively binds to sialic acid, to identify mature (post-Golgi) SCO secretory material containing sialic acid. Thus, by the combined use of A F R U and L F A in adjacent sections we had an idea of the amount of immature and mature secretory materials inside the SCO in a given situation. On the other hand, electron microscopy led the identification of the organules involved in the synthesis (RER) and those containing mature secretory materials (secretory granules).
Fig. 1. (1-6). Transverse sections through the subcommissural organ of snakes (1,4) and turtles (2,3,5,6) sacrificed in summer (1-3) and winter (4-6) immunostained with an antiserum against the bovine Reissner's fiber (1,2,4,5) (semithin methacrylate sections) and the lectin LFA (3,6) (paraffin sections). Summer animals show immunoreactive secretory materials homogeneously distributed throughout apical (a), perinuclear (p) and basal (b) cytoplasms whereas winter animals show patches in the apical cytoplasm. Note immunonegativeapical band in winter snakes (arrows in (4)). LFA stained the secretory materials and the glycocalix (arrowheads). Summer turtles displayed scarce LFA-positive granules (small arrow in (3)) whereas winter turtles showed many in the apical cytoplasm. Large arrows, Reissner's fiber; V, third ventricle. (7-10) Electron microscopic views of the apical (7,8) and perinuclear (9,10) cytoplasms of SCO cells of snakes sacrificed in summer (7,9) and winter (8,10). In the apical cytoplasm, summer snakes show different types of secretory granules (sg) whereas, in the winter snakes, secretory granules (sg) accumulate beneath an apical region devoid of organules (bar). In the perinuclear cytoplasm, summer snakes show many distended cisternae of RER that were absent in winter snakes. N, Nuclei of SCO cells. Scale bar, (1,2,4,5) x 850; (3,6) x 350; (7) × 19800; (8) x 23000; (9,10) x 22000.
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M. Ci[uentes et al. / Neuroscience Letters 219 (1996) 9-12
O u r results s h o w e d s e a s o n a l d i f f e r e n c e s in the r e p t i l i a n S C O . In s u m m e r , d i s t e n d e d c i s t e r n a e o f R E R a n d few mature (LFA-positive) secretory granules indicated an a c t i v e s y n t h e s i s a n d release. In winter, a b s e n c e o f distended RER and accumulation of mature secretory granules in the apical c y t o p l a s m s u g g e s t e d a n i m p a i r e d s y n t h e s i s a n d release. In c o n c l u s i o n , o u r results s u g g e s t t h a t in s n a k e s a n d turtles the S C O d e c r e a s e s its activity (synthesis and release) during winter, a season of lethargy for b o t h species. A m o n g the v e r t e b r a t e s p e c i e s studied, d i f f e r e n c e s e x i s t w i t h r e s p e c t to the a m o u n t o f s e c r e t o r y m a t e r i a l s i n s i d e S C O cells b e t w e e n a c t i v e a n d t o r p i d a n i m a l s . W h e r e a s t o r p i d p o i q u i l o t h e r m i c v e r t e b r a t e s s h o w e d , in g e n e r a l , s e c r e t o r y m a t e r i a l s in t h e S C O ([1,4,11] p r e s e n t results), h i b e r n a t i n g m a m m a l s s h o w e d v e r y little s e c r e t o r y m a t e r i als d e t e c t a b l e b y i m m u n o h i s t o c h e m i c a l p r o c e d u r e s [10]. In b o t h cases, h o w e v e r , i r r e s p e c t i v e o f the a m o u n t o f s e c r e t o r y m a t e r i a l p r e s e n t , the S C O h a s b e e n s h o w n to b e less a c t i v e d u r i n g torpor. I n h i b i t i o n o f the S C O activity was d e m o n s t r a t e d to b e a r e s p o n s e in a c t i v e frogs subm i t t e d to low t e m p e r a t u r e [1,3]. If arrest o f S C O activity is a m e r e r e s p o n s e to l o w t e m p e r a t u r e or if it is a true s e a s o n a l b i o r r i t h m c o n t r o l l e d b y central i n f l u e n c e s is not yet k n o w n . Supported by grants DGICYT PB93-0979 Madrid, and FIS 9 5 - 1 5 9 1 M a d r i d , Spain. ll] Diederen, J.H.B. and Vullings, H.G.B., Comparison of several parameters related to the secretory activity of the subcommissural organ in European green frogs, Cell Tissue Res., 212 (1980) 383394. [2] Diederen, J.H.B., Vullings, H.G.B. and Legerstee-Oostveen, G.G., Autoradiographic study of the production of secretory material by the subcommissural organ of frogs (Rana temporaria) after injection of several radioactive precursors, with special reference to the glycosylation and turnover rate of the secretory material, Cell Tissue Res., 248 (1987) 215-222. [3] Diederen, J.H.B. and Vullings, H.G.B., Dynamic aspects of the secretory process in the amphibian subcommissural organ. In A. Oksche, E.M. Rodrfguez and P. Fem~indez-Llebrez (Eds.), The Subcommissural Organ. An Ependymal Brain Gland, SpringerVerlag, Berlin, 1993, pp. 111-120. [4] D'Uva, V., Ciarcia, G. and Ciarletta, A., The subcommissural organ of the lizard Lacerta s. sicula Raf, Ultrastructure and secretory cycle, J. Submicrosc. Cytol., 8 (1976) 175-191.
[5] Femfindez-Llebrez, P., Pdrez-Figares, J.M., Becerra, J., P6rez, J. and Matin Gir6n, F., Morphological evidence for the presence of two cell types in the ependyma of the subcommissural organ of the snake Natrix maura, Cell Tissue Res., 238 (1984) 407-409. [6] Fern~indez-Llebrez, P., P6rez, J., Nadales, A.E., Pdrez-Ffgares, J.M. and Rodrfguez, E.M., Vascular and leptomeningeal projections of the subcommissural organ of reptiles, Lectirl histochemical, immunocytochemical and ultrastructural studies, Histochemistry, 87 (1987) 607-614. [7] Grondona, J.M., P6rez, J., Cifuentes, M., L6pez-Avalos, M.D., Nualart, F.J., Peruzzo, B., Fernfindez-LLebrez, P. and Rodrfguez, E.M., Analysis of the secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula), Mol. Brain Res., 26 (1994) 299 308. [8] L6pez-Avalos, M.D., Pdrez, J., P6rez-Ffgares, J.M., Peruzzo, B., Grondona, J.M. and Rodrfguez, E.M., Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms, Cell Tissue Res., 283 (1996) 75-84. [9] Nualart, F., Hein, S., Rodrfguez, E.M. and Oksche, A., Identification and partial characterization of the secretory glycoproteins of the bovine subcommissural organ-Reissner's fiber complex. Evidence for the existence of two precursor forms, Mol. Brain Res., 11 (1991) 227-238. [10] Ntirberger, F. and Pena Salazar, P.A., Effects of hibernation and hypotermia on the secretory activity of the subcommissural organ. In A. Oksche, E.M. Rodrfguez and P. Femfindez-Llebrez (Eds.), The Subcommissural Organ. An Ependymal Brain Gland, Springer-Verlag, Berlin, 1993, pp. 257-264. [11] Oksche, A., Histologische, histochemische und experimentelle Studien am Subkommissuralorgan von Anuren (mit Hinweisen auf den Epiphysenkomplex), Z. Zellforsch., 57 (1962) 240-326. [12] Oksche, A., Rodrfguez, E.M. and Fernfindez-Llebrcz, P., The Subcommissural Organ. An Ependymal Brain Gland, Springer-Verlag, Berlin, 1993, 333 p. [13] Peruzzo, B., P6rez, J., Fern~indez-Llebrez, P., P6rez-Ffgares, J.M., Rodrfguez, E.M. and Oksche, A., Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections, Histochemistry, 93 (1990) 269278. [14] Rodrfguez, E.M., Oksche, A., Hein, S., Rodriguez, S. and Yulis, R., Comparative immunocytochemical study of the subcommissural organ, Cell Tissue Res., 237 (1984) 427-441. [15] Rodriguez, E.M., Herrera, H., Peruzzo, B., Rodriguez, S., Hein, S. and Oksche, A., Light- and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: evidence for processing of secretory material, Cell Tissue Res., 243 (1986) 545-559. [16] Rodriguez, E.M., Oksche, A., Hein, S. and Yulis, C.R., Cell biology of the subcommissural organ, Int. Rev. Cytol., 135 (1992) 39121.