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The centrifugal visual system in Vipera aspis. An experimental study using retrograde axonal transport of HRP and [3H]adenosine
Brain Research, 183 (1980) 435--441 © Elsevier/North-Holland Biomedical Press
435
Short Communications
The centrifugal visual system in Vipero ospis. An experimental study using retrograde axonal transport of HRP and pH]adenosine
J. REPI~RANT, J. PEYRICHOUX, C. WEIDNER, D. MICELI and J. P. RIO Laboratoire de Neuromorphologie, I N S E R M U 106, H6pital Foch, Suresnes, 92150, Laboratoire de Psychophysiologie sensorielle, Universitd Paris VI, 75005, Paris, Laboratoire d',4natomie compar~e, M N H N , Paris (France)
(Accepted October llth, 1979) Key words: centrifugal visual system -- HRP -- autoradiography - - Vipera aspis
The existence of a centrifugal visual pathway in vertebrates is one of the most controversial issues in the literature. Although this pathway was first demonstrated in birds towards the end of the last century and thereafter received extensive confirmation (see ref. 4), its presence in other vertebrate groups has been subject to dispute: mammals (see ref. 20), amphibians (see ref. 23) and fishes (see ref. 17). In reptiles evidence for the existence of such a system appears to be provided by various data, both anatomical2,7,9,12 and physiological 15. Using the H R P method with intraocular injection of the enzyme, Halpern et alp and Ferguson et al. 7 observed in different reptiles, peroxidase-positive neurons in various regions of the central nervous system. These were considered to constitute cell bodies at the origin of fibers of retinal destination. Although the H R P method has provided a very precise localization of the sites of origin of centrifugal fibers in birds 14, the technique has been found to be inadequate for demonstrating analogous systems in teleostean fishes 17. In fact, both intraocular injection of H R P (without simultaneous occlusion of the ocular blood supply) and injection into the general circulation in teleosts give rise to heavy neuronal labeling in a number of structures having direct projections to the hypophysis. This cannot be attributed to retrograde axonal transport of H R P by the centrifugal visual system but rather reflects retrograde transport of the enzyme in the hypothalamo-hypophyseal tract after being taken up at the hypophysis which lacks the blood-brain barrier 17. In the light of these findings the data of Halpern et alp and Ferguson et al. 7 were here reconsidered and re-investigated in the snake Vipera aspis using, in addition to the H R P method, the radioautographic technique employing tritiated adenosine, which, under certain experimental circumstances, is reputed to be transported by the retrograde axonal flowl~,26, 30.
436
H R P studies. I n the first g r o u p o f snakes (12 specimens) 5 - 1 0 / A o f a 35-40 ~ solution o f H R P (Sigma type VI) dissolved in distilled water was injected unilaterally into the eye. Snakes o f the second g r o u p (7 specimens) received an intracardial inject i o n o f 25 mg H R P dissolved in physiological saline. In the third g r o u p (3 specimens) H R P was injected into the orbital cavity. F i n a l l y 5 c o n t r o l vipers either received or did n o t receive i n t r a o c u l a r or i n t r a c a r d i a l injections o f physiological saline solution. F o l l o w i n g various e x p e r i m e n t a l p r o c e d u r e s the animals were kept for 24-72 h at r o o m
OM
A
B
TOM
Fig. 1. Schematic representation of transverse sections taken from the rostral (A) to the caudal thalamus (D). The labeling was obtained in both the primary visual system and in neurons of the centrifugal optic thalamic nucleus after intraocular injection of either HRP (Colman et al. procedure) or [3H]adenosine. The optic endings appear as small dots, the primary optic fibers as dashed lines, labeled neurons at the origin of centrifugal fibres as large dots. BTP, telencephalic basal peduncle; COTN, centrifugal optic thalamic nucleus; HB, habenular nuclei; G Ld, nucleus geniculatus lateralis pars dorsalis; GLvs, nucleus geniculatus lateralis pars ventralis magnocellularis; GLvm, nucleus geniculatus lateralis pars ventralis molecularis; NS, nucleus suprapeduncularis; OV, nucleus ovalis; TOM, tractus opticus marginalis; VL, nucleus ventrolateralis.
437 temperature and perfused, under anesthesia through the single carotid with 25 Karnovsky solution. Brain sections of 40 #m were obtained on a freezing microtome and processed by either of two methods for the HRP reaction: (1) the Graham and Karnovsky technique with an incubation medium containing phosphate buffer, 3-3' diaminobenzidine tetrahydrochloride and H20~ (see ref. 14); and (2) the Colman et al. procedure using an unbuffered incubation medium containing O-dianisidine sodium nitroprusside and H202 (see ref. 9). Sections were then counterstained with cresyl violet, After intraocular injection of HRP, retrograde labeled cells were found to be distributed bilaterally but predominantly contralaterally in a small triangular cell field extending approximately 950 #m rostrocaudally within the ventro-lateral thalamus. Situated rostrally to the nucleus ovalis it continued caudally up until the boundary of the posterior thalamus beneath the nucleus geniculatus lateralis pars ventralis. Laterally it was partially in contact with the tractus opticus marginalis and medially was adjacent to the telencephalic basal peduncle (Fig. 1). This labeled cell field appears to correspond topographically (in reptiles) to the nucleus referred to as both the nucleus geniculatus medialis by Shanklin27 and Papez t6 and the nucleus of the ventral supraoptic commissure by Halpern and Frumins. The labeled area may also correspond to what has been termed in reptiles as either the corpus geniculatum internum3, the corpus geniculatum posticumS, 6 and the nucleus tracti tecto-thalamic cruciati together with the nucleus Z (see ref. 10). A more optimal cell labeling was obtained with the O-dianisidine incubation medium. Elsewhere the Colman et al. method demonstrated the presence of HRP product within the optic tracts (tractus opticus marginalis and basal optic root) and the optic neuropil of the thalamus, pretectum, tectum and tegmentum. In contrast and as previously reported 19 the Karnovsky method proved less sensitive by not providing any orthograde enzyme labeling of the primary visual system. No HRP-labeled neurons were identified within this same thalamic region either following intravascular injection of the enzyme or in the control animals. Furthermore, retrograde labeling of cells in this region was not observed when the HRP injection was confined to the orbital cavity, although in these cases labeled cell bodies were identified in the extraocular motor nuclei. Thus the peroxidase-positive labeling within the ventro-lateral thalamus cannot be attributed to retrograde transport of the enzyme from either the orbital musculature or any gland within the orbit. Moreover, it would appear not to result from HRP transport from regions lacking a blood-brain barrier. Lastly, the results exclude the presence of endogenous proteins susceptible to giving a peroxidase-positive reaction within this thalamic field. This labeled thalamic cell field in Vipera aspis would thus appear to represent the origin of a true retinopetal system. From many points of view, this nucleus is comparable to that identified by Halpern et alp in the snake Thamnophis using the HRP method and referred to as the nucleus of the ventral supraoptic commissure. Considering that this nucleus represents the origin of the retinopetal pathway in ophidians and that no link between this nucleus and the ventral supraoptic decussation has as yet been demonstrated experimentally, it would seem
4~o~
439 preferable to refer to this nucleus in snakes as the centrifugal optic thalamic nucleus. [3H]Adenosine studies. Four snakes were given unilateral eye injection of 2,8[3H] adenosine (30 #Ci in 5/A, NEN, spec. act. 30.5 Ci/mmol. The vipers were kept for 24-96 h at 22-24 °C and then perfused through the single carotid with 1 0 ~ formol saline. The brains were embedded in paraffin and sectioned serially at 12 /~m in the frontal plane. Sections were coated with K 5 llford emulsion and stored in light-tight boxes at 4 °C for a period of 4-5 weeks. They were then developed in D 19, fixed in Kodak hypofixer and counterstained with cresyl violet. Silver grains were found throughout the primary visual system (Fig. 2A) and particularly in the contralateral optic centers previously identified using either the Fink-Heimer technique 18 or following intraocular injection of tritiated amino acids21 : nuclei ovalis, geniculatus lateralis pars dorsalis and pars ventralis, geniculatus pretectalis, griseus tectalis, lentiformis mesencephali, posterodorsalis, opticus tegmenti, and the superficial layers of the tectum. The grain density was however considerably lower than that seen after intraocular injection of [aH]proline. Elsewhere neurons of the centrifugal optic thalamic nucleus showed heavy labeling (Fig. 2B and C) bilaterally although more so on the contralateral side. Cell counts generated a total of 660 labeled neurons of which 82.5 ~ were to be found in the nucleus contralateral to the injection. These proportions are comparable to those obtained with counts of peroxidase-posirive neurons after intraocular injection of the enzyme. No other adenosin heavily labeled cells were detected in any other brain regions. The anterograde transport of adenosine material is at present a well established phenomenon11,13,~4,~8, 29,~°. However, its retrograde transport has been contested in several reports13,24, 25. As a matter of fact, the adenosine neuronal labeling was initially interpreted as arising solely from transneuronal transport24,2L Although the occurrence of this type of labeling was subsequently confirmed (notably using long survival periods) in different systems11,22, 80, other investigations have clearly demonstrated that adenosine labeling could more simply result from retrograde axonal transport of the nucleoside compounds 11,26,30. The present results are in good agreement with the latter data. Whether using either the H R P or the adenosine methods, the area of distribution of labeled ipsilateral and contralateral neurons was essentially the same. Moreover, following intraocular injection of [3H]adenosine heavily labeled cells were localized exclusively within the centrifugal optic thalamic nucleus. If such labeling were due to transynaptic leakage of adenoside material from retinal endings, then labeled neurons should also have been observed particularly among the neuronal
Fig. 2. A: dark-field micrograph showing labeling in the primary visual system (GL, nucleus geniculatus lateralis; TOM, tractus opticus marginalis) and neurons (arrow) at the origin of the centrifugal visual system (COTN, centrifugal optic thalamus nucleus) in the rostral thalamus as observed 30 h after contralateral ocular injection of [all]adenosine. × 125. B: light-field micrograph sbowing labeling of neurons (arrow) at the origin of the centrifugal visual system (COTN, centrifugal optic thalamic nucleus) in the posterior thalamus as observed 72 h after contralateral intraocular injections of [all]adenosine. GL, nucleus geniculatus lateralis; TOM, tractus opticus marginalis; BTP, telencephalic basal peduncle. × 125. C: higher magnification ( × 800) of labeled neurons of the centrifugal optic thalamic nucleus, 72 h after intraocular injection of [3Hladenosine.
440 elements which are p o s t s y n a p t i c to the optic fibers. I n conclusion the present d a t a are in accord with those o b t a i n e d e m p l o y i n g the H R P m e t h o d a n d thus confirming the existence o f a centrifugal visual system in the viper. I n m o s t birds (see ref. 1), in the c r o c o d i l i a n Caiman erocodilus 7, the turtle Pseudemys scripta (Cervetto, referred to in ref. 15) a n d some lizards such as Gerrhonotus coeroleus coeruleus 9, the nucleus o f origin o f centrifugal fibers is situated in the mesencephalic t e g m e n t u m a n d m o r e precisely in the isthmic region. By contrast, in Vipera, as in the snakes e x a m i n e d by H a l p e r n et alP, the centrifugal optic nucleus is located within the ventral thalamus. This a r r a n g e m e n t does n o t a p p e a r to be a characteristic o f snakes as the latter investigators have also identified a centrifugal optic nucleus in the d i e n c e p h a l o n a d j a c e n t to the optic tract in the lizard Cordylus cordylus. Despite such t o p o g r a p h i c a l interspecies variations, preliminary studies r e g a r d i n g the a n a t o m i c a l connections o f this nucleus p e r f o r m e d on a n u m b e r o f species indicate several similarities. F o r example, in the pigeon 1,4, the crocodile 7, a n d in the snakes T h a m n o p h i s ~8 a n d Vipera (unpublished observations), the centrifugal optic nucleus receives afferents f r o m the optic tectum. However, the c o m p a r a t i v e d a t a regarding the centrifugal p a t h w a y o f s a u r o p s i d i a n s based on the criteria o f connectivity, ultrastructure a n d function are at present t o o few to p e r m i t any affirmation t h a t the systems which are involved are h o m o l o g o u s . W e would like to t h a n k F. R o g e r a n d C. Pages for excellent technical s u p p o r t , D. Le Cren for skillful p h o t o g r a p h i c assistance a n d S. A r n o l d for secretarial help. This w o r k was p a r t i a l l y s u p p o r t e d by I N S E R M , C N R S a n d D G R S T .
1 Angaut, P. and Rep6rant, J., A light and electron microscopic study of the nucleus isthmo-opticus in the pigeon, Arch. ,4nat. micr. Morph. exp., 67 (1978) 63-78. 2 Armstrong, J. A., An experimental study of the visual pathways in a snake (Natrix natrix), J. Anat. (Lond.), 84 0951) 275--289. 3 Bellonci, J., ~ber die centrale Endigung des Nervus opticus bei den Vertebraten, Z. wiss. Zool., 47 (1888) 1-46. 4 Cowan, W. M., Centrifugal fibres to the avian retina, Brit. reed. Bull., 26 0970) 112-118. 5 De Lange, S. J., Das Zwischenhirn und das Mittelhirn der Reptilien, Folia neuro-bioL (Lpz.), 7 0913) 67-138. 6 Edinger, L., Studien fiber das Zwiscbenhirn der Reptilien, Abh. Senk. Naturf Ges., 20 (1899) 160-202. 7 Ferguson, J. L., Mulvanny, P. J. and Brauth, S. E., Distribution of neurons projecting to the retina of Caiman crocodilus, Brain Behav. Evok, 15 (1978) 294-306. 8 Halpern, M. and Frumin, N., Retinal projections in a snake Thamnophis sirtalis, J. Morph., 14 (1973) 359-382. 9 Halpern, M., Wang, R. T. and Colman, D. R., Centrifugal fibers to the eye in a non-avian vertebrate: source revealed by horseradish peroxidase studies, Science, 194 (1976) I 185-1188. 10 Huber, G. C. and Crosby, E. C., On thalamic and tectal nuclei and fiber paths in the brain of the American alligator, J. comp. NeuroL, 40 0926) 97-227. l l Hunt, S. P. and Kfinzle, H., Bidirectional movement of label and transneuronal transport phenomena after injection of [all]adenosine into the central nervous system, Brain Research, 112 (1976) 127-132. 12 Kruger, L. and Maxwell, D. S., Wallerian degeneration in the optic nerve of a reptile: an electron microscopic study, Amer. J. Anat., 125 (1969) 247-270. 13 Kruger, L. and Saporta, S., Axonal transport of [all]adenosine in visual and somatosensory pathways, Brain Research, 122 (1977) 132-136.
441 14 La Vail, J. H. and La Vail, M. M., Retrograde axonal transport in the central nervous system, Sience, 176 (1972) 1416-1417. 15 Marchiafava, P. L., Centrifugal actions on amacrine and ganglion cells in the retina of the turtle, J. Physiol. (Lond.), 255 (1976) 137-155. 16 Papez, J. N., Thalamus of turtles and thalamic evolution, J. comp. Neurol., 61 (1935) 433-475. 17 Peyrichoux, J., Weidner, C., Rel~rant, J. and Miceli, D., An experimental study of the visual system of cyprinid fish using the HRP method, Brain Research, 130 (1977) 531-537. 18 Rep6rant, J., Les voies et les centres optiques primaires chez la vip6re (Vipera aspis), Arch. Anat. micr. Morph. exp., 62 (1973) 323-352. 19 Rep6rant, J., The orthograde transport of horseradish peroxidase in the visual system, Brain Research, 85 (1975) 307-312. 20 Rep6rant, J. and Gallego, A., Fibres centrifuges dans la r6tine humaine, Arch. Anat. mier. Morph. exp., 65 (1976) 103-120. 21 Rep6rant, J. and Rio, J. P., Retinal projections in Vipera aspis. A reinvestigation using light radioautographic and electron microscopic degeneration techniques, Brain Research, 107 (1976) 603609. 22 Rose, G. and Schubert, P., Release and transfer of [3H]adenosine derivatives in the cholinergic septal system, Brain Research, 121 (1977) 353-357. 23 Scalia, F. and Teitelbaum, 1., Absence of efferents to the retina in the frog and toad, Brain Research, 153 (1978) 340-344. 24 Schubert, P. and Kreutzberg, G. W., Axonal transport of adenosine and uridine and transfer to postsynaptic neurons, Brain Research, 76 (1974) 526-530. 25 Schubert, P. and Kreutzberg, G. W., [all]adenosine, a tracer for neuronal connectivity, Brain Research, 85 (1975) 317-319. 26 Schwarz, D. W. F., Schwarz, I. E. and Tomlinson, R. D., Avian efferent vestibular neurons identified by axonal transport of Jail]adenosine and horseradish peroxidase, Brain Research, 155 (1978) 103-107. 27 Shanklin, W. M., The central nervous system of Chameleon vulgaris, Aeta Zool., 11 (1930) 425-490. 28 Wang, R. T. and Halpern, M., Afferent and efferent connections of thalamic nuclei of the visual system of garter snake, Anat. Rec., 187 (1977) 741-742. 29 Wise, S. P. and Jones, E. G., Transneuronal or retrograde transport of [3H]adenosine in the rat somatic sensory system, Brain Research, 107 (1976) 127-131. 30 Wise, S. P., Jones, E. G. and Berman, N., Direction and specificity of axonal and transcellular transport of nucleosides, Brain Research, 139 (1978) 197-217.
435
Short Communications
The centrifugal visual system in Vipero ospis. An experimental study using retrograde axonal transport of HRP and pH]adenosine
J. REPI~RANT, J. PEYRICHOUX, C. WEIDNER, D. MICELI and J. P. RIO Laboratoire de Neuromorphologie, I N S E R M U 106, H6pital Foch, Suresnes, 92150, Laboratoire de Psychophysiologie sensorielle, Universitd Paris VI, 75005, Paris, Laboratoire d',4natomie compar~e, M N H N , Paris (France)
(Accepted October llth, 1979) Key words: centrifugal visual system -- HRP -- autoradiography - - Vipera aspis
The existence of a centrifugal visual pathway in vertebrates is one of the most controversial issues in the literature. Although this pathway was first demonstrated in birds towards the end of the last century and thereafter received extensive confirmation (see ref. 4), its presence in other vertebrate groups has been subject to dispute: mammals (see ref. 20), amphibians (see ref. 23) and fishes (see ref. 17). In reptiles evidence for the existence of such a system appears to be provided by various data, both anatomical2,7,9,12 and physiological 15. Using the H R P method with intraocular injection of the enzyme, Halpern et alp and Ferguson et al. 7 observed in different reptiles, peroxidase-positive neurons in various regions of the central nervous system. These were considered to constitute cell bodies at the origin of fibers of retinal destination. Although the H R P method has provided a very precise localization of the sites of origin of centrifugal fibers in birds 14, the technique has been found to be inadequate for demonstrating analogous systems in teleostean fishes 17. In fact, both intraocular injection of H R P (without simultaneous occlusion of the ocular blood supply) and injection into the general circulation in teleosts give rise to heavy neuronal labeling in a number of structures having direct projections to the hypophysis. This cannot be attributed to retrograde axonal transport of H R P by the centrifugal visual system but rather reflects retrograde transport of the enzyme in the hypothalamo-hypophyseal tract after being taken up at the hypophysis which lacks the blood-brain barrier 17. In the light of these findings the data of Halpern et alp and Ferguson et al. 7 were here reconsidered and re-investigated in the snake Vipera aspis using, in addition to the H R P method, the radioautographic technique employing tritiated adenosine, which, under certain experimental circumstances, is reputed to be transported by the retrograde axonal flowl~,26, 30.
436
H R P studies. I n the first g r o u p o f snakes (12 specimens) 5 - 1 0 / A o f a 35-40 ~ solution o f H R P (Sigma type VI) dissolved in distilled water was injected unilaterally into the eye. Snakes o f the second g r o u p (7 specimens) received an intracardial inject i o n o f 25 mg H R P dissolved in physiological saline. In the third g r o u p (3 specimens) H R P was injected into the orbital cavity. F i n a l l y 5 c o n t r o l vipers either received or did n o t receive i n t r a o c u l a r or i n t r a c a r d i a l injections o f physiological saline solution. F o l l o w i n g various e x p e r i m e n t a l p r o c e d u r e s the animals were kept for 24-72 h at r o o m
OM
A
B
TOM
Fig. 1. Schematic representation of transverse sections taken from the rostral (A) to the caudal thalamus (D). The labeling was obtained in both the primary visual system and in neurons of the centrifugal optic thalamic nucleus after intraocular injection of either HRP (Colman et al. procedure) or [3H]adenosine. The optic endings appear as small dots, the primary optic fibers as dashed lines, labeled neurons at the origin of centrifugal fibres as large dots. BTP, telencephalic basal peduncle; COTN, centrifugal optic thalamic nucleus; HB, habenular nuclei; G Ld, nucleus geniculatus lateralis pars dorsalis; GLvs, nucleus geniculatus lateralis pars ventralis magnocellularis; GLvm, nucleus geniculatus lateralis pars ventralis molecularis; NS, nucleus suprapeduncularis; OV, nucleus ovalis; TOM, tractus opticus marginalis; VL, nucleus ventrolateralis.
437 temperature and perfused, under anesthesia through the single carotid with 25 Karnovsky solution. Brain sections of 40 #m were obtained on a freezing microtome and processed by either of two methods for the HRP reaction: (1) the Graham and Karnovsky technique with an incubation medium containing phosphate buffer, 3-3' diaminobenzidine tetrahydrochloride and H20~ (see ref. 14); and (2) the Colman et al. procedure using an unbuffered incubation medium containing O-dianisidine sodium nitroprusside and H202 (see ref. 9). Sections were then counterstained with cresyl violet, After intraocular injection of HRP, retrograde labeled cells were found to be distributed bilaterally but predominantly contralaterally in a small triangular cell field extending approximately 950 #m rostrocaudally within the ventro-lateral thalamus. Situated rostrally to the nucleus ovalis it continued caudally up until the boundary of the posterior thalamus beneath the nucleus geniculatus lateralis pars ventralis. Laterally it was partially in contact with the tractus opticus marginalis and medially was adjacent to the telencephalic basal peduncle (Fig. 1). This labeled cell field appears to correspond topographically (in reptiles) to the nucleus referred to as both the nucleus geniculatus medialis by Shanklin27 and Papez t6 and the nucleus of the ventral supraoptic commissure by Halpern and Frumins. The labeled area may also correspond to what has been termed in reptiles as either the corpus geniculatum internum3, the corpus geniculatum posticumS, 6 and the nucleus tracti tecto-thalamic cruciati together with the nucleus Z (see ref. 10). A more optimal cell labeling was obtained with the O-dianisidine incubation medium. Elsewhere the Colman et al. method demonstrated the presence of HRP product within the optic tracts (tractus opticus marginalis and basal optic root) and the optic neuropil of the thalamus, pretectum, tectum and tegmentum. In contrast and as previously reported 19 the Karnovsky method proved less sensitive by not providing any orthograde enzyme labeling of the primary visual system. No HRP-labeled neurons were identified within this same thalamic region either following intravascular injection of the enzyme or in the control animals. Furthermore, retrograde labeling of cells in this region was not observed when the HRP injection was confined to the orbital cavity, although in these cases labeled cell bodies were identified in the extraocular motor nuclei. Thus the peroxidase-positive labeling within the ventro-lateral thalamus cannot be attributed to retrograde transport of the enzyme from either the orbital musculature or any gland within the orbit. Moreover, it would appear not to result from HRP transport from regions lacking a blood-brain barrier. Lastly, the results exclude the presence of endogenous proteins susceptible to giving a peroxidase-positive reaction within this thalamic field. This labeled thalamic cell field in Vipera aspis would thus appear to represent the origin of a true retinopetal system. From many points of view, this nucleus is comparable to that identified by Halpern et alp in the snake Thamnophis using the HRP method and referred to as the nucleus of the ventral supraoptic commissure. Considering that this nucleus represents the origin of the retinopetal pathway in ophidians and that no link between this nucleus and the ventral supraoptic decussation has as yet been demonstrated experimentally, it would seem
4~o~
439 preferable to refer to this nucleus in snakes as the centrifugal optic thalamic nucleus. [3H]Adenosine studies. Four snakes were given unilateral eye injection of 2,8[3H] adenosine (30 #Ci in 5/A, NEN, spec. act. 30.5 Ci/mmol. The vipers were kept for 24-96 h at 22-24 °C and then perfused through the single carotid with 1 0 ~ formol saline. The brains were embedded in paraffin and sectioned serially at 12 /~m in the frontal plane. Sections were coated with K 5 llford emulsion and stored in light-tight boxes at 4 °C for a period of 4-5 weeks. They were then developed in D 19, fixed in Kodak hypofixer and counterstained with cresyl violet. Silver grains were found throughout the primary visual system (Fig. 2A) and particularly in the contralateral optic centers previously identified using either the Fink-Heimer technique 18 or following intraocular injection of tritiated amino acids21 : nuclei ovalis, geniculatus lateralis pars dorsalis and pars ventralis, geniculatus pretectalis, griseus tectalis, lentiformis mesencephali, posterodorsalis, opticus tegmenti, and the superficial layers of the tectum. The grain density was however considerably lower than that seen after intraocular injection of [aH]proline. Elsewhere neurons of the centrifugal optic thalamic nucleus showed heavy labeling (Fig. 2B and C) bilaterally although more so on the contralateral side. Cell counts generated a total of 660 labeled neurons of which 82.5 ~ were to be found in the nucleus contralateral to the injection. These proportions are comparable to those obtained with counts of peroxidase-posirive neurons after intraocular injection of the enzyme. No other adenosin heavily labeled cells were detected in any other brain regions. The anterograde transport of adenosine material is at present a well established phenomenon11,13,~4,~8, 29,~°. However, its retrograde transport has been contested in several reports13,24, 25. As a matter of fact, the adenosine neuronal labeling was initially interpreted as arising solely from transneuronal transport24,2L Although the occurrence of this type of labeling was subsequently confirmed (notably using long survival periods) in different systems11,22, 80, other investigations have clearly demonstrated that adenosine labeling could more simply result from retrograde axonal transport of the nucleoside compounds 11,26,30. The present results are in good agreement with the latter data. Whether using either the H R P or the adenosine methods, the area of distribution of labeled ipsilateral and contralateral neurons was essentially the same. Moreover, following intraocular injection of [3H]adenosine heavily labeled cells were localized exclusively within the centrifugal optic thalamic nucleus. If such labeling were due to transynaptic leakage of adenoside material from retinal endings, then labeled neurons should also have been observed particularly among the neuronal
Fig. 2. A: dark-field micrograph showing labeling in the primary visual system (GL, nucleus geniculatus lateralis; TOM, tractus opticus marginalis) and neurons (arrow) at the origin of the centrifugal visual system (COTN, centrifugal optic thalamus nucleus) in the rostral thalamus as observed 30 h after contralateral ocular injection of [all]adenosine. × 125. B: light-field micrograph sbowing labeling of neurons (arrow) at the origin of the centrifugal visual system (COTN, centrifugal optic thalamic nucleus) in the posterior thalamus as observed 72 h after contralateral intraocular injections of [all]adenosine. GL, nucleus geniculatus lateralis; TOM, tractus opticus marginalis; BTP, telencephalic basal peduncle. × 125. C: higher magnification ( × 800) of labeled neurons of the centrifugal optic thalamic nucleus, 72 h after intraocular injection of [3Hladenosine.
440 elements which are p o s t s y n a p t i c to the optic fibers. I n conclusion the present d a t a are in accord with those o b t a i n e d e m p l o y i n g the H R P m e t h o d a n d thus confirming the existence o f a centrifugal visual system in the viper. I n m o s t birds (see ref. 1), in the c r o c o d i l i a n Caiman erocodilus 7, the turtle Pseudemys scripta (Cervetto, referred to in ref. 15) a n d some lizards such as Gerrhonotus coeroleus coeruleus 9, the nucleus o f origin o f centrifugal fibers is situated in the mesencephalic t e g m e n t u m a n d m o r e precisely in the isthmic region. By contrast, in Vipera, as in the snakes e x a m i n e d by H a l p e r n et alP, the centrifugal optic nucleus is located within the ventral thalamus. This a r r a n g e m e n t does n o t a p p e a r to be a characteristic o f snakes as the latter investigators have also identified a centrifugal optic nucleus in the d i e n c e p h a l o n a d j a c e n t to the optic tract in the lizard Cordylus cordylus. Despite such t o p o g r a p h i c a l interspecies variations, preliminary studies r e g a r d i n g the a n a t o m i c a l connections o f this nucleus p e r f o r m e d on a n u m b e r o f species indicate several similarities. F o r example, in the pigeon 1,4, the crocodile 7, a n d in the snakes T h a m n o p h i s ~8 a n d Vipera (unpublished observations), the centrifugal optic nucleus receives afferents f r o m the optic tectum. However, the c o m p a r a t i v e d a t a regarding the centrifugal p a t h w a y o f s a u r o p s i d i a n s based on the criteria o f connectivity, ultrastructure a n d function are at present t o o few to p e r m i t any affirmation t h a t the systems which are involved are h o m o l o g o u s . W e would like to t h a n k F. R o g e r a n d C. Pages for excellent technical s u p p o r t , D. Le Cren for skillful p h o t o g r a p h i c assistance a n d S. A r n o l d for secretarial help. This w o r k was p a r t i a l l y s u p p o r t e d by I N S E R M , C N R S a n d D G R S T .
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