- Email: [email protected]
A new tectal afferent nucleus of the infrared sensory system in the medulla oblongata of crotaline snakes
Brain Research, 195 (1980) 271-279 © Elsevier/North-Holland Biomedical Press
271
A NEW T E C T A L A F F E R E N T N U C L E U S OF T H E I N F R A R E D SENSORY SYSTEM IN T H E M E D U L L A O B L O N G A T A OF C R O T A L I N E SNAKES
REUI KISHIDA, FUMIAKI AMEMIYA, TOYOKAZU KUSUNOKI and SHIN-ICHI TERASHIMA Department of Anatomy, Yokohama City University School of Medicine, Yokohama and (S. T.) Department of Physiology, Tokyo Medical and Dental University School of Medicine, Tokyo (Japan)
(Accepted February 14th, 1980) Key words: tectal afferent - - new nucleus - - infrared sensory - - Crotaline snakes --horseradish
peroxidase - - histological comparison -- electrophysiology
SUMMARY The existence of an infrared sensory neuron group with ascending fibers which directly reach the optic tectum in Crotaline snakes was confirmed with three methods. (1) With the retrograde horseradish peroxidase (HRP) method, labeled neurons were not found within the nucleus descendens lateralis nervi trigemini (DLV), but in an unnamed cell group located immediately ventral to the DLV of the contralateral side at the transitional portion between the nucleus oralis (DVo) and the nucleus interpolaris (DVi). This unnamed cell group, which was seen only in the Crotalinae, was provisionally called the 'new nucleus'. (2) Normal brain series of 15 species were stained by the methods of Bodian-Otsuka, Klfiver-Barrera and Nissl staining to compare the cytoarchitecture of the medulla oblongata. The 'new nucleus' was found only in species belonging to the Crotalinae. This nucleus was situated in fiber tracts which appeared to correspond to the lemniscus spinalis and tractus spino-cerebellaris of the reptilian medulla oblongata, and contained medium-sized multipolar or fusiform neurons. (3) In an electrophysiological study 16 single units responding urtimodally to an infrared stimulus were recorded. Three of these recording sites were determined with Pontamine sky blue marking to be near or within the 'new nucleus'.
INTRODUCTION The optic tectum of all vertebrates receives fibers not only from the retina but from the non-optic exteroceptive nuclei as well 17. In non-mammalian vertebrates, the volume of visual and non-optic exteroceptive afferent fibers projecting to the optic tectum is intimately correlated with the life habits of the speciesS, 10.
272 The optic tectum of boas and pythons (Boidae) and of pit vipers (Crotalinae), both groups which can detect prey with infrared information, is an integrative center of mainly infrared and visual input4,.~,7,19. The first relay nucleus of the infrared sensory system in the central nervous system of the Boidae and Crotalinae is the nucleus descendens lateralis nervi trigemini (DLV) 12,14,1s. Molenaar reported that the neurons of the DLV projected to the contralateral optic tectum in Python reticulatus (Boidae) H, but Hartline et al., using tectal injection of HRP, found no evidence of a direct projection from the DLV to the optic tectum in rattlesnakes (CrotalinaeJ.L We attempted to clarify whether a direct ascending pathway from the DLV to the optic tectum exists, and if not, to discover where the relay nucleus of the infraredsensory system is located and whether that nucleus is present in all snakes possessing infrared receptors. MATERIALS AND METHODS Three different methods were employed to pursue our present goal: (!) H RP methods applied to the optic tectum, (2) histological comparison of the normal brain stems stained by Bodian-Otsuka, Klfiver-Barrera and Nissl methods, and (3) electrophysiological recording.
HRP method The experiments in the H R P study were performed on two subspecies of pit vipers (5 Agkistrodon blomhoffi blomhoffi and 10 Agkistrodon blomhoffi brevieaudus, all measuring about 0.6 m in total length) and one species possessing neither infrared receptors nor the DLV (8 Elaphe quadrivirgata, measuring about 1.2 m in total length). Snakes anesthetized with halothane or ethyl ether were fastened to boards with adhesive tape and injected with tubocurarine at a dose of 1-2 mg/kg of body weight for immobilization during the experiment. The snakes were given artificial respiration throughout the experiment. Brains were exposed with a dental drill and 0.2-0.5 /~1 of saline solution (0.75%) containing 35% HRP was injected with a microsyringe driven by a micromotor for 30 rain into the left optic tectum under visual guidance. Two to five days after the injection, the animals were perfused from the right aortic arch with 0.75 % saline solution containing 5-10 I.U. heparin/ml followed by a mixed solution of 1.6 or 4 % paraformaldehyde and 10 or 5 % glutaraldehyde in 0. ! M phosphate buffer (pH 7.4). The brains were removed from the skull, postfixed in the same fixative for 5-12 h and rinsed for 12-24 h in 0.1 M phosphate buffer (pH 7.4) containing 5 % sucrose. After embedding in Ames O. C. T. Compound, frontal serial sections were cut at 40 #m with a cryostat (--11 °C), and mounted alternatively on two series of slides which had previously been dipped into a 2-3 % gelatin solution and dried. After mounting, sections were dried with a fan at room temperature for 2 h, fixed again in the same fixative for 10 min, rinsed for 10 min in 0.2 M phosphate buffer (pH 7.4), and reacted with 3,3'-diaminobenzidine 9 or benzidine dihydrochloride 11. Alternate slides were counterstained with cresyl violet for histological identification.
273 TABLE I C o m p a r i s o n o f the p r e s e n c e ( 4 - ) or a b s e n c e ( - - ) o f p i t o r g a n s ( P i t s ) , the n u c l e u s d e s c e n d e n s lateralis n. t r i g e m i n i ( D L V) a n d the ' n e w nucleus' ( N e w nucL ) in 15 species o f s n a k e s
Note that the 'new nucleus' is present only in species belonging to the Crotalinae. Underorder
Family
Subfamily
Species
Pits
DLV
New nucl.
Henophidia
Boidae
Pythoninae
Python molurus P y t h o n spilotes E l a p h e quadrivirgata Elaphe climacophora E l a p h e conspicillata Elaphe rulbdorsata R a b d o p h i s tigrinus Xenochrophis piscator Vipera aspis Vipera a m m o d y t e s Agkistrodon blomhoffi blomhoffi Agkistrodon blomhoffi brevicaudus d g k i s t r o d o n caliginosus Trimeresurus flavoviridis S i s t r u r u s miliarius
+ + --------+ ÷ 444-
+ + --------qJr 444-
----------+ + 444-
Caenophidia Colubridae
Colubrinae
Natricinae Viperidae
Viperinae Crotalinae
Methods of Bodian-Otsuka, Kliiver-Barrera and Nissl Additional normal brain series were made of 15 species of snakes (Table I), which were perfused from the right aortic arch with 0.75 ~ saline solution containing 5-10 I.U. heparin/ml followed by a mixed solution of Bodian II or 10~o formalin under halothane or ethyl ether anesthesia. Brains were embedded in paraffin and sectioned serially at 15/zm in the transverse, parasagittal, or horizontal plane. The sections were stained by the methods of Bodian-Otsuka 15, KKiver-Barrera, or Nissl staining to compare the cytoarchitecture of the medulla oblongata.
Electrophysiological recording and marking Thirteen Agkistrodon blomhoffi brevicaudus were anesthetized with the same method as in the H R P study. A hole was drilled in the skull to expose the medulla oblongata through the inner ear at the posterior border of the VIII cranial nerve. Glass microelectrodes filled with 2 ~ Pontamine sky blue 6 in 2 M NaC1 solution were used for extracellular recording and marking (resistance 40 Mf~, tip diameter 0.5 /~m). Recording equipment was conventional. The recording site was marked using a current of 0.2/~A for 10 min with the electrode negative. RESULTS
HRP study The H R P was found to be confined within the left optic tectum and extended through all tectal layers (Figs. la and 2a). Labeled neurons were not found within the DLV (Fig. 2e), but were found in an unnamed cell group (Figs. 2c and d) located
( <;oo;
~'( • "L>.
tr,~., '~ "
Ac~
-,
IIII
k2. U ,'~',
~h(£
1,,".~...X/ .L/_tv~.....; "
],.~x
.~
J "-"., .-L l~
~ Y
,
19
~'LE
275 immediately ventral to the nucleus descendens nervi trigemini o f the contralateral side at the level o f the transitional portion between the nucleus oralis (DVo) and the nucleus interpolaris (DVi). This u n n a m e d cell group, which was seen only in pit vipers, will be provisionally called the 'new nucleus' in this paper (Figs. 2c, d, 3 ,4 and 5a). Other labeled neurons within the medullary regions were found within 3 cell groups in all animals studied: the bilateral reticular nucleus (R) at a level between the DVi and the midbrain, the contralateral nucleus vestibularis ventromedialis (VVM), and the contralateral D V o (Figs. l b - d and 2b-d). Only a few small neurons (10-20 #m) were labeled in these nuclei. Ascending fibers f r o m the 'new nucleus' immediately decussate, ascend in the lemniscus spinalis o f Molenaar 13, and invade lamina 7 of R a m 6 n TM (sublayers 7a and 7b of Butlar and Ebbesson 2) o f the optic tectum (Figs. 2a-d). Other labeled fibers from the medullary region to the optic tectum were distinguished in 3 fiber tracts in all animals studied: one which decussates at the level of the transitional portion between the D V o and DVi, ascends in the lemniscus spinalis of Molenaar 13, and invades lamina 7 o f Ram6nl~; a second which ascends in the ipsilateral tractus mesencephalicus nervi trigemini and enters lamina 6 of R a m 6 n 16; and a third which ascends in the tractus tecto-bulbo-spinalis dorsalis o f Molenaar 18 of the opposite side, along the ventral side of the fasciculus longitudinalis medialis (tim), and enters lamina 6 o f R a m 6 n ~6 after a decussation near the optic tectum.
Histological comparison The 'new nucleus' was found only in species belonging to the subfamily Crotalinae (Table I). This nucleus was situated in the fiber tracts which appeared to correspond to the lemniscus spinalis and tractus spino-cerebellaris o f previous reports on reptilian medulla oblongatal,3,13, and contained medium-sized (20-30 /~m in diameter) multipolar or fusiform neurons (Figs. 3 and 4). In most cases, the D L V was two or more times larger and more demarcated than the 'new nucleus', although the cell bodies were deeper stained by Kl~iver-Barrera and Nissl in the 'new nucleus' than in the D L V in the same series o f preparations. Figs. 1 and 2. Semi-schematic drawings of transverse sections through the brain stem of 1, Elaphe quadrivirgata, and 2, Agkistrodon blomhqlfiblomhoffi, showing representative sites of HRP injection into the optic tectum and the distribution pattern of HRP-labeled medullary cells and fibers, indicated by triangles and dashes respectively, a: at the level of the optic tectum, b: at the level of the trigeminal root. c: at the level of the VIIIth nerve root. d: just caudal to the VIIIth nerve root. e: at the level of the DVc. Labeled fibers were distinguished in three fiber tracts (1, 2 and 3). Labeled neurons within the 'new nucleus' were found only in the Crotalinae studied, and in these species labeled neurons were not found within the DLV. Other labeled neurons in all species studied were found in three medullary cell groups: the bilateral R, the contralateral DVo, and the contralateral VVM. 6, lamina 6; 7a, lamina 7a; 7b, lamina 7b. Abbreviations in these and subsequent figures: C, nucleus cochlearis; CE, cerebellum; DLV, nucleus descendens lateralis n. trigemini; DVo, nucleus oralis of the nucleus descendens n. trigemini; DVi, nucleus interpolaris of the nucleus descendens n. trigemini; DVc, nucleus caudalis of the nucleus descendens n. trigemini; IP, nucleus interpeduncularis; M, nucleus motorius n. trigemini ; 'N', new nucleus; OT, optic tectum; PrV, nucleus sensorius principalis n. trigemini; R, nucleus reticularis; VVM, nucleus vestibularis ventromedialis; dlv, tractus descendens lateralis n. trigemini; dv, tractus descendens n. trigemini; tim, fasciculus longitudinalis medialis; V, root of the Vth nerve; VIII root of the VIIIth nerve.
276
Fig. 3. a: transverse section of the HRP-labeled cell group in the 'new nucleus' contralateral to an injection side in the optic tectum of Agkistrodon blomhoffibrevicaudus. ?' 130. b : high power view of the same labeled cell group. × 400. Fig. 4. a: transverse section through the 'new nucleus" of Agkistrodon blomhoffibrevicaudusstained by the Kl0ver-Barrera method, x 65. 1, tractus descendens n. trigemini and tractus descendens lateralis n. trigemini; 2, nucleus oralis of the nucleus descendens n. trigemini; 3, 'new nucleus', b: high power view of the 'new nucleus' of the same section, x 400. F u r t h e r m o r e , the capillary network a n d the well convoluted neuropil were more developed in the DLV t h a n in the 'new nucleus'. But we could n o t find any difference in cell configurations between these two nuclei by the three methods used. The rostral pole of the DLV was situated caudal to the 'new nucleus' at the level of the nucleus caudalis of the nucleus descendens nervi trigemini (DVc) in the subfamily Crotalinae, b u t in Python molurus a n d Python spilotus the rostral pole of the DLV was located more rostral, at the level of the caudal part of the DVo, w i t h o u t the 'new nucleus'.
Electrophysiological stud). We were able to record 16 infrared-sensitive single units (Fig. 5b) from the location of the 'new nucleus' in 13 Crotaline snakes. W h e n single unit activity was recorded, its infrared-sensitive character was first tested by waving a h a n d 20 cm from the pit. These units responded well to a moving h a n d a n d also to a laser beam zl. The
277
. 1,,
A
I 1_1 11 l l l t l l l l l l l l l [ I l l ~ l •
~
g
v i i i
- - - v l v l v ~ - ~ v l ~
[ |
I
5b Fig. 5. a: left, transverse section stained with cresylviolet showinga lesion (arrow) made after recording the infrared response; right, a drawing showing the individual locations of three lesions (triangles) reconstructed from 3 Agkistrodon blomhoffi brevicaudus. × 20. Note that all 3 lesions were within or near the 'new nucleus', b: response of an infrared-sensitive single unit to moderate stimulation at room temperature of 23.3 °C. Lower trace indicates duration of stimulus (raised portion). Vertical bar indicates 2 mV and horizontal bar 200 msee. response to a moving hand was unchanged in light and complete darkness. This fact excludes the possibility of visual stimulus acting on the retina or light stimulus acting on the infrared receptor. Other possible stimuli were also tested, such as vibration (tapping the table on which the experimental animal was lying), sound (clapping the hands), and touch on the face (with cotton fibers). Infrared units did not respond to any of these stimuli. It is unlikely that the recordings were from the primary afferent fibers which run in the neighborhood, because the receptive areas were larger than those of primary fibers 22, although the size was not measured precisely. The sensitivity to a moving laser spot in these receptive areas was not homogeneous, in contrast to that of other central units 2°,21. One of these units was found to respond with directional selectivity to a laser spot moving on the pit membrane. These units in the 'new nucleus' seemed to be functionally heterogeneous. When the infrared nature of a unit had been established, the recording site was marked with Pontamine sky blue 6. One single unit was recorded from each of the 3 snakes and the 3 sites were marked (Fig. 5a). The lesions were all within or near the 'new nucleus'.
278 DISCUSSION Kass et al. 7, in an electrophysiological study, reported that infrared-sensitive cells were found in lamina 7 of Ram6n 16 throughout the optic tectum of pit vipers". We have been able to record infrared-sensitive activity from a 'new nucleus' in two subspecies of pit vipers. H R P studies show that this nucleus has ascending fibers which invade lamina 7 of Ram6n 16 in the contralateral optic tectum. Therefore we can safely conclude that in the Crotalinae the 'new nucleus' is a relay nucleus sending infrared information to the optic tectum. Although the present H R P study showed only contralateral projection from the 'new nucleus' to the optic tectum, Goris and Terashima reported that whereas most units responded only to contralateral stimulation, some responded to both ipsi- and contralateral stimulation 4. HRP was injected into the central region of the optic tectum in the present study, so it is possible that some parts of the marginal tectal region may also receive ipsilateral projection from the 'new nucleus'. The fact that the 'new nucleus' exists only in the Crotalinae and not in the Boidae seems to be related to species differences. The Boidae, belonging to the underorder Henophidia, possess more generalized infrared receptors than the Crotalinae, beloning to the underorder Caenophidia. The Crotalinae seems to be more specialized than the Boidae not only in the peripheral but also in the central infrared sensory nervous system, although we had only two examples from the subfamily Pyhoninae of the Boidae for comparative histological studies. This hypothesis could explain the apparent contradiction in the reports of Molenaar and Hartline et al. Using a degeneration method, Molenaar reported that neurons of the DLV projected to the contralateral optic tectum in Python reticulatus (Boidae) 14, but Hartline et al., using tectal injection of HRP, found no evidence of a direct projection from the DLV to the optic tectum in rattlesnakes (Crotatinae) 5. We believe that this is not due to the difference in the investigation techniques. The 'new nucleus' is not so demarcated from the surrounding reticular formation in normal Nissl preparations. However the 'new nucleus' might be highly differentiated in physiological function from the surrounding brain tissue, because we have observed strong histochemical activity of succinate dehydrogenase in the perikarya of this nucleus, and there is high contrast to the surrounding tissue (unpublished datum). We could not determine whether the 'new nucleus' is of secondary or higher order as a relay nucleus of the infrared sensory system in the central nervous system of Crotalinae. However, any additional relay nucleus between the DLV and the 'new nucleus' is unthinkable, because there is a direct projection from the DLV to the optic tectum in Boidae ~4. Further work is needed to make clear the afferent and efferent fiber connections of the 'new nucleus'. ACKNOWLEDGEMENTS We wish to thank Dr. Richard C. Goris for editing the manuscript. We express our thanks to Mr. Mikio Takaoka for collecting snakes and assistance, and to Miss
279 M a s a m i Y o s h i m o t o for her excellent help in preparing the histological materials a n d illustrations. A p a r t of this research was supported by G r a n t 477043 for scientific research from the Ministry of E d u c a t i o n of Japan.
REFERENCES 1 Anthony, J., Le n6vraxe des reptiles. In P. P. Grass6 (Ed.), Traitdde Zoologic, Reptiles, Tom XIV, Masson, Paris, 1970, pp. 202-332. 2 Butler, A. B. and Ebbesson, S. O. E., A Golgi study of the optic tectum of the Tegu lizard, Tupinambis nigropunctatus, J. Morph., 146 (1975) 215-228. 3 Ebbesson, S. O. E., Ascending axon degeneration following hemisection of the spinal cord in the Tegu lizard (Tupinambis nigropunctatus), Brain Research, 5 (1967) 178-206. 4 Goris, R. C. and Terashima, S., Central response to infrared stimulation of the pit receptors in Crotaline snake, Trimeresurusflavoviridis, J. exp. BioL, 58 (1973) 59-76. 5 Hartline, P. H., Kass, L. and Loop, M. S., Merging of modalities in the optic rectum: infrared and visual integration in rattlesnakes, Science, 199 (1978) 1125-1129. 6 HeUon, R. F., The marking of electrode tip positions in nervous tissue, J. Physiol. (Lond.), 214 (1971) 12P. 7 Kass, L., Loop, M. S. and Hartline, P. H., Anatomical and physiological localization of visual and infrared cell layers in the tectum of pit viper, J. eomp. Neurol., 182 (1978) 811-820. 8 Kishida, R., Comparative study on the teleostean optic rectum. Lamination and Cytoarchitecture, J. Hirnforseh., 20 (1979) 57-67. 9 Luiten, P. G. M., The horseradish peroxidase technique applied to the teleostean nervous system, Brain Research, 89 (1975) 181-186. 10 Masai, H., Structural patterns of the optic tectum in Japanese snakes of the family Colubridae, in relation to habit, J. Hirnforseh., 14 (1973) 367-374. 11 Mesulam, M.-M., The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility, J. Histochem. Cytoehem., 24 (1976) 1273-1280. 12 Molenaar, G. J., An additional trigeminal system in certain snakes possessing infrared receptors, Brain Research, 78 (1974) 340-344. 13 M••enaar• G. J.• The rh•mbencepha••n •f Pyth•n reti•ulatus• a snake p•ssessing infrared recept•rs• Neth. J. Zool., 27 (1977) 133-180. 14 Molenaar, G. J., The sensory trigeminal system of a snake in the possession of infrared receptors. II. The central projection of the trigeminal nerve, J. comp. Neurol., 179 (1978) 137-152. 15 Otsuka, N., Miyanaga, A., Tanaka, F. and Kimura, A., Neue Silberimpr~ignationsversuche zur Darstellung der Neurofibrillen an Paraffinschnitten, J. Kyoto Pref Univ. Med., 68 (1960) 1125-1128. 16 Ram6n, P., Estructura del encefalo del Camele6n, Rev. trimest. Micrograf, 1 (1896) 46-82. 17 Sarnat, H. B. and Netsky, M. G., Evolution of the Nervous System, Oxford Univ. Press, New York, 1974, pp. 162-164. 18 Schroeder, D. M. and Loop, M. S., Trigeminal projections in snakes possessing infrared sensitivity, J. comp. Neurol., 169 (1976) 1-14. 19 Terashima, S. and Goris, R. C., Tectal organization of pit viper infrared reception, Brain Research, 83 (1975) 490~94. 20 Terashima, S. and Goris, R. C., Receptive area of an infrared tectal unit, Brain Research, 101 (1976) 155-159. 21 Terashima, S. and Goris, R. C., Infrared bulbar units in Crotaline snakes, Proc. Jap. Acad. B, 53 (1977) 292-296. 22 Terashima, S. and Goris, R. C., Receptive area of primary infrared afferent neurons in crotaline snakes, Neuroseience, 4 (1979) 1137-1144. NOTE ADDED IN PROOF After preparation of this manuscript, a report by Gruberg et al. appeared in J. comp. Neurol., 188 (1979) 31-42, entitled 'Connections of the tectum of the rattlesnake Crotalus viridis: an HRP study', in which the nucleus reticularis caloris projects to the optic tectum. The 'new nucleus' of our report may be the same as their nucleus reticularis caloris, since its location in the medulla oblongata and the appearance of their Fig. 4 are very similar to our findings.
271
A NEW T E C T A L A F F E R E N T N U C L E U S OF T H E I N F R A R E D SENSORY SYSTEM IN T H E M E D U L L A O B L O N G A T A OF C R O T A L I N E SNAKES
REUI KISHIDA, FUMIAKI AMEMIYA, TOYOKAZU KUSUNOKI and SHIN-ICHI TERASHIMA Department of Anatomy, Yokohama City University School of Medicine, Yokohama and (S. T.) Department of Physiology, Tokyo Medical and Dental University School of Medicine, Tokyo (Japan)
(Accepted February 14th, 1980) Key words: tectal afferent - - new nucleus - - infrared sensory - - Crotaline snakes --horseradish
peroxidase - - histological comparison -- electrophysiology
SUMMARY The existence of an infrared sensory neuron group with ascending fibers which directly reach the optic tectum in Crotaline snakes was confirmed with three methods. (1) With the retrograde horseradish peroxidase (HRP) method, labeled neurons were not found within the nucleus descendens lateralis nervi trigemini (DLV), but in an unnamed cell group located immediately ventral to the DLV of the contralateral side at the transitional portion between the nucleus oralis (DVo) and the nucleus interpolaris (DVi). This unnamed cell group, which was seen only in the Crotalinae, was provisionally called the 'new nucleus'. (2) Normal brain series of 15 species were stained by the methods of Bodian-Otsuka, Klfiver-Barrera and Nissl staining to compare the cytoarchitecture of the medulla oblongata. The 'new nucleus' was found only in species belonging to the Crotalinae. This nucleus was situated in fiber tracts which appeared to correspond to the lemniscus spinalis and tractus spino-cerebellaris of the reptilian medulla oblongata, and contained medium-sized multipolar or fusiform neurons. (3) In an electrophysiological study 16 single units responding urtimodally to an infrared stimulus were recorded. Three of these recording sites were determined with Pontamine sky blue marking to be near or within the 'new nucleus'.
INTRODUCTION The optic tectum of all vertebrates receives fibers not only from the retina but from the non-optic exteroceptive nuclei as well 17. In non-mammalian vertebrates, the volume of visual and non-optic exteroceptive afferent fibers projecting to the optic tectum is intimately correlated with the life habits of the speciesS, 10.
272 The optic tectum of boas and pythons (Boidae) and of pit vipers (Crotalinae), both groups which can detect prey with infrared information, is an integrative center of mainly infrared and visual input4,.~,7,19. The first relay nucleus of the infrared sensory system in the central nervous system of the Boidae and Crotalinae is the nucleus descendens lateralis nervi trigemini (DLV) 12,14,1s. Molenaar reported that the neurons of the DLV projected to the contralateral optic tectum in Python reticulatus (Boidae) H, but Hartline et al., using tectal injection of HRP, found no evidence of a direct projection from the DLV to the optic tectum in rattlesnakes (CrotalinaeJ.L We attempted to clarify whether a direct ascending pathway from the DLV to the optic tectum exists, and if not, to discover where the relay nucleus of the infraredsensory system is located and whether that nucleus is present in all snakes possessing infrared receptors. MATERIALS AND METHODS Three different methods were employed to pursue our present goal: (!) H RP methods applied to the optic tectum, (2) histological comparison of the normal brain stems stained by Bodian-Otsuka, Klfiver-Barrera and Nissl methods, and (3) electrophysiological recording.
HRP method The experiments in the H R P study were performed on two subspecies of pit vipers (5 Agkistrodon blomhoffi blomhoffi and 10 Agkistrodon blomhoffi brevieaudus, all measuring about 0.6 m in total length) and one species possessing neither infrared receptors nor the DLV (8 Elaphe quadrivirgata, measuring about 1.2 m in total length). Snakes anesthetized with halothane or ethyl ether were fastened to boards with adhesive tape and injected with tubocurarine at a dose of 1-2 mg/kg of body weight for immobilization during the experiment. The snakes were given artificial respiration throughout the experiment. Brains were exposed with a dental drill and 0.2-0.5 /~1 of saline solution (0.75%) containing 35% HRP was injected with a microsyringe driven by a micromotor for 30 rain into the left optic tectum under visual guidance. Two to five days after the injection, the animals were perfused from the right aortic arch with 0.75 % saline solution containing 5-10 I.U. heparin/ml followed by a mixed solution of 1.6 or 4 % paraformaldehyde and 10 or 5 % glutaraldehyde in 0. ! M phosphate buffer (pH 7.4). The brains were removed from the skull, postfixed in the same fixative for 5-12 h and rinsed for 12-24 h in 0.1 M phosphate buffer (pH 7.4) containing 5 % sucrose. After embedding in Ames O. C. T. Compound, frontal serial sections were cut at 40 #m with a cryostat (--11 °C), and mounted alternatively on two series of slides which had previously been dipped into a 2-3 % gelatin solution and dried. After mounting, sections were dried with a fan at room temperature for 2 h, fixed again in the same fixative for 10 min, rinsed for 10 min in 0.2 M phosphate buffer (pH 7.4), and reacted with 3,3'-diaminobenzidine 9 or benzidine dihydrochloride 11. Alternate slides were counterstained with cresyl violet for histological identification.
273 TABLE I C o m p a r i s o n o f the p r e s e n c e ( 4 - ) or a b s e n c e ( - - ) o f p i t o r g a n s ( P i t s ) , the n u c l e u s d e s c e n d e n s lateralis n. t r i g e m i n i ( D L V) a n d the ' n e w nucleus' ( N e w nucL ) in 15 species o f s n a k e s
Note that the 'new nucleus' is present only in species belonging to the Crotalinae. Underorder
Family
Subfamily
Species
Pits
DLV
New nucl.
Henophidia
Boidae
Pythoninae
Python molurus P y t h o n spilotes E l a p h e quadrivirgata Elaphe climacophora E l a p h e conspicillata Elaphe rulbdorsata R a b d o p h i s tigrinus Xenochrophis piscator Vipera aspis Vipera a m m o d y t e s Agkistrodon blomhoffi blomhoffi Agkistrodon blomhoffi brevicaudus d g k i s t r o d o n caliginosus Trimeresurus flavoviridis S i s t r u r u s miliarius
+ + --------+ ÷ 444-
+ + --------qJr 444-
----------+ + 444-
Caenophidia Colubridae
Colubrinae
Natricinae Viperidae
Viperinae Crotalinae
Methods of Bodian-Otsuka, Kliiver-Barrera and Nissl Additional normal brain series were made of 15 species of snakes (Table I), which were perfused from the right aortic arch with 0.75 ~ saline solution containing 5-10 I.U. heparin/ml followed by a mixed solution of Bodian II or 10~o formalin under halothane or ethyl ether anesthesia. Brains were embedded in paraffin and sectioned serially at 15/zm in the transverse, parasagittal, or horizontal plane. The sections were stained by the methods of Bodian-Otsuka 15, KKiver-Barrera, or Nissl staining to compare the cytoarchitecture of the medulla oblongata.
Electrophysiological recording and marking Thirteen Agkistrodon blomhoffi brevicaudus were anesthetized with the same method as in the H R P study. A hole was drilled in the skull to expose the medulla oblongata through the inner ear at the posterior border of the VIII cranial nerve. Glass microelectrodes filled with 2 ~ Pontamine sky blue 6 in 2 M NaC1 solution were used for extracellular recording and marking (resistance 40 Mf~, tip diameter 0.5 /~m). Recording equipment was conventional. The recording site was marked using a current of 0.2/~A for 10 min with the electrode negative. RESULTS
HRP study The H R P was found to be confined within the left optic tectum and extended through all tectal layers (Figs. la and 2a). Labeled neurons were not found within the DLV (Fig. 2e), but were found in an unnamed cell group (Figs. 2c and d) located
( <;oo;
~'( • "L>.
tr,~., '~ "
Ac~
-,
IIII
k2. U ,'~',
~h(£
1,,".~...X/ .L/_tv~.....; "
],.~x
.~
J "-"., .-L l~
~ Y
,
19
~'LE
275 immediately ventral to the nucleus descendens nervi trigemini o f the contralateral side at the level o f the transitional portion between the nucleus oralis (DVo) and the nucleus interpolaris (DVi). This u n n a m e d cell group, which was seen only in pit vipers, will be provisionally called the 'new nucleus' in this paper (Figs. 2c, d, 3 ,4 and 5a). Other labeled neurons within the medullary regions were found within 3 cell groups in all animals studied: the bilateral reticular nucleus (R) at a level between the DVi and the midbrain, the contralateral nucleus vestibularis ventromedialis (VVM), and the contralateral D V o (Figs. l b - d and 2b-d). Only a few small neurons (10-20 #m) were labeled in these nuclei. Ascending fibers f r o m the 'new nucleus' immediately decussate, ascend in the lemniscus spinalis o f Molenaar 13, and invade lamina 7 of R a m 6 n TM (sublayers 7a and 7b of Butlar and Ebbesson 2) o f the optic tectum (Figs. 2a-d). Other labeled fibers from the medullary region to the optic tectum were distinguished in 3 fiber tracts in all animals studied: one which decussates at the level of the transitional portion between the D V o and DVi, ascends in the lemniscus spinalis of Molenaar 13, and invades lamina 7 o f Ram6nl~; a second which ascends in the ipsilateral tractus mesencephalicus nervi trigemini and enters lamina 6 of R a m 6 n 16; and a third which ascends in the tractus tecto-bulbo-spinalis dorsalis o f Molenaar 18 of the opposite side, along the ventral side of the fasciculus longitudinalis medialis (tim), and enters lamina 6 o f R a m 6 n ~6 after a decussation near the optic tectum.
Histological comparison The 'new nucleus' was found only in species belonging to the subfamily Crotalinae (Table I). This nucleus was situated in the fiber tracts which appeared to correspond to the lemniscus spinalis and tractus spino-cerebellaris o f previous reports on reptilian medulla oblongatal,3,13, and contained medium-sized (20-30 /~m in diameter) multipolar or fusiform neurons (Figs. 3 and 4). In most cases, the D L V was two or more times larger and more demarcated than the 'new nucleus', although the cell bodies were deeper stained by Kl~iver-Barrera and Nissl in the 'new nucleus' than in the D L V in the same series o f preparations. Figs. 1 and 2. Semi-schematic drawings of transverse sections through the brain stem of 1, Elaphe quadrivirgata, and 2, Agkistrodon blomhqlfiblomhoffi, showing representative sites of HRP injection into the optic tectum and the distribution pattern of HRP-labeled medullary cells and fibers, indicated by triangles and dashes respectively, a: at the level of the optic tectum, b: at the level of the trigeminal root. c: at the level of the VIIIth nerve root. d: just caudal to the VIIIth nerve root. e: at the level of the DVc. Labeled fibers were distinguished in three fiber tracts (1, 2 and 3). Labeled neurons within the 'new nucleus' were found only in the Crotalinae studied, and in these species labeled neurons were not found within the DLV. Other labeled neurons in all species studied were found in three medullary cell groups: the bilateral R, the contralateral DVo, and the contralateral VVM. 6, lamina 6; 7a, lamina 7a; 7b, lamina 7b. Abbreviations in these and subsequent figures: C, nucleus cochlearis; CE, cerebellum; DLV, nucleus descendens lateralis n. trigemini; DVo, nucleus oralis of the nucleus descendens n. trigemini; DVi, nucleus interpolaris of the nucleus descendens n. trigemini; DVc, nucleus caudalis of the nucleus descendens n. trigemini; IP, nucleus interpeduncularis; M, nucleus motorius n. trigemini ; 'N', new nucleus; OT, optic tectum; PrV, nucleus sensorius principalis n. trigemini; R, nucleus reticularis; VVM, nucleus vestibularis ventromedialis; dlv, tractus descendens lateralis n. trigemini; dv, tractus descendens n. trigemini; tim, fasciculus longitudinalis medialis; V, root of the Vth nerve; VIII root of the VIIIth nerve.
276
Fig. 3. a: transverse section of the HRP-labeled cell group in the 'new nucleus' contralateral to an injection side in the optic tectum of Agkistrodon blomhoffibrevicaudus. ?' 130. b : high power view of the same labeled cell group. × 400. Fig. 4. a: transverse section through the 'new nucleus" of Agkistrodon blomhoffibrevicaudusstained by the Kl0ver-Barrera method, x 65. 1, tractus descendens n. trigemini and tractus descendens lateralis n. trigemini; 2, nucleus oralis of the nucleus descendens n. trigemini; 3, 'new nucleus', b: high power view of the 'new nucleus' of the same section, x 400. F u r t h e r m o r e , the capillary network a n d the well convoluted neuropil were more developed in the DLV t h a n in the 'new nucleus'. But we could n o t find any difference in cell configurations between these two nuclei by the three methods used. The rostral pole of the DLV was situated caudal to the 'new nucleus' at the level of the nucleus caudalis of the nucleus descendens nervi trigemini (DVc) in the subfamily Crotalinae, b u t in Python molurus a n d Python spilotus the rostral pole of the DLV was located more rostral, at the level of the caudal part of the DVo, w i t h o u t the 'new nucleus'.
Electrophysiological stud). We were able to record 16 infrared-sensitive single units (Fig. 5b) from the location of the 'new nucleus' in 13 Crotaline snakes. W h e n single unit activity was recorded, its infrared-sensitive character was first tested by waving a h a n d 20 cm from the pit. These units responded well to a moving h a n d a n d also to a laser beam zl. The
277
. 1,,
A
I 1_1 11 l l l t l l l l l l l l l [ I l l ~ l •
~
g
v i i i
- - - v l v l v ~ - ~ v l ~
[ |
I
5b Fig. 5. a: left, transverse section stained with cresylviolet showinga lesion (arrow) made after recording the infrared response; right, a drawing showing the individual locations of three lesions (triangles) reconstructed from 3 Agkistrodon blomhoffi brevicaudus. × 20. Note that all 3 lesions were within or near the 'new nucleus', b: response of an infrared-sensitive single unit to moderate stimulation at room temperature of 23.3 °C. Lower trace indicates duration of stimulus (raised portion). Vertical bar indicates 2 mV and horizontal bar 200 msee. response to a moving hand was unchanged in light and complete darkness. This fact excludes the possibility of visual stimulus acting on the retina or light stimulus acting on the infrared receptor. Other possible stimuli were also tested, such as vibration (tapping the table on which the experimental animal was lying), sound (clapping the hands), and touch on the face (with cotton fibers). Infrared units did not respond to any of these stimuli. It is unlikely that the recordings were from the primary afferent fibers which run in the neighborhood, because the receptive areas were larger than those of primary fibers 22, although the size was not measured precisely. The sensitivity to a moving laser spot in these receptive areas was not homogeneous, in contrast to that of other central units 2°,21. One of these units was found to respond with directional selectivity to a laser spot moving on the pit membrane. These units in the 'new nucleus' seemed to be functionally heterogeneous. When the infrared nature of a unit had been established, the recording site was marked with Pontamine sky blue 6. One single unit was recorded from each of the 3 snakes and the 3 sites were marked (Fig. 5a). The lesions were all within or near the 'new nucleus'.
278 DISCUSSION Kass et al. 7, in an electrophysiological study, reported that infrared-sensitive cells were found in lamina 7 of Ram6n 16 throughout the optic tectum of pit vipers". We have been able to record infrared-sensitive activity from a 'new nucleus' in two subspecies of pit vipers. H R P studies show that this nucleus has ascending fibers which invade lamina 7 of Ram6n 16 in the contralateral optic tectum. Therefore we can safely conclude that in the Crotalinae the 'new nucleus' is a relay nucleus sending infrared information to the optic tectum. Although the present H R P study showed only contralateral projection from the 'new nucleus' to the optic tectum, Goris and Terashima reported that whereas most units responded only to contralateral stimulation, some responded to both ipsi- and contralateral stimulation 4. HRP was injected into the central region of the optic tectum in the present study, so it is possible that some parts of the marginal tectal region may also receive ipsilateral projection from the 'new nucleus'. The fact that the 'new nucleus' exists only in the Crotalinae and not in the Boidae seems to be related to species differences. The Boidae, belonging to the underorder Henophidia, possess more generalized infrared receptors than the Crotalinae, beloning to the underorder Caenophidia. The Crotalinae seems to be more specialized than the Boidae not only in the peripheral but also in the central infrared sensory nervous system, although we had only two examples from the subfamily Pyhoninae of the Boidae for comparative histological studies. This hypothesis could explain the apparent contradiction in the reports of Molenaar and Hartline et al. Using a degeneration method, Molenaar reported that neurons of the DLV projected to the contralateral optic tectum in Python reticulatus (Boidae) 14, but Hartline et al., using tectal injection of HRP, found no evidence of a direct projection from the DLV to the optic tectum in rattlesnakes (Crotatinae) 5. We believe that this is not due to the difference in the investigation techniques. The 'new nucleus' is not so demarcated from the surrounding reticular formation in normal Nissl preparations. However the 'new nucleus' might be highly differentiated in physiological function from the surrounding brain tissue, because we have observed strong histochemical activity of succinate dehydrogenase in the perikarya of this nucleus, and there is high contrast to the surrounding tissue (unpublished datum). We could not determine whether the 'new nucleus' is of secondary or higher order as a relay nucleus of the infrared sensory system in the central nervous system of Crotalinae. However, any additional relay nucleus between the DLV and the 'new nucleus' is unthinkable, because there is a direct projection from the DLV to the optic tectum in Boidae ~4. Further work is needed to make clear the afferent and efferent fiber connections of the 'new nucleus'. ACKNOWLEDGEMENTS We wish to thank Dr. Richard C. Goris for editing the manuscript. We express our thanks to Mr. Mikio Takaoka for collecting snakes and assistance, and to Miss
279 M a s a m i Y o s h i m o t o for her excellent help in preparing the histological materials a n d illustrations. A p a r t of this research was supported by G r a n t 477043 for scientific research from the Ministry of E d u c a t i o n of Japan.
REFERENCES 1 Anthony, J., Le n6vraxe des reptiles. In P. P. Grass6 (Ed.), Traitdde Zoologic, Reptiles, Tom XIV, Masson, Paris, 1970, pp. 202-332. 2 Butler, A. B. and Ebbesson, S. O. E., A Golgi study of the optic tectum of the Tegu lizard, Tupinambis nigropunctatus, J. Morph., 146 (1975) 215-228. 3 Ebbesson, S. O. E., Ascending axon degeneration following hemisection of the spinal cord in the Tegu lizard (Tupinambis nigropunctatus), Brain Research, 5 (1967) 178-206. 4 Goris, R. C. and Terashima, S., Central response to infrared stimulation of the pit receptors in Crotaline snake, Trimeresurusflavoviridis, J. exp. BioL, 58 (1973) 59-76. 5 Hartline, P. H., Kass, L. and Loop, M. S., Merging of modalities in the optic rectum: infrared and visual integration in rattlesnakes, Science, 199 (1978) 1125-1129. 6 HeUon, R. F., The marking of electrode tip positions in nervous tissue, J. Physiol. (Lond.), 214 (1971) 12P. 7 Kass, L., Loop, M. S. and Hartline, P. H., Anatomical and physiological localization of visual and infrared cell layers in the tectum of pit viper, J. eomp. Neurol., 182 (1978) 811-820. 8 Kishida, R., Comparative study on the teleostean optic rectum. Lamination and Cytoarchitecture, J. Hirnforseh., 20 (1979) 57-67. 9 Luiten, P. G. M., The horseradish peroxidase technique applied to the teleostean nervous system, Brain Research, 89 (1975) 181-186. 10 Masai, H., Structural patterns of the optic tectum in Japanese snakes of the family Colubridae, in relation to habit, J. Hirnforseh., 14 (1973) 367-374. 11 Mesulam, M.-M., The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility, J. Histochem. Cytoehem., 24 (1976) 1273-1280. 12 Molenaar, G. J., An additional trigeminal system in certain snakes possessing infrared receptors, Brain Research, 78 (1974) 340-344. 13 M••enaar• G. J.• The rh•mbencepha••n •f Pyth•n reti•ulatus• a snake p•ssessing infrared recept•rs• Neth. J. Zool., 27 (1977) 133-180. 14 Molenaar, G. J., The sensory trigeminal system of a snake in the possession of infrared receptors. II. The central projection of the trigeminal nerve, J. comp. Neurol., 179 (1978) 137-152. 15 Otsuka, N., Miyanaga, A., Tanaka, F. and Kimura, A., Neue Silberimpr~ignationsversuche zur Darstellung der Neurofibrillen an Paraffinschnitten, J. Kyoto Pref Univ. Med., 68 (1960) 1125-1128. 16 Ram6n, P., Estructura del encefalo del Camele6n, Rev. trimest. Micrograf, 1 (1896) 46-82. 17 Sarnat, H. B. and Netsky, M. G., Evolution of the Nervous System, Oxford Univ. Press, New York, 1974, pp. 162-164. 18 Schroeder, D. M. and Loop, M. S., Trigeminal projections in snakes possessing infrared sensitivity, J. comp. Neurol., 169 (1976) 1-14. 19 Terashima, S. and Goris, R. C., Tectal organization of pit viper infrared reception, Brain Research, 83 (1975) 490~94. 20 Terashima, S. and Goris, R. C., Receptive area of an infrared tectal unit, Brain Research, 101 (1976) 155-159. 21 Terashima, S. and Goris, R. C., Infrared bulbar units in Crotaline snakes, Proc. Jap. Acad. B, 53 (1977) 292-296. 22 Terashima, S. and Goris, R. C., Receptive area of primary infrared afferent neurons in crotaline snakes, Neuroseience, 4 (1979) 1137-1144. NOTE ADDED IN PROOF After preparation of this manuscript, a report by Gruberg et al. appeared in J. comp. Neurol., 188 (1979) 31-42, entitled 'Connections of the tectum of the rattlesnake Crotalus viridis: an HRP study', in which the nucleus reticularis caloris projects to the optic tectum. The 'new nucleus' of our report may be the same as their nucleus reticularis caloris, since its location in the medulla oblongata and the appearance of their Fig. 4 are very similar to our findings.