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A corticotectal projection in the lizard Agama agama
Neuroscience Letters, 18 (1980) 251-256
251
© Elsevier/North-Holland Scientific Publishers Ltd.
A C O R T I C O T E C T A L P R O J E C T I O N IN T H E LIZARD AGAMA AGAMA
D. ELPRANA, F.G. WOUTERLOOD* and V.E. ALONES
Department of Anatomy, Vrije Universiteit, Amsterdam (The Netherlands) (Received March 24th, 1980) (Revised version received April 9th, 1980) (Accepted April 9th, 1980)
SUMMARY
Following lesions in the dorsal cerebral cortex of the lizard Agama agama, terminal degeneration was observed by the F i n k - H e i m e r technique in the mediodorsal cortex, septal area, nucleus periventricularis and area lateralis of the hypothalamus after 7 days survival time. Following longer survival times (up to 28 days) diffuse terminal degeneration was found in layers 7 to 13 laterally and medially in the optic tectum. Terminal degeneration of the electron-dense type was found in electron micrographs of the same areas of the optic tectum.
Projections from the cerebral cortex to the superior colliculus have been reported in various mammals such as the tree shrew [2], the cat [7, 12] and the monkey [5]. In non-mammalian vertebrates connections between telencephalic structures and the optic tectum have been demonstrated in several species of fish [1, 3, 8] and in one amphibian [9]. The only reptilian species in which a corticotectal projection has been observed is the turtle Pseudemys scripta [6]. The purpose of the present study was to investigate whether a corticotectal projection is also present in lizards. Surgical lesions were made in the dorsal cerebral cortex of 14 lizards (Agama agama). Following postsurgical survival times of 6-28 days, 7 animals were anesthetized and perfused transcardially with 10°70 formalin. After two weeks the brains were embedded in gelatin-albumin, and 25 t~m frozen sections were cut in the transverse plane and stained with the F i n k - H e i m e r [4] and Nauta and Gygax [13] techniques. The other agamas were used for electron microscopy. Two to 15 days
* To whom correspondence should be addressed at: Department of Anatomy, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
252 after the surgery they were perfused transcardially with 150 mM phosphate buffer with 2°7o sucrose, p H 7.3, followed immediately by 150 ml of a freshly prepared mixture of 4°70 paraformaldehyde, 5°70 glutaraldehyde and 4°7o sucrose in buffer. Sections of 100 txm thickness containing the optic tectum were postfixed for 1 h in 2°70 OsO4 in buffer with 2°7o sucrose at 4°C, block-stained in maleate-buffered uranyl acetate, dehydrated rapidly in ethanol and embedded in Araldite. Thin sections were cut on a Reichert OMU-2 ultramicrotome and investigated with a Philips EM 301 electron microscope. Two unoperated agamas served as controls for EM. Light microscopy (Fig. 1). Following a lesion destroying the dorsal cortex (DC), degenerating fibers can be traced in the fiber layer towards the mediodorsal cortex (MDC). At the dorsomedial angle of the hemisphere a small group of degenerating fibers traverse the cellular layer of M D C and reach the superficial plexiform layer (SPL). Here they turn in the ventral as well as in the dorsal direction. Dense terminal degeneration is found in the SPL of the small-celled part of the MDC. In the largecelled part of the M D C terminal degeneration is less extensive. The majority of the degenerating fibers proceed ventrally in the fiber layer of the M D C and upon reaching the septum separate into lateral and medial fascicles. Most of the fibers of the lateral fascicle distribute to the septal area and dense degeneration can be identified in the central part of this area. The medial fascicle courses in the medial wall of the septum and enters the fimbria fornicis. Some degenerating fibers join the medial (MFB) and lateral (LFB) forebrain bundles. Other fibers cross the midline in the anterior pallial commissure and run dorsally in the fiber layers of the contralateral M D C and DC. Contralaterally in the septum and in the SPL of the MDC and DC, terminal degeneration is seen. The degenerating fibers in the fimbria fornicis curve in a ventrocaudal direction over the dorsal aspect of the anterior commissure and proceed in the postcommissural fornix towards the periventricular nuclei of the hypothalamus in which terminal degeneration is found. A few fibers of the lateral fascicle pass through the central septal area and run to the lateral hypothalamic area by way of the precommissural fornix. Terminal degeneration can be seen in this area. In the midbrain degenerating fibers leave the MFB and LFB as single fibers and course in a dorsal direction. A few fibers can be observed in the deep layers of the optic tectum. Diffuse terminal degeneration is present in the superficial layers (7 to 13) of both the ipsilateral and contralateral optic tectum. The highest density of terminal degeneration is present in the dorsolateral and ventrolateral areas. The site of crossing of the degenerating fibers remains obscure. Electron microscopy. In a normal tectum 4 types of boutons can be observed: profiles have either an electron-lucent or moderately dense matrix with either spherical or pleiomorphic vesicles and an occasional dense-cored vesicle. In the tecta of lesioned agamas, degenerating terminals (Fig. 2) are characterized by a conspicuously darker matrix with round swollen vesicles. In some dendritic profiles,
253
Mdc
Mdc
Dc
/ Sep
ff
mfb
Ifb
C
~
ca cl~a " ~ ~
A
~
~
fx fx
\~.'prec \ m f b Ifb
B
1-5 Hab Dm
~
7
stc fx
I~en
-13
u
\. :Peh Vmh
Fig. 1. Fiber and terminal degeneration following a lesion (black) in the dorsal cortex. Broken lines show fibers and dots terminals. Transverse sections: A, rostral part of the telencephalon; B, midtelencephalic level; C, diencephalic level; D, mesencephalic level. Abbreviations: Alv, alveus; ca, anterior commissure; Ch.op, optic chiasm; cpa, anterior pallial commissure; Dc, dorsal cortex; Dm, dorsomedial nucleus; dp, dorsal peduncle of lateral forebrain bundle; ff, fimbria fornicis; Hab, habenula; Lc, lateral cortex; lifo, lateral forebrain bundle; Mdc, mediodorsal cortex; mfb, medial forebrain bundle; Peh, periventricular nucleus of the hypothalamus; prec.fx, precommissural fornix; postc.fx, postcommissural fornix; Sd, dorsal striatum; Sep, septal area; Sv, ventral striatum; Vmh, ventromedial nucleus of the hypothalamus; Vlh, ventrolateral nucleus of the hypothalamus; vp, ventral peduncle of lateral forebrain bundle; Tec, optic tectum; 1-5, 6, 7-13, respective layers of the optic tectum. which are postsynaptic to degenerating b o u t o n s , enlarged or darkened m i t o c h o n d r i a were seen. A second type o f degenerating b o u t o n (Fig. 2) appears as markedly electron-dense profiles containing large scattered vesicles and distorted mitochondria. The m e m b r a n e is c o m m o n l y invaginated, resulting in a highly irregular profile, but synaptic specializations can still be seen. Glial processes are c o m m o n l y observed in the vicinity o f degenerating terminals. Clumps o f electrondense material, which could be identified by serial reconstruction as b o u t o n s , often
254
Fig. 2. Terminal degeneration patterns (A and B) in the optic rectum of agamas 15 days postsurgical to a cortical lesion, a: an electron-dense degenerating bouton with round swollen synaptic vesicles making synaptic contact with three dendritic profiles. × 53,500. B: a degenerating bouton with irregular and swollen vesicles synapsing on a dendritic spine. The bouton and the spine are surrounded by an aslroglial process, gf, gliofibrils. × 40,660. C: electron-dense body containing a large vesicle, in the cytoplasm of an astrocyte, gf, gliofibrils. × 40,660.
255 appear to be engulfed by glia. Both types of degenerating boutons appear predominantly in groups. They are present in the same layers of the optic tectum in which terminal degeneration was observed in the Fink-Heimer-stained preparations. Both types of degenerating bouton are found after relatively long survival periods (12-15 days). Lysosome-like electron-dense bodies commonly having one or two large vesicles (Fig. 2) appear in profiles identified as astrocytic processes: no membrane specializations, synaptic vesicles or mitochondrial debris could be observed. These bodies, which are rarely seen in the controls, are present during the entire survival period under study in the optic tectum of lesioned animals. They may consist of entire boutons ingested by glia but, more likely, they represent increased lysosomal activity caused by the trauma of the surgery. The results of the present study demonstrate a projection from the dorsal cortex to the optic tectum in a lizard. This observation could only be made following relatively long postsurgical survival periods. When shorter survival times were used, the fiber distribution of the dorsal cortex was found to be similar to that in the Tegu lizard, i.e. limited to the telencephalon and the diencephalon [11]. The only reptilian species in which a connection from the dorsal cortex to the optic tectum has thus far been described in is the turtle Pseudemys scripta. Although in both Pseudemys and Agama the corticotectal fibers originate from the dorsal cortex, in the former species the site of termination is restricted to the deep layers of the tectum, whereas in Agama the fibers distribute to its superficial layers. In Pseudemys it has been found that the dorsal cortex receives an input from the dorsal lateral geniculate nucleus [6] and the suggestion has been made that, at least in this species, the corticotectal projection is comparable to the connection between the visual cortex and the superior colliculus in the mammalian brain. In view of the fact that previous experiments in lizards have led to regard the dorsal cortex of squamata to be associated with hippocampal structures [10], it may seriously be questioned whether the above suggestion applies to reptiles in general.
ACKNOWLEDGEMENTS
We wish to thank Prof. Dr. A.H.M. Lohman for his advice and criticism on this paper.
REFERENCES 1 2 3
Airhart, M.J., Telencephalotectal projections in the goldfish C. auratus: a light and electron microscopic study, Anat. Rec., 193 (1979) 468. Casseday, J.H., Jones, D.R. and Diamond, I.T., Projections from cortex to tectum in the tree shrew, Tupaia glis, J. comp. Neurol., 185 (1979) 253-292. Ebbesson, S.O.E. and Schroeder, D.M., Connections of the Nurse shark's telencephalon, Science, 173 (1971) 254-256.
256
4 5
6 7 8 9 10 11 12 13
Fink, R.P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Res., 4 (1967) 369-374. Goldman, P.S. and Nauta, W . H . J . , Autoradiographic demonstration of a projection from prefrontal association cortex to the superior colliculus in the rhesus monkey, Brain R e s , 116 (1976) 145-149. Hall, J.A., Foster, R.E., Ebner, F.F. and Hall, W.C., Visual cortex in a reptile, the turtle (Pseudemys scripta and Chrysemys picta), Brain Res., 130 (1977) 197-216. Hollander, H., On the origin of the corticotectal projections in the cat, Exp. Brain Res., 21 (1974) 433 439. lto, H.I. and Kishida, R., Tectal afferent neurons identified by the retrograde H R P method in the carp telencephalon, Brain Res., 130 (1977) 142-145. Kokoros, J.J. and Northcutt, R.G., Telencephalic efferents of the Tiger salamander Ambystoma tigrinum tigrinum (green), J. comp. Neurol., 173 (1977) 613-628. Lacey, D.J., The organization of the hippocampus of the Fence lizard: a light and electron microscopic study, J. comp. Neurol., 182 (1978) 247 264. L o h m a n , A . H . M . and Mentink, G.M., Some cortical connections of the Tegu lizard (Yupinarnbis teguixin), Brain Res., 45 (1972) 325 344. MagalhS.es-Castro, H.H., Saraiva, P.E.S. and Magalhb.es-Castro, B., Identification of corticotectal cells of the visual cortex of cats by means of horseradish peroxidase, Brain Res., 83 (1975) 474-479. Nauta, W . J . H . and Gygax, P.A., Silver impregnation of degenerating axons in the central nervous system: a modified technique, Stain Yechnol., 29 (1954) 91-93.
251
© Elsevier/North-Holland Scientific Publishers Ltd.
A C O R T I C O T E C T A L P R O J E C T I O N IN T H E LIZARD AGAMA AGAMA
D. ELPRANA, F.G. WOUTERLOOD* and V.E. ALONES
Department of Anatomy, Vrije Universiteit, Amsterdam (The Netherlands) (Received March 24th, 1980) (Revised version received April 9th, 1980) (Accepted April 9th, 1980)
SUMMARY
Following lesions in the dorsal cerebral cortex of the lizard Agama agama, terminal degeneration was observed by the F i n k - H e i m e r technique in the mediodorsal cortex, septal area, nucleus periventricularis and area lateralis of the hypothalamus after 7 days survival time. Following longer survival times (up to 28 days) diffuse terminal degeneration was found in layers 7 to 13 laterally and medially in the optic tectum. Terminal degeneration of the electron-dense type was found in electron micrographs of the same areas of the optic tectum.
Projections from the cerebral cortex to the superior colliculus have been reported in various mammals such as the tree shrew [2], the cat [7, 12] and the monkey [5]. In non-mammalian vertebrates connections between telencephalic structures and the optic tectum have been demonstrated in several species of fish [1, 3, 8] and in one amphibian [9]. The only reptilian species in which a corticotectal projection has been observed is the turtle Pseudemys scripta [6]. The purpose of the present study was to investigate whether a corticotectal projection is also present in lizards. Surgical lesions were made in the dorsal cerebral cortex of 14 lizards (Agama agama). Following postsurgical survival times of 6-28 days, 7 animals were anesthetized and perfused transcardially with 10°70 formalin. After two weeks the brains were embedded in gelatin-albumin, and 25 t~m frozen sections were cut in the transverse plane and stained with the F i n k - H e i m e r [4] and Nauta and Gygax [13] techniques. The other agamas were used for electron microscopy. Two to 15 days
* To whom correspondence should be addressed at: Department of Anatomy, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
252 after the surgery they were perfused transcardially with 150 mM phosphate buffer with 2°7o sucrose, p H 7.3, followed immediately by 150 ml of a freshly prepared mixture of 4°70 paraformaldehyde, 5°70 glutaraldehyde and 4°7o sucrose in buffer. Sections of 100 txm thickness containing the optic tectum were postfixed for 1 h in 2°70 OsO4 in buffer with 2°7o sucrose at 4°C, block-stained in maleate-buffered uranyl acetate, dehydrated rapidly in ethanol and embedded in Araldite. Thin sections were cut on a Reichert OMU-2 ultramicrotome and investigated with a Philips EM 301 electron microscope. Two unoperated agamas served as controls for EM. Light microscopy (Fig. 1). Following a lesion destroying the dorsal cortex (DC), degenerating fibers can be traced in the fiber layer towards the mediodorsal cortex (MDC). At the dorsomedial angle of the hemisphere a small group of degenerating fibers traverse the cellular layer of M D C and reach the superficial plexiform layer (SPL). Here they turn in the ventral as well as in the dorsal direction. Dense terminal degeneration is found in the SPL of the small-celled part of the MDC. In the largecelled part of the M D C terminal degeneration is less extensive. The majority of the degenerating fibers proceed ventrally in the fiber layer of the M D C and upon reaching the septum separate into lateral and medial fascicles. Most of the fibers of the lateral fascicle distribute to the septal area and dense degeneration can be identified in the central part of this area. The medial fascicle courses in the medial wall of the septum and enters the fimbria fornicis. Some degenerating fibers join the medial (MFB) and lateral (LFB) forebrain bundles. Other fibers cross the midline in the anterior pallial commissure and run dorsally in the fiber layers of the contralateral M D C and DC. Contralaterally in the septum and in the SPL of the MDC and DC, terminal degeneration is seen. The degenerating fibers in the fimbria fornicis curve in a ventrocaudal direction over the dorsal aspect of the anterior commissure and proceed in the postcommissural fornix towards the periventricular nuclei of the hypothalamus in which terminal degeneration is found. A few fibers of the lateral fascicle pass through the central septal area and run to the lateral hypothalamic area by way of the precommissural fornix. Terminal degeneration can be seen in this area. In the midbrain degenerating fibers leave the MFB and LFB as single fibers and course in a dorsal direction. A few fibers can be observed in the deep layers of the optic tectum. Diffuse terminal degeneration is present in the superficial layers (7 to 13) of both the ipsilateral and contralateral optic tectum. The highest density of terminal degeneration is present in the dorsolateral and ventrolateral areas. The site of crossing of the degenerating fibers remains obscure. Electron microscopy. In a normal tectum 4 types of boutons can be observed: profiles have either an electron-lucent or moderately dense matrix with either spherical or pleiomorphic vesicles and an occasional dense-cored vesicle. In the tecta of lesioned agamas, degenerating terminals (Fig. 2) are characterized by a conspicuously darker matrix with round swollen vesicles. In some dendritic profiles,
253
Mdc
Mdc
Dc
/ Sep
ff
mfb
Ifb
C
~
ca cl~a " ~ ~
A
~
~
fx fx
\~.'prec \ m f b Ifb
B
1-5 Hab Dm
~
7
stc fx
I~en
-13
u
\. :Peh Vmh
Fig. 1. Fiber and terminal degeneration following a lesion (black) in the dorsal cortex. Broken lines show fibers and dots terminals. Transverse sections: A, rostral part of the telencephalon; B, midtelencephalic level; C, diencephalic level; D, mesencephalic level. Abbreviations: Alv, alveus; ca, anterior commissure; Ch.op, optic chiasm; cpa, anterior pallial commissure; Dc, dorsal cortex; Dm, dorsomedial nucleus; dp, dorsal peduncle of lateral forebrain bundle; ff, fimbria fornicis; Hab, habenula; Lc, lateral cortex; lifo, lateral forebrain bundle; Mdc, mediodorsal cortex; mfb, medial forebrain bundle; Peh, periventricular nucleus of the hypothalamus; prec.fx, precommissural fornix; postc.fx, postcommissural fornix; Sd, dorsal striatum; Sep, septal area; Sv, ventral striatum; Vmh, ventromedial nucleus of the hypothalamus; Vlh, ventrolateral nucleus of the hypothalamus; vp, ventral peduncle of lateral forebrain bundle; Tec, optic tectum; 1-5, 6, 7-13, respective layers of the optic tectum. which are postsynaptic to degenerating b o u t o n s , enlarged or darkened m i t o c h o n d r i a were seen. A second type o f degenerating b o u t o n (Fig. 2) appears as markedly electron-dense profiles containing large scattered vesicles and distorted mitochondria. The m e m b r a n e is c o m m o n l y invaginated, resulting in a highly irregular profile, but synaptic specializations can still be seen. Glial processes are c o m m o n l y observed in the vicinity o f degenerating terminals. Clumps o f electrondense material, which could be identified by serial reconstruction as b o u t o n s , often
254
Fig. 2. Terminal degeneration patterns (A and B) in the optic rectum of agamas 15 days postsurgical to a cortical lesion, a: an electron-dense degenerating bouton with round swollen synaptic vesicles making synaptic contact with three dendritic profiles. × 53,500. B: a degenerating bouton with irregular and swollen vesicles synapsing on a dendritic spine. The bouton and the spine are surrounded by an aslroglial process, gf, gliofibrils. × 40,660. C: electron-dense body containing a large vesicle, in the cytoplasm of an astrocyte, gf, gliofibrils. × 40,660.
255 appear to be engulfed by glia. Both types of degenerating boutons appear predominantly in groups. They are present in the same layers of the optic tectum in which terminal degeneration was observed in the Fink-Heimer-stained preparations. Both types of degenerating bouton are found after relatively long survival periods (12-15 days). Lysosome-like electron-dense bodies commonly having one or two large vesicles (Fig. 2) appear in profiles identified as astrocytic processes: no membrane specializations, synaptic vesicles or mitochondrial debris could be observed. These bodies, which are rarely seen in the controls, are present during the entire survival period under study in the optic tectum of lesioned animals. They may consist of entire boutons ingested by glia but, more likely, they represent increased lysosomal activity caused by the trauma of the surgery. The results of the present study demonstrate a projection from the dorsal cortex to the optic tectum in a lizard. This observation could only be made following relatively long postsurgical survival periods. When shorter survival times were used, the fiber distribution of the dorsal cortex was found to be similar to that in the Tegu lizard, i.e. limited to the telencephalon and the diencephalon [11]. The only reptilian species in which a connection from the dorsal cortex to the optic tectum has thus far been described in is the turtle Pseudemys scripta. Although in both Pseudemys and Agama the corticotectal fibers originate from the dorsal cortex, in the former species the site of termination is restricted to the deep layers of the tectum, whereas in Agama the fibers distribute to its superficial layers. In Pseudemys it has been found that the dorsal cortex receives an input from the dorsal lateral geniculate nucleus [6] and the suggestion has been made that, at least in this species, the corticotectal projection is comparable to the connection between the visual cortex and the superior colliculus in the mammalian brain. In view of the fact that previous experiments in lizards have led to regard the dorsal cortex of squamata to be associated with hippocampal structures [10], it may seriously be questioned whether the above suggestion applies to reptiles in general.
ACKNOWLEDGEMENTS
We wish to thank Prof. Dr. A.H.M. Lohman for his advice and criticism on this paper.
REFERENCES 1 2 3
Airhart, M.J., Telencephalotectal projections in the goldfish C. auratus: a light and electron microscopic study, Anat. Rec., 193 (1979) 468. Casseday, J.H., Jones, D.R. and Diamond, I.T., Projections from cortex to tectum in the tree shrew, Tupaia glis, J. comp. Neurol., 185 (1979) 253-292. Ebbesson, S.O.E. and Schroeder, D.M., Connections of the Nurse shark's telencephalon, Science, 173 (1971) 254-256.
256
4 5
6 7 8 9 10 11 12 13
Fink, R.P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Res., 4 (1967) 369-374. Goldman, P.S. and Nauta, W . H . J . , Autoradiographic demonstration of a projection from prefrontal association cortex to the superior colliculus in the rhesus monkey, Brain R e s , 116 (1976) 145-149. Hall, J.A., Foster, R.E., Ebner, F.F. and Hall, W.C., Visual cortex in a reptile, the turtle (Pseudemys scripta and Chrysemys picta), Brain Res., 130 (1977) 197-216. Hollander, H., On the origin of the corticotectal projections in the cat, Exp. Brain Res., 21 (1974) 433 439. lto, H.I. and Kishida, R., Tectal afferent neurons identified by the retrograde H R P method in the carp telencephalon, Brain Res., 130 (1977) 142-145. Kokoros, J.J. and Northcutt, R.G., Telencephalic efferents of the Tiger salamander Ambystoma tigrinum tigrinum (green), J. comp. Neurol., 173 (1977) 613-628. Lacey, D.J., The organization of the hippocampus of the Fence lizard: a light and electron microscopic study, J. comp. Neurol., 182 (1978) 247 264. L o h m a n , A . H . M . and Mentink, G.M., Some cortical connections of the Tegu lizard (Yupinarnbis teguixin), Brain Res., 45 (1972) 325 344. MagalhS.es-Castro, H.H., Saraiva, P.E.S. and Magalhb.es-Castro, B., Identification of corticotectal cells of the visual cortex of cats by means of horseradish peroxidase, Brain Res., 83 (1975) 474-479. Nauta, W . J . H . and Gygax, P.A., Silver impregnation of degenerating axons in the central nervous system: a modified technique, Stain Yechnol., 29 (1954) 91-93.