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New insights into the organization of the shark brain
Camp. Biochem. Physiol., 1972, Vol. 42A, pp. 121to 129.Pergamon Press. Printed in Great Britain
NEW INSIGHTS
INTO THE ORGANIZATION SHARK BRAIN
OF THE
SVEN 0. E. EBBESSON* Department of Neurosurgery, University of Virginia Medical School, Charlottesville, Virginia 22903 Abstract-l. Accurate tracing of distant neuronal connections in sharks have been made possible with techniques for the selective silver impregnation of degenerating axons. 2. These studies indicate that gross misconceptions have existed about shark brain organization. 3. This paper reviews studies on olfactory, retinal, tectal, telencephalic, thalamic, cerebellar and spinal projections in the nurse shark. INTRODUCTION behavioral or biochemical studies of the brain depend to a great extent upon an accurate definition of its structure. The recently developed Nauta method has provided greater insights into brain organization than any other of this century, and because of this technique, we now have an entirely new view of the shark brain. At the time of the 1958 Conference on the Basic Research Approaches to the Development of Shark Repellents, these new methods had not as yet been applied to fish material, and Dr. Aronson’s excellent review, at that conference, of the then voluminous available information on shark brain organization, revealed the existence of numerous descrepancies and uncertainties. In light of our own experiments since then, employing modifications of the Nauta-Gygax (1954) and Fink & Heimer (1967) methods, it is evident that the shark brain must be considered as poorly understood up to the advent of the silver impregnation techniques for tracing degenerating pathways.
PHYSIOLOGICAL,
MATERIALS
AND METHODS
The degeneration studies on sharks were started in 1965, in Puerto Rico, and were carried out there until 1970 when we shifted our base of operation to the Lemer Marine Laboratories in Bimini. Here behavioral studies were also initiated. Although the data reported below were obtained from the nurse shark (GingZymostoma ci~~atum), additional experiments have also been carried out on the tiger shark (Galeocerdo cuoieri) and the lemon shark (Negaprion brwirostris). The shark handling facilities at the Marine Biology Station in La Parquera, Puerto Rico, are unique in that they were designed for surgical procedures of large as well as small sharks. A surgical platform was added to an already existing pen that measured 30 x 60 ft (Fig. 1). By guiding sharks as long as 10 ft into a tray (Fig. 2) suspended from a 2-ton hoist in the well located to the left in Fig. 1, it is possible to lift * Supported by Career Development Institute lROlEY00154-Ol Al.
Award lK04NS46292-OlAl 121
and National Eye
122
SVEN 0.
E. EBBESSON
a shark, still submerged in water, to 30 in. above the platform. The tray is then stabilized for surgery. By controlling the amount of tricaine methyl sulfonate in the tray water (slightly less than 1 part per lO,OOO),it is possible to obtain surgical anesthesia concomitant with good gilling movements. The dorsal surface of the head is kept out of the water during surgery when a given part of the brain is lesioned (Fig. 3). The skin is then sutured and the animal returned to the large pen where it quickly recovers from the anesthesia. It is noteworthy that we have never lost a shark from anesthesia nor have we ever seen generalized axonal degeneration that could be interpreted as resulting from anoxia during the surgical procedure. The animals are usually killed after 2-7 weeks, since the selective argyrophilia of degenerating axons and terminals in sharks is most pronounced at that time (Ebbesson, 1970a). After transcardiac perfusion with formalin, the brain is removed and stored in the fixative until further processing (Ebbesson, 1970a). The results of these procedures are thin slices of brain in which the degenerating axons and terminals have been selectively impregnated with silver. By charting the location of such debris in serial sections through the brain, it is possible to accurately locate distant courses and terminations of a given lesioned neuronal aggregate (Figs. 4 and 5). RESULTS
AND DISCUSSION
The telencephalon
Conceptions of telencephalic organization and function have perhaps been most erroneous. Aronson (1963) and Nieuwenhuys (1967) expressed the then prevailing view that the forebrain of elasmobranchs serves primarily the organization of a very sensitive and effective olfactory system. The principal reason for the belief that the entire telencephalon was olfactory in nature stemmed from observations in “normal material” that indicated tertiary if not secondary olfactory projections to every telencephalic cell aggregate (Backstrom, 1924). The elasmotranch telencephalon has hence often been referred to as the “olfactory lobes”. Our studies with the Nauta and Fink & Heimer methods provide striking evidence against such a view and indicate that only a relatively modest portion is directly related to olfaction. The olfactory tracts in the nurse shark project to a restricted ventrolateral region (Fig. 6 and Ebbesson & Heimer, 1968, 1970). No evidence of contralateral projections, as Backstrom (1924) and others have observed in other species, were noted. Only when large lesions of the lateral olfactory area (OL) were made, was it possible to observe a contralateral olfactory connection. In this series of experiments (Ebbesson, in preparation), the lateral olfactory area was found to project heavily to the lateral portion of area superficialis basalis ipsilaterally with a few fibers extending caudally to cross at the level of the optic chiasm and terminate in the contralateral area superficialis basalis and the lateral olfactory area (Fig. 7). A few degenerating axons could also be traced into the contralateral olfactory tract. Since the tertiary olfactory projections also do not involve large portions of the telencephalon, the question arose whether the thalamus might not also project here as it does in mammals. We had previously determined that the thalamus has considerable inputs from the retina (Ebbesson, 1967; Ebbesson & Ramsey, 1968),
FIG.
1.
FIG. 2.
Aerial photograph of the shark pen in La Parquera, working platform is shown in the upper left-hand
Tray
for lifting
sharks
onto the platform. 2-ton hoist.
The
Puerto corner.
Rico.
tray is suspended
The
from
a
FIG.
3.
A tiger
shark in position for surgery, resting in the tray dorsal surface of the head out of the water.
with
only
the
FIG. 4. Photomicrograph showing debris of degenerating axons and terminals in the mesencephalic tegmentum in the nurse shark following hemicerebellectomy on the contralateral side. Modified Fink & Heimer method (see Ebbesson, 1970a).
FIG. 5. Photomicrograph from the same preparation and region as Fig. 4, but ipsilateral to the lesion. Note absence of silver impregnated debris of degenerating axons and terminals. The large black profiles are siIver impregnated blood vessels.
SHARK BRAIN ORGANIZATION
TO
AB
123
C
FIG. 6. Following transection of the olfactory tract (TO) in the nurse shark, degenerating fibers can be traced into the olfactory peduncle (PO), the lateral olfactory area (OL) and the lateral portion of area superficialis basalis (SB), as shown in these frontal sections (A-C) through the telencephalon. Dots and interrupted lines indicate the location of degenerating fibers of passage and open circles show loci of terminal degeneration.
FIG. 7. The lateral olfactory area (OL) projects to the ipsilateral area superficialis basalis (SB) and the contralateral lateral olfactory area (OL). The commissural fibers cross ventral to the third ventricle (III) and the decussation of the thalamotelencephalic tract (DTrTT). The lesion is shown in black in frontal section A.
feature of all vertebrates (Ebbesson, 1970b) and the possibility of a thalamo-telencephalic projection, therefore, did not seem unlikely. Johnston (1911) described such a pathway to a caudal telencephalic cell group which he labeled “the somatic area”, but Ariens Kappers et al. (1936), were doubtful about the existence of this tract. It is now evident that such a pathway does exist, in the nurse shark at least. However, it is strikingly different from those of any other vertebrate so far examined in that the fibers cross the midline and terminate in contralateral telencephalic cell aggregates (Fig. 8 and Ebbesson & Schroeder, 1971; Schroeder & Ebbesson, 1971). The latter cell groups are located medial to the lateral olfactory area. a common
5
324
SVEN 0. E. EBBESSON
Another smaller bilateral ventromedial large diencephalic lesions. This pathway diencephalon, as cases with small restricted pathway is reminiscent of the quinto-frontal additional experiments need to be made in of this tract.
ascending pathway was also seen after originates from levels caudal to the mesencephalic lesions revealed. This tract of birds (Wallenberg, 1907), but the shark to determine the exact origin
FIG. 8. Degenerating axons are traced from a lesion in the thalamus (shown in frontal section C) to the contralateral telencephalon via the ~alamo-telenceph~iG tract (TrTT) which crosses in the decussation of the thalamo-telencephalic tract (DTr’IT) ventral to the third ventricle (III). A bilateral ventromedial tract (see large arrow), terminating in the medial portion of the area superficialis basalis (SIB), also degenerates following such a lesion, but this tract has a more caudal origin than the thalamus (see text). A few ipsilateral thalamo-telencephalic fibers are also shown in the tractus pallii (TrP).
Connections of the telencephalon with caudal centers have also been misunderstood and thought to be primarily related to the epithalamus and the hypothalamus (Aronson, 1963). Contradiction and confusion about these pathways is evident everywhere in the literature because the data available could not provide definitive answers, and bias of one sort or another swayed interpretations. The tractus pallii of Edinger, for example, has been believed to be principally an ascending pathway by some (Catois, 1901) while others (AriCns Kappers, 1906; Wallenberg, 1907) held the opposite point of view. Our findings about telencephalic projections in the nurse shark differ in almost every respect from previous descriptions (Ari&ns Kappers et al., 1936). In an experiment involving some fifty sharks and telencephalic lesions of various sizes and locations (Ebbesson & Schroeder, 1971), connections with both epithalamic and hypothalamic centers, as shown by earlier workers, were found; but in addition massive connections with thalamus, optic tectum and select neuronal aggregates at every level of the brainstem were also discovered (Fig. 9). Although telencephalic projections to such levels are well known in mammals, the unique feature in the nurse shark is that most of the fibers cross at the level of the optic chiasm and distribute to contralateral cell groups. These studies reveal that the telencephalon of the nurse shark has some uniquely organized connections but is in some ways more directly comparable to that of
125
SHARK BRAIN ORGANIZATION
FIG. 9. A large lesion in the telencephalon (shown in frontal section A) results in massive degeneration of several descending fiber systems. One fasciculus decussates in the habenular commissure (CH) and terminates in a epithalamus; another bundle is bilateral and reaches the mesencephalic tegmentum (TM), and the fibers of a third tract decussates almost completely, to terminate profusely in the contralateral diencephalon (see frontal section B) including the lateral geniculate nucelus (GL). This tract also issues fibers to the optic tectum (TeO), the inferior lobe (LI) of the hypothalamus and more caudal brain stem nuclei (see frontal sections D and E).
ABCD
D
FIG. 10. The degenerating fibers following unilateral eye enucleation can be traced to the contralateral hypothalamus (not shown), lateral geniculate nucleus (GL), the pretectal area (AP) and the optic tectum (TeO). (TO) optic tract; III, third ventricle.
126
SVEN 0. E. EBBESSON
mammals and considerably less completely monopolized by the olfactory system than earlier studies by other methods had seemed to indicate. The diencephalon
The connectional organization of the shark hypothalamus remains unknown except for the information about a telencephalic input shown above (Fig. 9) and a retinal input revealed for the first time in elasmobranchs by means of the Nauta method (Ebbesson, 1967; Ebbesson & Ramsey, 1968). Considerable information has been obtained about the thalamus which can only be briefly described here. The retinal input was found to involve a relatively large portion (Fig. 10 and Ebbesson, 1967; Ebbesson & Ramsey, 1968) but other inputs have also recently been identified that add a whole new dimension to our understanding of thalamic organization. Contrary to common belief (see Aronson’s review, 1963) there are indeed inputs to the thalamus from spinal cord (Ebbesson, manuscript in preparation), optic tectum (Ebbesson, in preparation) and cerebellum (Ebbesson & Campbell, in preparation). Since such connections are known to exist in other vertebrates, it is now evident that the thalamus of the shark also has more in common with other vertebrates than hitherto believed. The mesencephalon
Although efferent projections of the tegmentum have not been studied as yet, the tectal projections have been studied in the nurse shark and are essentially similar to those of other vertebrates (Fig. 11). Our findings differ from earlier observations (Aronson, 1963) in that we were not able to confirm a tectal projection to the motor nuclei of the extraocular muscles, but observed a hitherto undescribed projection to the thalamus (Ebbesson, unpublished information).
FIG. 11. Projections of the optic tectum (TeO) are to the mesencephalic tegmenturn (TM), lateral geniculate nucleus (GL) and rhombencephalic reticular nuclei (see frontal section C). (Cb) cerebellum; (LI) inferior lobe of the hypothalamus; (Tel) telencephalon.
SHARK
BRAIN
127
ORGANIZATION
The cerebellum Cerebellar connections in the nurse shark also differ somewhat from earlier descriptions. In contrast to earlier workers, we found no evidence of a direct projection from cerebellar cortex to reticular and motor nuclei of the medulla and spinal cord. Only when the lateral cerebellar nucleus was lesioned were fibers Such lesions resulted in degeneration of traced outside the cerebellum proper. pathways reaching the motor nuclei of the extrinsic muscles of the eye, reticular cell groups, and a few fibers extending as far as the thalamus (Ebbesson & Campbell, manuscript in preparation). In many respects these connections are reminiscent of those found in other vertebrates although they give the appearance of greater diffuseness (Fig. 12).
c FIG. 12. Lesions of the cerebellum (Cb) involving the cerebellar nuclei (NCb) result in the degeneration of pathways that issue fibers to the contralateral rhombencephalic reticular formation (RR) via the brachium conjunctivum (bc). lateral geniculate nucleus; III, third ventricle.
(GL)
13. Degenerating fibers can be traced from the spinal cord (after a hemisection of the cord at the second spinal segment) to the dorsal column nuclei (DC); rhombencephalic reticular nuclei (RR); mesencephalon (M), and the thalamus (see frontal section A). (GL) lateral geniculate nucleus; III, third ventricle. FIG.
128
SVEN 0. E. EBBESSON
The spinal cord
The only available information about the spinal cord obtained from Nauta material relates to the ascending spinal projections (Fig. 13 and Ebbesson, manuscript in preparation). These appear similar to those described in other elasmobranchs with other methods, except for the finding of a small diffuse fascicle of spinal fibers that reach the thalamus. Earlier data had suggested a direct pathway only as far as the mesencephalon. SUMMARY
The studies reported here, in which the accurate tracing of distant neuronal connections was made possible by the Nauta method, are preliminary in nature and provide only information about general patterns of organization. Even so they do indicate that gross misconceptions about shark brain organization have existed and that only now, with recent tools like the Nauta method, electron microscopic techniques and histochemicaf methods, will we begin to understand the structural matrix underlying shark behavior. Acknow~dg~t-Tie the illustrations.
author wishes to thank Dr. Dolores Schroeder for hefping with
REFERENCES A&NS KAPPERSC. U. (1906) The structure of the teieostean and selachian brain. J. coomp. Neural. 16, l-112. AI&NS KAPPERSC. U., HUBERG. C. & CROSBY E. C. (1936) The Compurutiwe Anatomy of the Nervous System of Vertebrates, Including Man. MacMillan, New York. ARONSONL. R. (1963) The centrai nervous system of sharks and bony fishes with special reference to sensory and integrative mechanism. In Sharks and Survival (Edited by GILBERTP. W.), pp. 165-241. Heath, Boston, Mass. BXCKSTRBM K. (1924) Contributions to the forebrain morphology in selachians. Acta. zool., Stockh. 5, 123-240. CATOISE. M. (1901) Recherches sur I’histologie et l’anatomie micros~opique de l’endphale chez les poissons. Bull. Scient. Fr. Belg. 36, l-166. EBBESSONS. 0. E. (1967) Retinal projections in two species of sharks (Guleocerdo cuvieri and Ginglymostoma ciwatum). Anat. Rec. 157, 238. EBEJZSSONS. 0. E. (1970a) The selective silver impregnation of degenerating axons and Research Meth~ their synaptic endings in non-mammalian species. In C~t~~oyayy in Neuroanutomy (Edited by NAUTA W. J. H. & EBBESSONS. 0. E.), pp. 132-161. Springer-Verlag, New York. EBBWON S. 0. E. (1970b) On the organization of central visual pathways in vertebrates. Brain Behav. & Evol. 3, 178-194. EBBESSONS. 0. E. & HEIMER L. (1968) OIfactory bulb projections in two species of sharks (Galeocerdo cuvieri and Ginglymostoma ciwutum). Anat. Rec. 160 (Abstr.). EBBESSONS. 0. E. & HEIMER L. (1970) Projections of the olfactory tract fibers in the nurse shark (GirsgZymostomaciwatum). Bruin Res. 17, 47-55. EBBESSONS. 0. E. & RAMSEYJ. S. (1968) The optic tracts of two species of sharks (Galeocerdo cuvieri and Ginglymostoma ciwutum). Bruin Res. 8, 36-53. EBBESSONS. 0. E. & SCHROEDBR D. (1971) Connections of the nurse shark’s telencephalon. Science 173, 254-256.
SHARKBRAINORGANIZATION
129
FINK R. P. 81 HEIMERL. (1967) Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4,369-374. JOHNSTONJ. B. (1911) The telencephalon of selachians. J. comp. Neural. 21, 1-113. NAUTA-GYGAX(1954) Silver impregnation of degenerating axons in the central nervous system: a modified technique. Stain Technol. 29, 91-93. NIEUWENHUYSR. (1967) Comparative anatomy of olfactory centres and tracts. In Progress in Brain Research (Edited by ZOTTERMAN Y.), Vol. 23, pp. l-64. Elsevier, Amsterdam. SCHROEDER D. M. & EBBESSONS. 0. E. (1971) Diencephalic projections to the telencephalon of the nurse shark (Gingbymostoma cirratum). Anat. Rec. 169, 421 (Abstr.). WALLENBERGA. (1907) Beitrage zur Kenntniss des Gehirns der Teleostier und Selachier. Anat. Anz., Bd. 31, 369. Key Word Index-Brain; olfactory lobes; retina; optic tectum; telencephalon; thalamus; cerebellum; spinal cord; Ginglymostoma cirratum.
NEW INSIGHTS
INTO THE ORGANIZATION SHARK BRAIN
OF THE
SVEN 0. E. EBBESSON* Department of Neurosurgery, University of Virginia Medical School, Charlottesville, Virginia 22903 Abstract-l. Accurate tracing of distant neuronal connections in sharks have been made possible with techniques for the selective silver impregnation of degenerating axons. 2. These studies indicate that gross misconceptions have existed about shark brain organization. 3. This paper reviews studies on olfactory, retinal, tectal, telencephalic, thalamic, cerebellar and spinal projections in the nurse shark. INTRODUCTION behavioral or biochemical studies of the brain depend to a great extent upon an accurate definition of its structure. The recently developed Nauta method has provided greater insights into brain organization than any other of this century, and because of this technique, we now have an entirely new view of the shark brain. At the time of the 1958 Conference on the Basic Research Approaches to the Development of Shark Repellents, these new methods had not as yet been applied to fish material, and Dr. Aronson’s excellent review, at that conference, of the then voluminous available information on shark brain organization, revealed the existence of numerous descrepancies and uncertainties. In light of our own experiments since then, employing modifications of the Nauta-Gygax (1954) and Fink & Heimer (1967) methods, it is evident that the shark brain must be considered as poorly understood up to the advent of the silver impregnation techniques for tracing degenerating pathways.
PHYSIOLOGICAL,
MATERIALS
AND METHODS
The degeneration studies on sharks were started in 1965, in Puerto Rico, and were carried out there until 1970 when we shifted our base of operation to the Lemer Marine Laboratories in Bimini. Here behavioral studies were also initiated. Although the data reported below were obtained from the nurse shark (GingZymostoma ci~~atum), additional experiments have also been carried out on the tiger shark (Galeocerdo cuoieri) and the lemon shark (Negaprion brwirostris). The shark handling facilities at the Marine Biology Station in La Parquera, Puerto Rico, are unique in that they were designed for surgical procedures of large as well as small sharks. A surgical platform was added to an already existing pen that measured 30 x 60 ft (Fig. 1). By guiding sharks as long as 10 ft into a tray (Fig. 2) suspended from a 2-ton hoist in the well located to the left in Fig. 1, it is possible to lift * Supported by Career Development Institute lROlEY00154-Ol Al.
Award lK04NS46292-OlAl 121
and National Eye
122
SVEN 0.
E. EBBESSON
a shark, still submerged in water, to 30 in. above the platform. The tray is then stabilized for surgery. By controlling the amount of tricaine methyl sulfonate in the tray water (slightly less than 1 part per lO,OOO),it is possible to obtain surgical anesthesia concomitant with good gilling movements. The dorsal surface of the head is kept out of the water during surgery when a given part of the brain is lesioned (Fig. 3). The skin is then sutured and the animal returned to the large pen where it quickly recovers from the anesthesia. It is noteworthy that we have never lost a shark from anesthesia nor have we ever seen generalized axonal degeneration that could be interpreted as resulting from anoxia during the surgical procedure. The animals are usually killed after 2-7 weeks, since the selective argyrophilia of degenerating axons and terminals in sharks is most pronounced at that time (Ebbesson, 1970a). After transcardiac perfusion with formalin, the brain is removed and stored in the fixative until further processing (Ebbesson, 1970a). The results of these procedures are thin slices of brain in which the degenerating axons and terminals have been selectively impregnated with silver. By charting the location of such debris in serial sections through the brain, it is possible to accurately locate distant courses and terminations of a given lesioned neuronal aggregate (Figs. 4 and 5). RESULTS
AND DISCUSSION
The telencephalon
Conceptions of telencephalic organization and function have perhaps been most erroneous. Aronson (1963) and Nieuwenhuys (1967) expressed the then prevailing view that the forebrain of elasmobranchs serves primarily the organization of a very sensitive and effective olfactory system. The principal reason for the belief that the entire telencephalon was olfactory in nature stemmed from observations in “normal material” that indicated tertiary if not secondary olfactory projections to every telencephalic cell aggregate (Backstrom, 1924). The elasmotranch telencephalon has hence often been referred to as the “olfactory lobes”. Our studies with the Nauta and Fink & Heimer methods provide striking evidence against such a view and indicate that only a relatively modest portion is directly related to olfaction. The olfactory tracts in the nurse shark project to a restricted ventrolateral region (Fig. 6 and Ebbesson & Heimer, 1968, 1970). No evidence of contralateral projections, as Backstrom (1924) and others have observed in other species, were noted. Only when large lesions of the lateral olfactory area (OL) were made, was it possible to observe a contralateral olfactory connection. In this series of experiments (Ebbesson, in preparation), the lateral olfactory area was found to project heavily to the lateral portion of area superficialis basalis ipsilaterally with a few fibers extending caudally to cross at the level of the optic chiasm and terminate in the contralateral area superficialis basalis and the lateral olfactory area (Fig. 7). A few degenerating axons could also be traced into the contralateral olfactory tract. Since the tertiary olfactory projections also do not involve large portions of the telencephalon, the question arose whether the thalamus might not also project here as it does in mammals. We had previously determined that the thalamus has considerable inputs from the retina (Ebbesson, 1967; Ebbesson & Ramsey, 1968),
FIG.
1.
FIG. 2.
Aerial photograph of the shark pen in La Parquera, working platform is shown in the upper left-hand
Tray
for lifting
sharks
onto the platform. 2-ton hoist.
The
Puerto corner.
Rico.
tray is suspended
The
from
a
FIG.
3.
A tiger
shark in position for surgery, resting in the tray dorsal surface of the head out of the water.
with
only
the
FIG. 4. Photomicrograph showing debris of degenerating axons and terminals in the mesencephalic tegmentum in the nurse shark following hemicerebellectomy on the contralateral side. Modified Fink & Heimer method (see Ebbesson, 1970a).
FIG. 5. Photomicrograph from the same preparation and region as Fig. 4, but ipsilateral to the lesion. Note absence of silver impregnated debris of degenerating axons and terminals. The large black profiles are siIver impregnated blood vessels.
SHARK BRAIN ORGANIZATION
TO
AB
123
C
FIG. 6. Following transection of the olfactory tract (TO) in the nurse shark, degenerating fibers can be traced into the olfactory peduncle (PO), the lateral olfactory area (OL) and the lateral portion of area superficialis basalis (SB), as shown in these frontal sections (A-C) through the telencephalon. Dots and interrupted lines indicate the location of degenerating fibers of passage and open circles show loci of terminal degeneration.
FIG. 7. The lateral olfactory area (OL) projects to the ipsilateral area superficialis basalis (SB) and the contralateral lateral olfactory area (OL). The commissural fibers cross ventral to the third ventricle (III) and the decussation of the thalamotelencephalic tract (DTrTT). The lesion is shown in black in frontal section A.
feature of all vertebrates (Ebbesson, 1970b) and the possibility of a thalamo-telencephalic projection, therefore, did not seem unlikely. Johnston (1911) described such a pathway to a caudal telencephalic cell group which he labeled “the somatic area”, but Ariens Kappers et al. (1936), were doubtful about the existence of this tract. It is now evident that such a pathway does exist, in the nurse shark at least. However, it is strikingly different from those of any other vertebrate so far examined in that the fibers cross the midline and terminate in contralateral telencephalic cell aggregates (Fig. 8 and Ebbesson & Schroeder, 1971; Schroeder & Ebbesson, 1971). The latter cell groups are located medial to the lateral olfactory area. a common
5
324
SVEN 0. E. EBBESSON
Another smaller bilateral ventromedial large diencephalic lesions. This pathway diencephalon, as cases with small restricted pathway is reminiscent of the quinto-frontal additional experiments need to be made in of this tract.
ascending pathway was also seen after originates from levels caudal to the mesencephalic lesions revealed. This tract of birds (Wallenberg, 1907), but the shark to determine the exact origin
FIG. 8. Degenerating axons are traced from a lesion in the thalamus (shown in frontal section C) to the contralateral telencephalon via the ~alamo-telenceph~iG tract (TrTT) which crosses in the decussation of the thalamo-telencephalic tract (DTr’IT) ventral to the third ventricle (III). A bilateral ventromedial tract (see large arrow), terminating in the medial portion of the area superficialis basalis (SIB), also degenerates following such a lesion, but this tract has a more caudal origin than the thalamus (see text). A few ipsilateral thalamo-telencephalic fibers are also shown in the tractus pallii (TrP).
Connections of the telencephalon with caudal centers have also been misunderstood and thought to be primarily related to the epithalamus and the hypothalamus (Aronson, 1963). Contradiction and confusion about these pathways is evident everywhere in the literature because the data available could not provide definitive answers, and bias of one sort or another swayed interpretations. The tractus pallii of Edinger, for example, has been believed to be principally an ascending pathway by some (Catois, 1901) while others (AriCns Kappers, 1906; Wallenberg, 1907) held the opposite point of view. Our findings about telencephalic projections in the nurse shark differ in almost every respect from previous descriptions (Ari&ns Kappers et al., 1936). In an experiment involving some fifty sharks and telencephalic lesions of various sizes and locations (Ebbesson & Schroeder, 1971), connections with both epithalamic and hypothalamic centers, as shown by earlier workers, were found; but in addition massive connections with thalamus, optic tectum and select neuronal aggregates at every level of the brainstem were also discovered (Fig. 9). Although telencephalic projections to such levels are well known in mammals, the unique feature in the nurse shark is that most of the fibers cross at the level of the optic chiasm and distribute to contralateral cell groups. These studies reveal that the telencephalon of the nurse shark has some uniquely organized connections but is in some ways more directly comparable to that of
125
SHARK BRAIN ORGANIZATION
FIG. 9. A large lesion in the telencephalon (shown in frontal section A) results in massive degeneration of several descending fiber systems. One fasciculus decussates in the habenular commissure (CH) and terminates in a epithalamus; another bundle is bilateral and reaches the mesencephalic tegmentum (TM), and the fibers of a third tract decussates almost completely, to terminate profusely in the contralateral diencephalon (see frontal section B) including the lateral geniculate nucelus (GL). This tract also issues fibers to the optic tectum (TeO), the inferior lobe (LI) of the hypothalamus and more caudal brain stem nuclei (see frontal sections D and E).
ABCD
D
FIG. 10. The degenerating fibers following unilateral eye enucleation can be traced to the contralateral hypothalamus (not shown), lateral geniculate nucleus (GL), the pretectal area (AP) and the optic tectum (TeO). (TO) optic tract; III, third ventricle.
126
SVEN 0. E. EBBESSON
mammals and considerably less completely monopolized by the olfactory system than earlier studies by other methods had seemed to indicate. The diencephalon
The connectional organization of the shark hypothalamus remains unknown except for the information about a telencephalic input shown above (Fig. 9) and a retinal input revealed for the first time in elasmobranchs by means of the Nauta method (Ebbesson, 1967; Ebbesson & Ramsey, 1968). Considerable information has been obtained about the thalamus which can only be briefly described here. The retinal input was found to involve a relatively large portion (Fig. 10 and Ebbesson, 1967; Ebbesson & Ramsey, 1968) but other inputs have also recently been identified that add a whole new dimension to our understanding of thalamic organization. Contrary to common belief (see Aronson’s review, 1963) there are indeed inputs to the thalamus from spinal cord (Ebbesson, manuscript in preparation), optic tectum (Ebbesson, in preparation) and cerebellum (Ebbesson & Campbell, in preparation). Since such connections are known to exist in other vertebrates, it is now evident that the thalamus of the shark also has more in common with other vertebrates than hitherto believed. The mesencephalon
Although efferent projections of the tegmentum have not been studied as yet, the tectal projections have been studied in the nurse shark and are essentially similar to those of other vertebrates (Fig. 11). Our findings differ from earlier observations (Aronson, 1963) in that we were not able to confirm a tectal projection to the motor nuclei of the extraocular muscles, but observed a hitherto undescribed projection to the thalamus (Ebbesson, unpublished information).
FIG. 11. Projections of the optic tectum (TeO) are to the mesencephalic tegmenturn (TM), lateral geniculate nucleus (GL) and rhombencephalic reticular nuclei (see frontal section C). (Cb) cerebellum; (LI) inferior lobe of the hypothalamus; (Tel) telencephalon.
SHARK
BRAIN
127
ORGANIZATION
The cerebellum Cerebellar connections in the nurse shark also differ somewhat from earlier descriptions. In contrast to earlier workers, we found no evidence of a direct projection from cerebellar cortex to reticular and motor nuclei of the medulla and spinal cord. Only when the lateral cerebellar nucleus was lesioned were fibers Such lesions resulted in degeneration of traced outside the cerebellum proper. pathways reaching the motor nuclei of the extrinsic muscles of the eye, reticular cell groups, and a few fibers extending as far as the thalamus (Ebbesson & Campbell, manuscript in preparation). In many respects these connections are reminiscent of those found in other vertebrates although they give the appearance of greater diffuseness (Fig. 12).
c FIG. 12. Lesions of the cerebellum (Cb) involving the cerebellar nuclei (NCb) result in the degeneration of pathways that issue fibers to the contralateral rhombencephalic reticular formation (RR) via the brachium conjunctivum (bc). lateral geniculate nucleus; III, third ventricle.
(GL)
13. Degenerating fibers can be traced from the spinal cord (after a hemisection of the cord at the second spinal segment) to the dorsal column nuclei (DC); rhombencephalic reticular nuclei (RR); mesencephalon (M), and the thalamus (see frontal section A). (GL) lateral geniculate nucleus; III, third ventricle. FIG.
128
SVEN 0. E. EBBESSON
The spinal cord
The only available information about the spinal cord obtained from Nauta material relates to the ascending spinal projections (Fig. 13 and Ebbesson, manuscript in preparation). These appear similar to those described in other elasmobranchs with other methods, except for the finding of a small diffuse fascicle of spinal fibers that reach the thalamus. Earlier data had suggested a direct pathway only as far as the mesencephalon. SUMMARY
The studies reported here, in which the accurate tracing of distant neuronal connections was made possible by the Nauta method, are preliminary in nature and provide only information about general patterns of organization. Even so they do indicate that gross misconceptions about shark brain organization have existed and that only now, with recent tools like the Nauta method, electron microscopic techniques and histochemicaf methods, will we begin to understand the structural matrix underlying shark behavior. Acknow~dg~t-Tie the illustrations.
author wishes to thank Dr. Dolores Schroeder for hefping with
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FINK R. P. 81 HEIMERL. (1967) Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4,369-374. JOHNSTONJ. B. (1911) The telencephalon of selachians. J. comp. Neural. 21, 1-113. NAUTA-GYGAX(1954) Silver impregnation of degenerating axons in the central nervous system: a modified technique. Stain Technol. 29, 91-93. NIEUWENHUYSR. (1967) Comparative anatomy of olfactory centres and tracts. In Progress in Brain Research (Edited by ZOTTERMAN Y.), Vol. 23, pp. l-64. Elsevier, Amsterdam. SCHROEDER D. M. & EBBESSONS. 0. E. (1971) Diencephalic projections to the telencephalon of the nurse shark (Gingbymostoma cirratum). Anat. Rec. 169, 421 (Abstr.). WALLENBERGA. (1907) Beitrage zur Kenntniss des Gehirns der Teleostier und Selachier. Anat. Anz., Bd. 31, 369. Key Word Index-Brain; olfactory lobes; retina; optic tectum; telencephalon; thalamus; cerebellum; spinal cord; Ginglymostoma cirratum.