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Visual cortex in a reptile, the turtle (Pseudem ys scripta and Chrysem ys picta)
Brain Research, 130 (1977) 197-216 ~:) Elsevier/North-Holland Biomedical Press
197
Research Reports
VISUAL CORTEX IN A REPTILE, T H E T U R T L E ( P S E U D E M YS SCRIPTA AND CHR YSEM YS PICTA)
JANET A. HALL, ROBERT E. FOSTER, FORD F. EBNER and WILLIAM C. HALL Departments of Anatotny and Psychology, Duke University Medical Center, Durham, N.C. 27710 and Neuroseiences Section, Division of Biological and Medical Sciences, Brown University, Providence, R.I. 02912 (U.S.A.)
(Accepted November 18th, 1976)
SUMMARY The afferent and efferent connections of general cortex were studied in two species of turtle, Pseudemys scripta and Chrysemys picta. The thalamic distribution of labeled cells following cortical applications of horseradish peroxidase (HRP) indicated that at least 3 dorsal thalamic nuclei, the dorsal lateral geniculate nucleus, nucleus ventralis and the nucleus dorsolateralis anterior, project to general cortex. When cortical applications of H R P were combined with intraocular injections of tritiated proline in the same animal, autoradiographically labeled retinal terminations were found among the dendrites of lateral geniculate neurons containing the H R P reaction product. These experiments demonstrated that the dorsal lateral geniculate nucleus not only projects to general cortex, but also receives retinal input. Thus, general cortex in the turtle is a target of a visual pathway which relays in the dorsal thalamus. Cortical lesions produced anterograde degeneration in the same thalamic nuclei which the HRP experiments demonstrated project to cortex, thereby indicating that the dorsal thalamus and general cortex have reciprocal connections in the turtle. These same experiments with cortical lesions demonstrated that general cortex also sends projections to the optic rectum and tegmentum of the midbrain. These afferent and efferent connections of general cortex in the turtle are compared with the connections of general cortex in other reptilian groups and with those of neocortex in mammals.
I NTROD UCT1ON The dorsal or general cortex of reptiles has long been of special interest to comparative neurobiologists because of its possible evolutionary relationships to the
198 neocortex of mammals. Since one of the most characteristic features of the neocortex is its reciprocal connections with the dorsal thalamus, the question of whether or not the general cortex is also interconnected with dorsal thalamic nuclei is crucial for determining how the general cortex and neocortex are related. Early in the last decade, experimental neuroanatomical evidence based on retrograde degeneration suggested that the dorsal thalamus of reptiles does not project to the general cortex since thalamic neurons did not undergo detectable retrograde changes following cortical lesions ~l,as. More recently, however, experimental neuroanatomical evidence based on anterograde degeneration was presemed ~hich suggested that the dorsal thalamic target of the retina in the turtle, Pseudcml~ ,scripta, projects to a restricted region in the general cortex ~s,~v. That is, eye enucleations produced terminal degeneration among dendrites of cells of the dorsal lateral geniculate nucleus in the thalamus and lesions which destroyed the dorsal lateral geniculate nucleus produced terminal degeneration among the apical dendrites of neurons in general cortex. In contrast to the studies which demonstrated a retino-geniculo-cortical pathway in the turtle, recent studies in other reptilian species which used both anterograde degeneration and axonal transport techniques s:-': failed to demonstrate a visual pathway to cortex which relays via the thalamus. Moreover, no efferent projections from the cortex were found to any dorsal thalamic nucleieS, zs. Because of the significance of these results for our views of cortical evolution, it seemed important to atternpt to confirm the existence of a visual pathway to cortex in the turtle with the newer techniques which employ axonal transport. Using methods based on both the retrograde transport of horseradish peroxidase (HRP) and the anterograde transport of tritiated amino acids, we have now confirmed the results of the earlier studies which described this pathway utilizing anterograde degeneration. In addition, we have obtained evidence from experiments based on anterograde degeneration which indicates that general cortex in the turtle sends a reciprocal projection back to the dorsal lateral geniculate nucleus. The results of these experiments are the subject of" the present report. METHODS Our experiments were performed on two species of turtle, Pseudem)s scripta and Chrysemys picta. The experimental procedures and results were similar for both species.
General surgical procedures Before surgery, the animal was anesthetized with sodium pentobarbital (37 mg/kg) and then placed in a warm, moist chamber until it reached a surgical level of anesthesia. The head was secured for surgery in a holder which allowed the head to be tilted in different angles. The brain was exposed in the following manner: a U-shaped skin flap was cut around the back o f the head and carefully retracted from the temporal muscles and skull, leaving the skin intact and attached rostrally between the
199 eyes. A dental burr was used to make the initial skull opening, which was widened with rongeurs to complete the exposure of the brain. After surgery, the cut edges of the skin were sutured together and the animal was placed in the warm, moist chamber until it completely recovered from the effects of the anesthetic. During the postoperative survival periods, the animals were maintained in an environmental temperature of approximately 25-30 °C.
Experiments with HRP Ten turtles received cortical applications of HRP. The H R P was applied to the general cortex by saturating a small piece of gelfoam with 30 ~ H R P (Sigma Type VI) in 0 . 6 9 ~ NaCI and placing it on the cortex over an area where a small cut in the pia had been made. Following survival periods of 2-5 days (we found 5 days to be best for retrograde transport when the animals were maintained at 25 °C), the animals were anesthetized and perfused with a phosphate-buffered solution of 0.5/o°/paraformaldehyde 2.50J/o glutaraldehyde. The brains were removed from the skull, stored in the fixative solution at 4 °C for 2 h, embedded in gelatine albumin, and stored overnight in a sucrose-phosphate buffer solution at 4 °C. After sectioning with a freezing microtome at 40 or 50 #m, the sections were incubated for 30 rain in a solution of 3,3'diaminobenzidine tetrahydrochloride according to the procedure of LaVail and LaVail 2a. The sections were then mounted on coated slides and lightly stained with cresyl violet acetate.
E.vperiments with autoradiography Four animals had in'~raocular injections of [aH]proline (a 20 #l injection concentrated in 0.691'o NaCI to 6.25 ktCi/ktl) made with a Hamilton syringe. Following survival periods which varied from 2 to 4 weeks, the animals were sacrificed and the brains removed and sectioned into 30 .urn thick sections on a freezing microtome. The sections were mounted on glass slides, defatted, coated with Kodak NTB-3 nuclear track emulsion and exposed in light tight boxes for 4 weeks at 4 °C. Following the exposure period, the slides were developed in D-19 at 14 c'C, fixed and counterstained with cresyl violet acetate 4.
Combination experiments In these experiments we used a combination of two techniques. Two turtles were used. The time course of the experiments was as follows: on day I each turtle received an intraocular injection of [aH]proline (a 20 #1 injection concentrated in 0.69 °/ooNaCI to 6.25 #Ci/ktl); on day 9 the same turtles received a cortical application of H R P in the manner described above; on day 14 the animals were sacrificed and the brains processed to the point of sectioning according to the procedure outlined above for the experiments utilizing HRP. The brains from the combination experiments were sectioned at 40 ~tm on a freezing microtome and the sections were collected in phosphate buffer. The sections then were processed by our standard procedure for producing the H R P reaction product. Every other section was also processed for autoradiography.
200
Anterograde degeneration experiments Twenty-six specimens of Pseudemys scripta received cortical lesions which were made under microscopic control by subpial aspiration with a fine glass pipette. Survival times ranged from 7 to 28 days. The optimal survival time was from 14 to 18 days. Following the survival period, the animals were perfused with 0.69 .°~i NaCI o~ forma li n. The brains were removed from the skull and stored in 10/,, ° followed by 10/o formalin for 4-7 days. Next, they were embedded in gelatin-albumin and placed in a 3 0 ~ sucrose-formalin solution for an additional 3-4 days. The brains were then sectioned at 30/~m on a freezing microtome. Every 10th section was stained with cresyl violet acetate and every 4th section was impregnated with silver by following procedure I of the Fink-Heimer technique 9. In selected cases, sections were also impregnated with silver by following method 7 described by EbbessonL
Golgi method The Cox-Golgi material described in the results was prepared according to Van der Loos' procedure4L
Analysis of the data In order to analyze and record the data, the distributions of degeneration and label were plotted on drawings of the sections with an X - Y plotter which was coupled to the stage of a Zeiss photomicroscope 10. The drawings of the Golgi material were made with the aid of a camera lucida. The photomicrographs were made with the Zeiss photomicroscope using Kodak Panatomic-X or High Contrast Copy film. RESULTS First, we will describe the results of the experiments designed to demonstrate that the dorsal lateral geniculate nucleus in Pseudemys seripta and Chrysemyspicta projects to general cortex. Next, we will describe the distribution of the efferent projections from general cortex to the brain stem. The intrahemispheric connections of the general cortex will be described in a later paper.
Geniculo-corticalprojections The results of an experiment in which a small localized application of H R P was placed in general cortex is illustrated in Fig. 1. The location of the application in this case is reconstructed on a dorsal view of the hemisphere at the top of the figure, with representative transverse sections through the application site illustrated just below. The application site is restricted to the general cortex with perhaps some extension into the adjacent pallial thickening. The application site does not involve either the subiculum medially, the pyriform cortex laterally or the dorsal ventricular ridge ventrally. In the lower part of Fig. 1 are drawings of 3 transverse sections through the diencephalon. The labeled cells are indicated by dots on each transverse section. The drawings illustrate that the cells in the thalamus which contain the H R P reaction
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Fig. 1. Case with an application of HRP in general cortex. The application site is shown in black on a dorsal view of the hemisphere in the upper part of the figure and in transverse sections immediately below the hemisphere. The stippled area surrounding the black area contains less dense reaction product, presumably due to spread. Selected sections through the thalamus are shown at the bottom of the figure. Cells which are labeled with HRP reaction product are depicted in each section by black dots. In this case, the labeled cells are restricted to the dorsal lateral geniculate nucleus. Survival time, 5 days. Scale, 1 ram. Abbreviations used in this and following figures: AT, area triangularis; AC, anterior commissure; BTN, basal telencephalic nuclei; DLA, nucleus dorsolateralis anterior; DM, nucleus dorsomedialis anterior; DP, dorsal peduncle of the lateral forebrain bundle; DVR, dorsal ventricular ridge; ExSt, extrastriate cortex; GC, general cortex; GLd, lateral geniculate nucleus, dorsal part; GLv, lateral geniculate nucleus, ventral part; H, habenula; H', hippocampus; Hy, hypothalamus; LFB, lateral forebrain bundle; MFB, medial forebrain bundle; OB, olfactory bulb; PaTh, pallial thickening; PT, pretectal area; Pul, pulvinar; PYR, pyriform cortex; Re, nucleus reuniens; Rh, rhinal fissure; Rt, nucleus rotundus; S, subiculum; SC, superior colliculus; SGAP, stratum griseum et album periventriculare; St, striate cortex; T, temporal cortex; Teg, tegmentum; TeO, optic rectum; TrO, optic tract; V, nucleus ventralis; VP, ventral peduncle of the lateral forebrain bundle; n.III, oculomotor nerve; 1II, oculomotor nucleus.
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203 product are restricted to one cell group, the dorsal lateral geniculate nucleus. In particular, note that no labeled cells are present in either nucleus reuniens or nucleus rotundus, both of which are cell groups of the reptilian dorsal thalamus known to project to the underlying dorsal ventricular ridgell,l
Fig. 2. Case with an application of H R P in general cortex. T h e locations of the labeled cells depicted by dots are plotted on outlines of transverse sections t h r o u g h the t h a l a m u s . The p h o t o m i c r o g r a p h s of the Nissl-stained sections are of the sections outlined in the figure, but were printed as their mirror image. Survival time, 5 days. Scale, 1 ram.
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Fig. 4. A: a drawing of the dorsal lateral geniculate nucleus from a section impregnated by the GolgiCox method. The dendrites of the lateral geniculate neurons extend into the neuropil between the cell bodies and the optic tract. Scale, 135/~m. B, C: dark-field photomicrographs of the autoradiographic signal (B) and degeneration (C) in the dorsal lateral geniculate nucleus following an injection (B) and lesion (C) in general cortex. The arrows indicate the medial border of the optic tract. Both techniques show that the corticofugal projection is focussed around the cell bodies rather than in the dendritic neuropil between the cell bodies and the optic tract• The photomicrograph in C is of section 20 in Fig. 5. Scale, 0.59 ram. optic tract a n d the cell perikarya of the dorsal lateral geniculate cells (see Fig. 3B, C a n d D middle column). The drawing of a Golgi-stained section t h r o u g h the lateral geniculate nucleus in Pseudemysscripta in Fig. 4A shows that the geniculate cells issue dendrites into the retinal terminal zone intercalated between the cell bodies and the optic tract. Finally, the dark-field photographs in the right c o l u m n of Fig. 3 illustrate that at each level through the dorsal lateral geniculate nucleus (Fig. 3B, C a n d D), dense H R P reaction p r o d u c t was present in the cell perikarya of the same cells which send their dendrites into the terminal zone of the retinal projections. In conclusion, cells in the dorsal lateral geniculate nucleus of the turtle receive projections from the retina and, in turn, project to general cortex. In Fig. 3 it should be noted that the autoradiographic signal extends slightly
Fig. 3. Photomicrographs of sections through the thalamus of a case which received both a cortical application of H RP and an intraocular injection of [3H]proline. This is the same case that was illustrated in Fig. 2. The photomicrographs in the left column are enlargements ( × 22) of the photomicrographs shown in Fig. 2 and are Nissl-counterstained sections which were processed for HRP. The areas within the small rectangles are shown in dark-field photomicrographs ( ~ 200) in the right column. The area within the rectangle in B, C and D is within the dorsal lateral geniculate nucleus and contains cells labeled with the HRP reaction product (B, C, D). The labeled cells in A are located in nucleus ventralis. The middle column shows adjacent sections ( x 22) through the same brain which were processed for autoradiography. The autoradiographic label is concentrated in the zone intercalated between the cell bodies of the lateral geniculate nucleus and the optic tract.
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Fig. 5. Case with a cortical lesion. The lesion in general cortex is shown in black at the top of the figure in transverse sections and on a dorsal view of the hemisphere. The axon degeneration is illustrated by short lines in the plots of cortical and brain stem sections. Terminal debris is illustrated by small dots. Survival time, 18 days, Scale, I mm.
m o r e rostral than did the a n t e r o g r a d e d e g e n e r a t i o n in a previous study ]7, a n d we now include this most rostral a r e a (see section 85 in Fig. 2 a n d the same section in Fig. 3B) in our definition o f the dorsal lateral geniculate nucleus. The ultimate justification for our definition is the c o r r e s p o n d e n c e between the d i s t r i b u t i o n o f retinal projections and the cells which project to general cortex.
CorticoJugal proiections The following cases will d e m o n s t r a t e that the m a j o r efferent conriections of general cortex in the turtle are with the d o r s a l t h a l a m u s , the optic rectum and the t e g m e n t u m o f the midbrain. Fig. 5 illustrates a lesion in general cortex a n d the resulting a n t e r o g r a d e d e g e n e r a t i o n which was t r a c e d to the b r a i n stem, D e g e n e r a t e d axons t h a t emerge from the d a m a g e d area are present in two separate pathways, one medial a n d one lateral (see Fig. 5, sections 9 and 13). The fibers in the medial p a t h w a y descend t o w a r d the brain stem in the medial wall o f the hemisphere j u s t medial to the cells of the subiculum a n d h i p p o c a m p u s . I n their course t o w a r d the d i e n c e p h a l o n
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207 these fibers pass through the parolfactory area and enter the diencephalon as part of the medial forebrain bundle (Fig. 5 sections 9 and 13, 20 28.5). The axons of the lateral pathway leave the general cortex between the pallial thickening and pyriform cortex and then descend through the basal telencephalic nuclei to form part of the lateral forebrain bundle. They exit the telencephalon in the lateral forebrain bundle and, by their location, can be clearly distinguished from the fibers in the medial forebrain bundle. When these lateral fibers enter the diencephalon, they form part of the ventral peduncle of the lateral forebrain bundle (Fig. 5, sections 20 28.5). The fibers in the medial forebrain bundle are separate from and medial to those in the ventral peduncle of the lateral forebrain bundle (Fig. 5, sections 20-28.5). In the diencephalon, fibers leave the ventral peduncle and ascend to the dorsal thalamus where they terminate among the cell bodies of the dorsal lateral geniculate nucleus (Fig. 5, sections 20-23). The plots of the terminal distribution of this pathway in Fig. 5 indicate that, in contrast to the retinal projections which terminate primarily in the dendritic zone intercalated between the cell bodies and the optic tract, the main terminal focus of the corticofugal projections is among the geniculate cell bodies. Much sl~arser degeneration is present in the dendritic zone of the dorsal lateral geniculate nucleus. This distribution of terminal degeneration in the lateral geniculate nucleus is illustrated by the dark-field photomicrograph of the degeneration in section 20 which is presented in Fig. 4C. The photomicrograph in Fig. 4B is from an autoradiographic experinaent and shows that the same distribution of terminations is found in the dorsal lateral geniculate nucleus after a cortical injection of tritiated proline. Following larger cortical lesions, such as the one illustrated in Fig. 6, terminations are found in several thalamic nuclei in addition to dorsal lateral geniculate, including nucleus rotundus, nucleus ventralis and the nucleus dorsolateralis anterior. In the case illustrated in Fig. 6, sparse degeneration is also present in nucleus dorsomedialis anterior but, on the basis of other cases, we attribute this sparse degeneration to a slight extension of the lesion into the subiculum or hippocampus on the medial wall of the hemisphere. In the mesencephalon, the degenerated fibers from the ventral peduncle divide into two bundles: one terminates in the midbrain tegmentum and the other travels dorsally to reach the deep layers of the optic tectum (see section 28.5 in Fig. 5). Althou~_h well-defined terminal fields were not found in the optic tectum following cortical lesions, in experiments utilizing anterograde transport of labeled amino acids, dense autoradiographic signal is present in the Feriventricular layers and less dense signal in the more suFerficial layers (unpublished experiments). The bundle of fibers which terminates in the midbrain tegmentum is confined to the central zone of the tegmentum. The degenerated fibers in the medial forebrain bundle travel through the diencephalon apparently without terminating and enter the midbrain where they terminate in the tegmentum (Fig. 5, section 28.5). This area of termination either overlaps or is immediately adjacent to the te~mental target of the lateral forebrain bundle, and, as a consequence, separate terminal zones could not be differentiated. No degenerating fibers from either the medial or lateral forebrain bundle pathways could be traced caudal to the midbrain.
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Fig. 7. Reconstruction of cortical lesions on a dorsal view of the hemisphere. The black areas indicate the locations of lesions which produced degeneration only in the medial forebrain bundle, while the parallel lines indicate the locations of lesions which produced degeneration in both the medial and the lateral forebrain bundle. The dashed line indicates the extent of the cortical target of the dorsal thalamus as it was estimated in an earlier study by the distribution of degeneration following hemithalamectomies ~6. The single lesion located caudal and lateral to the thalamic receptive zone may have undercut the descending pathways. Lesions in various regions o f the turtle's cortex suggest that the medial forebrain bundle fibers and the lateral forebrain bundle fibers arise from different cortical areas. F o r example, fig. 7 illustrates the locations o f 11 small cortical lesions. The 7 lesions indicated in black in Fig. 7 are all restricted to the medial half o f general cortex a n d p r o d u c e d d e g e n e r a t i o n only in the medial f o r e b r a i n bundle. The 4 lesions which are indicated by d i a g o n a l lines in Fig. 7 all include d a m a g e to the lateral half o f general cortex a n d all p r o d u c e d d e g e n e r a t i o n in the lateral as well as in the medial forebrain p a t h w a y . Finally, medial lesions which invaded the h i p p o c a m p u s p r o d u c e d degeneration o f axons which descended a l o n g the medial wall o f the hemisphere a n d t e r m i n a ted in the h y p o t h a l a m u s . This projection was only seen following survival times s h o r t e r t h a n the 21 day p e r i o d for the case illustrated in Fig. 6. DISCUSSION The results o f the present study are consistent with the view that general cortex in at least the species o f turtle we have studied shares h o m o l o g o u s features with the n e o c o r t e x o f m a m m a l s . First o f all, general cortex, like neocortex, receives projections from the dorsal thalamus. Thus, our results d e m o n s t r a t e d that general cortex in the turtle is a target o f at least one ascending sensory p a t h w a y which relays in the dorsal t h a l a m u s . T h a t is, i n t r a o c u l a r injections o f tritiated a m i n o acids p r o d u c e d label
Fig. 6. Case with a large cortical lesion. In the upper part of the figure the lesion is shown in black on a dorsal view of the hemisphere and in transverse sections. The plots in the lower part of the figure are of sections through the thalamus and midbrain. In these plots axon degeneration is illustrated by short lines and terminal debris by small dots. Survival time, 21 days. Scale, 1 ram.
210 among the dendrites of the same dorsal thalamic cells which contained reaction product following cortical applications of HRP. Moreover, since in our cases with larger cortical applications of HRP labeled cells were present in additional thalamic cell groups including nucleus ventralis and nucleus dorsolateralis anterior, it seems probable that the dorsal lateral geniculate nucleus is not the only thalamic nucleus which projects to general cortex in turtles. The second feature of general cortex demonstrated by the present experiments is that it sends reciprocal projeciions back to the thalamus. That is, cortical lesions produced degeneration in the same cell groups which the HRP experiments demonstrated project to cortex. Finally, the same cortical lesions which demonstrated reciprocal projections to the thalamus also revealed corticofugal projections to the optic tectum of the midbrain. All of these connections of general cortex are also shared by the neocortex of mammals. The afJerent connections of general cortex in the turtle and other reptilian ,~roups The present study together with the previous anterograde degeneration studiesl6,17 comprise two lines oF evidence for thalamocortical projections in turtles which utilized techniques that rely on very different types of neuronal processes, anterograde degeneration and retrograde axonal transport. Degenerating axon terminals in the turtle's general cortex have also been observed with the electron microscope following thalamic lesions s. On the other hand, the results of similar studies in two species of a second reptilian group, the lizards, suggest that general cortex is not a target of a visual pathway which relays through the dorsal thalamus in all reptiles. For example, following numerous injections of HRP in various parts of general cortex in the Tegu lizard, Tupinambis nigropunctatus, labeled cells were found in nucleus dorsolateralis anterior but not in the dorsal lateral geniculate nucleus~7~ Similarly, Distel and Ebbesson 6 were unable to demonstrate significant projections from the dorsal lateral geniculate nucleus to general cortex in the monitor lizard, Varanus benegah, nsis, with anterograde degeneration techniques, but in the same series of experiments were able to demonstrate a cortical projection from the nucleus dorsolateralis anterior. These differences from our results in the turtle do not simply reflect a difference in nomenclature for thalamic nuclei in these two reptilian groups since other experiments 2,5J~ have shown that nucleus dorsolateralis anterior is ~ot a maior target of the retina in lizards, and furthermore, the dorsal thalamic target of the retina in the Tegu lizard does not contain labeled cells after cortical injections of HRW 7. In comparing the organization of cortex in the turtle and other reptiles, it is important to emphasize that our larger injections of H R P in the turtle's general cortex did produce label in cells in nucleus dorsolateralis anterior as well as in the dorsal lateral geniculate nucleus. This result suggests that at least the pathway from the nucleus dorsolateralis anterior to cortex is shared by both reptilian groups, Although the further connections of the nucleus dorsolateralis anterior are unknown for any reptilian species, the efferent connections of its cortical target in the Tegu lizard (see below) suggest that it may have close associations with the limbic system. In addition to the dorsal lateral geniculate nucleus and nucleus dorsolateralis anterior, a third nucleus, nucleus ventralis, contained labeled cells following the larger
211 injections of HRP in general cortex of the turtle. This result is of special interest since several lines of evidence suggest that nucleus ventralis may constitute a dorsal thalamic relay for the somatic pathway which physiologists have reported ascends to general cortex 'q,a°. For example, Distel and Ebbesson 6 have traced projections from the spinal cord and dorsal column nuclei to this general region in the monitor lizard. Moreover, Belekhova and Kosareva 1 reported that four-fifths of the cells they recorded from in this region in the turtle responded to somatic sensory stimulation, and that tetanizing stimulation of any part of this zone blocked the cortical evoked potentials to either ipsilateral or contralateral somatic stimulation. In summary, we have presented evidence that at least 3 dorsal thalamic nuclei have connections with the general cortex of the turtle. Two of these nuclei, the dorsal lateral geniculate and nucleus ventralis, may be relays for ascending sensory pathways. The visual pathway may not be present in all reptiles, since it has not been demonstrated in studies of" thalamocortical pathways in two species of lizard 6,27. The somatic pathway may be absent in at least one species of lizard, the Tegu, Tupinambis nigropunctatus ~7. The third dorsal thalamic nucleus, nucleus dorsolateralis anterior, appears to be present in both lizards and turtles and may have close associations with the limbic system.
The e/5]kJrent connection of general cortex in the turth, and other reptilian groups General cortex in the turtles we have studied not only receives projections from the dorsal thalamus but also sends projections back to the thalamus. Thus, lesions in the general cortex result in anterograde degeneration in the dorsal thalamus. In particular, projections were found to the same cell group, the dorsal lateral geniculate nucleus, which contains cells with reaction product following cortical applications of H RP. Following large cortical lesions, terminal fields are also present in several nuclei in addition to the dorsal lateral geniculate nucleus, including nucleus rotundus, nucleus dorsolateralis anterior and nucleus ventralis in the dorsal thalamus. The only other major brain stem targets of general cortex found were the optic tectum and the underlying tegmentum of the midbrain. These corticofugal projections provide another line of evidence that general cortex is not organized in the same manner in all reptilian groups, since cortical lesions in the Tegu lezard, Tupinambis nigropunctatus do not reveal projections to either the dorsal thalamus or the midbrain. Instead, the major non-telencephalic target of general cortex in the Tegu lizard is the hypothalamus2~, 26. In our experiments in Pseudeno's scripta, projections to the hypothalamus were found only after lesions or injections of [aH]proline (unpublished experiments) which extended far enough medially to invade the hippocampus. Although degenerated fibers could be traced caudally in the medial as well as in the lateral forebrain bundle following lateral lesions in the turtle's general cortex, in our material terminations contributed by the medial fibers could only be found in the midbrain tegmentum. Whether this medial system of fibers reflects limbic as well as exteroceptive sensory associations for the lateral general cortex in turtles cannot be answered by the present experiments. The tegmental target of this pathway could
2t2 conceivably be a pretectal area with associations with the visual system~ a midbrain limbic area, or a source of decending reticulo-brain stem and spinal motor pathways. The question of whether the same cell populations in general cortex give rise to both the lateral and medial pathways to the brain stem might be answered in further experiments by injecting H R P in the targets of these two pathways. The possibility remains, for example, that the degenerated fibers in the medial forebrain bundle, which we find after lateral lesions in the general cortex, arise from another structure such as the pyrifrom cortex and travel through general cortex in route to the medial wall of the hemisphere. On the other hand, the close spatial association in the midbrain tegmentum between the targets of the medial and lateral pathways from the lateral parts of general cortex suggests that they may arise from functionally similar cell groups. Finally, it should be emphasized that projections from the general cortex to the dorsal thalamus are not unique to Pseudemys scripta among reptiles since corticofugal pathways to the thalamus have also been described in Chrysemys picta 2~. in the sidenecked turtle, Podoenemis unifilis 43, and in garter snakes of the genus ThamnophislS~ In the garter snake, the thalamic target of general cortex is a cell group with unknown relationships to nuclei in the thalamus of turtles, but in Chrysemys pieta the target apparently includes the lateral geniculate nucleus. In Podoenernis unifilis, the thalamic targets include both the dorsal and ventral lateral geniculate nuclei and nucleus rotundus. These efferent connections from the cortex to the thalamus in turtles and snakes are clearly different from the total absence of efferent projections to the thalamus which has been reported for general cortex in Tegu lizards. Either these connections between the cortex and the thalamus do not exist in the Tegu lizard or they are so much less well-developed that available techniques have failed to reveal them. In summary, we have shown that corticofucal fibers from the turtle's general cortex innervate several brain stem structures including the dorsal lateral geniculate nucleus, nucleus rotundus, nucleus ventralis, nucleus dorsolateralis anterior, the optic tectum and the rostral midbrain tegmentum. Similar corticofugal projections are seen in a distantly related species of turtle as well as in the garter snake. Once again there appears to be a difference in cortical organization between turtles and at [east one species of lizard, the Tegu, since the only brain-stem target of the Tegu lizard's general cortex is the hypothalamus. However, not all members of the order Squamata have this organization of efferent connections from general cortex since the garter snake's corticofugal pathways include a thalamic target.
Visual pathways to the telencephalon in the turtle and mammals The main result of the present study together with the results of previous studies of the connections of nucleus rotundus in the turtle are summarized diagrammatically in the left half of Fig. 8. Two separate visual pathways can be traced to the telencephalon in at least the two reptilian species we have studied, Pseudemys scripta and Chrysemyspieta. The first is the retino-geniculo-cortical system which sends visual information to general cortex by means of a relay in the dorsal thalamus. In the turtle,
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Fig. 8. Diagram of visual pathways in the turtle, Pseudemys scripta and in a mammal, Tupaia g/is. Two pathways to the telencephalon are illustrated for both groups, one which projects to cortex after a relay in the dorsal thalamus (GLd) and one which projects to the telencephalon after relaying first in the optic tectum of the midbrain and then in the dorsal thalamus (Rt or Pul). In both groups, the projections to the telencephalon from the thalamus are reciprocated by descending connections. The cortex in both groups also gives rise to projections to the optic rectum.
214 this pathway may provide the anatomical basis for the potentials which can be evoked in general cortex by visual stimulationl,~a, a0. This pathway in the turtle resembles the mammalian geniculo-striate system which is diagrammed in the right half of Fig. 8. As Fig. 8 illustrates, both in mammals and in the turtle, the cortical target of this visual pathway also sends reciprocal projections back to the dorsal lateral geniculate nucleus. In most vertebrates, the superficial layers of the optic tectum are also major targets of the retina, and in reptiles, the optic tectum projects heavily to nucleus rotundus in the dorsal thalamus a,1°,17. Nucleus rotundus in turn hab reciprocal connections with the dorsal ventricular ridge in the telencephalon 1a,16,:~:~, A similar visual pathway in mammals which relays in the superficial layers of the superior colliculus and the putvinar in route to extrastriate and temporal cortex is diagrammed on the right in Fig. 8. As previous papers have pointed out 'm, a very similar second visual pathway to the telencephalon is also present in birds, and consequently, il seems probable that at least two separate visual pathways to the telencephalon were established by the time these 3 classes diverged during the evolution of vertebrates. Finally, both general cortex in the turtle and the mammalian neocortex project to the optic tectum. In mammals, these projections are very prominent and in physiological experiments have been demonstrated to play an important role in establishing the receptive field properties of tectal neurons "s,'~1,4°,41,44. In the turtle. these projections are also prominent but their functions are unknown. Cortical organization in turtles and mammals In comparing our results with the results of similar studies in mammals, it is evident that the reciprocal connections between the thalamus and general cortex resemble the interconnections between the mammalian thalamus and neocortex. It is also evident that the overall distribution of corticofugal projections to the thalamus and midbrain in the turtle is more reminiscent of the efferent connections of neocortical sensory areas than of mammalian motor cortex. Thus, none of the efferent projections to brain stem nuclei caudal to the midbrain or to the spinal cord which are characteristic of mammalian motor cortex ss,s4 were found in the turtle, even after very large cortical lesions which extended from the medial wall of the hemisphere laterally to include the pallial thickening. The lack of efferent projections from the pallial thickening to the lower brain stem and spinal cord is of special interest since this is the telencephalic area of turtles which Johnston 19 proposed, on the basis of electrical stimulation, was the reptilian motor cortex. Whether other telencephalic areas in the turtle give rise to these pathways in unknown, but the motor responses which can be evoked by cortical stimulation in this group 19 might be mediated by corticoreticular pathways which our results indicate project from general cortex to the midbrain tegmentum. In conclusion, our evidence indicates that in turtles there is a region in cortex which receives projections from a visual thalamic area and sends reciprocal projections back. This same cortical area apparently sends projections to the optic tectum. Thus, general cortex shares several features with the 6-layered mammalian neocortex. At the same time, general cortex in the turtle consists of only a single layer of cells and may
215 correspond more closely to one neocortical cell layer, such as layer V1, which receives thalamocortical projections a n d gives rise to corticofugal projections in m a m m a l s ~', 34,z7-39, t h a n to a composite of all the neocortical layers. In any case, the reduced n u m b e r and complexity of cortical connections in turtles, taken together with the similarities in brain stem targets which they share with mammals, support the view that general cortex in turtles approximates an early stage in the evolution of the m a m m a l i a n neocortex. It is our hope that further studies of this much more simple cortex will eventually shed new light on the mechanisms a n d functional significance of the neocortex. ACKNOWLEDGEMENTS Supported by: N I N D S G r a n t NS09623 to W. C. Hall a n d U.S.P.H.S. G r a n t NB06551 to F. F. Ebner. N I H Predoctoral Fellowship 5 F01 M H 58576 to Robert E. Foster. N I M H Research Scientist Development Award M H 25734 to W. C. Hall.
REFERENCES 1 Belekhova, M. G., and Kosareva, A. A., Organization of the turtle thalamus: visual, somatic and tectal zones, Brain Behav. Evol., 4 (1971) 337 375. 2 Butler, A. B. and Northcutt, R. G., Retinal projections in Iguana iguana and Anolis carolinensis, Brain Research, 26 ( 1971) 1-13. 3 Butler, A. B. and Northcutt, R. G., Ascending tectal efferent projections in the lizard Iguana iguana, Brahl Research, 35 (1971) 597-601. 4 Cowan, W. M., Gottlieb, D. 1., Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brahz Research, 37 (1972) 21-51. 5 Cruce, W. L. R. and Cruce, J. A. F., Projections from the retina to the lateral geniculate nucleus and mesencephalic tectum in a reptile (Tupinambis nigropunctatus): a comparison of anterograde transport and anterograde degeneration, Brain Research, 85 (1975) 221 228. 6 Distel, H. and Ebbesson, S. O. E., Connections of the thalamus in the monitor lizard, Neurosci. Abstr., 1 (1975) 559. 7 Ebbesson, S. O. E., The selective silver impregnation of degenerating axoplasm in non-mammalian species. In W. J. Nauta and S. O. E. Ebbesson (Eds.), Contemporao, Research Methods h~ Neuroamttomy, Springer, New York, 1970, pp. 132-161. 8 Ebner, F. F. and Colonnier, M., lnterneuronal and neuroependymal synaptic relationships in "visual" cortex of adult turtle (Pseudemys scripta), Anat. Rec., 175 (1973) 312. 9 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic ends in the central nervous system, Brain Research, 4 (1967) 369 374. 10 Foster, R. E. and Hall, W. C., The connections and laminar organization of the optic rectum in a reptile (Iguana iguana), J. comp. NeuroL, 163 (1975) 397 426. 11 Foster, R. E. and Peele, T. L., Thalamotelencephalic auditory pathways in the lizard (Iguana iguana), Anat. Rec., 18l (1975) 530. 12 Gilbert, C. D. and LeVay, S., Laminar patterns of genicu[ocortical projections in cat, Neurosci. Abstr., 1 (1975)63. 13 Gusel'nikov, V. 1., Morenkov, E. D. and Pivovarov, A. S., Unit responses of the turtle forebrain to visual stimuli, Neurosci. Behav. Physiol., 5 (1971) 235 242. 14 Hall, J. A. and Ebner, F. F., Unpublished experiments. 15 Hall, W. C., Visual pathways to the telencephalon in reptiles and mammals, Brain Behav. Evol., 5(1972) 95 113. 16 Hall, W. C. and Ebner, F. F., Thalamotelencephalic projections in the turtle (Pseudemys scripta), J. comp. NeuroL, 140 (1970) 101-122.
2t6 17 Hall, W. C. and Ebner, F. F., Parallels in the visual afferent projections of the thalamus in the hedgehog (Paraeehinus hypomelas) and the turtle (Pseudemys scripta), Brain Behav. EvoL, (1970) 135-154. 18 Halpern, M., Efferent connections of the lateral and dorsal cortices of snakes of the genus I'hamnophis, Anat. Rec., 184 (1976) 421. 19 Johnston, J. B., Evidence of a motor pallium in the lbrebrain of reptiles, J. comp. Neuro[., 26 (1916~ 475~,79. 20 Karten, H. J., The organization of the avian te[encephalon and some speculations on the phylogeny of the amniote telencephalon, Ann. N. Y. Acad. Sci., 167 (1969) 164-179. 2l Kruger, L. and Berkowitz, E. C., The main afferent connections of the reptilian telencephalon as determined by degeneration and electrophysiological methods, J. comp. Neurol.: 115 (1960) 125-141. 22 Kuypers, H. G. J. M. and Lawrence, D. G., Cortical projections to the red nucleus and the brainstem in the rhesus monkey, Brain Research, 4 (1967) 151-188. 23 LaVail, J. H. and LaVail, M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. Neuro/.. 157 (1974) 303-357. 24 Lawrence, D. G. and Kuypers, H. G. J. M., Pyramidal and non-pyramidal pathways in monkeys: anatomical and functional correlation, Science, 148 (1965) 973-975. 25 Lohman, A. H. M. and Mentink, G. M., Some cortical connections of the tegu lizard (Tupinambi.v teguixin), Brain Research, 45 (1972) 325- 344. 26 Lohman, A. H. M. and Van Woerden-Verkley, 1., kurthe[ studies on the cortical connections of the tegu lizard, Brain Research, 103 (1976) 9-28. 27 Lohman, A. H. M. and Van Woerden-Verkley, I., Thatamocortical relationships in the brain of a lizard, Exp. Brain Res., in press. 28 Michael, C. R., Integration of retinal and cortical information in the superior colliculus of the ground squirrel, Brain Behav. Evol., 3 (1970) 205--209. 29 Northcutt, R. G., The Telencephalon of the Western Painted Turtle ( Chrysemys picta belli ), Illinois Biol. Monogr., Vol. 43. Univ. of lllinois Press, Urbana, 111., 1970, pp. 1-113. 30 Orrego, F., The reptilian forebrain. I. The olfactory pathways and cortical areas in the turtle, Arch. ital. BioL, 99 (1961) 425 445. 31 Palmer, A. L. and Rosenquist, A. C., Visual receptive fields of single striate cortical neurons projecting to the superior colliculus in the cat, Brain Research, 67 (1974) 27-42. 32 Papez, J. W., Thalamus of turtles and thalamic evolution, J. comp. Neurol., 61 (1935) 433-475. 33 Parent, A., Striatal afferent connections in the turtle (Chryseno,s picta) as revealed by retrograde axonal transport of horseradish peroxidase, Brain Research, 108 (1976) 25-36. 34 Peters, A. and Feldman, M. L., The projections of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. I. General description, J. NeurocytoL, 5 (1976) 63-84. 35 Powell, T. P. S. and Kruger, L., The thalamic projection upon the telencephalon in Lacerta viridis, J. Anat. (Lond.), 94 (1960) 528-542. 36 Pritz, M. B., Ascending connections of a thalamic auditory area in a crocodile, Caim~m crococfih~s, J. comp. Neurol., 153 (1974) 199-214. 37 Ribak, C. E. and Peters, A., An autoradiographic study of the projections from the lateral geniculate body in the rat, Brain Research, 92 (1975) 341-368. 38 Robson, J. A. and Hall, W. C., Connections of layer VI in striate cortex of the grey squirrel (Sciurus carolinensis), Brahl Research, 93 (1975) 133 139. 39 Rosenquist, A. C., Edwards, S. B. and Palmer, L. A., An autoradiographic study of the projections of the dorsal lateral geniculate nucleus in the cat, Brain Research, 80 (1974) 71-93. 40 Rosenquist, A. C. and Palmer, L. A., Visual receptive field properties of cells of the superior collicutus after cortical lesions in the cat, L~p. Neurol., 33 (1971) 629-652. 4l Schiller, P. H., Stryker, M., Cynader, M. and Berman, N., Response characteristics of single cells in the monkey superior colliculus following ablation or cooling of visual cortex, J. Neurophysiol., 37 (1974) 181-194. 42 Van der Loos, H., Dendrodendritische Verbindingen in de Schors der Grote Hersenen, Doctoral Thesis, University of Amsterdam, 1956. 43 Ware, C. B., Projections of dorsal cortex in the side necked turtle (Podocnemis unifilis'), Neurosci. Abstr., (1974) 466. 44 Wickelgren, B. G. and Sterling, P., Influence of visual cortex on receptive fields in the superior colliculus of the cat, J. Neurophysiol., 32 (1969) 16-23.
197
Research Reports
VISUAL CORTEX IN A REPTILE, T H E T U R T L E ( P S E U D E M YS SCRIPTA AND CHR YSEM YS PICTA)
JANET A. HALL, ROBERT E. FOSTER, FORD F. EBNER and WILLIAM C. HALL Departments of Anatotny and Psychology, Duke University Medical Center, Durham, N.C. 27710 and Neuroseiences Section, Division of Biological and Medical Sciences, Brown University, Providence, R.I. 02912 (U.S.A.)
(Accepted November 18th, 1976)
SUMMARY The afferent and efferent connections of general cortex were studied in two species of turtle, Pseudemys scripta and Chrysemys picta. The thalamic distribution of labeled cells following cortical applications of horseradish peroxidase (HRP) indicated that at least 3 dorsal thalamic nuclei, the dorsal lateral geniculate nucleus, nucleus ventralis and the nucleus dorsolateralis anterior, project to general cortex. When cortical applications of H R P were combined with intraocular injections of tritiated proline in the same animal, autoradiographically labeled retinal terminations were found among the dendrites of lateral geniculate neurons containing the H R P reaction product. These experiments demonstrated that the dorsal lateral geniculate nucleus not only projects to general cortex, but also receives retinal input. Thus, general cortex in the turtle is a target of a visual pathway which relays in the dorsal thalamus. Cortical lesions produced anterograde degeneration in the same thalamic nuclei which the HRP experiments demonstrated project to cortex, thereby indicating that the dorsal thalamus and general cortex have reciprocal connections in the turtle. These same experiments with cortical lesions demonstrated that general cortex also sends projections to the optic rectum and tegmentum of the midbrain. These afferent and efferent connections of general cortex in the turtle are compared with the connections of general cortex in other reptilian groups and with those of neocortex in mammals.
I NTROD UCT1ON The dorsal or general cortex of reptiles has long been of special interest to comparative neurobiologists because of its possible evolutionary relationships to the
198 neocortex of mammals. Since one of the most characteristic features of the neocortex is its reciprocal connections with the dorsal thalamus, the question of whether or not the general cortex is also interconnected with dorsal thalamic nuclei is crucial for determining how the general cortex and neocortex are related. Early in the last decade, experimental neuroanatomical evidence based on retrograde degeneration suggested that the dorsal thalamus of reptiles does not project to the general cortex since thalamic neurons did not undergo detectable retrograde changes following cortical lesions ~l,as. More recently, however, experimental neuroanatomical evidence based on anterograde degeneration was presemed ~hich suggested that the dorsal thalamic target of the retina in the turtle, Pseudcml~ ,scripta, projects to a restricted region in the general cortex ~s,~v. That is, eye enucleations produced terminal degeneration among dendrites of cells of the dorsal lateral geniculate nucleus in the thalamus and lesions which destroyed the dorsal lateral geniculate nucleus produced terminal degeneration among the apical dendrites of neurons in general cortex. In contrast to the studies which demonstrated a retino-geniculo-cortical pathway in the turtle, recent studies in other reptilian species which used both anterograde degeneration and axonal transport techniques s:-': failed to demonstrate a visual pathway to cortex which relays via the thalamus. Moreover, no efferent projections from the cortex were found to any dorsal thalamic nucleieS, zs. Because of the significance of these results for our views of cortical evolution, it seemed important to atternpt to confirm the existence of a visual pathway to cortex in the turtle with the newer techniques which employ axonal transport. Using methods based on both the retrograde transport of horseradish peroxidase (HRP) and the anterograde transport of tritiated amino acids, we have now confirmed the results of the earlier studies which described this pathway utilizing anterograde degeneration. In addition, we have obtained evidence from experiments based on anterograde degeneration which indicates that general cortex in the turtle sends a reciprocal projection back to the dorsal lateral geniculate nucleus. The results of these experiments are the subject of" the present report. METHODS Our experiments were performed on two species of turtle, Pseudem)s scripta and Chrysemys picta. The experimental procedures and results were similar for both species.
General surgical procedures Before surgery, the animal was anesthetized with sodium pentobarbital (37 mg/kg) and then placed in a warm, moist chamber until it reached a surgical level of anesthesia. The head was secured for surgery in a holder which allowed the head to be tilted in different angles. The brain was exposed in the following manner: a U-shaped skin flap was cut around the back o f the head and carefully retracted from the temporal muscles and skull, leaving the skin intact and attached rostrally between the
199 eyes. A dental burr was used to make the initial skull opening, which was widened with rongeurs to complete the exposure of the brain. After surgery, the cut edges of the skin were sutured together and the animal was placed in the warm, moist chamber until it completely recovered from the effects of the anesthetic. During the postoperative survival periods, the animals were maintained in an environmental temperature of approximately 25-30 °C.
Experiments with HRP Ten turtles received cortical applications of HRP. The H R P was applied to the general cortex by saturating a small piece of gelfoam with 30 ~ H R P (Sigma Type VI) in 0 . 6 9 ~ NaCI and placing it on the cortex over an area where a small cut in the pia had been made. Following survival periods of 2-5 days (we found 5 days to be best for retrograde transport when the animals were maintained at 25 °C), the animals were anesthetized and perfused with a phosphate-buffered solution of 0.5/o°/paraformaldehyde 2.50J/o glutaraldehyde. The brains were removed from the skull, stored in the fixative solution at 4 °C for 2 h, embedded in gelatine albumin, and stored overnight in a sucrose-phosphate buffer solution at 4 °C. After sectioning with a freezing microtome at 40 or 50 #m, the sections were incubated for 30 rain in a solution of 3,3'diaminobenzidine tetrahydrochloride according to the procedure of LaVail and LaVail 2a. The sections were then mounted on coated slides and lightly stained with cresyl violet acetate.
E.vperiments with autoradiography Four animals had in'~raocular injections of [aH]proline (a 20 #l injection concentrated in 0.691'o NaCI to 6.25 ktCi/ktl) made with a Hamilton syringe. Following survival periods which varied from 2 to 4 weeks, the animals were sacrificed and the brains removed and sectioned into 30 .urn thick sections on a freezing microtome. The sections were mounted on glass slides, defatted, coated with Kodak NTB-3 nuclear track emulsion and exposed in light tight boxes for 4 weeks at 4 °C. Following the exposure period, the slides were developed in D-19 at 14 c'C, fixed and counterstained with cresyl violet acetate 4.
Combination experiments In these experiments we used a combination of two techniques. Two turtles were used. The time course of the experiments was as follows: on day I each turtle received an intraocular injection of [aH]proline (a 20 #1 injection concentrated in 0.69 °/ooNaCI to 6.25 #Ci/ktl); on day 9 the same turtles received a cortical application of H R P in the manner described above; on day 14 the animals were sacrificed and the brains processed to the point of sectioning according to the procedure outlined above for the experiments utilizing HRP. The brains from the combination experiments were sectioned at 40 ~tm on a freezing microtome and the sections were collected in phosphate buffer. The sections then were processed by our standard procedure for producing the H R P reaction product. Every other section was also processed for autoradiography.
200
Anterograde degeneration experiments Twenty-six specimens of Pseudemys scripta received cortical lesions which were made under microscopic control by subpial aspiration with a fine glass pipette. Survival times ranged from 7 to 28 days. The optimal survival time was from 14 to 18 days. Following the survival period, the animals were perfused with 0.69 .°~i NaCI o~ forma li n. The brains were removed from the skull and stored in 10/,, ° followed by 10/o formalin for 4-7 days. Next, they were embedded in gelatin-albumin and placed in a 3 0 ~ sucrose-formalin solution for an additional 3-4 days. The brains were then sectioned at 30/~m on a freezing microtome. Every 10th section was stained with cresyl violet acetate and every 4th section was impregnated with silver by following procedure I of the Fink-Heimer technique 9. In selected cases, sections were also impregnated with silver by following method 7 described by EbbessonL
Golgi method The Cox-Golgi material described in the results was prepared according to Van der Loos' procedure4L
Analysis of the data In order to analyze and record the data, the distributions of degeneration and label were plotted on drawings of the sections with an X - Y plotter which was coupled to the stage of a Zeiss photomicroscope 10. The drawings of the Golgi material were made with the aid of a camera lucida. The photomicrographs were made with the Zeiss photomicroscope using Kodak Panatomic-X or High Contrast Copy film. RESULTS First, we will describe the results of the experiments designed to demonstrate that the dorsal lateral geniculate nucleus in Pseudemys seripta and Chrysemyspicta projects to general cortex. Next, we will describe the distribution of the efferent projections from general cortex to the brain stem. The intrahemispheric connections of the general cortex will be described in a later paper.
Geniculo-corticalprojections The results of an experiment in which a small localized application of H R P was placed in general cortex is illustrated in Fig. 1. The location of the application in this case is reconstructed on a dorsal view of the hemisphere at the top of the figure, with representative transverse sections through the application site illustrated just below. The application site is restricted to the general cortex with perhaps some extension into the adjacent pallial thickening. The application site does not involve either the subiculum medially, the pyriform cortex laterally or the dorsal ventricular ridge ventrally. In the lower part of Fig. 1 are drawings of 3 transverse sections through the diencephalon. The labeled cells are indicated by dots on each transverse section. The drawings illustrate that the cells in the thalamus which contain the H R P reaction
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Fig. 1. Case with an application of HRP in general cortex. The application site is shown in black on a dorsal view of the hemisphere in the upper part of the figure and in transverse sections immediately below the hemisphere. The stippled area surrounding the black area contains less dense reaction product, presumably due to spread. Selected sections through the thalamus are shown at the bottom of the figure. Cells which are labeled with HRP reaction product are depicted in each section by black dots. In this case, the labeled cells are restricted to the dorsal lateral geniculate nucleus. Survival time, 5 days. Scale, 1 ram. Abbreviations used in this and following figures: AT, area triangularis; AC, anterior commissure; BTN, basal telencephalic nuclei; DLA, nucleus dorsolateralis anterior; DM, nucleus dorsomedialis anterior; DP, dorsal peduncle of the lateral forebrain bundle; DVR, dorsal ventricular ridge; ExSt, extrastriate cortex; GC, general cortex; GLd, lateral geniculate nucleus, dorsal part; GLv, lateral geniculate nucleus, ventral part; H, habenula; H', hippocampus; Hy, hypothalamus; LFB, lateral forebrain bundle; MFB, medial forebrain bundle; OB, olfactory bulb; PaTh, pallial thickening; PT, pretectal area; Pul, pulvinar; PYR, pyriform cortex; Re, nucleus reuniens; Rh, rhinal fissure; Rt, nucleus rotundus; S, subiculum; SC, superior colliculus; SGAP, stratum griseum et album periventriculare; St, striate cortex; T, temporal cortex; Teg, tegmentum; TeO, optic rectum; TrO, optic tract; V, nucleus ventralis; VP, ventral peduncle of the lateral forebrain bundle; n.III, oculomotor nerve; 1II, oculomotor nucleus.
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203 product are restricted to one cell group, the dorsal lateral geniculate nucleus. In particular, note that no labeled cells are present in either nucleus reuniens or nucleus rotundus, both of which are cell groups of the reptilian dorsal thalamus known to project to the underlying dorsal ventricular ridgell,l
Fig. 2. Case with an application of H R P in general cortex. T h e locations of the labeled cells depicted by dots are plotted on outlines of transverse sections t h r o u g h the t h a l a m u s . The p h o t o m i c r o g r a p h s of the Nissl-stained sections are of the sections outlined in the figure, but were printed as their mirror image. Survival time, 5 days. Scale, 1 ram.
205
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Fig. 4. A: a drawing of the dorsal lateral geniculate nucleus from a section impregnated by the GolgiCox method. The dendrites of the lateral geniculate neurons extend into the neuropil between the cell bodies and the optic tract. Scale, 135/~m. B, C: dark-field photomicrographs of the autoradiographic signal (B) and degeneration (C) in the dorsal lateral geniculate nucleus following an injection (B) and lesion (C) in general cortex. The arrows indicate the medial border of the optic tract. Both techniques show that the corticofugal projection is focussed around the cell bodies rather than in the dendritic neuropil between the cell bodies and the optic tract• The photomicrograph in C is of section 20 in Fig. 5. Scale, 0.59 ram. optic tract a n d the cell perikarya of the dorsal lateral geniculate cells (see Fig. 3B, C a n d D middle column). The drawing of a Golgi-stained section t h r o u g h the lateral geniculate nucleus in Pseudemysscripta in Fig. 4A shows that the geniculate cells issue dendrites into the retinal terminal zone intercalated between the cell bodies and the optic tract. Finally, the dark-field photographs in the right c o l u m n of Fig. 3 illustrate that at each level through the dorsal lateral geniculate nucleus (Fig. 3B, C a n d D), dense H R P reaction p r o d u c t was present in the cell perikarya of the same cells which send their dendrites into the terminal zone of the retinal projections. In conclusion, cells in the dorsal lateral geniculate nucleus of the turtle receive projections from the retina and, in turn, project to general cortex. In Fig. 3 it should be noted that the autoradiographic signal extends slightly
Fig. 3. Photomicrographs of sections through the thalamus of a case which received both a cortical application of H RP and an intraocular injection of [3H]proline. This is the same case that was illustrated in Fig. 2. The photomicrographs in the left column are enlargements ( × 22) of the photomicrographs shown in Fig. 2 and are Nissl-counterstained sections which were processed for HRP. The areas within the small rectangles are shown in dark-field photomicrographs ( ~ 200) in the right column. The area within the rectangle in B, C and D is within the dorsal lateral geniculate nucleus and contains cells labeled with the HRP reaction product (B, C, D). The labeled cells in A are located in nucleus ventralis. The middle column shows adjacent sections ( x 22) through the same brain which were processed for autoradiography. The autoradiographic label is concentrated in the zone intercalated between the cell bodies of the lateral geniculate nucleus and the optic tract.
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Fig. 5. Case with a cortical lesion. The lesion in general cortex is shown in black at the top of the figure in transverse sections and on a dorsal view of the hemisphere. The axon degeneration is illustrated by short lines in the plots of cortical and brain stem sections. Terminal debris is illustrated by small dots. Survival time, 18 days, Scale, I mm.
m o r e rostral than did the a n t e r o g r a d e d e g e n e r a t i o n in a previous study ]7, a n d we now include this most rostral a r e a (see section 85 in Fig. 2 a n d the same section in Fig. 3B) in our definition o f the dorsal lateral geniculate nucleus. The ultimate justification for our definition is the c o r r e s p o n d e n c e between the d i s t r i b u t i o n o f retinal projections and the cells which project to general cortex.
CorticoJugal proiections The following cases will d e m o n s t r a t e that the m a j o r efferent conriections of general cortex in the turtle are with the d o r s a l t h a l a m u s , the optic rectum and the t e g m e n t u m o f the midbrain. Fig. 5 illustrates a lesion in general cortex a n d the resulting a n t e r o g r a d e d e g e n e r a t i o n which was t r a c e d to the b r a i n stem, D e g e n e r a t e d axons t h a t emerge from the d a m a g e d area are present in two separate pathways, one medial a n d one lateral (see Fig. 5, sections 9 and 13). The fibers in the medial p a t h w a y descend t o w a r d the brain stem in the medial wall o f the hemisphere j u s t medial to the cells of the subiculum a n d h i p p o c a m p u s . I n their course t o w a r d the d i e n c e p h a l o n
i
207 these fibers pass through the parolfactory area and enter the diencephalon as part of the medial forebrain bundle (Fig. 5 sections 9 and 13, 20 28.5). The axons of the lateral pathway leave the general cortex between the pallial thickening and pyriform cortex and then descend through the basal telencephalic nuclei to form part of the lateral forebrain bundle. They exit the telencephalon in the lateral forebrain bundle and, by their location, can be clearly distinguished from the fibers in the medial forebrain bundle. When these lateral fibers enter the diencephalon, they form part of the ventral peduncle of the lateral forebrain bundle (Fig. 5, sections 20 28.5). The fibers in the medial forebrain bundle are separate from and medial to those in the ventral peduncle of the lateral forebrain bundle (Fig. 5, sections 20-28.5). In the diencephalon, fibers leave the ventral peduncle and ascend to the dorsal thalamus where they terminate among the cell bodies of the dorsal lateral geniculate nucleus (Fig. 5, sections 20-23). The plots of the terminal distribution of this pathway in Fig. 5 indicate that, in contrast to the retinal projections which terminate primarily in the dendritic zone intercalated between the cell bodies and the optic tract, the main terminal focus of the corticofugal projections is among the geniculate cell bodies. Much sl~arser degeneration is present in the dendritic zone of the dorsal lateral geniculate nucleus. This distribution of terminal degeneration in the lateral geniculate nucleus is illustrated by the dark-field photomicrograph of the degeneration in section 20 which is presented in Fig. 4C. The photomicrograph in Fig. 4B is from an autoradiographic experinaent and shows that the same distribution of terminations is found in the dorsal lateral geniculate nucleus after a cortical injection of tritiated proline. Following larger cortical lesions, such as the one illustrated in Fig. 6, terminations are found in several thalamic nuclei in addition to dorsal lateral geniculate, including nucleus rotundus, nucleus ventralis and the nucleus dorsolateralis anterior. In the case illustrated in Fig. 6, sparse degeneration is also present in nucleus dorsomedialis anterior but, on the basis of other cases, we attribute this sparse degeneration to a slight extension of the lesion into the subiculum or hippocampus on the medial wall of the hemisphere. In the mesencephalon, the degenerated fibers from the ventral peduncle divide into two bundles: one terminates in the midbrain tegmentum and the other travels dorsally to reach the deep layers of the optic tectum (see section 28.5 in Fig. 5). Althou~_h well-defined terminal fields were not found in the optic tectum following cortical lesions, in experiments utilizing anterograde transport of labeled amino acids, dense autoradiographic signal is present in the Feriventricular layers and less dense signal in the more suFerficial layers (unpublished experiments). The bundle of fibers which terminates in the midbrain tegmentum is confined to the central zone of the tegmentum. The degenerated fibers in the medial forebrain bundle travel through the diencephalon apparently without terminating and enter the midbrain where they terminate in the tegmentum (Fig. 5, section 28.5). This area of termination either overlaps or is immediately adjacent to the te~mental target of the lateral forebrain bundle, and, as a consequence, separate terminal zones could not be differentiated. No degenerating fibers from either the medial or lateral forebrain bundle pathways could be traced caudal to the midbrain.
~j
~w o
209
Fig. 7. Reconstruction of cortical lesions on a dorsal view of the hemisphere. The black areas indicate the locations of lesions which produced degeneration only in the medial forebrain bundle, while the parallel lines indicate the locations of lesions which produced degeneration in both the medial and the lateral forebrain bundle. The dashed line indicates the extent of the cortical target of the dorsal thalamus as it was estimated in an earlier study by the distribution of degeneration following hemithalamectomies ~6. The single lesion located caudal and lateral to the thalamic receptive zone may have undercut the descending pathways. Lesions in various regions o f the turtle's cortex suggest that the medial forebrain bundle fibers and the lateral forebrain bundle fibers arise from different cortical areas. F o r example, fig. 7 illustrates the locations o f 11 small cortical lesions. The 7 lesions indicated in black in Fig. 7 are all restricted to the medial half o f general cortex a n d p r o d u c e d d e g e n e r a t i o n only in the medial f o r e b r a i n bundle. The 4 lesions which are indicated by d i a g o n a l lines in Fig. 7 all include d a m a g e to the lateral half o f general cortex a n d all p r o d u c e d d e g e n e r a t i o n in the lateral as well as in the medial forebrain p a t h w a y . Finally, medial lesions which invaded the h i p p o c a m p u s p r o d u c e d degeneration o f axons which descended a l o n g the medial wall o f the hemisphere a n d t e r m i n a ted in the h y p o t h a l a m u s . This projection was only seen following survival times s h o r t e r t h a n the 21 day p e r i o d for the case illustrated in Fig. 6. DISCUSSION The results o f the present study are consistent with the view that general cortex in at least the species o f turtle we have studied shares h o m o l o g o u s features with the n e o c o r t e x o f m a m m a l s . First o f all, general cortex, like neocortex, receives projections from the dorsal thalamus. Thus, our results d e m o n s t r a t e d that general cortex in the turtle is a target o f at least one ascending sensory p a t h w a y which relays in the dorsal t h a l a m u s . T h a t is, i n t r a o c u l a r injections o f tritiated a m i n o acids p r o d u c e d label
Fig. 6. Case with a large cortical lesion. In the upper part of the figure the lesion is shown in black on a dorsal view of the hemisphere and in transverse sections. The plots in the lower part of the figure are of sections through the thalamus and midbrain. In these plots axon degeneration is illustrated by short lines and terminal debris by small dots. Survival time, 21 days. Scale, 1 ram.
210 among the dendrites of the same dorsal thalamic cells which contained reaction product following cortical applications of HRP. Moreover, since in our cases with larger cortical applications of HRP labeled cells were present in additional thalamic cell groups including nucleus ventralis and nucleus dorsolateralis anterior, it seems probable that the dorsal lateral geniculate nucleus is not the only thalamic nucleus which projects to general cortex in turtles. The second feature of general cortex demonstrated by the present experiments is that it sends reciprocal projeciions back to the thalamus. That is, cortical lesions produced degeneration in the same cell groups which the HRP experiments demonstrated project to cortex. Finally, the same cortical lesions which demonstrated reciprocal projections to the thalamus also revealed corticofugal projections to the optic tectum of the midbrain. All of these connections of general cortex are also shared by the neocortex of mammals. The afJerent connections of general cortex in the turtle and other reptilian ,~roups The present study together with the previous anterograde degeneration studiesl6,17 comprise two lines oF evidence for thalamocortical projections in turtles which utilized techniques that rely on very different types of neuronal processes, anterograde degeneration and retrograde axonal transport. Degenerating axon terminals in the turtle's general cortex have also been observed with the electron microscope following thalamic lesions s. On the other hand, the results of similar studies in two species of a second reptilian group, the lizards, suggest that general cortex is not a target of a visual pathway which relays through the dorsal thalamus in all reptiles. For example, following numerous injections of HRP in various parts of general cortex in the Tegu lizard, Tupinambis nigropunctatus, labeled cells were found in nucleus dorsolateralis anterior but not in the dorsal lateral geniculate nucleus~7~ Similarly, Distel and Ebbesson 6 were unable to demonstrate significant projections from the dorsal lateral geniculate nucleus to general cortex in the monitor lizard, Varanus benegah, nsis, with anterograde degeneration techniques, but in the same series of experiments were able to demonstrate a cortical projection from the nucleus dorsolateralis anterior. These differences from our results in the turtle do not simply reflect a difference in nomenclature for thalamic nuclei in these two reptilian groups since other experiments 2,5J~ have shown that nucleus dorsolateralis anterior is ~ot a maior target of the retina in lizards, and furthermore, the dorsal thalamic target of the retina in the Tegu lizard does not contain labeled cells after cortical injections of HRW 7. In comparing the organization of cortex in the turtle and other reptiles, it is important to emphasize that our larger injections of H R P in the turtle's general cortex did produce label in cells in nucleus dorsolateralis anterior as well as in the dorsal lateral geniculate nucleus. This result suggests that at least the pathway from the nucleus dorsolateralis anterior to cortex is shared by both reptilian groups, Although the further connections of the nucleus dorsolateralis anterior are unknown for any reptilian species, the efferent connections of its cortical target in the Tegu lizard (see below) suggest that it may have close associations with the limbic system. In addition to the dorsal lateral geniculate nucleus and nucleus dorsolateralis anterior, a third nucleus, nucleus ventralis, contained labeled cells following the larger
211 injections of HRP in general cortex of the turtle. This result is of special interest since several lines of evidence suggest that nucleus ventralis may constitute a dorsal thalamic relay for the somatic pathway which physiologists have reported ascends to general cortex 'q,a°. For example, Distel and Ebbesson 6 have traced projections from the spinal cord and dorsal column nuclei to this general region in the monitor lizard. Moreover, Belekhova and Kosareva 1 reported that four-fifths of the cells they recorded from in this region in the turtle responded to somatic sensory stimulation, and that tetanizing stimulation of any part of this zone blocked the cortical evoked potentials to either ipsilateral or contralateral somatic stimulation. In summary, we have presented evidence that at least 3 dorsal thalamic nuclei have connections with the general cortex of the turtle. Two of these nuclei, the dorsal lateral geniculate and nucleus ventralis, may be relays for ascending sensory pathways. The visual pathway may not be present in all reptiles, since it has not been demonstrated in studies of" thalamocortical pathways in two species of lizard 6,27. The somatic pathway may be absent in at least one species of lizard, the Tegu, Tupinambis nigropunctatus ~7. The third dorsal thalamic nucleus, nucleus dorsolateralis anterior, appears to be present in both lizards and turtles and may have close associations with the limbic system.
The e/5]kJrent connection of general cortex in the turth, and other reptilian groups General cortex in the turtles we have studied not only receives projections from the dorsal thalamus but also sends projections back to the thalamus. Thus, lesions in the general cortex result in anterograde degeneration in the dorsal thalamus. In particular, projections were found to the same cell group, the dorsal lateral geniculate nucleus, which contains cells with reaction product following cortical applications of H RP. Following large cortical lesions, terminal fields are also present in several nuclei in addition to the dorsal lateral geniculate nucleus, including nucleus rotundus, nucleus dorsolateralis anterior and nucleus ventralis in the dorsal thalamus. The only other major brain stem targets of general cortex found were the optic tectum and the underlying tegmentum of the midbrain. These corticofugal projections provide another line of evidence that general cortex is not organized in the same manner in all reptilian groups, since cortical lesions in the Tegu lezard, Tupinambis nigropunctatus do not reveal projections to either the dorsal thalamus or the midbrain. Instead, the major non-telencephalic target of general cortex in the Tegu lizard is the hypothalamus2~, 26. In our experiments in Pseudeno's scripta, projections to the hypothalamus were found only after lesions or injections of [aH]proline (unpublished experiments) which extended far enough medially to invade the hippocampus. Although degenerated fibers could be traced caudally in the medial as well as in the lateral forebrain bundle following lateral lesions in the turtle's general cortex, in our material terminations contributed by the medial fibers could only be found in the midbrain tegmentum. Whether this medial system of fibers reflects limbic as well as exteroceptive sensory associations for the lateral general cortex in turtles cannot be answered by the present experiments. The tegmental target of this pathway could
2t2 conceivably be a pretectal area with associations with the visual system~ a midbrain limbic area, or a source of decending reticulo-brain stem and spinal motor pathways. The question of whether the same cell populations in general cortex give rise to both the lateral and medial pathways to the brain stem might be answered in further experiments by injecting H R P in the targets of these two pathways. The possibility remains, for example, that the degenerated fibers in the medial forebrain bundle, which we find after lateral lesions in the general cortex, arise from another structure such as the pyrifrom cortex and travel through general cortex in route to the medial wall of the hemisphere. On the other hand, the close spatial association in the midbrain tegmentum between the targets of the medial and lateral pathways from the lateral parts of general cortex suggests that they may arise from functionally similar cell groups. Finally, it should be emphasized that projections from the general cortex to the dorsal thalamus are not unique to Pseudemys scripta among reptiles since corticofugal pathways to the thalamus have also been described in Chrysemys picta 2~. in the sidenecked turtle, Podoenemis unifilis 43, and in garter snakes of the genus ThamnophislS~ In the garter snake, the thalamic target of general cortex is a cell group with unknown relationships to nuclei in the thalamus of turtles, but in Chrysemys pieta the target apparently includes the lateral geniculate nucleus. In Podoenernis unifilis, the thalamic targets include both the dorsal and ventral lateral geniculate nuclei and nucleus rotundus. These efferent connections from the cortex to the thalamus in turtles and snakes are clearly different from the total absence of efferent projections to the thalamus which has been reported for general cortex in Tegu lizards. Either these connections between the cortex and the thalamus do not exist in the Tegu lizard or they are so much less well-developed that available techniques have failed to reveal them. In summary, we have shown that corticofucal fibers from the turtle's general cortex innervate several brain stem structures including the dorsal lateral geniculate nucleus, nucleus rotundus, nucleus ventralis, nucleus dorsolateralis anterior, the optic tectum and the rostral midbrain tegmentum. Similar corticofugal projections are seen in a distantly related species of turtle as well as in the garter snake. Once again there appears to be a difference in cortical organization between turtles and at [east one species of lizard, the Tegu, since the only brain-stem target of the Tegu lizard's general cortex is the hypothalamus. However, not all members of the order Squamata have this organization of efferent connections from general cortex since the garter snake's corticofugal pathways include a thalamic target.
Visual pathways to the telencephalon in the turtle and mammals The main result of the present study together with the results of previous studies of the connections of nucleus rotundus in the turtle are summarized diagrammatically in the left half of Fig. 8. Two separate visual pathways can be traced to the telencephalon in at least the two reptilian species we have studied, Pseudemys scripta and Chrysemyspieta. The first is the retino-geniculo-cortical system which sends visual information to general cortex by means of a relay in the dorsal thalamus. In the turtle,
213
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~
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Fig. 8. Diagram of visual pathways in the turtle, Pseudemys scripta and in a mammal, Tupaia g/is. Two pathways to the telencephalon are illustrated for both groups, one which projects to cortex after a relay in the dorsal thalamus (GLd) and one which projects to the telencephalon after relaying first in the optic tectum of the midbrain and then in the dorsal thalamus (Rt or Pul). In both groups, the projections to the telencephalon from the thalamus are reciprocated by descending connections. The cortex in both groups also gives rise to projections to the optic rectum.
214 this pathway may provide the anatomical basis for the potentials which can be evoked in general cortex by visual stimulationl,~a, a0. This pathway in the turtle resembles the mammalian geniculo-striate system which is diagrammed in the right half of Fig. 8. As Fig. 8 illustrates, both in mammals and in the turtle, the cortical target of this visual pathway also sends reciprocal projections back to the dorsal lateral geniculate nucleus. In most vertebrates, the superficial layers of the optic tectum are also major targets of the retina, and in reptiles, the optic tectum projects heavily to nucleus rotundus in the dorsal thalamus a,1°,17. Nucleus rotundus in turn hab reciprocal connections with the dorsal ventricular ridge in the telencephalon 1a,16,:~:~, A similar visual pathway in mammals which relays in the superficial layers of the superior colliculus and the putvinar in route to extrastriate and temporal cortex is diagrammed on the right in Fig. 8. As previous papers have pointed out 'm, a very similar second visual pathway to the telencephalon is also present in birds, and consequently, il seems probable that at least two separate visual pathways to the telencephalon were established by the time these 3 classes diverged during the evolution of vertebrates. Finally, both general cortex in the turtle and the mammalian neocortex project to the optic tectum. In mammals, these projections are very prominent and in physiological experiments have been demonstrated to play an important role in establishing the receptive field properties of tectal neurons "s,'~1,4°,41,44. In the turtle. these projections are also prominent but their functions are unknown. Cortical organization in turtles and mammals In comparing our results with the results of similar studies in mammals, it is evident that the reciprocal connections between the thalamus and general cortex resemble the interconnections between the mammalian thalamus and neocortex. It is also evident that the overall distribution of corticofugal projections to the thalamus and midbrain in the turtle is more reminiscent of the efferent connections of neocortical sensory areas than of mammalian motor cortex. Thus, none of the efferent projections to brain stem nuclei caudal to the midbrain or to the spinal cord which are characteristic of mammalian motor cortex ss,s4 were found in the turtle, even after very large cortical lesions which extended from the medial wall of the hemisphere laterally to include the pallial thickening. The lack of efferent projections from the pallial thickening to the lower brain stem and spinal cord is of special interest since this is the telencephalic area of turtles which Johnston 19 proposed, on the basis of electrical stimulation, was the reptilian motor cortex. Whether other telencephalic areas in the turtle give rise to these pathways in unknown, but the motor responses which can be evoked by cortical stimulation in this group 19 might be mediated by corticoreticular pathways which our results indicate project from general cortex to the midbrain tegmentum. In conclusion, our evidence indicates that in turtles there is a region in cortex which receives projections from a visual thalamic area and sends reciprocal projections back. This same cortical area apparently sends projections to the optic tectum. Thus, general cortex shares several features with the 6-layered mammalian neocortex. At the same time, general cortex in the turtle consists of only a single layer of cells and may
215 correspond more closely to one neocortical cell layer, such as layer V1, which receives thalamocortical projections a n d gives rise to corticofugal projections in m a m m a l s ~', 34,z7-39, t h a n to a composite of all the neocortical layers. In any case, the reduced n u m b e r and complexity of cortical connections in turtles, taken together with the similarities in brain stem targets which they share with mammals, support the view that general cortex in turtles approximates an early stage in the evolution of the m a m m a l i a n neocortex. It is our hope that further studies of this much more simple cortex will eventually shed new light on the mechanisms a n d functional significance of the neocortex. ACKNOWLEDGEMENTS Supported by: N I N D S G r a n t NS09623 to W. C. Hall a n d U.S.P.H.S. G r a n t NB06551 to F. F. Ebner. N I H Predoctoral Fellowship 5 F01 M H 58576 to Robert E. Foster. N I M H Research Scientist Development Award M H 25734 to W. C. Hall.
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