Afferent Connexions of the Allocortex by B

J. Anat. (1965), 99, 2, pp. 339-357 339 With 2 plates and 6 text-figures Printed in Great Britain

Afferent connexions of the allocortex By B. G. CRAGG Department of Anatomy, University College, London

INTRODUCTION The cingulate, entorhinal and retrosplenial areas are major sources of projections to the hippocampus, but little is known of any interconnexions between the neocortex and these allocortical areas. Adey & Meyer (1952a) described one monkey brain in which a massive temporal lesion had caused the degeneration of some fibres in the entorhinal cortex stained by the method of Glees. This result was con- firmed and refined by Whitlock & Nauta (1956) who made lesions in the inferior temporal gyrus in four monkeys, and stained degenerating fibres in the entorhinal area with the Nauta method. Another source was found by Adey & Meyer (1952b) in two monkeys with large lesions in the frontal cortex. Degenerating fibres were stained in the presubiculum by the method of Glees. After similar lesions, Showers (1958) stained degenerating fibres with the Marchi method in the cingulate and hippocampal gyri in four monkey brains. In rabbits, rats and cats, the olfactory tubercle and prepiriform cortex are sources of afferents to the entorhinal area and the presubiculum (Cragg, 1961 b). Following lesions made in the olfactory cortex, degenerating fibres were stained by the method of Nauta & Gygax (1954) in the entorhinal area and presubiculum. The dorsal and posterior part of the entorhinal area also receives a projection from the contra-lateral hemisphere by way of the dorsal hippocampal commissure (Cragg & Hamlyn, 1957). The origin of this projection is unknown. In 1951 Dr P. K. Thomas (personal communication) suggested recording the electrical potentials of the entorhinal cortex while exploring the neocortex system- atically with a stimulating electrode. One experiment, on an anaesthetized cat, was done with Dr Thomas, and a small region in the mid-suprasylvian gyrus was found to evoke electrical responses in the ipsilateral entorhinal area when stimulated electrically, or when strychnine was applied topically. A similar result was found by Niemer, Goodfellow & Speaker (1963). Small lesions have now been made in the mid-suprasylvian gyrus and degenerating fibres traced by the Nauta method to the cingulate cortex. The latter in turn has been found to give rise to some fibres that end in the entorhinal cortex, as well as to the larger projection to the presubiculum. Frontal and temporal lesions have also been made in the neocortex to test the results found in the monkey. Lesions made in several other neocortical areas did not cause the degeneration of projections to the allocortex. It appears that there are three areas of neocortex that project to the allocortex, that there are two-way interconnexions within the areas of allocortex, and that there is an important projection from the septum to allocortex. 340 B. G. CRAGG

MATERIALS AND METHODS Cats and a small number of rabbits were anaesthetized with pentobarbital sodium, and given penicillin. Brain lesions were made under sterile conditions with suction or with a coagulating current passed through an electrode. After survival periods of 7-24 days, the brains were perfused and subsequently stained by the method of Nauta & Gygax (1954) with the minor modifications set out previously (Cragg, 1961 a). The most important modification was that the fixation period in buffered neutral formol saline was not allowed to exceed 2 weeks. With this precaution satisfactory preparations were obtained from more than forty consecutive brains during the course of this study. The time of treatment with potassium perman- ganate was kept fixed at 12 min. If the intensity of staining of the first section of any batch was too weak, the ammoniacal silver bath was adjusted for the rest of the batch by adding more silver nitrate solution. Many of the fibres involved in this study were extremely fine, and did not appear in under-impregnated sections. The brains of one cat and one rabbit were fixed by immersion a few hours after death, and gave good preparations. One rabbit was perfused with Bouin's fixative for Nissl's method, and the brain was then further fixed by immersion in formol saline. This brain, too, gave satisfactory results with the Nauta method. The variability and occasional staining failures encountered with the original method are probably due to the prolonged action of the fixative rather than to the conditions at the initial fixation. In order to be able to put loose frozen sections taken at close intervals through the thalamus into strict serial order, three consecutive sections were taken at each interval (0-25 -05 mm.) and placed in a separate compartment in a plastic tray filled with formol saline solution. When the sectioning was finished, the three sections from each compartment were taken out into a large dish of water and each snicked with scissors in some particular gyrus or area irrelevant of the final analysis. The snicks were placed in different positions in the sections from the other compart- ments. One section from each compartment was then mounted in order and stained with cresyl violet after fat extraction in hot pyridine, while another section from each compartment was stained loose by the Nauta method, leaving a third series in reserve. When the Nauta sections were mounted, thev were numbered by comparing the snicks with the Nissl series.

RESULT S Projections froin the sujprasylvian gyrus Lesions were made in the middle part of the suprasvlvian gcyrus in five cat brains, unilaterally in three (B1R3) and bilaterally in two (B4, 5). One brain (B 1) was cut frontally, two parasagittally (B 2, 3) and the two with bilateral lesions were cut frontally on one side and parasagittally on the other. The lesions were all similar to that in B 3 shown in Text-fig. 1, and the resulting fibrc degeneration can be described for the group as a whole. Degenerating fibres passed from the lesions through the subeallosal fasciculus to the caudate nucleus, where terminal degeneration was seen in the dorso-medial part of the head of the nucleus. A small amount of fibre degeneration was also seen in the Afferent connexiof8 of the allocortex 341 claustrum. Many degenerating fibres entered the thalamus from the dorsal part of the internal capsule and projected to no less than seven thalamic nuclei. These were the ventral parts of the nucleus lateralis dorsalis, the dorsal part of the nucleus ventralis anterior, the dorsal part of the nucleus centralis lateralis, the anterior and dorso-lateral parts of the pulvinar nucleus, the nucleus lateralis posterior, the reticular nucleus, and the pars ventralis of the lateral geniculate nucleus. No sign of

A:'~~~~~~~~~~~~~~~PSSG B 8 B 9 B7 B11 X(<~~~~~~~~~~~~~~~33

Text-fig. 1. The course of the degenerating fibres from a lesion in the middle part of the suprasylvian gyrus (B3) to the cingulate cortex. The lesions (B6-11) surrounding the mid-suprasylvian gyrus caused little or no fibre degeneration in the allocortex. All text- figures and plates refer to cat brains unless otherwise stated. retrograde thalamic degeneration was recognized in these Nauta preparations. Further back there was dense fibre degeneration in the pretectum, and deep in the superior colliculus, and also in the anterior pons, especially in the nucleus pontis lateralis of Winkler & Potter (1914, plate XXI). In addition to these widespread projections, there were degenerating fibres distributed in an arc in the white matter lateral and ventral to the splenial sulcus. Some of these fibres ended in the peristriate cortex (area 18) and others in the adjacent cingulate cortex, but there were none in the retrosplenial area of Rose & Woolsey (1948 a). Within the cortex the degeneration was most concentrated at the dorso-medial lip of the ventral bank of the splenial sulcus, where the cingulate and peristriate areas adjoin (Text-fig. 1; PI. 1, fig. 1). The degeneration surrounding the splenial sulcus extended posteriorly from the level of the lesion. Beneath the posterior end of the splenial sulcus, there is a small gyrus separated from the 342 B. G. CRAGG entorhinal area below by the posterior end of the rhinal fissure (see Text-fig. 3; P1. 1, fig. 3). This gyrus does not appear to be separately named, but forms part of the posterior splenial gyrus of Winkler & Potter (1914, plate XX), who treat it as part of area 29 (post-splenialis). It is not, however, included in the retrosplenial area of Rose & Woolsey (1948 a), and has a rather homogeneous structure that is difficult to characterize (PI. 1, fig. 3). The degenerating fibres surrounding the splenial sulcus continued into this part of the posterior splenial gyrus (P1. 1, fig. 2). Only a very small number of fibres entered the dorsal entorhinal cortex. In the brains with unilateral lesions no fibre degeneration was seen in the allocortex of the contra- lateral hemispheres. Lesions in the mid-suprasylvian gyrus thus caused the degeneration of a projection to the cingulate cortex, and lesions have therefore been made in neighbouring areas to define the limits of the neocortex contributing to this projection. Two brains (B 6, 7) with lesions of the posterior suprasylvian gyrus (Text-fig. 1) showed only a very small number of degenerating fibres in part of the cingulate cortex, and none in the posterior splenial gyrus. In another brain (B 8) a lesion was made in the anterior part of the lateral gyrus (Text-fig. 1), but although there were many degenerating fibres in the cortex surrounding the anterior end of the splenial sulcus, few penetrated as far back as the cingulate cortex. As shown in Text-fig. 1, the anterior part of the suprasylvian gyrus was damaged in B 9. No degeneration was found in the posterior cingulate cortex or posterior splenial gyrus, but some degenerating fibres were present in a small anterior part of the cingulate cortex. In one further brain (B 1O) lesions were made in the middle part of the lateral gyrus medial to the effective mid-suprasylvian region, and also in the Sylvian gyrus lateral to the critical region. No degenerating fibres were seen in the allocortex. Finally, lesions were made close to the splenial sulcus at the medial margin of the lateral gyrus on one side, and at the posterior margin of the lateral gyrus on the other side, in brain B I1 (Text-fig. 1), without causing the degeneration of fibres in the allocortex. In all these brains (B16-11), in which there were substantially negative findings in the allocortex, there was satisfactory staining of degenerating fibres near the lesion and elsewhere in the brain. The origin of this projection to the cingulate cortex was thus the middle part of the suprasylvian gyrus. Since the mid-suprasylvian lesions caused the degeneration of few fibres in the entorhinal area, a brain, B 12, with a large neocortical lesion was studied to test the possibility of other neocortical connexions to the entorhinal area. Almost all the neocortex had been removed from one hemisphere of this brain, B 12, by Dr J. L. de C. Downer for another purpose. The only regions of neocortex remaining were the anterior part of the orbital gyrus, the gyrus proreus, and the postero- ventral ends of the ectosylvian and suprasylvian gyri. There was some damage to the white matter behind the cingulate gyrus, and degenerating fibres were seen running centrally in the presubiculum just anterior to the entorhinal area. The entorhinal cortex was substantially free from degeneration except at the dorsal margin, where some degenerating fibres entered in layer 1, and in the deeper white matter, but penetrated only 2 mm. in the ventral direction. In the contra-lateral hemisphere no degenerating fibres were seen in allocortical areas. Most of the entorhinal area is thus without neocortical afferent connexions unless the small Afferent connexions of the allocortex 343 areas of neocortex spared by the lesion in brain B 12 have projections to the entorhinal cortex. This possibility was tested because the areas spared happen to correspond in part to the temporal and frontal regions that project to allocortex in the monkey. Projectionsfront temporal sieocortex Lesions in temporal neocortex were studied in eight cat brains together with one cat brain containing an extensive lesion of the orbital gyrus. In four brains, projections to the entorhinal area and to the posterior splenial gyrus were found. In a

a , 1 B41 F

B 14 Text-fig. 2. Lesions of thec temporal or orbital neocortex (B 13 10) that caused thle degeneration of a small number of fibres to the entorhinal area, and of a projection to the cortex surrounding the posterior end of the splenial sulcus. The latter projection weas degenerated in B17 19, but in these brains entorhinal degeneration was not detected. Neither projection wras degenerated in B20 or 21. The course of the degenerating fibres in the frontal plane is indicated in the righlt-hand diagram, in which the solid line shows the orientation of the mid-line.

further three brains the projection to the posterior splenial gyrus was present, but degenerating fibres could not be stained in the entorhinal area. Finally, in two of the brains, there was no degeneration in either the entorhinal area or in the posterior splenial gymus. In the first group of brains showing both projections, two hemispheres (B 13, 14) had similar lesions in the posterior ectosylvial gymus that stopped 1 mm lateral to the rhinal fissure (see Text-fig. 2). A third hemisphere (B 15) had a lesion in the ventral part of the posterior Sylvian gymus, and a fourth (B 16) had an extensive lesion in the orbital gyrus. This last lesion extended into the white matter, and must have interrupted nearly all the fibres leaving the cortex of the orbital gymus, and possibly fibres of neighbouring origin-as well. In each of these four hemispheres a 344 B. G. CRAGG few degenerating fibres were seen in the ventral entorhinal area (PI. 1, fig. 4). Although sparse, this fibre degeneration was quite distinct from the almost clear background. The fibres appeared from the underlying white matter of the internal capsule and ended in the cortex in layers 5 and 6. In brains B 14 and B 15 some sections showed a small number of fine fibres in layer1 which ran round the rhinal fissure from the neocortical lesion to themost lateral and ventral part of the entorhinal area. In agreement with the findings of Whitlock & Nauta (1956) in the monkey this temporal neocortical projection to the entorhinal area, as demonstrated by the Nauta method, constitutes a very small connexion. The other degenerated projection, to the posterior splenial gyrus, was larger in these brains.The degenerating fibres appeared from the underlying white matter of the internal capsule, and formed moderate or sparse ramifications around the posterior end of the splenial sulcus in the deeper layers of the cortex (the peristriate and post-splenial areas of Winkler & Potter, 1914). The three further brains (B 17- 19) that showed this projection but not the entorhinal connexion had similar small lesions in the ventral temporal neocortex (Text-fig. 2). In brainB 17 the lesion damaged the anterior Sylvian and posterior orbital gyri. The lesion in brain B 18 affected the posterior Sylvian gyrus with small extensions into the posterior ectosylvian and anterior Sylvian gyri. The superficial lesion in the posterior Sylvian and ectosylvian gyri in B 19 extended a short distance into the white matter beneath the cortical lesion. In this last brain the fibre degeneration in the posterior splenial gyrus formed a dense band around the posterior splenial sulcus (P1. 1, fig. 5). All these lesions in the temporal neocortex had been made close to the rhinal fissure because in the monkey it is the inferior temporal gyrus that gives rise to an allocortical projection (Whitlock & Nauta, 1956). For comparison, two brains (B 20, 21) with more dorsal lesions of temporal neocortex were studied (Text-fig. 2). Both these lesions were in the posterior Sylvian gyrus, but were separated by 5 and 10 mm. respectively from the rhinal fissure. No degenerating fibres were found in the entorhinal area or posterior splenial gyrus in either brain. Thus, in the cat, the most ventral part only of the temporal neocortex has a sparse connexion with the entorhinal area, and also gives rise to a more substantial projection to the posterior splenial gyrus and to the adjacent peristriate cortex. A projection from frontal neocortex The gyrus proreus was damaged in five cat brains (B 22-26). These lesions, two of which are illustrated in Text-fig. 3, involved mainly the precentral agranular area of Rose & Woolsey (1948a). No degenerating fibres could be stained by the Nauta method in the entorhinal cortex, retrosplenial area, or posterior cingulate cortex. There was, however, a projection from the lesion to the anterior limbic area of Rose & Woolsey (1948a) in the ventral bank of the cruciate sulcus on the medial surface of the hemisphere (see Text-fig. 3). This projection was distinguished by the occurrence of tight bundles of fine degenerating fibres (P1. 2, figs. 6 and 7), which appeared in frontal section as dorso-ventrally elongated areas of closely packed Nauta degeneration in the cortex of the anterior limbic area. These bundles of fibres terminated abruptly and a number of objects, one of which can be clearly seen in PI. 2, fig. 7, that might be terminal boutons were stained by the Nauta method. Afferent connexions of the allocortex 345 These bundles of fibres were accompanied by other degenerating fibres of more normal appearance. While some of the latter spread back along the ventral bank of the cruciate sulcus to reach the anterior part of the cingulate cortex, the bundles of fibres were not found posterior to the anterior limbic area.

Text-fig. 3. A diagram of the medial surface of the cat's brain showing lesions in the precentral agranular cortex in brains B 22 and 24 that caused the degeneration of a projection to the anterior limbic area.

Prag

Text-fig. 4. A diagram of the medial surface of the cat's brain showing lesions affecting the anterior allocortex or septum that caused the degeneration of projections to the more posterior parts of the allocortex. Since lesions in the preccntral agranular area in the cat did not cause the degeneration of fibres passing back as far as the presubiculum or entorhinal area as described by Adev & Mever (1952b) and Showers (1958) in the monkey some more lesions were made in the anterior parts of the allocortex.

Projections within the allocortex The anterior limbic area was damaged in five cat brains (B127-31), in one case (B 27) bilaterally. In each brain some inadvertent damage to neocortex occurred 346 B. G. CRAGG (rext-fig. 4). In two brains (B128, 29) the neocortex of area 5 directly antero- dorsal to the anterior limbic area was damaged, so in another brain (B132) a lesion was made in this area alone. In one brain (B 27) the dorsal lips of the cruciate sulcus on the medial surface of the hemisphere were damaged, so a similar lesion of this neocortex alone was made in another brain (B 33). A fourth brain (B 30) had a small track of damage in the precentral agranular area, but the lesion spread back to involve the infralimbic area immediately under the genu of the corpus callosum, and the most anterior part of the septum, which will be considered in the next section. The fifth brain (B 31) had a lesion in the anterior limbic area that spread back into the infralimbic cortex, but did not involve the septum. Finally, in three more brains (B 34-36) lesions were made behind the anterior limbic area to involve the cingulate cortex. When these brains were sectioned and stained by the Nauta method, it was found that the two control lesions confined to neocortex (B132, 33) gave rise to no degenerating fibres within the allocortex, but that the hemispheres with allocortical damage all contained degenerating fibres in parts of the posterior allocortex. The majority of the degenerating fibres followed the same route in each of the eight brains with allocortical lesions (B 27-31, B 34-36). Posterior to the lesions, degenerating fibres passed back in the ventral bank of the splenial sulcus, some ending in the cingulate cortex while others joined the cingulum. At the level of the splenium of the corpus callosumi, degenerating fibres from the cingulum turned centrally and ran down through the deeper layers of the retrosplenial cortex, where many of them ended. Some fibres, however, penetrated as far as the presubiculum, where they formed two sparse groups, one deep in the cortex and one superficial in layer 1. This general description of the degeneration common to the eight brains must be supplemented by the consideration of some special cases. The two brains B 34 and B 36 with small lesions of the cingulate cortex are interesting because the lesions affect the dorso-medial lip of the ventral bank of the splenial sulcus (Text-fig. 4). This is the region where the afferent fibres from the mid- suprasylvian gyrus are most concentrated. Although the ventral boundary of the cingulate cortex where it meets the retrosplenial cortex is sharp, the dorsal boundary with the peristriate cortex is ill-defined and lies somewhere in the ventral bank of the splenial sulcus (Rose & Woolsev, 1948a, p. 295). The small lesions in B34 and B36 did cause the degeneration of fibres to the posterior cingulate region, the retrosplenial cortex and a few to the presubiculum. It is useful to have this positive indication that the impulses carried by the fibres from the mid-suprasylvian gyrus could be related to the hippocampus. An additional projection was seen in brain B 35 with a large cingulate lesion and was also present but less prominent in B 36. This consisted of a small number of degenerating fibres that entered the anterior and ventral part of the entorhinal area from the underlying internal capsule (stratum sagittale externum of Winkler & Potter, 1914, plate XIII), and ended deep in the entorhinal cortex just medial to the rhinal fissure (PI. 2, fig. 8). These fibres passed ventrally from the white matter lateral to the cingulate lesion as a thin band lining the lateral side of the lateral ventricle. In brain B 35 these fibres extended back into the most posterior part of the entorhinal cortex, and into the posterior splenial gyrus. Afferent connexions of the allocortex 347 Finally, in the brain B 30, in which the lesion of the anterior limbic area extended back through the infralimbic area to involve the most anterior part of the septum, there was a substantial number of fine degenerating fibres in the presubiculum. These fibres were not degenerated in brain B 31 which had a similar limbic lesion without involvement of the septum. Some of the degenerating fibres reached the presubiculum by running over the alveus of the hippocampus, while others passed through the cingulate and retrosplenial cortex as in the brains without septal involvement. The fibre degeneration was present throughout the length of the presubiculum, including the ventral extremity from which impulses would be relayed into the antero-ventral end of the hippocampus. In this region there were also a number of fine degenerating fibres in the entorhinal area just medial to the rhinal fissure. These fibres came out of the white matter forming the floor of the lateral ventricle in the pole of the temporal lobe. In this region the alveus of the hippocampus and the angular tract (Cajal, 1955) are continuous with the internal capsule, and each of these structures appeared to contribute some of the degenerating fibres to the entorhinal cortex. A presubicular projection has been described previously by Cragg & Hamlvn (1957) after lesions affecting the alveus of the hippocampus in the rabbit. Some more septal lesions were therefore made in three cats to assess the importance of the septum as a source of fibres to the allocortex.

Septal projections Lesions in the septum were made in three brains (B 37- 39) by inserting an insula- ted needle electrode between the two hemispheres to measured co-ordinates. The lesion in B 37 (Text-fig. 5) affected the infralimbic area, the diagonal band, and the posterior and medial part of the septum including probably the dorsal fornix. Some of the degenerating fibres passing back in the latter penetrated the corpus callosum to join the cingulum and ended in the posterior cingulate and retrosplenial cortex. Other fibres from the septum entered the fimbria and passed back over the alveus of the hippocampus, some ending in the presubiculum and others turning ventro- laterally to run along the angular tract of Cajal. At the bottom of the lateral ventricle, fibres left the angular tract to end in the entorhinal cortex (PI. 2, fig. 9) in larger numbers than after the small septal lesion described above (B 30). There again appeared to be a contribution from the internal capsule of degenerating fibres to the entorhinal area just medial to the rhinal fissure, but it is possible that the fibres were derived from the adjacent angular tract. Further posteriorly, some fibres passed back in the white matter overlying the presubiculum to end in the posterior splenial gvrus. The other two lesions in the septum (B138, 39) were smaller and more anterior than that in B37. The degenerating fibres had the same distribution as in the two brains previously described (B130, 37), but the degeneration in the entorhinal area was sparse as in B 30.

The origin of the dorsal hippocampal cominissure One of the larger afferent projections to the allocortex is that carried by the dorsal hippocampal commissure (which is continuous with the angular tract of Cajal) to 348 B. G. CRAGG the dorsal posterior part of the entorhinal area (Cragg & Hamlyn, 1957). Cajal (1955) thought that this connexion ran in the opposite direction, from the entorhinal cortex to the contralateral hippocampus. Although the Nauta method has demonstrated that the termination is in fact in the dorsal entorhinal area, the origin of the tract is still unknown. Lesions have now been made in six rabbit brains and one cat

Text-fig. 3. Three sections through the septal and infralimbie lesion in B 37 which caused fibre degeneration in the presubiculum and entorhinal area. The course of the degenerating fibres in the frontal plane is shown in the right-hand diagram in which the solid line indicates the orientation of the mid-line. brain in an attempt to determine the origin of this important projection to the allocortex. The rabbit was used because the region of the angular tract is more accessible in this species. Repeated efforts to damage the presubiculum without involving the overlaying angular tract were unsuccessful. The angular tract was then cut on one side ill a rabbit brain (B 40), and the opposite hemisphere inspected for retrograde chronla- tolysis or cell loss in Nissl preparations after a survival period of 18 days. No cell loss or gliosis could be detected, and the number of cells with dispersed Nissl sub- stance in the subicular and hippocampal regions did not appear to be greater than the considerable number found in normal control brains. Since retrograde chroma- tolv-sis has not been detected in the hippocampus after section of the fornix (Daitz & Afferent connexions of the allocortex 349 Powell, 1954), this approach did not seem to be worth pursuing. Lesions were therefore made just in front of, or just behind, the angular tract, and the contra- lateral entorhinal area studied in Nauta preparations to detect any contribution to the dorsal hippocampal commissure damaged by the lesion. The positions of the lesions in five rabbit brains (B 41-45) is indicated in Text-fig. 6. The lesion in B 41 damaged much of the posterior cingulate cortex and the radia- tion of part of the retrosplenial cortex without involving the angular tract itself. The

B 43 b 41

F ~~~B45 PrL

Text-fig. 6. Diagrams of parasagittal sections through three lesions (B43-45) affecting the hippocamipus that caused the degeneration of fibres passing through the dorsal hippocampal commissure to end in the contra-lateral dorsal entorhinal area. The two posterior lesions (B 41, 42) did not cause degeneration in the confra-lateral entorhinal area. (B41-45 are rabbit brains.) contra-lateral entorhinal area and angular tract were free of degeneration. There was however a massive ipsi-lateral projection to the presubiculum, and a band of fine fibres passed in layer 1 from the lesion to the dorsal entorhinal cortex where it terminated superficially. Exactly the same result was found in B 42 which had a lesion of the posterior cingulate cortex. The other three brains (B 43-45) had lesions affecting the hippocampus which would interrupt fibres passing over the alveus from the septum. All three of these 350 B. G. CRAGG brains showed a large number of fine degenerating fibres in the medial part of the contra-lateral dorsal entorhinal area. These fibres were derived from the angular tract and the dorsal hippocampal commissure. On the ipsi-lateral side all three brains showed the degeneration of a massive projection to the presubiculum and also some degenerating fibres that passed back from the presubiculum to end in the dorsal entorhinal area. It is probable that the degeneration in the contra-lateral entorhinal area (P1. 2, fig. 10) was due to the hippocampal damage rather than to the interruption of fibres passing over the alveus of the hippocampus from the septum. The four septal lesions (B 30, B 37-39) were substantially unilateral, and the contra- lateral entorhinal areas contained very few or no degenerating fibres. Thus these results indicate that a considerable part of the dorsal hippocampal commissure is derived from the hippocampus, but very little from the retrosplenial or cingulate cortex, while the subiculum and presubiculum, which are most intimately related to the angular tract, remain untested. Finally, one cat brain (B 46) with a lesion of the entorhinal area was searched for a contribution to the dorsal hippocampal commissure, or a projection back into the more dorsal and anterior allocortical areas. The lesion in this brain was made by inserting a hooked sucker tube into the entorhinal area, and frontal sections showed that while neocortex had been spared, there was extensive damage to the whole extent of the entorhinal area spreading forwards to involve the antero-ventral end of the hippocampus, the medial part of the amygdala, and part of the prepiriform cortex at the level of the optic chiasma. There was heavy degeneration in the per- forant pathway of the hippocampus, and degenerating fibres ran in the fimbria to the septum, fornix and mamillary body. The findings of most interest, however, were that a number of fibres passed up the angular tract and through the dorsal hippocampal commissure to end in the contra-lateral ventral entorhinal area. These fibres may have been derived from the damaged ventral hippocampus or from the entorhinal area. Other degenerating fibres ran dorsally in the presubiculum, some ending in the dorsal part of the presubiculum and at the junction of the latter with the retrosplenial cortex, while others ascended past the splenium of the corpus callosum and joined the cingulum, from which they entered the retrosplenial cortex at more anterior levels. A small number of degenerating fibres passed from the entorhinal lesion towards the neocortex. A few of these fibres came round the indentation of the rhinal fissure in laver 1 and ended superficially, while a small number of others entered the neocortex more deeply at the margin with the white matter. The whole projection was sparse and did not penetrate more than 2 mm. from the depth of the rhinal fissure into the neocortex. These results indicate that a two-way system of inter-allocortical connexions exists, and would repay further study.

DISC U SSIONN In the cat, three areas of neocortex have been shown to send axons to allocortical areas, and it is interesting to consider what these regions have in common. The mid- suprasylvian gyrus is well known as an association area. Secondary responses to visual, auditory and somatic sensory stimuli have been recorded here in cats anaesthetized with chloralose (Buser, Borenstein & Bruner, 1959; Thompson & Afferent connexions of the allocortex 351 Sindberg, 1960; Thompson, Johnson & Hoopes, 1963), and single units respond to both visual and auditory stimulation in unanaesthetized cats (Bental & Bihari, 1963). Bilateral lesions destroying the mid-suprasylvian gyrus in cats have been shown to cause an inferior performance in the Hebb-Williams maze (Warren, Warren & Akert, 1961) without interfering with the discrimination of visual patterns (Hara & Warren, 1961). The mid-suprasylvian gyrus receives thalamic projections from the pulvinar nucleus and from part of the nucleus lateralis dorsalis (Waller & Barris, 1937; Warren et al. 1961), though Vastola (1961) has described electrical responses to optic nerve stimulation which are interpretable as due to a direct projection from the lateral geniculate nucleus to the medial lip of the middle part of the mid-suprasylvian gyrus. The part of the temporal neocortex with allocortical connexions is ventral to the three auditory areas described by Rose & Woolsey (1949). In monkeys, lesions of the neocortex alone in this region have been found to produce the Kluver-Bucy syndrome including deficiencies of visual discrimination (Akert, Gruesen, Woolsey & Meyer, 1961), but such visual defects do not appear to be well known in cats. However, ablation of the neocortex between the rhinal fissure and the auditory areas AII and Ep has been found to cause a deficiency in sound pattern recognition in cats (Goldberg, Diamond & Neff, 1957; Diamond & Neff, 1957). Conditioned responses to simple stimuli that differed in pitch were learned as rapidly as by intact cats, but there was a profound loss of the ability to discriminate between two patterns of tones such as high-low-high and low-high-low. This auditory defect may well be analogous to the defect in maze learning, with preservation of visual pattern recognition, that is seen after mid-suprasylvian lesions. In both cases the defect appears only when a temporal sequence of discriminations is involved. The same is probably true of the defects produced in experimental animals by hippocampal lesions (Kimble & Pribram, 1963; Drachman & Ommaya, 1964; Kaada, Rasmussen & Kveim, 1961). The ventral temporal neocortical lesions described by Goldberg et al. (1957) caused retrograde thalamic cell degeneration in the extreme caudal end of the medial geniculate nucleus. As well as this auditory connexion, there may be visual associa- tions in this area, for the projection from the temporal neocortex to the posterior end of the splenial gyrus ends partly in peristriate cortex. An analogous connexion in the monkey from the middle temporal gyrus (area TE) to the prestriate cortex (areas OA and OB) has been found by Kuypers & Szwarcbart (1960). The functions of the third area of neocortex, in the frontal region, are less easily defined, but again defects appear when a temporal sequence of discriminations is required. The well-known deficit in the performance of delayed responses in primates was originally attributed to a loss of short-term memory, or to an increase in distractability (Crawford, Fulton, Jacobsen & Wolfe, 1948). More recent studies (Ihawicka & Konorski, 1963; Brutkowski, Mishkin & Rosvold, 1963) have stressed the perseveration of previously established behaviour. Thus there is difficulty in responding appropriately to two stimuli that alternate in spatial position in succes- sive trials. This is due to a tendency to adopt a stereotyped motor response to either stimulus, and to persist with it. It is not certain that lesions in the precentral agranular cortex alone would produce the typical symptoms of a frontal lobe lesion, 22e Anat. 99 352 B. G. CRAGG but the more medial frontal lesions in cats would probably involve the anterior limbic area as well. The thalamie projections to the frontal cortex in the cat are derived from the dorso-medial nucleus (Waller, 1940; Rose & Woolsey, 1948b). Thus the three areas of neocortex connected to the allocortex appear to have nothing in common as regards the projections they receive from the thalamus. MIoreover, the three areas do not all belong to those described as giving secondary electrical responses to peripheral stimulation in cats anaesthetized with chloralose. Two such areas that were described by Buser et al. (1959), in addition to the mid- suprasylvial gyrus, lie on the anterior lateral gyrus and on the posterior suprasylvian gyrus. Lesions in these regions caused few fibres to the allocortex to degenerate (Text-fig. 1). Nor do the three areas of neocortex correspond to the alleged suppressor areas (Garol, 1942), although the frontal region may be in a suppressor area, and the part of the suprasylvian gyrus just behind the critical middle region was thought to be a suppressor area. The three areas of neocortex connected to the allocortex do however correspond closely to those mapped in fig. 7 of Morison & Dempsey (1942). These authors found that the dorsal part of the gyrus proreus, the mid-suprasylvian gyrus and the extreme postero-ventral end of the suprasylvian gyrus in the cat showed particularly prominent recruiting responses to electrical stimulation of the mid-line and intralaminar nuclei of the thalamus, and also a particularly high amplitude of spontane- ous electrical rhythms at 8-12/sec. This result may mean that the electrical activity of these three areas of neocortex is particularly influenced by the reticular formation. The recruiting responses have the long latency of 20-35 msec., and the pathway by which the reticular formation, or the mid-line and intralaminar nuclei of the thalamus, influence the cortex is unknown (see Powell, 1958). Although stimulation of the mid-suprasylvial gyrtms was found to elicit electrical responses in the hippocampus by Niemer et al. (1963), these authors do not indicate that any responses were observed on stimulating the frontal cortex. On the other hand, the greatest concentration of points where stimulation elicited hippocampal responses is shown in the temporal neocortex. The allocortical connexions from this region were found to be sparse in the present work, and in that of Whitlock & Nauta (1956), except for the projection to the posterior splenial gyrus. Whether the latter projects to the hippocampus is unknown, and repeated attempts to damage the gvrus in the present study always lead to involvement of the underlying white matter carrying hippocampal afferents from the entorhinal area. In view of the apparent importance of the hippocampus in the formation of new memories (see Drachman & Onmmaya, 1964) it is surprising that the neocortical connexions of the allocortex are not more prominent. One possibility is that there are short fibre links with the neocortex all round the margin of the allocortex, as suggested by Krnjevic & Silver (1963) on the basis of normal preparations stained for cholinesterase. The direction of these short fibres cannot be discerned in normal preparations, and in making lesions it has been found difficult to get close enough to the rhinal or splenial sulci without doing any direct damage to the allocortex. The largest afferent connexions of the hippocampal system seem to arise within the allocortex. Thus the connexion from the cingulate cortex through the cingulum Afferent connexions of the allocortex 353 to the presubiculum that was described by Adey (1951) and White (1959) is probably larger than any other projection received by the allocortex. A more anterior link in the same chain, from the anterior limbic area to the cingulate and retrosplenial cortex, has been found in the present study, as well as projections from the cingulate cortex and the septum to the entorhinal area. Another large projection is the cruciate connexion to the entorhinal area carried by the dorsal hippocampal commissure and the angular tract, part of which has now been found to arise in the contralateral hippocampus. The projection of the olfactory tubercle and prepiriform cortex into the entorhinal area and presubiculum (Cragg, 1961 b) is also substantial, and another ascending intra-allocortical connexion has now been found from the entorhinal area to the more dorsal presubiculum and retrosplenial cortex (brain B 46). The density of these intra-allocortical connexions is presumably the reason why the hippocampus in rabbits, rats and cats responds so much more strongly to olfactory stimulation than to stimulation of other modalities (Cragg, 1960). It is possible that in primates a reduction in the olfactory connexions of the hippocampus together with the increase in size of the neocortex is associated with a strengthening of the neocortical connexions with the allocortex, but as yet only the frontal and temporal regions appear to have been tested.

SU MM1ARY 1. In the cat, lesions in three areas of the neocortex cause the degeneration of fibres to the allocortex that can be stained by the Nauta method. 2. The mid-suprasylvian gyrus projects to the cingulate and peristriate cortex and to the posterior splenial gyrus. 3. The ventral temporal neocortex projects to the entorhinal area and to the posterior splenial gyrus. 4. The precentral agranular cortex projects to the anterior limbic area with peculiar tight bundles of fibres. 5. There are strong interconnexions within the allocortical areas. 6. The anterior limbic area projects to the cingulate and retrosplenial cortex and to the presubiculum. 7. The septum and the cingulate cortex both project to the presubiculum and to the entorhinal cortex. 8. In the rabbit, some of the fibres carried by the dorsal hippocampal commissure to the entorhinal area arise in the contralateral hippocampus. 9. The behavioural and physiological properties attributed to the three areas of neocortex have certain aspects in common.

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In Peripheral and Central Mechanisms of Motor Functions (ed. E. Gutmann). Prague: Czecho- slovak Acad. Sci. BUSER, P., BORENSTEIN, P. & BRUNER, J. (1959). Etude de systbmes 'associatifs' visuels et auditifs chiez le chat anesthesia au chloralose. Electroenceph. clin. lNeurophysiol. 11, 305-324. CAJAL, S. RAMON Y, (1955). Studies on the Cerebral Cortex (Limbic Structures) (translated by L. M. Kraft). London: Lloyd-Luke. CRAGG, B. G. (1960). Responses of the hippocampus to stimulation of the olfactory bulb and of various afferent nerves in five mammals. Expl Neurol. 2, 547-572. CRAGG, B. G. (1961a). The connections of the habenula in the rabbit. Expl. Neurol. 3, 388-409. CRAGG, B. G. (1961b). Olfactory and other afferent connections of the hippocampus in the rabbit, rat and cat. Expl Neurol. 3, 588-600. CRAGG, B. G. & HAMILYN, L. H. (1957). Some commissural and septal connexions of the hippocampus in the rabbit. A combined histological and electrical study. J. Physiol. 135, 460-485. 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A 11131t E VIA TI ON S I Alveus iL Infralimbic area AL Anterior limnbic area Prag Precentral agranular cortex A T Angular tract PrSu Presubiculumi (2 Cingulatc cortex PSpG Posterior splenial gyrus cc Corpus callosum R1 Retrosplenial cortex C(rS Cruciate sulcus RS Rhinal sulcus Dl? Diagonal band S Septum E Entorhiinal area SpS Splenial sulcus F Fimbria SSG Suprasylvian gyrus II Hippocampus Su Subiculum

EXPLANATION OF PLATES All figures show degenerating fibres stained by the Nauta method and photographed at the same magnification, except fig. 3 which is a Nissl preparation.

PLATE 1 Fig. 1. Cingulate cortex after a lesion in the mid-suprasylvian gyrus. Fig. 2. Cortex in the posterior splenial gyrus after a lesion in the mid-suprasylvian gyrus. Fig. 3. The rather uniform structure of the posterior splenial gyrus between the splenial and rhinal sulci in parasagittal section. Fig. 4. A very few degenerating fibres in the entorhinal cortex after the temporal lesion in B 14 shown in Text-fig. 2. Fig. 5. The posterior splenial gyrus after the temporal lesions in B 19 shown in Text-fig. 2.

PLATE 2 Fig. 6. A frontal section of the bundles of degenerating fibres in the anterior limbic area after a lesion in the precentral agranular cortex in B 24 (Text-fig. 3). Fig. 7. A parasagittal section of the fibre bundles, running in the upward direction, in B25. Fig. 8. Entorhinal cortex after the large cingulate lesion in B35 (Text-fig. 4). Fig. 9. Entorhinal cortex after the septal lesion in B37 (Text-fig. 5). Fig. 10. Entorhinal cortex after a lesion in the contra-lateral hippocampus in rabbit B45 (Text- fig. 6), perfumed with Bouin's fluid. 356 Plate 1

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