EXPERIMENTAL
NEUROLOGY
47, 433-441 (1975)
Anatomical Evidence for a Projection Cortex to the Contralateral Dentate DAN
GOLDOWITZ, GARY
Dejwrtrmvt
of
Psychohiolo~y,
W,
FROST WHITE,
LYNCH,
AND
U)tiwrsity Rrcciwd
Janarary
CARL
of
from the Entorhinal Gyrus of the Rat OSWALD COTMAN
California,
STEWARD, 1
Iwine,
California
92664
10. 197.5
A relatively sparse direct projection from the entorhinal cortex to the rostra1 part of the contralateral dentate gyrus of the hippocampus has been demonstrated by the Fink-Heimer and autoradiographic techniques in normal rats. The fibers terminate in the outer two-thirds of the molecular layer, similar to the projection to the ipsilateral dentate gyrus. Thus extrinsic cortical input is bilaterally directed, further illustrating the principle of the entorhinal probilaterality of hippocampal projections. A contralateral jection has been reported previously only as a consequence of apparent axon sprouting following a unilateral entorhinal lesion. The demonstration of this projection in normal rats indicates the changes following recovery from unilateral entorhinal lesions need not involve extensive new fiber growth as had been previously hypothesized.
INTRODUCTION The entorhinal cortex is the primary source of extrinsic input to the hippocampal formation, and its projections have been extensively investigated (2, 3, 4, 8, 10, 12, 13). It has been known from the time of Cajal that this paleocortical ‘structure richly innervates the molecular layer of the dentate gyrus and stratum lacunosum-moleculare of hippocampus regio superior. Cajal, and Lorente de Ni, after him, speculated upon the possibility that entorhinal fibers cross in the dorsal psalterium and enter the contralateral hippocampal formation via the angular bundle (3, 4, 10). More recent work has revealed a crossed projection from the entorhinal cortex to stratum lacunosum-moleculare of hippocampus regio superior (2, 13). No crossed projection to the contralateral dentate gyrus from the 1 This study was supported by NIMH grant MH 19691 to Dr. Cotman and by grant MH 19793 to Dr. Lynch. 0. Stewart presently at University of Virginia, Charlottesville. NIMH
CoDyright .411 sights
s
1975 by Academic Press. Inc. reproduction in any form reserved.
434
GOLDOWITZ
ET
AL.
entorhinal cortex has been reported in normal animals, However, following a unilateral lesion of the entorhinal cortex, we have reported a projection from the remaining entorhinal cortex to the contralateral dentate gyrus (14, 15). There are two possible interpretations of this result: (i) The crossed temporoammonic fibers to CA1 send axon sprouts into the dentate gyrus, or (ii) a previously undetected entorhinal projection to the contralateral dentate gyrus proliferates as a result of the lesion. In the present study we used both the Fink-Heimer stain and autoradiography, two independent methods of demonstrating axon terminals, to determine whether the entorhinal cortex innervates the contralateral dentate gyrus of normal rats. By optimizing the Fink-Heimer staining conditions and survival time after lesion, and by the use of dark field microscopy to reveal autoradigraphic grains, we have obtained evidence for a direct projection to the contralateral dentate gyrus. METHODS Male adult Sprague-Dawley rats (Simonsen Labs, Gilroy, Ca.) 90 days or older were used. Unilateral lesions of the entorhinal cortex were made electrolytically. At various postlesion survival times, the rats were perfused with 10% formalin in normal saline and the brains postfixed in a 30% sucrose (w/v) 10% formalin (w/v) solution. Sections 25 pm thick were cut with a freezing microtome and placed in 2% (w/v) formalin. After storage at 4 C for at least 1 week, sections were stained for degeneration by a modified Fink-Heimer procedure and the staining conditions were systematically varied in order to maximize the appearance of degeneration and minimize background artifact (1, 11). A total of 14 animals, which survived from l-10 days after lesion, were used. In autoradiographic studies, 1 ~1 containing 12 &i of [4-SH]proline (16 Ci/mmol, New England Nuclear Corp., Boston, MA) was injected into the entorhinal cortex of naive rats under stereotaxic guidance. The animals (n = 6) were killed 1, 2, 4, 5, or 6 days later, and the brains were processed as previously described (14). The lesion and injection sites were reconstructed from horizontal sections as previously described ( 14). RESULTS Following a unilateral lesion of the entorhinal cortex, rats were killed after l-10 days and the brains stained by the Fink-Heimer method for degenerating fibers and boutons. The detection of degeneration in the contralateral dentate gyrus was very dependent on the survival time after lesion, being most evident at very short times. At a survival time of about 38 hr we observed maximal terminal degeneration products contralateral
ENTORIIINAL
Ipsilateral
435
CORTEX
Contralateral
FIG. 1. Degeneration products in the ventral leaves of the dentate ,gyrus ipsilateral and contralateral to an entorhinal lesion after 38 hr postlesion survival time (case R49d). GC marks the position of the granule cells. Ipsilateral to the lesion (,4), very fine grained, terminal degeneration (deg) is densely distributed in the outer zone of the molecular layer (ML). Contralateral to lesion (B), terminal degeneration products are much less densely distributed in the outer zone of the ML. Sections are from a coronal plane through the dorsal hippocampal formation, intermediate to levels b and c of Steward et al. (14). The approximate outer one-third of the molecular layer is not shown in order to achieve appropriate magnification. Three other animals with short survival times gave similar results. (initial magnification X ZOO).
to the lesion in the outer two-thirds of the molecular layer (Fig. 1) . They on the ipsilateral side. were much less dense than those in the same locus The Degeneration generation products in the contralateral molecular layer showed both mediolateral and rostrocaudal gradients. Degeneration was most dense in the ventral leaf. In the dorsal leaf degeneration products
436
GOLDOWITZ
ET
AL.
FIG. 2. Reconstruction of the entorhinal lesion (case R49-6) and the [4-‘H] proline injection site (case R3%1) shown at various levels [II, IV, VI from HjosrthSimonsen and Jeune (S)]. The lesion (A) included the majority of the entorhinal cortex with the exception of the most lateral and ventral portions. As shown in B, the injection site most heavily labelled the medial entorhinal cortex.
were most abundant at the medial end, but in the ventral leaf there was no such mediolateral gradient. Degeneration was restricted to the rostra1 portion of the dentate gyrus when the lesion destroyed all but the most lateral and ventral portions of the entorhinal cortex (Fig. Za). It was not clearly detectable in the posterior hippocampal formation where it curves ventrally [in coronal sections level d, Ref. (14) 1. At this point the ispilateral projection was still evident. Within 5 days after lesion, the terminal degeneration products in the contralateral dentate were not detectable and the remaining degeneration products were larger and coarser in appearance, indicative of axonal debris (Fig. 3). They were most abundant at the medial tip of the dentate gyrus. By contrast, in the ipsilateral molecular layer abundant terminal degeneration was still present. Axonal degeneration products in the contralateral molecular layer were less evident at survival times beyond 5 days. It was evident the type and even the presence of degeneration products in the hippocampal formation after entorhinal lesion depended markedly on survival time. This was true for the hippocampal subfield CAl, as well as the dentate gyrus. At survival times of 1.5-2.0 days, the stratum la-
ENTORHINAL
437
CORTEX
cunosum-moleculare on the ipsilateral side contained axonal-like degeneration products. However, only sparse degeneration products if any were present in the corresponding contralateral layer. By 4 days after lesion, degeneration in the ipsilateral side remained coarse and axonal-like whereas on the contralateral side, it now appeared fme and terminal-like. Autoradiographic analysis was performed as an independent means of studying the entorhinal projection to the dentate gyrus and to exclude the possibility that the degeneration products arose solely from the transection of fibers passing through the entorhinal cortex. When the injection of [4-3H]proline labelled the entorhinal cortex very heavily, analysis by dark field microscopy revealed a significant, but relatively sparse, population of silver grains in the outer two-thirds of the molecular layer in the contralateral dentate gyrus. Ipsilateral to the injection there was extremely heavy Iabelling in this area (Fig. 4). The topography of the contralateral
Contralateral
bilateral
GC
ML deg
FIG. 3. Degeneration products in the ventral leaves of the dentate gyrus ipsilateral and contralateral to entorhinal lesion after 4 days postlesion survival time (case HA39, level as in Fig. 1). Ipsilateral to lesion (A), terminal degeneration (de,g) is most prevalent in the outer two-thirds of the molecular layer (ML) below the granule cells (GC). Some axonal-like debris is also present. Contralateral to lesion (B), axonal degeneration products are sparsely distributed in the outer two-thirds of the molecular layer. Very little, if any, terminal degeneration is apparent. Four other animals with survival times of either 3, 4, or 5 days gave similar results. (initial magnificaticn X XD).
438
GOLDOWITZ
ET
AL.
A. lpsilateral
FIG. 4. Autoradiographic evidence for a bilateral projection from the entorhinal cortex to the dentate gyrus. [4-‘H] proline was injected into the entorhinal cortex and the animal killed 24 hours later (case R3%1). As shown in the dark field photomicrograph (A) of the medial tip of the dentate gyrus ipsilateral to the injection, the outer two-thirds of the molecular layer is labelled. Grains are mainly concentrated over the middle one-third of the molecular layer as characteristic of a medial entorhinal injection. Above the fissure, the CA1 field is also heavily labelled. (initial magnification X 100.) In B a schematic drawing of label over the entire hippocampal formation ipsilateral to injection is shown. The granule cells are designated by solid circles, pyramidal cells by triangles, and the hippocampal fissure by connected, open circles. Contralateral to the injection (C), the dark field photomicrograph shows light label over the middle part of the molecular layer. Above the fissure the CA1 field is more heavily labelled than the dentate molecular layer. D is a schematic drawing of label over the entire hippocampal formation contralateral to the injection. Grains are slightly more abundant in the ventral leaf of the dentate gyrus; a medio-lateral gradient is apparent in the dorsal leaf of the dentate. A few grains are present in the deep CA3 field (moleculare) which corresponds to ipsilateral label in the same area. However, Fink-Heimer degeneration products were never observed in this area contralaterally, following an entorhinal lesion. The section is at coronal level b of Steward et al. (14). Five other animals with survival times of 1, 2, 4, 5 or 6 days gave similar results.
dentate projection was similar to that demonstrated by Fink-Heimer stain, The silver grains over the contralateral dentate gyrus were restricted to the rostra1 portion of this region, with a medial injection of the dorsal entorhinal cortex (Fig. 2b). In addition, grains were slightly more abundant
ENTORHIKAL
CORTEX
439
and more uniformly distributed in the ventral than in the dorsal leaf, which showed a mediolateral gradient. This pattern was similar to that seen by the Fink-Heimer technique. We attempted to determine if the presence of silver grains over the contralateral molecular layer might have been due to the transneuronal transport of label. This possible source of artifact appeared unlikely because the contralateral molecular layer was labelled even at such short survival times as 24 hr (Fig. 4) when transneuronal labelling should be minimal. For example, the mossy fiber terminal field in area CA3 was labelled transneuronally at 6 days after injection of the entorhinal cortex but was not labelled at 24 hr after injection. Thus sliver grains over the contralateral dentate gyrus molecular layer probably did not arise from transneuronal transport of radioactivity, but it signifies a direct projection from the entorhinal cortex. DISCUSSION Our findings indicate that the entorhinal cortex projects directly to the molecular layer of the contralateral dentate gyrus. Similar results were obtained both by autoradiography and the Fink-Heimer technique. The projection was localized to the outer two-thirds of the molecular layer, and it was most prominent in the rostra1 dentate gyrus, especially in the ventral leaf. The projection to the molecular layer on the contralateral side was much less dense than that to the corresponding ipsilateral side. Both the relative sparseness of the projection and the stringent requirements for demonstrating it by the Fink-Heimer technique probably account for the reason it has heretofore escaped detection. We found it necessary to use survival times as short as 24 hr and to optimize the Fink-Heimer staining conditions in order to obtain clear evidence for terminal degeneration contralaterally. In contrast, the degeneration in the ipsilateral molecular layer is demonstrable after longer survival times and under less optimized staining conditions. The use of such short survival times is relatively unusual in Fink-Heimer studies. However, the necessity of using short survival times has been shown in one study on neuronal connections of immature golden hamsters (9) and has been noted in a few studies on adult animals as well (7). S imilarly, in autoradigraphic analysis only very heavy labelling of the proteins in the entorhinal cortex provided sufficient radioactivity at the terminal region to detect the projection. We have shown previously that connections from the contralateral entorhinal cortex to the dentate gyrus are readily demonstrable after an ipsilateral entorhinal lesion (14, 15). Since no such projection had been previously reported, nor was one detected in our studies, we hypothesized that a new fiber pathway to the dentate gyrus had been created by sprouting
440
GQLDOWITZ
ET
AL.
from the crossed temporo-ammonic fibers which innervate area, CAl, a phenomenon unprecedented in the adult CNS. The demonstration of a normal contralateral projection to the dentate gyrus provides a more plausible explanation for our earlier observations. Rather than the growth of a new fiber system, it is possible that the fibers already present form additional boutons. However, in order to determine the exact nature of the alterations, quantitative data are required on the number of new terminals and the nature of fiber changes as a function of time after denervation. The finding that the entorhinal cortex projects bilaterally to the dentate gyrus seems to be yet another example of a principle that ipsilateral projections in the hippocampal formation often have a contralateral counterpart (5). For example, hippocampal subfield CA3 gives rise to both an ipsilateral associational projection and a contralateral commissural projection (5). The bilaterality of the entorhinal projection extends this principle to the major extrinsic input to the hippocampal formation. Thus it appears that extrinsic, as well as intrinsic, pathways may play a vital role in the coordination and integration of neural information. REFERENCES L. A., and F. F. EBNER. 1971. The areas and layers o,f corticocortical terminations in the visual cortex of the Viriginia Opposum. J. Comp. Newel. 141: 157-190. 2. BLACKSTAD, T. W. 1956. Commissural connections of the hippccampal region in the rat, with special reference to their mode of termination. J. Camp. Neural. 105 : 417-537. 3. CAJAL, S. RAM~N Y. 1901. “Studies on the Cerebral Cortex (Limbic Structures).” Lloyd-Luke, London, 1955 (Trans. L. M. &aft). 4. CAJAL, S. RAM~N Y. 1911. “Histologie du Systeme Nerveux de 1’Homme & des VertCbr&. T. 2.” Instituto Ramon y Cajal, Madrid, 1955 (Trans. L. Azoulay). 5. FINK, R. P., and L. HEIMER. 1967. Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4: 369-374. 6. GOTTLIEB, D. I., and W. M. COWAN. 1973. Autoradiographic studies of the commissural and ipsilateral association connections of the hippocampus and dentate gyrus of the rat. J. Camp. Neural. 149: 393-422. 7. HEIMER, L. 1970. Selective silver-impregnation of degenerating axoplasm, pp. 106129. 11t “Contemporary Research Methods in Neurcanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.], Springer-Verlag, New York. 8. HJORTH-SIMONSEN, A., and B. JEUNE. 1972. Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J. Camp. Neural. 144 : 215-232. 0. LEONARD, C. M. 1973. A method for assessing stages of neural maturation. Braitt Res. 53 : 412416. 10. LORENTE DE N6. R. 1934. Studies on the structure of the cerebral cortex. II. Continuation of the study of the Ammonic system. J. Psycltol. Neural. (Lpz). 46 : 113-177. 1. BENEVENTO,
ENTORHINAL
CORTEX
441
11. LYNCH, G., and H. KILLACKEY. 1974. Neuroanatomical techniques for neuroof Aggression.” R. E. behavioral research, pp. 99-123. 1~ “Neuropsychology Whalen [Ed.]. Plenum, New York. 2. NAFSTAD, P. H. 1967. An electron microscope study on the termination of the perforant path fibres in the hippocampus and the fascia dentata. 2. Zel[forsclt. 76 : 532-542. 13. RAISMAN, G., W. M. COWAN, and T. P. S. POWELL. 1965. The extrinsic afferent, commissural and association fihres of the hippocampus. Bruirz 88: 963-996. 14. STEWARD, O., C. W. COTMAN, and G. S. LYNCH. 1973. Re-establishment of electrophysiologically functional entorhinal cortical input to the dentate gyrus by the contralateral deafferented by ipsilateral entorhinal lesions : Innervation entorhinal cortex. Exp. Brain Res. 18: 396-414. 15. STEWARD, O., C. W. COTMAN, and G. L. LYNCH. 1974. Growth of a new fiber projection in the brain of adult rats: Re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions. Exfi. Brain Res. 30: 4546.
NEUROLOGY
47, 433-441 (1975)
Anatomical Evidence for a Projection Cortex to the Contralateral Dentate DAN
GOLDOWITZ, GARY
Dejwrtrmvt
of
Psychohiolo~y,
W,
FROST WHITE,
LYNCH,
AND
U)tiwrsity Rrcciwd
Janarary
CARL
of
from the Entorhinal Gyrus of the Rat OSWALD COTMAN
California,
STEWARD, 1
Iwine,
California
92664
10. 197.5
A relatively sparse direct projection from the entorhinal cortex to the rostra1 part of the contralateral dentate gyrus of the hippocampus has been demonstrated by the Fink-Heimer and autoradiographic techniques in normal rats. The fibers terminate in the outer two-thirds of the molecular layer, similar to the projection to the ipsilateral dentate gyrus. Thus extrinsic cortical input is bilaterally directed, further illustrating the principle of the entorhinal probilaterality of hippocampal projections. A contralateral jection has been reported previously only as a consequence of apparent axon sprouting following a unilateral entorhinal lesion. The demonstration of this projection in normal rats indicates the changes following recovery from unilateral entorhinal lesions need not involve extensive new fiber growth as had been previously hypothesized.
INTRODUCTION The entorhinal cortex is the primary source of extrinsic input to the hippocampal formation, and its projections have been extensively investigated (2, 3, 4, 8, 10, 12, 13). It has been known from the time of Cajal that this paleocortical ‘structure richly innervates the molecular layer of the dentate gyrus and stratum lacunosum-moleculare of hippocampus regio superior. Cajal, and Lorente de Ni, after him, speculated upon the possibility that entorhinal fibers cross in the dorsal psalterium and enter the contralateral hippocampal formation via the angular bundle (3, 4, 10). More recent work has revealed a crossed projection from the entorhinal cortex to stratum lacunosum-moleculare of hippocampus regio superior (2, 13). No crossed projection to the contralateral dentate gyrus from the 1 This study was supported by NIMH grant MH 19691 to Dr. Cotman and by grant MH 19793 to Dr. Lynch. 0. Stewart presently at University of Virginia, Charlottesville. NIMH
CoDyright .411 sights
s
1975 by Academic Press. Inc. reproduction in any form reserved.
434
GOLDOWITZ
ET
AL.
entorhinal cortex has been reported in normal animals, However, following a unilateral lesion of the entorhinal cortex, we have reported a projection from the remaining entorhinal cortex to the contralateral dentate gyrus (14, 15). There are two possible interpretations of this result: (i) The crossed temporoammonic fibers to CA1 send axon sprouts into the dentate gyrus, or (ii) a previously undetected entorhinal projection to the contralateral dentate gyrus proliferates as a result of the lesion. In the present study we used both the Fink-Heimer stain and autoradiography, two independent methods of demonstrating axon terminals, to determine whether the entorhinal cortex innervates the contralateral dentate gyrus of normal rats. By optimizing the Fink-Heimer staining conditions and survival time after lesion, and by the use of dark field microscopy to reveal autoradigraphic grains, we have obtained evidence for a direct projection to the contralateral dentate gyrus. METHODS Male adult Sprague-Dawley rats (Simonsen Labs, Gilroy, Ca.) 90 days or older were used. Unilateral lesions of the entorhinal cortex were made electrolytically. At various postlesion survival times, the rats were perfused with 10% formalin in normal saline and the brains postfixed in a 30% sucrose (w/v) 10% formalin (w/v) solution. Sections 25 pm thick were cut with a freezing microtome and placed in 2% (w/v) formalin. After storage at 4 C for at least 1 week, sections were stained for degeneration by a modified Fink-Heimer procedure and the staining conditions were systematically varied in order to maximize the appearance of degeneration and minimize background artifact (1, 11). A total of 14 animals, which survived from l-10 days after lesion, were used. In autoradiographic studies, 1 ~1 containing 12 &i of [4-SH]proline (16 Ci/mmol, New England Nuclear Corp., Boston, MA) was injected into the entorhinal cortex of naive rats under stereotaxic guidance. The animals (n = 6) were killed 1, 2, 4, 5, or 6 days later, and the brains were processed as previously described (14). The lesion and injection sites were reconstructed from horizontal sections as previously described ( 14). RESULTS Following a unilateral lesion of the entorhinal cortex, rats were killed after l-10 days and the brains stained by the Fink-Heimer method for degenerating fibers and boutons. The detection of degeneration in the contralateral dentate gyrus was very dependent on the survival time after lesion, being most evident at very short times. At a survival time of about 38 hr we observed maximal terminal degeneration products contralateral
ENTORIIINAL
Ipsilateral
435
CORTEX
Contralateral
FIG. 1. Degeneration products in the ventral leaves of the dentate ,gyrus ipsilateral and contralateral to an entorhinal lesion after 38 hr postlesion survival time (case R49d). GC marks the position of the granule cells. Ipsilateral to the lesion (,4), very fine grained, terminal degeneration (deg) is densely distributed in the outer zone of the molecular layer (ML). Contralateral to lesion (B), terminal degeneration products are much less densely distributed in the outer zone of the ML. Sections are from a coronal plane through the dorsal hippocampal formation, intermediate to levels b and c of Steward et al. (14). The approximate outer one-third of the molecular layer is not shown in order to achieve appropriate magnification. Three other animals with short survival times gave similar results. (initial magnification X ZOO).
to the lesion in the outer two-thirds of the molecular layer (Fig. 1) . They on the ipsilateral side. were much less dense than those in the same locus The Degeneration generation products in the contralateral molecular layer showed both mediolateral and rostrocaudal gradients. Degeneration was most dense in the ventral leaf. In the dorsal leaf degeneration products
436
GOLDOWITZ
ET
AL.
FIG. 2. Reconstruction of the entorhinal lesion (case R49-6) and the [4-‘H] proline injection site (case R3%1) shown at various levels [II, IV, VI from HjosrthSimonsen and Jeune (S)]. The lesion (A) included the majority of the entorhinal cortex with the exception of the most lateral and ventral portions. As shown in B, the injection site most heavily labelled the medial entorhinal cortex.
were most abundant at the medial end, but in the ventral leaf there was no such mediolateral gradient. Degeneration was restricted to the rostra1 portion of the dentate gyrus when the lesion destroyed all but the most lateral and ventral portions of the entorhinal cortex (Fig. Za). It was not clearly detectable in the posterior hippocampal formation where it curves ventrally [in coronal sections level d, Ref. (14) 1. At this point the ispilateral projection was still evident. Within 5 days after lesion, the terminal degeneration products in the contralateral dentate were not detectable and the remaining degeneration products were larger and coarser in appearance, indicative of axonal debris (Fig. 3). They were most abundant at the medial tip of the dentate gyrus. By contrast, in the ipsilateral molecular layer abundant terminal degeneration was still present. Axonal degeneration products in the contralateral molecular layer were less evident at survival times beyond 5 days. It was evident the type and even the presence of degeneration products in the hippocampal formation after entorhinal lesion depended markedly on survival time. This was true for the hippocampal subfield CAl, as well as the dentate gyrus. At survival times of 1.5-2.0 days, the stratum la-
ENTORHINAL
437
CORTEX
cunosum-moleculare on the ipsilateral side contained axonal-like degeneration products. However, only sparse degeneration products if any were present in the corresponding contralateral layer. By 4 days after lesion, degeneration in the ipsilateral side remained coarse and axonal-like whereas on the contralateral side, it now appeared fme and terminal-like. Autoradiographic analysis was performed as an independent means of studying the entorhinal projection to the dentate gyrus and to exclude the possibility that the degeneration products arose solely from the transection of fibers passing through the entorhinal cortex. When the injection of [4-3H]proline labelled the entorhinal cortex very heavily, analysis by dark field microscopy revealed a significant, but relatively sparse, population of silver grains in the outer two-thirds of the molecular layer in the contralateral dentate gyrus. Ipsilateral to the injection there was extremely heavy Iabelling in this area (Fig. 4). The topography of the contralateral
Contralateral
bilateral
GC
ML deg
FIG. 3. Degeneration products in the ventral leaves of the dentate gyrus ipsilateral and contralateral to entorhinal lesion after 4 days postlesion survival time (case HA39, level as in Fig. 1). Ipsilateral to lesion (A), terminal degeneration (de,g) is most prevalent in the outer two-thirds of the molecular layer (ML) below the granule cells (GC). Some axonal-like debris is also present. Contralateral to lesion (B), axonal degeneration products are sparsely distributed in the outer two-thirds of the molecular layer. Very little, if any, terminal degeneration is apparent. Four other animals with survival times of either 3, 4, or 5 days gave similar results. (initial magnificaticn X XD).
438
GOLDOWITZ
ET
AL.
A. lpsilateral
FIG. 4. Autoradiographic evidence for a bilateral projection from the entorhinal cortex to the dentate gyrus. [4-‘H] proline was injected into the entorhinal cortex and the animal killed 24 hours later (case R3%1). As shown in the dark field photomicrograph (A) of the medial tip of the dentate gyrus ipsilateral to the injection, the outer two-thirds of the molecular layer is labelled. Grains are mainly concentrated over the middle one-third of the molecular layer as characteristic of a medial entorhinal injection. Above the fissure, the CA1 field is also heavily labelled. (initial magnification X 100.) In B a schematic drawing of label over the entire hippocampal formation ipsilateral to injection is shown. The granule cells are designated by solid circles, pyramidal cells by triangles, and the hippocampal fissure by connected, open circles. Contralateral to the injection (C), the dark field photomicrograph shows light label over the middle part of the molecular layer. Above the fissure the CA1 field is more heavily labelled than the dentate molecular layer. D is a schematic drawing of label over the entire hippocampal formation contralateral to the injection. Grains are slightly more abundant in the ventral leaf of the dentate gyrus; a medio-lateral gradient is apparent in the dorsal leaf of the dentate. A few grains are present in the deep CA3 field (moleculare) which corresponds to ipsilateral label in the same area. However, Fink-Heimer degeneration products were never observed in this area contralaterally, following an entorhinal lesion. The section is at coronal level b of Steward et al. (14). Five other animals with survival times of 1, 2, 4, 5 or 6 days gave similar results.
dentate projection was similar to that demonstrated by Fink-Heimer stain, The silver grains over the contralateral dentate gyrus were restricted to the rostra1 portion of this region, with a medial injection of the dorsal entorhinal cortex (Fig. 2b). In addition, grains were slightly more abundant
ENTORHIKAL
CORTEX
439
and more uniformly distributed in the ventral than in the dorsal leaf, which showed a mediolateral gradient. This pattern was similar to that seen by the Fink-Heimer technique. We attempted to determine if the presence of silver grains over the contralateral molecular layer might have been due to the transneuronal transport of label. This possible source of artifact appeared unlikely because the contralateral molecular layer was labelled even at such short survival times as 24 hr (Fig. 4) when transneuronal labelling should be minimal. For example, the mossy fiber terminal field in area CA3 was labelled transneuronally at 6 days after injection of the entorhinal cortex but was not labelled at 24 hr after injection. Thus sliver grains over the contralateral dentate gyrus molecular layer probably did not arise from transneuronal transport of radioactivity, but it signifies a direct projection from the entorhinal cortex. DISCUSSION Our findings indicate that the entorhinal cortex projects directly to the molecular layer of the contralateral dentate gyrus. Similar results were obtained both by autoradiography and the Fink-Heimer technique. The projection was localized to the outer two-thirds of the molecular layer, and it was most prominent in the rostra1 dentate gyrus, especially in the ventral leaf. The projection to the molecular layer on the contralateral side was much less dense than that to the corresponding ipsilateral side. Both the relative sparseness of the projection and the stringent requirements for demonstrating it by the Fink-Heimer technique probably account for the reason it has heretofore escaped detection. We found it necessary to use survival times as short as 24 hr and to optimize the Fink-Heimer staining conditions in order to obtain clear evidence for terminal degeneration contralaterally. In contrast, the degeneration in the ipsilateral molecular layer is demonstrable after longer survival times and under less optimized staining conditions. The use of such short survival times is relatively unusual in Fink-Heimer studies. However, the necessity of using short survival times has been shown in one study on neuronal connections of immature golden hamsters (9) and has been noted in a few studies on adult animals as well (7). S imilarly, in autoradigraphic analysis only very heavy labelling of the proteins in the entorhinal cortex provided sufficient radioactivity at the terminal region to detect the projection. We have shown previously that connections from the contralateral entorhinal cortex to the dentate gyrus are readily demonstrable after an ipsilateral entorhinal lesion (14, 15). Since no such projection had been previously reported, nor was one detected in our studies, we hypothesized that a new fiber pathway to the dentate gyrus had been created by sprouting
440
GQLDOWITZ
ET
AL.
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