EXPERIMENTAL

Subicular

NEUROLOGY

5?,264-274

Projections RICHARD

(1977)

to the Posterior C. MEIBACH

AND ALLAN

Department of Neurology, Albert Einstein Bronx, New York 10461, and Department New Jersey Medical School, Newark, Received

Cingulate

Agril

Cortex

in Rats

SIEGEL l

College

of Medicine,

of Neurosciences, New

Jersey

07103

13, 1977

The subicular cortex and not Ammon’s horn has been previously shown to receive afferents from the posterior cingulate cortex. Utilizing the techniques of horseradish peroxidase histochemistry and amino acid autoradiography we present evidence that in the rat the dorsal subicular cortex has a reciprocal connection with the posterior cingulate cortex. The prosubiculum has a small projeotion to the ventral portion of the retrosplenialis granularis. The presubiculum has the most massive projection to layers III (IV) of the retrosplenial is granularis which extends dorsally up to the border of the retrosplenialis agranularis. Relations of the presubicular component of the hippocampal formation with other components of the Papez circuit are discussed.

INTRODUCTION Since Papez (11) first proposed an anatomical circuit for emotional behavior whose focus included the hippocampus, much attention has been given to the anatomical and functional characteristics of this structure. However, it has become increasingly apparent that the subicular cortex and not the cornu ammonis is the true nodal point for the limbic connections suggestedby Papez. For example, there is no evidence in the literature of direct connections from the cingulate cortex to the hippocampus. Instead, fibers arising from cingulate gyrus supply only the subicular component of the hippocampal formation (1, 3-5, 10). Furthermore, the postcommissural fornix, which forms a major link of the Papez circuit by connecting the hippocampal formation with the anterior thalamus and mammillary bodies, has been found to arise from the subicular cortex (2, 7-9, 17). In addition, we have also recently demonstrated that even the majority of fibers which 1 This by Grant

project was supported MH 06418 to R.C.M.

by U.S.

Public

Health

Service

Grant

NS

07941

07 and

264 1977 by Academic Press, Inc. Copyri tit All r&s o4 reproduction in any form reserved.

ISSN 0014-4886

SUBICULAR-CINGULATE

265

PROJECTIONS

comprise the precommissural fornix (i.e., the connections between hippocampal formation and septum) arise from subicular cortex (7). The present report attempts to analyze still another connection of this remarkable region of hippocampal formation : namely, the topographical organization of the subicular projections to the cingulate gyrus. METHODS The present investigation utilizes both retrograde and anterograde transport procedures. We found that a combination of [3H]leucine injections in the regions under question followed by horseradish peroxidase (HRP) injections in terminal distribution sites determined by autoradiography is the most effective method at the present time to map out the precise origin, course, and distribution of neuronal pathways. In this study, injections of [3H]leucine (specific activity 30 to 50 Ci/ mmole, New England Nuclear, Boston, Massachusetts) concentrated to

A

B

c

FIG. 1. Sagittal view indicating the components of the cingulate cortex and its relationship with the hippocampal formation. A, B, and C indicate levels at which coronal sections were viewed in the photomicrographs of Fig. 2 (from M. Rose). Abbreviations in this and subsequent figures: ACG-anterior cingulate gyrus, CC-corpus callosum, DF-dorsal fornix, DH-dorsal hi,ppocampal formastion, ENT-entorhinal cortex, Ffimbria, IRa-infraradiata anterior, IRp-infraradiata posterior, PARA-parasubiculum, PCG-posterior cingulate cortex, PH-posterior hippocampal formation, PREpresubiculum, PRECAG-area precentralis agranularis,, PRO-prosubiculum, RSAG -retrosplenialis agranularis, RSGd-retrosplenialis granularis dorsalis, RSGv-retrosplenialis granularis ventralis, SUB-subiculum, and VH-ventral hippocampal forma-

tion.

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10 &i/p1 were made into the rat subicular cortex and adjacent hippocampus throughout the rostrocaudal extent of the hippocampal formation. After 24 h, the animals were killed with 0.9% saline followed by 10% formalin. The brains were removed and postfixed 1 week. Paraffin blocks were cut at 8 pm, mounted, deparaffinized, dipped into Kodak nuclear track emulsion NTB-2, and stored 2 weeks in light-tight boxes at 4°C. The slides were developed in Kodak developer D19 and counterstained lightly with cresyl violet. For HRP experiments animals were injected with 0.0s to 0.1 ,J of 30% to 35% HRP (type VI, Sigma Chemical Co., St. Louis, Missouri). Injections were made into the anterior and posterior cingulate cortices in 14 male Sprague-Dawley rats. The animals were killed after 24 h by intracardiac perfusion of 0.9% saline followed by 1% paraformaldehyde-2% glutaraldehyde in 0.1 11 cacodylate buffer of pH 7.4. The brains were removed, postfixed 4 h, blocked, and soaked overnight in cold buffer containing 5% sucrose. Serial sections were cut at 60 pm on a freezing microtome and processed in an incubation medium (3,3’-diaminobenzidine-tetrahydrochloride in Tris buffer of pH 7.6) for the presence of reaction product. RESULTS Nomenclature The hippocampal formation (hippocampus, dentate gyrus, subicular cortex) was divided into five regions located along its septal-temporal axis. Each region was further subdivided into five components: regio inferior, regio superior, prosubiculum, subiculum, and presubiculum. For a complete description of the components of the subicular cortex refer to a previous communication (7). The cingulate gyrus has been subdivided according to the terminology of M. Rose (12) ( see Fig. 1). The region which lies dorsal to the caudal subicular cortex is referred to as the retrosplenialis granularis ventralis (RSGv). Rostra1 to it, and immediately dorsal to the corpus callosum lies the retrosplenialis granularis dorsalis. The retrosplenialis agranularis lies dorsal to the retrosplenialis granularis dorsalis. The entire retrosplenial FIG. 2. Photomicrographs taken at three different levels of the cingulate cortex showing the cytoarchitectural distinctions. A-Section through the anterior cingulate cortex (area infraradiata) demonstrating absence of granular layer. B-Section through the posterior cingulate cortex at the level of the anterior dorsal hippocampus indicating the retrosplenialis granularis. The retrosplenialis agranularis lies dorsal to the RSG (arrow). C-Section through the posterior dorsal hippocampal formation indicating the juxtaposition of the subicular cortex with the retrosplenialis granularis. X 16.

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SIEGEL

cortex has also been described as the posterior cingulate cortex (4). At approximately the midlevel of the fornix bundle a pronounced transition takes place in the cortical cells which defines the posterior border of the anterior cingulate cortex or area infraradiata, which is recognized by the loss of compactness of the granular layer III (IV). We have used the classification III (IV) and not II-IV because layer II of the neocortex is not continuous with the granular layer of the retrosplenial cortex. [See Vogt (21) for a complete description of the cytoarchitecture of the retrosplenial cortex.] Figure 2 demonstrates the morphological components of the anterior and posterior cingulate gyrus. Autoradiographic

Findings

Prosubiculuna Injections. Case 5. An injection of [3H]leucine was made into the prosubiculum at a central locus along the longitudinal axis of the dorsal hippocampal formation (Fig. 3). Labeled fibers were traced through the superficial layers of the subicular cortex and into the posterior cingulate gyrus. At the level of the corpus callosum these fibers were observed to enter layers III (IV) of the retrosplenialis granularis dorsalis. The distribution of silver grains was confined to the most ventral part of the granular cortex immediately dorsal to the corpus callosum (Fig. 4B). In addition, these fibers extended as far rostrally as the border with the area infraradiata. The injections were repeated with similar results obtained in three additional cases (cases 6-8). Prosubiculum injections were also made at posterior levels of the dorsal subicular cortex and similar results were obtained (cases 23-26). However, injections of the prosubiculum at the level of the ventral subicular cortex did not label the pathway to the cingulate cortex (cases 30-34). Instead, fibers arising from this region of subicular cortex form the medial corticohypothalamic tract. Subiculuna Injections. Case 35. An injection was made into the subiculum at the same central level within the dorsal subicular cortex as demonstrated in case 5. Labeled fibers again could be traced through the presubiculum, around the splenium of the corpus callosum, and into the retrosplenialis granularis. The distribution of silver grains differed slightly from that of prosubiculum injections in that the label was distributed to more dorsal portions of the posterior cingulate cortex. In cases 36-39 injections were made into the subiculum at similar levels and in cases 40-45 injections were made at more posterior levels of the dorsal subicular cortex. In each of these instances projections identical to that described in case 35 were observed. Presubiculum Injections. Case 19. An injection was made into the region of the subicular cortex which borders upon the retrosplenial cortex (Fig. 3). From the level of the central dorsal subicular cortex and extending in a caudal direction this region is referred to as the presubiculum. Labeled

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FIG. 3. Coronal sections from rostra1 (top) th.rough caudal levels indicating position of injection sites of [‘Hlleucine placed into the prosubiculum (lefit side) and presubiculum (right side) and the resultant distribution of radioactive label in the cingulate cortex.

fibers could be traced ventrally and medially into layer III (IV) of the retrosplenialis granularis. Within the retrosplenial cortex radioactive label was distributed in a dorsal-ventral plane throughout the entire granular

270

MEIBACH

AND

SIEGEL

FIG. 4. Dark-field photograph of distribution of silver grains in retrosplenialis notice abrupt granularis. A-Case in which injecmtion was placed into the presubiculum, end of fibers at border with RSAg; B-case in which injection was placed into prosubiculum, notice decreasing amount of label dorsal to the corpus callosum. X135.

region (Fig. 5). However, no label was observed in the retrosplenialis agranularis (Fig. 4). In all casesin which [3H]leucine was injected into the presubiculum, the most massive projection to the posterior cingulate cortex was demonstrated. Control Injections. Injections were made into all cytoarchitectonic fields of the hippocampus and dentate gyrus at each level studied along the longitudinal axis. In none of these caseswere any labeled grains observed in the cingulate cortex. Horseradish Peroxidase Injections. Case CGl (and Repeated in Five Additional Cases). In this case an injection was made along the midline into the dorsal portion of the retrosplenialis granularis. Within the hippocampal formation, HRP-positive cells were confined solely to the subicular cortex. The majority of cells were present in the presubiculum with fewer cells noted in the subiculum. No labeled cells were observed in the prosubiculum nor in the cornu ammonis (Fig. 6). This pattern of labeling was observed in anterior levels of the dorsal hippocampal formation and extended to posterior levels of the dorsal hippocampal formation as well. No labeled cells were observed in the ventral subicular cortex.

SVBICULAK-CINGULATE

FIG. 5. Bright field photomicrograph granular cell layer III ( IV). A4-I,ow cells (X360).

PROJECTIONS

showing power

distribution of radioactive (X180) ; B-high power

271

label in of granule

Horseradish Pcvoxidase Injections. Case CG7 (and Repcatcd in TK~O Additional Cases). An injection was made into the dorsal portion of the area infraradiata posterior. h’o labeled cells were observed in either subicular cortex or cornu ammonis. Horseradisll Per&&se Jnjectitms. Case CGlO. Following an injection into the ventral portion of the area infraradiata posterior, no label was observed in any subfield of the hippocampal formation. Horseradish Pcvon-idase Injections. Case CGIZ. An injection was made in the lateral portion of the retrosplenialis granularis. The enzyme did not spread to layers III (IV) of the granular cortex. HRP-positive cells were not observed in any subfield of the hippocampal formation. DISCUSSION The results of the present experiments clearly demonstrate the existence of a direct projection from the subicular cortex to the granular layer of the posterior cingulate cortex, This projection arises from all divisions of the dorsal subicular cortex. However, it is the medial portion (i.e., presubiculum) lying adjacent to the retrosplenialis granularis which contains the

272

MEIBACH

FIG.

resultant

AND

SIEGEL

6. Outlines of coronal sections demonstrating pattern of labeling in subicular cortex.

position

of injection

CINGULATE

ENTORHINAL CORTEX

l-4

and

CORTEX

PRESUBICULUM

r

CORNU AMMONIS

-

1

1

?j DENTATE GYRUS

of HRP

t

+

I

ANTEROVENTRAL THALAMIC NUCLEUS

J

t -

4-

t +

MAMMILLARY BODIES

FIG. 7. Summary diagram illustrating the connections of the presubiculum. the presubiculum is reciprocally connected with both (the posterior cingulate anteroventral thalamic nucleus and projects heavily to the mammillary entorhinal cortex as well.

Note cortex bodies

that and and

SUBICULAR-CINGULATE

PROJECTIOKS

273

most massive projection to both dorsal and ventral levels of the granular cortex. In previous studies, we were able to demonstrate a projection to the mammillary bodies from all components of the subicular cortex (7, 9). It is interesting to note that, in regard to the postcommissural fornix, the most extensive projection throughout the entire extent of the pars posterior of the medial mammillary nucleus also arises from the presubiculum. However, this same region of the hippocampal formation does not contribute fibers to the precommissural fornix. In a study of hippocampal projections to the anterior thalamus, we found that the presubiculum is the principal source of fornix fibers which project to the anteroventral thalamic nucleus (8). This finding, coupled with the observations of Shipley and Sorensen (14) who observed a thalamopresubicular pathway, indicates that the anteroventral nucleus and presubiculum are reciprocally connected. The anteroventral nucleus has also been shown to receive fibers from the retrosplenial cortex in addition to the presubiculum (3, 8, 15). Our finding of direct presubicular-cingulate projections provides further evidence that the presubiculum is the major site within the hippocampal formation which gives rise to periallocortical connections (Fig. 7). It has previously been reported (6, 13, 16) that the presubiculum projects to the entorhinal cortex as well. The entorhinal cortex, in turn, receives input directly from the neocortex (IS-20). Thus, these facts suggest the possibility that the presubicular component of the hippocampal formation is a critical region involved in the integration of cortical and thalamic signals within the limbic system. REFERENCES 1. ;\DEY,

R. 1951. An experimental study of the hippocampal connections of the cortex in the rabbit. Brain 74 : 233-247. 2. CHRONISTER, R. B., R. W. SIKE, AND L. E. WHITE. 1975. Post-commissural fornix. Origin and distribution in rodent. Neurosci. Mt. 1: 199-202. 3. DOMESICK, V. B. 1969. ,4 new look at the “Papez circuit.” Pages 193-194 i Procecdiugs of the i?tll Am~71 Convmtion of tllc Amcricm Psychological As.sociutioe. 4. DOMESICK, V. B. 1969. Projections from the cingulate cortex in the rat. Brain RPS. 12 : 296-320. 5. DOMESICK, V. B. 1972. Thalamic relationships of the medial cortex in the rat. Brain Bchav. Evol. 6: 457-483. 6. MARTEN, H. J. 1963. P,rojections of the parahippocampal gyrus of the cat. dnut. Rec. 145 : 247-248. 7. MEIBACH, R. C., AND A. SIEGEL. 1977. Efferent connections of the hippocampal formation in the rat. Brain Res. 124 : 197-224. 8. MEIBACH, R. C., AND A. SIEGEL. 1977. Thalamic projections of the hippocampal formation: Evidence for an alternate pathway involving the internal capsule. Brain Rrs. In press. W. cingulate

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9. MEIBACH, R. C., AND ,4. SIEGEL. 1975. The origin of fornix fibers which project to the mammillary bodies in the rat: A horseradish peroxidase study. B&t KLIS. 88 : 518522. 10. PANDYA, D. N., G. W. VANHOESEN, AND V. B. DOMESICK. 1973. A cinguloamygdaloid projection in the rhesus monkey. Brain Res. 61: 369-373. 11. PAPEZ, J. W. 1937. -4 proposed mechanism of emotion. Adz. Neztrol. Psychiafry 38: 725-734. 12. ROSE, M. 1929. Zytoarchitektonischer atlas der grobhirnrinde der maus. J. Psyclzol. Neural. 40 : 1-51. 13. SHIPLEY, M. T. 1974. Presubiculum afferents to the entorhinal area and the Papez circuit. Brain Res. 67 : 162-168. 14. SHIPLEY, M. T., AND K. E. SORENSEN. 1975. On the laminar organization of the anterior thalamus projections to the presubiculum in the guinea pig. Brailz Res. 86: 473-477. 15. SIEGEL, A., R. TROIANO, AND A. ROYCE. 1973. Differential projections of the anterior and posterior cingulate gyrus to the thalamus in the cat. Exp. Newol. 38: 192-201. 16. STEWARD, O., AND S. A. SCOVILLE. 1976. Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentafta of the rat. J. Co+?zp. Ncrlvnl. 169: 347-370. 17. SWANSON, L., AND W. M. COWAN. 1975. Hippocampal hypothalamic connections: Origin in subicular cortex, not ammon’s horn. Science 189: 303304. 18. VANHOESEN, G. W., AND D. N. PANDYA. 1975. Some connections of the ‘entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. I. temporal lobe afferents. Brain Res. 95 : l-24. 19. VANHOESEN, G. W., D. N. PANDYA, AND N. BUTTERS. 1972. Cortical afferents to the entorhinal cortex of the rhesus monkey. Science 175 : 1471-1473. 20. VANHOESEN, G. W., D. N. PANDYA, AND N. BUTTERS. 1975. Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. II. frontal lobe afferents. BY&Z Res. 95 : 25-38. 21. VET, B. A. 1976. Retrosplenial cortex in the rhesus monkey: A cytoarchitectonic and golgi study. J. Camp. Nezrrol. 169 : 63-98.

Subicular projections to the posterior cingulate cortex in rats.

EXPERIMENTAL Subicular NEUROLOGY 5?,264-274 Projections RICHARD (1977) to the Posterior C. MEIBACH AND ALLAN Department of Neurology, Albert E...
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