THE JOURNAL OF COMPARATNE NEUROLOGY

300593-606 (1990)

Connections of the Retrosplenial Granular A Cortex in the Rat THOMAS VAN GROEN AND J. MICHAEL WYSS Department of Cell Biology and Anatomy, University of Alabama a t Birmingham, UAB Station, Birmingham, Alabama 35294

Although the retrosplenial granular a cortex (Rga) is situated in a critical position between the hippocampal formation and the neocortex, few studies have examined its connections. The present experiments use both retrograde and anterograde tracing techniques to characterize the afferent and efferent connections of Rga. Cortical projections to Rga originate in the ipsilateral area infraradiata, the retrosplenial agranular and granular b cortices, the ventral subiculum, and the contralateral Rga. Subcortical projections originate in the claustrum, the diagonal band of Broca, the thalamus, the midbrain raphe nuclei, and the locus coeruleus. The thalamic projections to Rga originate mainly in the anterodorsal (AD) and laterodorsal (LD) nuclei, with sparse projections arising in the anteroventral (AV) and reuniens nuclei. Each projection to Rga terminates in distinct layers of the cortex. The thalamic projection from AD terminates primarily in layers I, 111, and IV of Rga, whereas the s o n s arising from the LD nucleus have a dense terminal plexus only in layer I. The projections arising from the subiculum end predominantly in layer 11, whereas the postsubiculum projects to layers I and 111-V. Axons from the contralateral Rga form a dense terminal plexus in layers IV and V, with a smaller number of terminals in layers I and VI. Rga projects ipsilaterally to the AV and LD nuclei of the thalamus and to the anterior cingulate, retrosplenial agranular, and postsubicular cortices. Contralaterally it projects to the retrosplenial agranular and Rga cortices. Rga projections to the thalamus terminate ipsilaterally in the dorsal part of LD and bilaterally in AV. Together, these data suggest that Rga integrates thalamic with limbic information. Key words: anterior thalamic nuclei, cingulate cortex, hippocampus, limbic system, Papez circuit

In 1937 Papez suggested that “the hypothalamus, the anterior thalamic nuclei, the gyrus cinguli, the hippocampus and their interconnections constitute a harmonious mechanism which may elaborate the functions of central emotion, as well as participate in emotional expression.” In this scheme, the cingulate (retrosplenial)cortex was considered to be involved in the emotional “experience” whereas the hypothalamus was thought to be involved in the “expression” of emotions. Although Papez’s hypothesis had support from behavioral studies, there were few anatomical data documenting these connections. Recent anatomical studies have begun to document these connections more fully, and have demonstrated that several feedback loops are contained within this “circuit’)(Domesick, ’69; Shipley, ’74). For instance, several studies have shown that reciprocal connections exist between the posterior cingulate (retrosplenial) and subicular cortices (Sqirensen, ’80; Vogt and Miller, ’83; Vogt et al., ’861, and between the cingulate cortex and the anterior nuclei of the thalamus (Domesick, ’69; Robertson and Kaitz, ’81; Sripanidkulchai and Wyss, ‘86b).

o 1990 WILEY-LISS, INC.

Other recent studies have demonstrated that the retrosplenial cortex contributes to the processes of learning and memory (Gabriel and Sparenborg, ’86, ’87; Valenstein et al., ’87; Sutherland et al., ’88; Sif et al., ’89; Matsunami et al., ’89), probably via its connections to the hippocampal formation, an area that has long been recognized as having an important role in these functions (e.g., O’Keefe and Nadel, ’78; Olton, ’83; McNaughton and Morris, ’87).The efferent projections from the hippocampal formation to subcortical regions and the entorhinal cortex have been investigated extensively (see, e.g., Swanson and Cowan, ’77; Meibach and Siegel, ’77b; Finch and Babb, ’80, ’81; Van Groen et al., ’86; Van Groen and Lopes da Silva, ’86; Groenewegen et al., ’87), and several studies have elucidated the cortical and thalamic connections of the retrosplenial cortex, especially the connections of the retrosplenial granular b cortex (Vogt and Miller, ‘83; Wyss and Sripanidkulchai, ’84; Vogt et al., ’86; Sripanidkulchai and Wyss, ’86b, ’87; Wyss et al., ’90). In contrast, only a few studies Accepted July 25,1990

594

T. VAN GROEN AND J.M. WYSS

have documented the connections between hippocampal formation and Rga (Meibach and Siegel, '77a; Finch and Babb, '81). In the present study we characterize the connections of Rga, a region that appears to bridge between the hippocampal formation and neocortical regions. The results of the present experiments demonstrate that the connections of Rga have similarities and significant differences from the connections of adjacent regions of "limbic cortex." Rga receives thalamic, hippocampal, and neocortical innervation and projects this information to the hippocampal formation (Segal and Landis, '74; Van Hoesen and Pandya, '75; Swanson and Cowan, '77) and other areas of the cortex. The interconnections between Rga and the hippocampal formation suggest that Rga plays a role in processing information involved in memory, learning, and emotional functions.

Midgard CS-3 constant current source. After a survival time of 7-10 days, the rats were reanesthetized, transcardially perfused with 100 ml phosphate-buffered (0.1 M) saline (pH 7.41, followed by 200 ml of an Na-acetate (0.1 MI-buffered 4% paraformaldehyde solution (pH 6.5) followed by 350 ml of a Na-borate (0.1 M)-buffered 4% paraformaldehyde, 0.1% glutaraldehyde solution (pH 9.5). Following 2-4 hours postfixation in the final fixative, the brains were stored overnight at 4°C in 0.1 M phosphate buffer (pH 7.4). Three (or two) series (one in six) of frozen sections (30 pm) were cut on a freezing microtome and collected in phosphate buffer (0.1 M, pH 7.4). The first series was stained with cresyl violet and coverslipped; the second series was rinsed overnight in a solution of 0.05 M Tris buffer (pH 8.6), 0.5 M NaCl, and 0.5% Triton X-100 (TBS-T). The next day the sections were transferred to a TBS-T solution containing a 1:1,000 concentration of the primary goat-anti-Phaseolus (Vector) antibody. The tissue was incubated on a rotation table for 18hours at 20°C in the MATERIALSANDMETHODS dark. The sections were rinsed 3 x 5 minutes in TBS-T and Thirty-four male Sprague-Dawley rats (275-400 g, incubated with rabbit-anti-goat whole serum (Sigma) in Charles River, Inc., Wilmington, MA) were deeply anesthe- TBS-T (1:400) for 2 hours. After washes (3 x 5 minutes) in tized with sodium pentobarbital prior to surgery (35 mgkg TBS-T, the sections were transferred to goat peroxidaseip). A glass pipette (10 p m tip diameter) or Hamilton 0.5 p1 antiperoxidase (PAP; Sigma) in TBS-T (1:400) for 4 hours. microsyringe needle with a 90 pm tip was lowered to the After rinsing (3 x 5 minutes) in TBS-T the sections were appropriate region by stereotaxic guidance. For autoradio- incubated for 1 hour with a solution containing 40 mg graphic, anterograde transport experiments, a mixture of diaminobenzidine in 100 ml TBS-T to which 0.9 ml H,O, [3Hl amino acids (20-100 KCi/pl; equal parts of proline, (1.5%) was added. The sections were washed thoroughly, lysine, and leucine or proline only; New England Nuclear, mounted on subbed slides, coverslipped, or counterstained Boston, MA) was injected (n = 10) either by iontophoresis and coverslipped. The (optional) third series was stained for (5-10 minutes, 1.0 pA positive-pulsed current) or by pres- acetylcholinesterase (AChE; Geneser-Jensen and Blacksure injection (20-100 nl) as previously described (Wyss, stad, '71), mounted, and coverslipped as previously de'81; Wyss and Sripanidkulchai, '84). After a 2-7 day scribed (Sripanidkulchai and Wyss, '86a). The resulting survival period the animals were reanesthetized and per- series were inspected by using brightfield microscopy. For retrograde transport experiments a small (10-30 nl) fused with 10%formalin, and the brains were removed and processed for autoradiography as described by Cowan et al. amount of a fluorescent dye (fast blue [FBI, Illing, FRG; or ('72). After paraffin embedding, the brains were sectioned fluorogold [FG], Fluorochrome, Inc. Englewood, CO; 4% in in the frontal plane (15 pm-thick sections), and a one in ten DH,O) was injected by pressure into a defined region of the series of sections from each brain was mounted on slides brain. After a 7-10 day survival period, the rats were and exposed to nuclear emulsion at 4°C for 2-15 weeks. The reanesthetized and transcardially perfused with 100 ml of resulting autoradiographs were subsequently inspected by phosphate-buffered (0.1 M, pH 7.4) saline followed by 250 using both bright- and darkfield microscopy. In 20 animals ml of 10% formalin in phosphate buffer. The brains were the anterograde transport of Phaseolus vulgaris leucoagglu- removed from the skulls and stored 48 hours at 4°C in a tinin (PHA-L; Vector, Burlingame, CA) was employed to 10% formalin, 25% sucrose solution. Three one in six series study the pattern of axonal terminals (Gerfen and of sections were cut in the frontal plane on a freezing Sawchenko, '84). Glass micropipettes (10-20 pm tip) were microtome (30 pm-thick sections) and collected in trays. filled with a 2.5%solution of PHA-L in 0.05 M Tris buffer Two series were mounted on gelatin-coated slides immediand stereotaxically positioned in the brain. A positive, ately; one was stained with methylene blue and coverpulsed (7 seconds on, 7 seconds o m DC current (5-6 PA) slipped; the other was coverslipped only. The third series was applied for 20-40 minutes to the pipette by using a was stained for acetylcholinesterase (AChE), mounted, and

Abbreviations

AD AV

AM cc CL

CM DBB DR EC

G IAM IR

LD MD PARA

POST

anterodorsal nucleus of the thalamus anteroventral nucleus of the thalamus anteromedial nucleus of the thalamus corpus callosum claustrum central medial nucleus of the thalamus diagonal band of Broca dorsal raphe nucleus entorhinal cortex nucleus gelatinosus of the thalamus interanteromedial nucleus of the thalamus area infraradiata (anterior cingulate cortex) laterodorsal nucleus of the thalamus medial dorsal nucleus of the thalamus parasubiculum postsubiculum

PRE PT PV Rag Re Rga Rgb Rt

sc

SUB

sm V VLG VPN VR

presubiculum parataenial nucleus of the thalamus paraventricular nucleus of the thalamus retrosplenial agranular cortex reuniens nucleus of the thalamus retrosplenial granular a cortex retrosplenial granular b cortex reticular nucleus of the thalamus superior colliculus subiculum stria medullaris ventral basal complex of the thalamus ventral lateral geniculate nucleus ventral pontine nuclei ventral raphe

CONNECTIONSOFRGACORTEX

Fig. 1. Two high-power photomicrographs of coronal sections of Rga to demonstrate the cytoarchitectonic divisions, A Nissl stained, B: AChE stained. Arrowheads indicate borders between adjacent areas. Scale bar equals 500 km.

595

596

T. VAN GROEN AND J.M. WYSS A

Fig. 2. Four line drawings to demonstrate the position of labeled axons following an injection of PHA-L into

Rga (PHA 34;crosshatching). Arrowheads indicate borders between adjacent areas. Scale bar equals 1,000km.

coverslipped as previously described (Sripanidkulchai and Wyss, '86a). All fluorescent material was inspected under brightfield and fluorescent illumination.

RESULTS Nomenclature The retrosplenial granular cortex was subdivided in two parts, granular a (Rga) and granular b (Rgb), according to the mapping of Wyss and Sripanidkulchai ('84) following the classification suggested by Rose ('27a,b). Rga corresponded t o areas 29a and 29b, and Rgb corresponds to area 29c of Vogt and Peters ('81), respectively. The retrosplenial granular cortex had a staining pattern both in Nissl and AChE preparations that distinguished it from the adjacent cortical areas (Fig. 1).The border between the Rga and the more dorsal Rgb was characterized by two changes in Nissl staining. First, in Rgb compared to Rga, layer II was wider and contained smaller and more darkly staining cells, and in Rgb these cells formed more prominent clumps. Second, in Rgb compared to Rga, layer I11 was thin and the pyramidal cell bodies were randomly spaced; in Rga the layer I11 neuronal cell bodies tended to be arranged in bands parallel to the pial surface. The border between Rga and

Rgb also was characterized by a change in AChE staining. Whereas layer IV in Rga was densely stained and thin, in Rgb the densely stained layer IV was wider. Ventrally and caudally Rga was bounded by the postsubiculum (Fig. 1); the border between Rga and postsubiculum was characterized by a change in Nissl staining. While layer N of Rga was occupied by granular cells, this layer in postsubiculum contained the lamina dissecans superficially and small pyramidal cells in its deeper half. The border between the retrosplenial cortex and the postsubiculum also was characterized by an abrupt change in AChE staining. First, layer I was evenly stained in the postsubiculum, but in the retrosplenial cortex layer I displayed patches of light and dark staining (Fig. 1B). Second, in layer I11 of postsubiculum AChE staining was dense, but in Rga (Fig. 1B) it was only sparse. Third, layer IV of the retrosplenial cortex was heavily stained (Fig. lB), but layer IV of postsubiculum was less densely stained.

Tracingexperiments Rga, efferent connections. Large injections of L3H] amino acids (e.g., CIR 109) into Rga resulted in anterograde labelingin the Rgb, Rag, IR, and entorhinal cortices, the LD and AV nuclei of the thalamus, and postsubiculum, presub-

CONNECTIONS OF RGA CORTEX

597

Fig. 3. Photomicrographs (A-E) demonstrating the pattern of labeling following a PHA-L injection into Rga (PHA 34;A). B demonstrates labeled fibers and terminals in contralateral Rga. C demonstrates labeled fibers and terminals in the AV nucleus of the thalamus. D demonstrates labeled fibers and terminals in the LD nucleus of the thalamus. E displays labeled fibers crossing midline in the ventral

hippocampal commissure. F demonstrates labeled fibers and terminals in layer I of Rga following a PHA-L injection (PHA 41) in the LD nucleus of the thalamus. Asterisks in C indicate the border between AD and AV, arrow in D denotes labeled fibers in the reticular nucleus of the thalamus. Arrows indicate borders between adjacent areas. Scale bar equals 100 wm.

iculum, and parasubiculum. Most contralateral labeling was in Rga, with lighter labeling in Rag, Rgb, subicular, and entorhinal cortices and in the AV nucleus of the anterior thalamus. To study these projections in more detail, several injections of PHA-L were made in Rga. Following an injection involving all layers (11-VI) in the middle part of Rga (PHA 34, Figs. 2C, 3A), the pattern of labeled fibers (Fig. 2) was similar to that observed in the [3H]amino acid experiments. A dense terminal field was labeled in the lateral third of the Rag cortex, primarily in layers 1-111 (Figs. 2B,C, 4C).In the caudal Rgb cortex near the injection, labeled axonal endings were present in all layers, but

most labeled fibers were present in layers 1-111. In more rostral segments of the Rgb cortex, fewer terminals were labeled, and these were found in layers I1 and I11 (Fig. 2A). More rostrally, in the IRbp and IRba cortices (see Wyss and Sripanidkulchai, '84)only a few fibers were labeled in these layers. These cortically projecting axons traversed the cingulum bundle en route to the anterior cingulate cortex. Two groups of labeled axons coursed ventrally into the caudal postsubiculum, presubiculum, parasubiculum, and entorhinal cortex. One group of labeled axons ran through layer I of the postsubiculum to the presubiculum, with the majority of these appearing to terminate in layer I of the

598

Fig. 4. Photomicrographs to demonstrate the pattern of labeling in the ventral lateral geniculate nucleus (A [Nissl stained] and B) and the Rag cortex ( C ) following a PHA-L injection into Rga (PHA 34). The boxed area in A designates the position of the labeled fibers in B. D

T. VAN GROEN AND J.M. WYSS

demonstrates labeled fibers and terminals in layers I1 and 111 of Rga following a PHA-L injection in the ventral subiculum (PHA 43). Scale bar equal in A equals 500 Fm; scale bar in B equals 50 Km; scale bars in C and D equal 100 Km.

CONNECTIONS OF RGA CORTEX

Fig. 5. Line drawings to demonstrate the position of labeled axons in the thalamus following a PHA-L injection into Rga (PHA 34).Injection site (insert at bottom of column) is shown on a unfolded map of the cortical region (see Wyss and Sripanidkulchai, '84). Scale bars equal 1,000 pm.

599

T. VAN GROEN AND J.M. WYSS

600 TABLE 1. Laminar Distribution of Cells of Origin of Projections From the Rga Cortex Projection areas Rga layer

Thal.

I1 111

N V

VI

Comm.

Assoc.

Ento

Coll.

+ + +

+ + +

+

+

+I

+

+

'+ = pmjection origmates in ths layer.

presubiculum (Fig. 2C). The second group of labeled axons gave rise to deeper terminal labeling in postsubiculum, presubiculum, and parasubiculum (Fig. 2C,D). Furthermore, over much of the caudal occipital cortex, axons were labeled in layer I (Fig. 2D). The Rga injections also labeled fibers which crossed midline in the ventral hippocampal commissure (Fig. 3E), and labeled terminals contralaterally in the Rga, Rgb, and Rag and in the postsubicular and presubicular cortices. The contralateral, homotopic Rga received a dense innervation in layers IV and V (Figs. 2, 3B), and labeled terminals were also present in the superficial layers, often in small patches (Fig. 2C). The contralatera1 Rgb cortex received only a sparse innervation of the deep layers. Compared to Rgb, the Rag cortex was densely innervated especially in layers V and VI. Dense labeling also was present in lateral Rag cortex in layers I-IV, in the same place as an ipsilateral patch of labeling in these layers (Fig. 2C). In the contralateral postsubiculum and presubiculum labeled axons terminated in layers V and VI (Fig. 2D). Subcortical projections from Rga coursed around the lateral ventricle, below the stria terminalis and into the rostral thalamus (Fig. 2A). In the thalamus these axons arborized extensively and terminated in the dorsal LD nucleus; rostral in LD in one patch (Figs. 2, 3D, 5E-G), more caudal in LD in two patches (one lateral and one medial [Fig. 5I,J]), and in the rostral half of the AV nucleus (both magno- and parvicellular segments [Figs. 3C, 5B-DI). Some of the labeled axons ( < 10%) in the AV nucleus extended into the anterior medial nucleus and from there crossed midline and terminated in a homotopic region of the contralateral AV nucleus (Fig. 5). No labeling was observed in the LD nucleus contralateral to the injection. Ipsilaterally, a small number of labeled fibers were labeled in the rostral tip of the AD nucleus, and contralaterally only a few fibers were labeled (Fig. 5A,B). A small patch of labeled axons was present in the ipsilateral reticular nucleus adjacent to the LD labeling (Fig. 3D) and a few terminals were labeled in the nucleus reuniens. The axons in the LD nucleus extended laterally to form a small terminal field in the rostroventral part of a ventral lateral geniculate nucleus primarily in the parvicellular division (Fig. 4A,B).The labeled axons in LD also extended caudally through the lateral posterior nucleus (in which sparse terminal labeling was present) and arborized extensively to form a terminal field in the medial intermediate gray of the superior colliculus (Fig. 2C). A number of labeled axons diverted from the thalamicdirected axons as the latter entered the rostral thalamus. The former axons displayed terminal labeling in the caudomedial part of the striatum and more caudally gave rise to a terminal field in the ventral pontine nuclei (Fig. 2D). A few labeled axons appeared to extend into adjacent regions of the ventral pons. A very small number of axons (five to

eight) were labeled in the descending fornix; these axons extended as far as the mamillary complex where they formed a very sparse, diffuse terminal plexus throughout the mamillary bodies. Retrograde labeling experiments demonstrated that the commissural projections of Rga originated in layers 11, 111, and V (Table 1).The association projections arose from neurons in layers 11,111,V, and VI, with the layer V and VI neurons providing the longer projections to the anterior segment of retrosplenial cortex and anterior cingulate (IR) cortex (Fig. 6C,D). The thalamic projections (e.g., CF 280, 286; Fig. 6A,B) originated in layer VI of Rga. The projections to the entorhinal and subicular cortices originated in layer V of Rga (Table 1). The long projections to the superior colliculus arose from neurons in both layers V and VI of Rga. Rga, afferent connections. Injections of the retrogradely transported tracers into Rga (e.g., CF 310, Figs. 7E, 8A)labeled neurons ipsilaterally in Rga and Rgb, area 18b, area infraradiata, postsubiculum, hippocampus, rostroventral subiculum (Fig. SD), presubiculum, and contralateral Rga (Fig. 7). Subcortically, cell bodies were labeled in the claustrum; diagonal band of Broca (both vertical and horizontal limbs); the reuniens; AD, AV, and LD nuclei of the thalamus; the midbrain raphe nuclei; and the locus coeruleus (Fig. 7). In contralateral Rga, labeled cell bodies were present in layers 11, 111, and V, with the greater proportion of labeled the cells in layer I1 and only very few in layer I11 (Fig. 8B). Bilaterally, in the caudal Rgb cortex near to the injections of Rga, labeled cell bodies were present only in layer V (Fig. 7F), but no neurons were labeled in the rostral Rgb. In IR, however, a significant number of labeled neurons were seen in deep layer V (Fig. 7A). In the dorsal hippocampus a small number of pyramidal and nonpyramidal neurons were labeled in field CAI. It should be noted that injections rostral in the Rga cortex (e.g., CF 384) resulted in a slightly different pattern of labeled neurons than more caudal injections in the Rga cortex (e.g., CF 245). Injections in the rostral Rga cortex resulted in a greater number of labeled neurons in caudal area infraradiata, in contrast to caudal Rga injections that labeled neurons in the rostral area infraradiata. Rostra1 Rga injections resulted in a greater number of labeled neurons in caudal Rgb, whereas caudal injections of Rga resulted in a greater number of labeled neurons in area 18b. Injections in rostral Rga labeled cell bodies in the intermediate third of the subiculum, in contrast t o more caudal injections that labeled neurons in the temporal third of the subiculum. In the thalamus, the rostral injection labeled neurons in the lateral and caudal part of the LD nucleus, but the caudal injection labeled a smaller number of neurons in the anterior medial region of the LD nucleus (Fig. 9). To confirm these projections to Rga and characterize the laminar pattern of their terminations, injections of anterogradely transported tracers were made in several regions projecting to Rga (Table 2). Injections of Rgb (e.g., PHA 38) labeled axons and terminals in layer I of Rga, Following injections in the postsubiculum (e.g., PHA 28) labeled axons could be seen in layers I, 111, IV, and V, with the greater part of the label in layer Ib and Ic. Injections of the ventral subiculum (e.g., PHA 43) resulted in labeled axons in layers I, 11, and 111 of Rga, with the greater part of the labeled fibers in layer I1 (Fig. 4D). Injections in the thalamus confirmed that the AD nucleus (e.g., CIR 56) provided

Fig. 6. Four photomicrographs to demonstrate the laminar position of retrogradely labeled neurons in Rga. A (Nissl) and B (fluorescent) demonstrate labeled neurons in Rga following an injection into the LD nucleus of the thalamus (CF 280). C (Nissl) and D (fluorescent) demonstrate labeled neurons in caudal Rga following an injection into rostral Rga (CF 348).Scale bar equals 250 pm.

T. VAN GROEN AND J.M. WYSS

602

n

Fig. 7. Six line drawings to demonstrate the position of labeled neurons (diamonds) following an injection of FG into Rga (CF 310; crosshatching). One symbol equals approximately five labeled neurons. Arrowheads indicate borders between adjacent areas. Scale bar equals 1,000 pm.

a major projection to layers I and 111, and IV of Rga (Fig. 8E,F). LD nucleus injections (e.g., PHA 41)labeled a dense terminal field in layer I of the cortex (Fig. 3F), and AV nucleus injections (e.g., CIR 141) resulted in only light labeling, primarily in layer I.

DISCUSSION The Rga is innervated predominantly by the AD and the LD thalamic nuclei, the postsubicular, the caudal Rgb, and the contralateral Rga cortices (Figs. 7, 10). The major efferent projections of Rga are to the AV and the LD

thalamic nuclei, the caudal Rag, the postsubiculum, and the contralateral Rga (Figs. 2, 10). These connections confirm that the Rga cortex is a part of the "Papez-circuit" (Papez, '37). Further, the differences in connections between Rga and Rgb (Fig. 10)corroborate the cytoarchitectonic subdivision of the retrosplenial cortex into Rga and Rgb (vide infra; Sripanidkulchai and Wyss, '86b, '87). The projection of the thalamic nuclei to these brain areas is strikingly different (vide infra; Van Groen and Wyss, '88). The AD nucleus of the thalamus projects primarily to Rga, Rgb, postsubiculum, and parasubiculum. In contrast, the AV nucleus predominantly projects to Rgb, postsubiculum, and presub-

CONNECTIONS OF RGA CORTEX

Fig. 8. Six photomicrographs to demonstrate the organization of cell bodies that project to Rga. A-D demonstrate labeled neurons following a Fluorogold injection into Rga (CF 310; A). B demonstrates labeled neurons in contralateral Rga. C demonstrates labeled neurons in the AD nucleus of the thalamus. D demonstrates labeled neurons in

603

the ventral subiculum. E (brightfield) and F (darkfield) demonstrate the anterograde labeling in Rga following a C3H1 amino acid injection into the AD nucleus of the thalamus (CIR 56). The arrowheads in E and F point to the same two pial cells. Arrowheads in A and B indicate the borders of Rga. Scale bar equals 100 km.

T. VAN GROEN AND J.M. WYSS

604

Fig. 9. Line drawings to demonstrate the position of labeled neurons in the thalamus following injections into the rostra1 (CF 348;filled circles, left) and caudal (CF 245; stars, right) Rga cortex. Scale bar equals 1,000 pm.

iculum. The LD nucleus of the thalamus has a large terminal field in the retrosplenial agranular cortex, with smaller terminal fields in Rga, postsubiculum, presubiculum, and parasubiculum (vide infra; Sripanidkulchai and Wyss, '86b; Van Groen and Wyss, '88, '90a,b). The cortical inputs of Rga and Rgb are also different. Whereas Rga is densely innervated by ventral subiculum, presubiculum, postsubiculum, and caudal Rgb, Rgb is innervated by axons from dorsal subiculum, postsubiculum, IR, and cortical areas 18a and 18b. Further, the commissural connections of the granular retrosplenial cortical regions predominantly are only to themselves, that is, Rga to Rga, and Rgb to Rgb. It should be noted that the commissural fibers of the presubiculum, postsubiculum, and Rga cross in the ventral hippocampal commissure, whereas the Rgb commissural fibers take a direct transcallosal route. This suggests that Rga (compared to Rgb) is related more closely to the hippocampal formation than to the neocortex (vide infra; Sripanidkulchai and Wyss, '87; Van Groen and Wyss, '88). Thalamic projections to Rgb and Rga terminate primarily in layers I, 111, and IV as would be expected from previous results (Vogt, '85; Sripanidkulchai and Wyss, '86b; Van

Groen and Wyss, '88). The projections to Rga from the AD nucleus are to layers I, 111, and n7; and in layer I the projections are confined to layer Ia with periodic spread into Ib and Ic (Fig. 8E,F). Conversely, the projection from the LD nucleus is distributed evenly in layer I (Fig. 3F1. It should be noted that a segregation of anterior thalamic inputs to layer I of the Rgb cortex has been documented previously (Vogt, '85; Sripanidkulchai and Wyss, '86b; Sripanidkulchai and Wyss, '871, and related to the bundling of apical dendrites in this layer (Wyss et al., '90); a similar segregation is present in Rga. Also, as is seen in Rgb, in Rga a patchy AChE pattern of staining is noted in layer I (Fig. 1B). Although our current data are incomplete, it seems likely that each of these inputs to Rga preferentially relates to different neuronal cell types, as is the case in the Rgb cortex in which layer I1 cells appear preferentially connected to the thalamic input, while the apical dendrites of deeper neurons receive predominantly cortical input (Sripanidkulchai and Wyss, '86b; Wyss et al., '90). The present data demonstrate that the topographically organized thalamic projections from Rga are different than those from Rgb (Domesick, '69; Seki and Zyo, '84; Thomp-

CONNECTIONS OF RGA CORTEX

605

TABLE 2. The Laminar Organization of IpsilateralMerents to the Rga Cortex Rgacortex layer I I1 I11

Rga

SUB

POST

Rgb

LD

AD

+'

+

++ + + +

++

++

+

++* ++ +

rv V

VI

++ +

+ t

'+ labeledaxons. '+ + higher ( > 3 x 1 density of labeled axons son and Robertson, '87b; Wyss and Van Groen, '88). Rga projects primarily to the dorsal LD and AV nuclei, while Rgb projects primarily to more ventral and medial portions of the LD nucleus and has a small projection to the AD nucleus. Rgb has only a very sparse projection to the AV nucleus (Wyss et al., '88). The lateral dorsal nucleus of the thalamus is known to project to posterior limbic cortex (e.g., Rose and Woolsey, '48; Locke et al., '64; Locke and Kerr, '73; Niimi, '78; Niimi et al., '78; Robertson and Kaitz, '81; Sripanidkulchai and Wyss, '86b; Thompson and Robertson, '87a), and the current data demonstrate that in the rat Rga, but not Rgb, receives this projection. In contrast, both regions project to the LD nucleus. The function of the LD nucleus is not well understood, but early studies indicate that it is connected with visual cortical regions and is involved in relaying visual information (Waller and Barris, '37; Walker, '38). In the rat, the LD nucleus also projects to area 18b of visual cortex (Sripanidkulchai and Wyss, '86b), which is itself reciprocally connected with Rga (vide infra; Vogt and Miller, '83; Vogt et al., '86). Further, Itaya et al. ('81) have demonstrated that retinal ganglion cells project into the anterior nuclei of the thalamus. Together these findings indicate that Rga may be involved in the processing of visual information possibly in relation to eye movements (Sikes et al., '85) or relative to external visual cues for learned behavior (Sutherland et al., '88, '89). Two of the present findings should be noted in this regard. First, as shown by us previously, the limbic cortex projects to the middle layers of the superior colliculus (Wyss and Sripanidkulchai, '84). Our PHA-L data demonstrate that Rga also projects to this region, The difficulty in demonstrating this projection previously likely reflects the lower sensitivity of the autoradiographic method compared to PHA-L techniques. Second, the PHA-L data clearly demonstrate that Rga projects to a small region of the lateral geniculate nucleus. We believe this is the first demonstration of this projection. These two findings lend further support to a role for Rga in integrating limbic and visual information. The Rga cortex has been hypothesized to be intimately involved in processing "limbic" information (Papez, '37) and the results of this study support the hypothesis that the

Fig. 10. Schematic summary of the main connections of Rga and Rgb.

Rga forms a part of the so-called "Papez-circuit" (Papez, '37); both the main inputs and outputs of Rga are to areas in the limbic system. Rga is interconnected with the cingulate cortex (Bassett and Berger, '82; Vogt and Miller, '83; Finch et al., '84a; Sripanidkulchai and Wyss, '871, Rga projects to and receive projections from the rostra1 thalamic nuclei, and Rga receives hippocampal (i.e., subicular) inputs (Fig. 10). Together with the studies of others, these data suggest that the retrosplenial cortex contributes to the role of the hippocampus in memory and learning (Sif et al., '89; Matsunami et al., '89). Gabriel and colleagues (Gabriel et al., '80, '89; Gabriel and Sparenborg, '86, '87) have demonstrated that the response of retrosplenial cortex neurons in a learning paradigm is in part dependent on intact connections from the hippocampal formation to the retrosplenial cortex. Sutherland et al. ('88, '89) have demonstrated that an intact retrosplenial cortex is essential for the ability to move accurately to points in space by using distal cues, ie., place navigation. Bilateral lesions of the retrosplenial cortex impair rabbits in their ability to reverse discrimination in a nictating membrane response paradigm (Berger et al., '86). Furthermore, Valenstein et al. ('87) have demonstrated the development of amnesia in a patient after lesions in the retrosplenial cortex. In light of these findings, the elucidation of the anatomy and physiology of the interconnections between the hippocampal formation and the retrosplenial cortex takes on an added importance.

ACKNOWLEDGMENTS We thank Mrs. Maxine Rudolph for her secretarial assistance in the preparation of this manuscript. This study was supported by NIH grants NS 16592 and HL 34315.

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