THE JOURNAL OF COMPARATIVE NEUROLOGY 315200-216 (1992)

Connections of the Retrosplenial Dysgranular Cortex in the Rat THOMAS VAN GROEN AND J. MICHAEL WYSS Department of Cell Biology, University of Alabama at Birmingham, UAB Station, Birmingham, Alabama 35294

ABSTRACT Although the retrosplenial dysgranular cortex (Rdg) is situated both physically and connectionally between the hippocampal formation and the neocortex, few studies have focused on the connections of Rdg. The present study employs retrograde and anterograde anatomical tracing methods to delineate the connections of Rdg. Each projection to Rdg terminates in distinct layers of the cortex. The thalamic projections to Rdg originate in the anterior (primarily the anteromedial), lateral (primarily the laterodorsal), and reuniens nuclei. Those from the anteromedial nucleus terminate predominately in layers I and IV-VI, whereas the axons arising from the laterodorsal nucleus have a dense terminal plexus in layers I and 111-IV. The cortical projections to Rdg originate primarily in the infraradiata, retrosplenial, postsubicular, and areas 17 and 18b cortices. The projections arising from visual areas 18b and 1 7 predominantly terminate in layer I of Rdg, axons from contralateral Rdg form a dense terminal plexus in layers I-IV, with a smaller number of terminals in layers V and VI, afferents from postsubiculum terminate in layers I and 111-V, and the projection from infraradiata cortex terminates in layers I and V-VI. The efferent projections from Rdg are widespread. The major cortical projections from Rdg are to infraradiata, retrosplenial granular, area 18b, and postsubicular cortices. Subcortical projections from Rdg terminate primarily in the ipsilateral caudate and lateral thalamic nuclei and bilaterally in the anterior thalamic nuclei. The efferent projections from Rdg are topographically organized. Rostra1 Rdg projects to the dorsal infraradiata cortex and the rostral postsubiculum, while caudal Rdg axons terminate predominately in the ventral infraradiata and the caudal postsubicular cortices. Caudal but not rostral Rdg projects to areas 17 and 18b of the cortex. The Rdg projections to the lateral and anterior nuclei also are organized along the rostral-caudal axis. Together, these data suggest that Rdg integrates thalamic, hippocampal, and neocortical information. Key words: thalamic nuclei, cingulate cortex, hippocampus, limbic system, visual cortex

Both physiological and anatomical evidence suggest that the retrosplenial cortex plays an important role in the integration of hippocampal and visual information (Vastola, '82; Vogt and Miller, '83; Thompson and Robertson, '87b; Murray et al., '89; Van Groen and Wyss, '90b). Behavioral studies show that the retrosplenial cortex contributes to learning and memory tasks, several of which have a visual component (Gabriel and Sparenborg, '86, '87; Valenstein et al., '87; Sutherland et al., '88; Markowska et al., '89; Murray et al., '89; Sif et al., '89; Matsunami et al., '89). Anatomical experiments demonstrate that the retrosplenial cortex has connections with the hippocampal formation (Meibach and Siegel, '77a,b; Swanson and Cowan, '77; Vogt and Miller, '83; Finch et al., '84a,b; S ~ r e n s e n '80; , Witter et al., 'go), an area known to be involved in learning and memory (e.g., O'Keefe and Nadel, '78; Olton, '83; McNaughton and Morris, '87). Further, previous research suggests that the retrosplenial cortex is connected to thaQ

1992 WILEY-LISS, INC.

lamic nuclei and cortical areas that are part of the visual system (Schober, '81; Vogt and Miller, '83; Vogt et al., '86). The connections between the granular part of the retrosplenial cortex, the thalamus, and the cortex have been extensively investigated (Vogt and Miller, '83; Wyss and Sripanidkulchai, '84; Vogt et al., '86; Sripanidkulchai and Wyss, '86b, '87; Van Groen and Wyss, '90b; Wyss et al., '901, but the connections of the dysgranular part of the retrosplenial cortex have not been studied extensively with the new anterograde and retrograde tracers that facilitate a detailed characterization of the precise origin and laminar termination of projections. In the present study we characterize the connections of Rdg. The results of the present experiments demonstrate that the connections of Rdg have similarities with and significant differences from the connections of adjacent Accepted October 4, 1991

CONNECTIONS OF RETROSPLENIAL DYSGRANULARCORTEX regions of limbic cortex (e.g., retrosplenial granular cortex). Rdg receives thalamic, hippocampal, and neocortical innervation and projects to the neocortex (Vogt and Miller, '83), the hippocampal formation (Segal and Landis, '74; Van Hoesen and Pandya, '75; Vogt and Miller, '831, and the thalamus (Domesick, '69; Thompson and Robertson, '87b). Thus, Rdg bridges between the hippocampal formation and the neocortex, and the interconnections between Rdg, the visual cortex, and the hippocampal formation indicate that Rdg may play an important role in processing visual information involved in learning and memory functions.

METHODS Forty-nine male Sprague-Dawley rats (275-400 g, Charles River, Inc., Wilmington, MA) were deeply anesthetized with sodium pentobarbital prior to surgery (40 mg/kg ip). A glass pipette (15 p.m tip diameter) or a Hamilton 0.5 pl microsyringe equipped with a 90 Km tipped needle was lowered to the appropriate region by stereotaxic guidance. In 27 animals the anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L; Vector, Burlingame, CA) was employed to study the pattern of axonal terminals (Gerfen and Sawchenko, '84). Glass micropipettes (10-20 pm tip) were filled with a 2.5% solution of PHA-L in 0.05 M Tris buffer, stereotaxically positioned in the brain, and the solution was iontophoretically injected by a positive, pulsed (7 second on, 7 second off) DC current (5-6 FA, 20-40 minutes), delivered from a Midgard CS 3 constant current source. After a survival time of 7-14 days, the rats were reanesthetized, transcardially perfused with 100 ml of a phosphate-buffered (0.1 M) saline (pH 7.41, followed by 200 ml of a Na-Acetate (0.1 M) buffered, 4% paraformaldehyde solution (final pH 6.51, followed by 350 ml of a Na-Borate (0.1 M) buffered, 4% paraformaldehyde, and 0.1% glutaraldehyde solution (final pH 9.5). Following 24 hours postfixation at 4°C in the final fixative, the brains were put in a 30% sucrose solution for 2 days (at 4°C). Two series (1in 6) of frozen sections (30 p.m) 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 NaC1, 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

201

primary goat-anti-Phaseolus (Vector, Burlingame, CA) antibody. The tissue was incubated on a rotation table for 18 hours at 20°C in the dark. The sections were rinsed 3 x 5 minutes in TBS-T and incubated with rabbit-anti-goat whole serum (Sigma Chemical Co., St. Louis, MO) in TBS-T (1:400) for 2 hours. After washes (3 x 5 minutes) in TBS-T, the sections were transferred to goat peroxidase-antiperoxidase (PAP; Sigma Chemical Co., St. Louis, MO) in TBS-T (1:400) for 4 hours. After rinsing (3 X 5 minutes) in TBS-T the sections were incubated for 1 hour with a solution containing 40 mg diaminobenzidine (DAB) to which 0.9 ml H,O, (1.5%) was added, or the sections were reacted with Ni-enhanced DAB (12.5 mg DAB in 25 mlO.1 M phosphate buffer, 0.9 ml H,O, [1.5%1, pH 7.4 with 1 ml of a 15% ammonium Ni-sulfate solution added). The sections were rinsed thoroughly, mounted on gelatin-coated slides, and coverslipped. The resulting series were inspected by brightfield microscopy. For retrograde transport experiments, a small (10-30 nl) amount of a fluorescent dye [fast blue (FBI, Illing, FRG; or fluorogold (FG), Fluorochrome, Inc., Englewood, CO; 4% in DH,Ol was injected by pressure into a defined region of the brain. After a 7-10 day survival period, the rats were reanesthetized, transcardially perfused with 100 ml of phosphate-buffered (0.1 M, pH 7.4) saline, and followed by 250 ml of a 4% paraformaldehyde solution in phosphate buffer (pH 7.4). The brains were removed from the skulls and stored 48 hours at 4°C in a 30% sucrose solution. Two (or three) 1 in 6 series of sections were cut in the frontal plane on a freezing microtome (30 pm thick sections) and collected in trays. Two series were mounted on gelatin coated slides immediately; one was stained with methylene blue and coverslipped and the other was coverslipped only. The (optional) third series was stained for acetylcholinesterase (AChE; Geneser-Jensen and Blackstad, '711, mounted, and coverslipped as previously described (Sripanidkulchai and Wyss, '86a). All fluorescent material was inspected under brightfield and fluorescent illumination. For combined anterograde and retrograde transport experiments, a small amount of the new, fluorescent dye Fluoro-Ruby [FR, a tetramethyl rhodamine-dextran-amine (10,000 MJ; Schmued et al., '901 was iontophoretically injected into a defined region of the brain with a positive, pulsed (7 second on, 7 second off) DC current (5-6 FA; 20 minutes). After a survival time of 7-14 days, the rats were

Abbreviations AD AGl

AM AV cc

c1

CM CP DBB DR FG G 1,11,111, IV, v, VI IAM IRa IRP LD LP MD

anterodorsal nucleus of the thalamus lateral agranular cortex anteromedial nucleus of the thalamus anteroventral nucleus of the thalamus corpus callosum claustrum central medial nucleus of the thalamus caudateiputamen diagonal band of Broca dorsal raphe nucleus Fluorogold nucleus gelatinosus of the thalamus Superficial to deep layers of cortex, respectively interanteromedial nucleus of the thalamus area infraradiata a (rostra1anterior cingulate cortex) area infraradiata p (caudal anterior cingulate cortex) laterodorsal nucleus of the thalamus lateroposterior nucleus of the thalamus medial dorsal nucleus of the thalamus

Orb PAG Post Preag PT PV Rdg Re Rga Rgb Rt

sc

sm SUB

VB WN

VR ZI 17.18b

orbital cortex periaqueductal gray nucleus of the midbrain postsubiculum precentral agranular cortex parataenial nucleus of the thalamus paraventricular nucleus of the thalamus retrosplenial dysgranular cortex reuniens nucleus of the thalamus retrosplenial granular a cortex retrosplenial granular b cortex reticular nucleus of the thalamus superior colliculus stria medullaris subiculum ventral basal complex of the thalamus ventral pontine nuclei ventral raphe zona incerta The Brodmann ('09) designation for areas of visual cortex

T.VAN GROEN AND J.M. WYSS

202 reanesthetized, transcardially perfused, and the sections were cut, mounted, and stained as above. All fluorescent material was inspected under brightfield and fluorescent illumination.

RESULTS Nomenclature The classification of the retrosplenial cortex suggested by Rose ('27a,b) is used. The dorsal portion of the retrosplenial cortex lacks a well-developed internal granular layer compared to homotypic areas of neocortex, but some multipolar neurons are present between layer I11 and layer V pyramidal neurons. Rose ('27a,b) labeled this region retrosplenial agranular cortex, even though he noted this small population of granule cells, and in previous accounts we and others have used this terminology. However, in the present and future reports we shall identify it as retrosplenial dysgranular cortex (Rdg), thus recognizing that this cortex contains a rudimentary layer IV in all mammals thus far studied (Vogt and Peters, '81). Rdg has been designated area 29d by Brodmann ('09) and Vogt and Peters ('81) and 29c by Krieg ('46). Rdg has a staining pattern both in Nissl and AChE preparations that distinguishes it from the adjacent cortical areas (Fig. 1).The border between Rdg and more ventral Rgb is characterized by two changes in Nissl staining. First, in Rdg compared to Rgb, layer 11-111 is wider, and in Rgb layer I1 cells are more darkly stained and are more densely packed. Second, in Rdg compared to Rgb, layer IV is wider, and the layer V neuronal cell bodies tend to be larger (Fig. 1A,C).The border between Rdg and Rgb also is characterized by a change in AChE staining. Whereas layers Ia and IV in Rgb are densely stained and layer I1 is very lightly stained, in Rdg AChE staining is relatively evenly distributed in layers I-IV (Fig. 1B,D).Laterally and rostrally Rdg is bordered by medial (precentral agranular; Preag) and lateral (AGl) agranular cortex (Donoghue and Wise, '82; Fig. 1A,B), the border between Rdg and Preag, and AGl is characterized by a change in Nissl staining. While layer IV of Rdg is occupied by few granule cells, in Preag and AGl there is no granule cell layer. The border between Rdg and AGl also is characterized by a change in AChE staining. Whereas layers I-IV in Rdg are relatively evenly stained, in AGl AChE staining is dense in layer Ia and layers Ib-I11 are relatively lightly stained (Fig. 1B). For most of its length, Rdg is bounded laterally by area 18b (Fig. 1C; Schober and Winkelmann, '75); the border between Rdg and area 18b is characterized by an abrupt change in Nissl staining. While layer IV of Rdg is occupied by few granule cells, this layer in 18b contains many granule cells. The border between Rdg and area 18b again is characterized by a change in AChE staining. Layers I-IV of Rdg are evenly and darkly stained, but in area 18b only layer I is darkly stained (Fig. 1D). Furthermore, in Vogt-silver stained material (Vogt, '74) Rdg is densely filled with vertically oriented axon bundles, whereas in area 18b substantially fewer axons are stained (not illustrated). The retrosplenial granular cortex is 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 corresponds to areas 29a and 29b, and Rgb corresponds to area 29c ofVogt and Peters ('811, respectively. The anterior cingulate cortex is designated as area infraradiata (IR; Rose, '27a,b). The rostral and caudal parts are

termed IRa and IRP, respectively. Each of these regions is further divided into dorsal (IRca, IRcP), middle (IRba, IRbP) and ventral (IRaa, IRap) segments (see WYSSand Sripanidkulchai, '84).

Tracing Experiments Rdg, afferent connections. An injection of FB into mid rostrocaudal level of Rdg (e.g., CFR 30; Figs. 2G, 3A), involving all layers of the cortex, retrogradely labeled cortical neuronal cell bodies in orbital, IR, retrosplenial, areas 18b, 17, and postsubicular cortices. Contralaterally, labeled neurons were present in Rdg, Rgb, and Rga cortices. Subcortically, labeled neuronal cell bodies were present in claustrum, diagonal band of Broca, medial septal nucleus, anterior, lateral, and reuniens nuclei of the thalamus, the raphe nuclei and the locus coeruleus (Fig. 2). Near the injection site most retrogradely labeled neurons were in layers 111-V of Rga, and some labeled neurons were in layer I11 of Rgb and the deep layers (V and VI) of area 18b (Figs. 2G, 3C) and area 17 (Fig. 2G). Rostral to the injection site, labeled neurons were present in layers 11-V of Rdg, with a few labeled neurons in layer V of Rgb (Fig. 2E). More rostrally, a few labeled neuronal cell bodies were present in caudal IRP cortex, however, most labeled neurons in IRP were in the rostral third. In rostral IRbP and IRcP a relatively large number of neurons was labeled in layers 111-V (Fig. 2C). Further rostrally, neurons were labeled in the superficial layers (i.e., layers I1 and 111) of the lateral orbital cortex (Fig. 2A). Caudal to the injection site neuronal cell bodies were labeled in the deep layer (VI)of Rdg, areas 18b, 17, Rga, and postsubicular cortices (Fig. 2H). Superficial neurons were labeled only in Rdg (i.e., layers 11-111; Fig. 2H). Contralateral to the injection site, neurons were labeled in layers 11-V of Rdg and Rga; most of these labeled neurons were in layers 11-111 of Rdg (Figs. 2G, 3B). Rostral to the injection site, neurons were labeled only in Rdg (Fig. 2F and G), and in the IRP cortex, where a few labeled neurons were in layer V of IRcp and IRbP (Fig. 2C). Subcortically, neuronal cell bodies were labeled in the claustrum along its full extent (Figs. 2A-2D, 3D). In the diagonal band of Broca neurons were labeled in the horizontal limb, and, to a lesser extent, in the vertical limb (Fig. 2 0 . The medial septal nucleus also contained a few labeled neurons. In the thalamus a few neurons were labeled in the anterior nuclei, i.e., anteroventral (AV) and anterodorsal (AD; Figs. 2D, 4A-D) and the nucleus reuniens (Fig. 4B-E); while many neurons were labeled in the anteromedial nucleus (AM; Figs. 2D, 4A-El. More caudally, many neurons were labeled in laterodorsal nucleus (LD), and in the adjacent, rostral part of lateroposterior nucleus (LP; Figs. 2E, 4E-M). Injections of FB and FG in the rostral part of Rdg (e.g., CFR 163) resulted in a somewhat different pattern of labeling than more caudal injections in Rdg (e.g.,CFR 164). Injections of rostral Rdg labeled a greater number of neurons in dorsal IRP, Preag and AGl, whereas caudal injections resulted in a greater number of labeled neurons in ventral IRP, and in areas 18b and 17. In the thalamus, rostral Rdg injections labeled a greater number of neurons in ventral and caudal AM, and in the ventral and medial parts of LD, whereas caudal injections labeled a greater number of neurons in dorsal and anterior parts of AM, and in lateral and dorsal parts of LD (Fig. 4).

CONNECTIONSOFRETROSPLENIAL DYSGRANULARCORTEX

203

Fig. 1. Four photomicrographs of coronal sections of Rdg demonstrate the cytoarchitectonic divisions. A, and C: Nissl stained; B, and D: AChE stained. The arrows in A indicate the borders between cortical layers. Scale bar equals 250 pm.

To confirm these projections to Rdg and to characterize the laminar pattern of their terminations, injections of anterogradely transported tracers were made in several regions that project to Rdg (Table 1).The projection from the contralateral Rdg terminated in layers I-IV (Fig. 5A). Injections of IRP (e.g., PL 63)predominantly labeled axons and terminals in layer I and V-VI (Fig. 5B). Injections of area 17 (e.g., PL 138) and area 18b (e.g., PL 139)labeled

axons and terminals in layer I of Rdg, with a few labeled axons in the deep layers (i.e., layers V-VI). Injections of Rgb (e.g., PHA 38)labeled axons and terminals in layer I of Rdg (not illustrated). Injections of Rga (e.g., PHA 34) mainly labeled axons and terminals in layer 111,with a few axons in layer I. Following injections in the postsubiculum (e.g., PHA 28) labeled axons could be seen in layers I, and 111-V, with the greater part of the label in layer Ib and Ic (not

T. VAN GROEN AND J.M. WYSS

204 Preag

-

2500 pm

Fig. 2. Eight line drawings demonstrate the position of retrogradely labeled neurons ( amonds) that followed an injection of FG into Rdg ICFR 30; stippling). One symbol equals approximately five labeled neurons.

illustrated). Injections in the thalamus demonstrated that the LD nucleus (e.g., PL 84) had a dense terminal field in layers I and 111-IV of the cortex (Fig. 5C) and that the AM nucleus (e.g.,PL 124) provided a major projection to layers

I and IV-VI of Rdg (Fig. 5D). Following injections into AD (e.g., PL 97) and AV (e.g., PHA 491, a very small number of axons and terminals were labeled in layers I and 111-IV of Rdg (not illustrated).

CONNECTIONS OF RETROSPLENIAL DYSGRANULAR CORTEX

Fig. 3. Four photomicrographs demonstrate labeled afferent neurons to Rdg. A demonstrates the injection site in Rdg (CFR 30) and B demonstrates labeled neurons in contralateral Rdg (layers of the cortex indicated on the right). C shows labeled neurons in layer V of area 18b

205

and D shows neurons in the claustrum that were labeled by an injection into Rdg. The arrowheads indicate the lateral and medial borders of Rdg in A and the edge of cortical layers I and I1 in B. Scale bars equal 100 pm.

206

T.VAN GROEN AND J.M. WYSS

A

1000 pm Fig. 4. Line drawings demonstrate the position of labeled neurons in the thalamus that followed an injection of FG into rostra1 Rdg (left side, triangles; CFR 163) and caudal Rdg (right side, filled circles; CFR 164). One symbol equals approximately five labeled neurons.

Rdg, efferent connections. A large injection of PHA-L into the middle (rostro-caudal) part of Rdg (e.g.,PL 70; Fig. 6E) resulted in anterogradely transported label in orbital, IR, retrosplenial, areas 18b, 17, and postsubicular cortices (Fig. 6). Subcortically, labeled axons were present in caudate putamen, anterior, lateral, reticular and reuniens

nuclei of the thalamus, zona incerta, superior colliculus, periaqueductal grey, and ventral pontine nuclei. Most contralateral labeling was present in Rdg, with lighter labeling in IR, Rgb, Rga, and postsubicular cortices (Fig. 6). Near the injection site, a dense terminal field was labeled in the ventral third of Rgb, primarily in layers I and 111-V

CONNECTIONSOFRETROSPLENIALDYSGRANULAR CORTEX TABLE 1. The Laminar Organization of Afferent6 to the Rdg Cortex' Source of Projection Rdg layer I

IRP

Rdg

Rgb

+

+ +

+

I1 111

IV V VI

+ +

+ +

Rga

18b

Post

LD

+

++ +

+ + +

++

++

+

+

+

++ +

AM ++

+ + +

'+,Labeled axons; ++, higherl>3x)densityoflabeledaxons

(Figs. 6E, 6F, 7C). A group of labeled axons extended from the injection site into the cingulum bundle, where a large number of fibers turned rostrally and terminated in rostral Rdg and Rgb. A dense terminal plexus was present in Rdg, predominantly in layers I and 111, with a smaller number of labeled fibers and terminals in layer V, whereas in AGl a few axons were labeled in layer I and in Rgb only a few labeled axons and terminals were present in layers I1 and V (Fig. 6D). Many labeled fibers in the cingulum bundle continued further rostrally to terminate in the IR cortex and caudate/ putamen. In the caudal IR cortex (i.e., IRP) a dense plexus of labeled axons and terminals was present, with the densest labeling in IRcp (Figs. 6C, 7A), a small number of labeled axons extended into the precentral agranular cortex (Preag). A small number of labeled axons extended further rostrally to rostral IR cortex (i.e., IRa) and orbital cortex (Fig. 6B,A). A second group of labeled axons left the injection site and turned caudally in the cingulum bundle to terminate in posterior retrosplenial, areas 18b, 17, and postsubicular cortices. In caudal Rdg a terminal plexus of axons and terminals was labeled in layers I-V (Fig. 6F). In caudal Rgb only a few labeled axons and terminals were present, whereas a denser terminal plexus was present in the splenial part of Rga, in layers I and 111-V (Fig. 6F). Labeled axons extended laterally from caudal Rdg to terminate in caudal parts of area 18b, primarily in layer I with a few labeled axons and terminals in layers 111-V (Figs. 6G, 7B), and very sparse labeling in layer I of area 17. A number of labeled axons extended ventrally to postsubiculum, where a dense terminal plexus was labeled in layers I and V (Figs. 6G, 7D). A small number of labeled axons extended further ventrally to end in the superficial layers of caudal parts of presubiculum, parasubiculum, and the caudal medial entorhinal cortex (not illustrated). A number of labeled axons crossed midline at the level of the injection site to form a dense terminal plexus in contralateral Rdg homotopic to the injection site (Fig. 6E). Close to the injection site axons and terminals predominantly were labeled in layers I and 111-IV, with a smaller number of labeled axons in layer I1 (Fig. 6D), while caudal to the injection site, most labeled axons and terminals were in layers 111-V. Rgb, contralateral to the injection site, displayed only a few labeled axons and terminals, and further rostrally labeled axons and terminals were in IRP (Fig. 6C). Caudal to the injection site labeled axons and terminals were present in Rdg, with fewer labeled axons in Rgb, Rga, and postsubiculum (Fig. 6F,G). One group of subcortical projections coursed around the third ventricle and formed terminal plexuses in the thalamus, in reticular, AV, AM, ventrobasal (VB), LD, LP, centrolateral, and reuniens nuclei and in the zona incerta (Figs. 6D,E, 8-10). The labeled axons arborized extensively and terminated primarily in AM and LD (Figs. 7E, 8, 9).

207

Some of the labeled axons in AM extended across the midline and terminated in the homotopic region of contralatera1AM (Figs. 8A-C, 9A,B). No labeling was observed in the LD nucleus contralateral to the injection. Caudally, the labeled, axonal plexus extended from LD into LP (Figs. 6D,E, 8G-I, 9H-J). A number of these labeled axons extended caudally through LP to form a small, dense terminal plexus in the middle layers of the superior colliculus (Figs. 6F, 6G). A very small number of labeled axons and terminals were present in the periaqueductal grey (Fig. 6F,G), and a few labeled axons gave rise to a terminal plexus in the ventral pontine (Fig. 6G), the interpeduncular, and the medial raphe nuclei. The second group of subcortically directed axons left the cingulum bundle near the genu of the corpus callosum to terminate in the dorsomedial part of the caudate/putamen (Fig. 6B, 6C). Injections into the rostral part of Rdg (e.g., PHA 69) resulted in a slightly different labeling pattern than more caudal injections into Rdg (e.g., PL 159). Injections into rostral Rdg predominantly labeled axons in dorsal parts of IRP (i.e., IRcP) and Preag, and rostral parts of postsubiculum, whereas injections into caudal Rdg predominantly labeled axons and terminals in more ventral parts of IRP (i.e., IRbP) and caudal parts of postsubiculum. Further, caudal injections of Rdg labeled axons in area 18b (Fig. 7B) and 17, whereas rostral injections did not label axons in these areas. In the thalamus, rostral Rdg injections labeled axons in the caudal part of AM and the ventral medial part of LD and the adjacent part of rostral LP (Fig. 8). Injections into caudal Rdg predominantly gave rise to labeled axons in rostral parts of AM, dorsal medial parts of LD, and the adjacent part of rostral LP (Fig. 9). Retrograde labeling experiments demonstrated that the commissural projections of Rdg predominantly originated in layers I1 and 111, with a smaller number of labeled neurons in layer V (Table 2). The association projections arose from neurons in layers 11,111,and V, with the layer V neurons providing the longer projections to the anterior segment of retrosplenial cortex and to anterior cingulate (IR) cortex (Table 2). The projections to the postsubiculum originated in layer V of Rdg (Table 2). The long projections to the superior colliculus arose from neurons in both layer V and VI of Rdg, and the thalamic projections (Table 2) originated in layer VI of Rdg.

DISCUSSION The results of this study demonstrate that Rdg forms an integral part of the limbic system cortex (MacLean, '52), being interconnected with the infraradiata cortex (Baleydier and Mauguiere, '80; Bassett and Berger, '82; Vogt and Miller, '83; Finch et al., '84b; Sripanidkulchai and Wyss, '871, the limbic thalamic nuclei, and the hippocampal formation he., postsubiculum; Van Groen and Wyss, '90a). Rdg is innervated predominantly by orbital, infraradiata, Rga, visual (area 18b and 17), and contralateral Rdg cortices (Figs. 2 and 111, and by claustrum, AM, and LD. The major efferent projections of Rdg are to Rdg, area 18b, postsubiculum, A M ,and LD (Figs. 6 and 11). Furthermore, the differences in connections between Rdg, Rgb, and Rga (Fig. 11)corroborate the present cytoarchitectonic subdivision of the retrosplenial cortex (vide infra and Sripanidkulchai and Wyss, '86b, '87; Van Groen and Wyss, '90b). The cortical, efferent projections of Rdg are significantly different from those of Rgb and Rga (Domesick, '69; Vogt

208

T.VAN GROEN AND J.M. WYSS

Fig. 5. Four photomicrographs (A-D) show labeled axons and terminals in Rdg that followed a PHAL injection into: A, contralateral Rdg (PL 70); B, IRbp (PL 63); C, the LD nucleus of the thalamus (PL 84); and D, the AM nucleus of the thalamus (PL 124). The arrowheads indicate the borders between adjacent cortical layers. Scale bars equal 100 Fm.

CONNECTIONS OF RETROSPLENIAL DYSGRANULAR CORTEX

209

Fig. 6. Seven line drawings demonstrate the position of labeled mans that followed an injection of PHAL. into Rdg (PL 70; stippling).

and Miller, '83; Vogt, '85; Van Groen and Wyss, '90b). Whereas Rdg projects densely to IRcP, Rga, postsubiculum, and area 18b, Rgb innervates IRP and postsubiculum. Rga projects densely to Rdg, Rgb, and postsubiculum. Also, the commissural connections of each retrosplenial cortical region are predominantly reciprocal, i.e., Rdg to Rdg, Rgb to Rgb, and Rga to Rga (Fig. 11). The present data further demonstrate that the topographically organized thalamic projections from Rdg are different than those from Rgb and Rga (Domesick, '69; Seki and Zyo, '84; Thompson and Robertson, '87b; Wyss et al., '88; Van Groen and Wyss, '90b). While Rdgprojects mainly to AM, to

medial parts of LD and adjacent LP, Rgb projects primarily to rostral AD and intermediate parts of LD, and Rga projects primarily to dorsal AV and LD nuclei. Rdg has only a rather sparse projection to AV and no projection to AD. The cortical projections to each subarea of retrosplenial cortex are also different (Vogt and Miller, '83; Finch et al., '84b; Van Groen and Wyss, '90b; Witter et al., '90). Rdg is densely innervated by caudal IR, Rga, and cortical area 18b; conversely Rgb is innervated by s o n s from dorsal subiculum, postsubiculum, and IRP. Rga is innervated by axons from Rdg, ventral subiculum, presubiculum, and postsubiculum (Fig. 11).The thalamic projections t o each of these

210

Fig. 7. Five photomicrographs demonstrate the pattern of axonal labeling that followed an injection of PHA-L into Rdg (PL 70). A demonstrates labeled fibers and axons in IRp, B demonstrates labeled axons and terminals in area 18b that followed an injection into caudal Rdg (PL 1591, C shows labeled axons and terminals in Rgb, D

T. VAN GKOEN AND J.M. WYSS

demonstrates labeled axons and terminals in postsubicular cortex, and E demonstrates labeled axons and terminals in the LD nucleus of the thalamus. The arrowheads indicate the borders between adjacent cortical layers. Scale bars equal 100 Frn.

CONNECTIONS OF RETROSPLENIAL DYSGRANULAR CORTEX

A

B

C

D

Fig. 8. Line drawings demonstrate the position of labeled axons in the thalamus that followed a PHA-L injection into rostra1 Rdg (e.g., PL 62).

211

T. VAN GROEN AND J.M. WYSS

212

e

A

I

B

Re

E

Fig. 9. Line drawings demonstrate the position of labeled axons in the thalamus that followed a PHA-L injection into caudal Rdg (e.g., PL 159).

CONNECTIONSOFRETROSPLENIALDYSGRANULARCORTEX

213

Fig. 10. A photomicrograph (B) demonstrates the pattern of axonal and terminal labeling in the ventral basal complex of the thalamus following a PHA-L injection into the Rdg cortex (PL 70). A is a photomicrograph of a Nissl-stained section adjacent to B, to demon-

strate the location of the labeling (area outlined). The arrowheads show the position of the same blood vessel in the two photomicrographs. Scale bar in A equals 100 pm and in B equals 50 pm.

cortical areas also differ (vide infra Sripanidkulchai and Wyss, '86b; Van Groen and Wyss, 'SS, 'gob, '91). AD projects primarily to Rga and Rgb; AV predominantly projects to Rgb, and LD has a dense terminal field in Rdg, but a more sparse terminal field in Rgb and Rga (vide infra; Rose and Woolsey, '48; Sripanidkulchai and Wyss, '86b; Van Groen and Wyss, '88, 'gob, '91). Further, only Rdg is innervated by the LP nucleus. Nucleus reuniens, dorsal and medial raphe, and locus coeruleus project to all three retrosplenial regions (Sripanidkulchai and Wyss, '86b;

TABLE 2 . Laminar Distribution of Cells of Origin of Projections From the Rdg Cortex' Projection areas Rdg layer

Comrn.

Assoc.

I1 I11

++ ++

+

+

+

TI I,7

v "I I+,

Projection ong~natesin this layer

Post

S.C.

+

+

Thal.

+

+

+

+

T.VAN GROEN AND J.M. WYSS

2 14

and the visual cortex projects to LD (Takahashi, '85). Together these anatomical findings suggest that LD is related t o the visual system (Thompson and Robertson, '87b). LP also is innervated by the superior colliculus (Donnelly et al., '83; Thompson and Robertson, '87b), and both LP and LD project to Rdg (Olavarria, '79; Schober, '81; Thompson and Robertson, '87; vide infra). Further, the limbic cortex, including Rdg (present data), projects to the middle layers of the superior colliculus (Wyssand Sripanidkulchai, '84; Van Groen and Wyss, '90). These anatomical data suggest that Rdg is a principal area where inputs from the visual and limbic systems interact. Functional studies demonstrate that large retrosplenial lesions that destroy Rdg and Rgb impair place learning of rats in a water maze; however the animals are not impaired in learning (including visual tasks) per se. Thus "posterior cingulate areas are essential to the ability to move accurately to points in space using the relationships among distal cues" (Sutherland et al., '88). Other recent studies demonstrate that Rdg contains "head direction cells," i.e., neurons that discharge as a function of the rat's head direction in the horizontal plane (Chen et al., '901, while the hippocampus proper contains "place cells" (i.e., neurons that fire when a rat is in a specific place; O'Keefe and Nadel, '78). It should be noted that the postsubiculum, which receives a dense projection from Rdg (Figs. 6G, 7D), also contains "head direction cells" (Taube et al., '90a,b). Studies by Sikes et al. ('85, '88) indicate that some Rdg neurons are sensitive to eye movements. Thus, in addition to the anatomical evidence there is functional evidence that Rdg is involved in the processing of information related to the visual system. Behavioral studies suggest that the retrosplenial cortex contributes to the role of the hippocampus in memory and learning (Markowskaet al., '89; Murray et al., '89; Sif et al., '89; Matsunami et al., '89). Gabriel and colleagues (Gabriel et al., '80; Gabriel and Sparenborg, '86; '87; Gabriel et al., '89) 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. In rabbits, bilateral lesions of the retrosplenial cortex impair reversal discrimination in a nictating membrane response paradigm (Berger et al., '86). Fig. 11. Schematic summary of the main connections of Rdg, Rgb, Further, Markowska et al. ('89) have demonstrated that and Rga. retrosplenial cortex lesions induce spatial memory impairments in rats, and using an identical experimental design, Murray et al. ('89) have demonstrated a similar deficit in Wouterlood et al., '90). As would be expected, the thalamic monkeys. Recently, Valenstein et al. ('87) have extended projections from LD (and AD and AV) to Rdg terminate in this finding by demonstrating the development of amnesia layers I, 111, and IV Wogt, '85; Sripanidkulchai and Wyss, in a patient with a localized lesion in the retrosplenial '86b; Van Groen and Wyss, '88; Wyss et al., '90; Van Groen cortex. Thus the retrosplenial cortex appears to be imporand Wyss, '91 (Fig. 4C)l; however, the projections to Rdg tant for learning and memory in a broad spectrum of from AM avoid layer 111 and terminate only in layers I, mammalian species. IV-VI (Niimi, '78; Fig. 4D). Our anatomical findings together with these functional Although the projections of LD to Rdg have been known studies suggest that Rdg may contribute t o the integration for over a decade (e.g., Locke et al., '64; Niimi et al., '78; Baleydier and Mauguiere, '80; Robertson and Kaitz, '81; of visual and hippocampal information in relation to learnSripanidkulchai and Wyss, '86b; Thompson and Robertson, ing and memory. In light of these findings, the elucidation '87a; Van Groen and Wyss, '91), the function of LD remains of the anatomy and physiology of the interconnections poorly understood. In the rat, LD is densely innervated by between these three cortical regions takes on an added the pretectal nuclei (Robertson, '83; Robertson et al., '83) importance. and is less densely innervated by the superior colliculus and the ventral lateral geniculate nucleus (Thompson and ACKNOWLEDGMENTS Robertson, '87b). LD projects to area 18b of visual cortex This study was supported by NIH grant HL 34315. (Sripanidkulchai and Wyss, '86b; Van Groen and Wyss, '91)

m

CONNECTIONS OF RETROSPLENIAL DYSGRANULAR CORTEX

LITERATURE CITED Baleydier, C. and F. Mauguiere (1980) The duality of the cingulate gyrus in monkey. Neuroanatomical study and functional hypothesis. Brain 103: 525-554. Bassett, J.L., and T.W. Berger (1982) Associational connections between the anterior and posterior cingulate gyrus in rabbit. Brain Res. 248:371-376. Berger, T.W., C.L. Weikart, J.L. Bassett, and W.B. Orr (1986) Lesions of the retrosplenial cortex produce deficits in reversal learning of the rabbit nictating membrane response: Implications for potential interactions between hippocampal and cerebellar brain systems. Behav. Neurosci. I00:802-809. Brodmann, K. (1909) Vergleichende Lokalisationslehre der Grosshirnrinde in ihren Prinzipien dargestellt auf Grund des Zellenbaues. Leipzig: Barth. Chen, L.L., B.L. McNaughton, C.A. Barnes, and E.R. Ortiz, (1990) Headdirectional and behavioral correlates of posterior cingulate and medial prestriate cortex neurons in freely-moving rats. Soc. Neurosci. Abs. 16:441. Domesick, V.B. (1969) Projections from the cingulate cortex in the rat. Brain Res. 12.296-320. Donnelly, J.F., S.M. Thompson, and R.T. Robertson (1983) Organization of projections from the superior colliculus to the thalamic lateral posterior nucleus of the rat. Brain Res. 288:315-319. Donoghue J.P., and S.P. Wise (1982) The motor cortex ofthe rat: Cytoarchitecture and microstimulation mapping. J. Comp. Neurol. 21276-88. Finch, D.M., E.L. Derian, and T.L. Babb (1984a) Excitatory projection of the rat subicular complex to the cingulate cortex and synaptic integration with thalamic afferents. Brain Res. 301:25-37. Finch, D.M., E.L. Derian, and T.L. Babb (198413) Afferent fibers to rat cingulate cortex. Exp. Neurol. 83:468-485. Gabriel, M., K. Foster, and E. Orona (1980) Interaction of the laminae of cingulate cortex and the anteroventral thalamus during behavioral learning. Science 208: 1050-1052. Gabriel, M., and S. Sparenborg (1986) Anterior thalamic discriminative neuronal responses enhanced during learning in rabbits with subicular and cingulate cortical lesions. Brain Res. 384:195-198. Gabriel, M., and S. Sparenborg (1987) Posterior cingulate cortical lesions eliminate learning-related unit activity in the anterior cingulate cortex. Brain Res. 409:151-157. Gabriel, M., S. Sparenborg, and Y. Kubota (1989) Anterior and medial thalamic lesions, discriminative avoidance learning, and cingulate cortical neuronal activity in rabbits. Exp. Brain Res. 76:441-457. Geneser-Jensen, F., and T.W. Blackstad (1971) Distribution of acetyl cholinesterase in the hippocampal region of the guinea pig. I Entorhinal area, parasubiculum and presubiculum. Z. Zellforsch. 114:460-481. Gerfen, C.R., and P.E. Sawchenko (1984) An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res. 290:219-238. Krieg, W.J.S. (1946) Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas. J. Comp. Neurol. 84:221-275. Locke, S.,J.B. Angevine, and P.I. Yakovlev (1964) Limbic nuclei ofthalamus and connections of limbic cortex. VI. Thalamocortical projection of lateral dorsal nucleus in cat and monkey. Arch. Neurol. 11:1-12. MacLean, P.D. (1952) Some psychiatric implications of physiological studies on frontotemporal portion of limbic system. Electroenceph. Clin. Neurophysiol. 4:407-418. Markowska, A.L., D.S. Olton, E.A. Murray, and D. Gaffan (1989) A comparative analysis of the role of fornix and cingulate cortex in memory: Rats. Exp. Brain Res. 74:187-201. Matsunami, K., T. Kawashima, and H. Satake (1989) Mode of [l4C12-deoxyD-glucose uptake into retrosplenial cortex and other memory-related structures of the monkey during a delayed response. Brain Res. Bull. 22829-838. McNaughton, B.L., and R.G.M. Morris (1987)Hippocampal synaptic enhancement and information storage within a distributed memory system. TINS 10t408-415. Meibach, R.C., and A. Siegel (1977a) Subicular projections to the posterior cingulate cortex in r8ts. Exp. Neurol. 572644274, Meibach, R.C., and A. Siegel (1977b) Efferent connections of the hippocampal formation in the rat. Brain Res. 124:197-224. Murray, E.A., M. Davidson, D. Gaffan, D.S. Olton, and S.Suomi (1989)

215

Effects of fornix transaction and cingulate cortical ablation on spatial memory in rhesus monkeys. Exp. Brain Res. 74: 173-186. Niimi, K., M. Niimi, and Y . Okada (1978) Thalamic afferents to the limbic cortex in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. Brain Res. 145.225-238. O’Keefe, J., and L. Nadel (1978) The hippocampus as a cognitive map. Oxford: Clarendon Press. Olavarria, J. (1979) A horseradish peroxidase study of the projections from the latero-posterior nucleus to three lateral peristriate areas in the rat. Brain a s . 173:137-141. Olton, D.S. (1983) Memory functions and the hippocampus. In W. Seifert (ed): Neurobiology of the Hippocampus. New York: Academic Press. Robertson, R.T. (1983) Efferents of the pretectal complex: separate populations of neurons project to lateral thalamus and to inferior olive. Brain Res. 258:91-95. Robertson, R.T., and S.S. Kaitz (1981) Thalamic connections with limbic cortex. I. Thalamocortical projections. J. Comp. Neurol. 195t501-525. Robertson, R.T., S.M. Thompson, and S.S.Kaitz (1983)Projections from the pretectal complex to the thalamic lateral dorsal nucleus of the cat. Exp. Brain Res. 51:157-171. Rose, M. (1927a) Der Allocortex bei Tier und Mensch. J. Psychol. Neurol. 34: 1-111. Rose, M. (1927133 Gyrus limbicus anterior und Regio retrosplenialis (Cortex holoprotoptychos quinquestratificatus) Vergleichende Architektonik bei Tier und Mensch. J. Psychol. Neurol. 35.65-173. Rose, J.E., and C.N. Woolsey (1948) Structure and relations of limbic cortex and anterior thalamic nuclei in rabbit and cat. J. Comp. Neurol. 89:219-347. Schmued, L., Kyriakidis, K., and Heimer, L. (1990) In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextranamine, Fluororuby, within the CNS. Brain Res. 526:127-134. Schober, W. (1981) Efferente und afferente Verbindungen des Nucleus lateralis posterior thalami (“Pulvinar”) der Albinoratte. Z. mikrosk. Anat. Forsch. 95827-844. Schober, W., and E. Winkelmann (1975) Der visuelle Kortex der Ratte: Cytoarchitektonik und stereotaktische Parameter. Z. Mikrosk. Anat. Forsch. 89,431-446. Segal, M., and S. Landis (1974) Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase. Brain Research 78:l-15. Seki, M., and K. Zyo (1984) Anterior thalamic afferents from the mammillary body and the limbic cortex in the rat. J. Comp. Neurol. 229:242-256. Sif, J., M. Meunier, C. Messier, A. Calas, and C. Destrade (19891 Quantitative [14C12-deoxyglucosestudy of a functional dissociation between anterior and posterior cingulate cortices in mice. Neurosci. Lett. 101:223228. Sikes, R.W., B.A. Vogt, and H.A. Swadlow (19851 Limbic cortex neuronal activity associated with saccadic eye movements in awake rabbits and possible underlying afferents. Soc. Neurosci. Abs. 1I :1042. Sikes, R.W., B.A. Vogt, and H.A. Swadlow (1988) Neuronal responses in rabbit cingulate cortex linked to quick-phase eye movements during nystagmus. J. Neurophysiol. 59:922-936. Sgrensen, K.E. (1980) Ipsilateral projection from the subiculum to the retrosplenial cortex in the guinea pig. J. Comp. Neurol. 19.3r893-911. Sripanidkulchai, K., and J.M. Wyss (1986a) Two rapid methods of counterstaining fluorescent dye tracer containing sections without reducing the fluorescence. Brain Res. 397:117-129. Sripanidkulchai, K., and J.M. Wyss (1986b) Thalamic projections to retrosplenial cortex in the rat. J. Comp. Neurol. 254t143-165. Sripanidkulchai, K., and J.M. Wyss (1987) The laminar organization of efferent neuronal cell bodies in the retrosplenial granular cortex. Brain Res. 406:255-269. Sutherland, R.J., I.&. Whishaw, and B. Kolb (1988) Contributions of cingulate cortex to two forms of spatial learning and memory. J. Neurosci. 8:1863-1872. Swanson, L.W., and W.M. Cowan (1977) An autoradiographic study of the organization of the efferent connections of the hippocampal formation in the rat. J. Comp. Neurol. 172t49-84. Takahashi, T. (1985) The organization of the lateral thalamus of the hooded rat. J. Comp. Neurol. 231:281-309. Taube, J.S., R.U. Muller, and J.B. Ranck, J r . (1990a) Head-direction cells recorded from the postsubiculum in freely moving rats. I. Description and quantitative analysis. J. Neurosci. IOt420-435. Taube, J.S., R.U. Muller, and J.B. Ranck, Jr. (1990b) Head-direction cells

216 recorded from the postsubiculum in freely moving rats. 11. Effects of environmental manipulations. J. Neurosci. 10:436447. Thompson, S.M., and R.T. Robertson (1987a) Organization of subcortical pathways for sensory projections to the limbic cortex I. Subcortical projections to the medial limbic cortex in the rat. J. Comp. Neurol. 265: 175-188. Thompson, S.M., and R.T. Robertson (1987b) Organization of subcortical pathways for sensory projections to the limbic cortex. 11. Afferent projections to the thalamic lateral dorsal nucleus in the rat. J. Comp. Neurol. 265:189-202. Valenstein, E., D. Bowers, M. Verfaellie, K.M. Heilman, A. Day, and R.T. Watson (1987) Retrosplenial amnesia. Brain 110:1631-1646. Van Groen, Th., and J.M. Wyss (1988) The distribution of projections from Neurosci. Abs. the anterior thalaniic nuclei to the subicular cortex. SOC. 14:921. Van Groen, Th., and J.M. Wyss (1990ai The postsubicular cortex in the rat: Characterization of the fourth region of the subicular cortex and its connections. Brain Res. 529:165-177. Van Groen, Th., and J.M. Wyss (1990bi Connections of the retrosplenial granular a cortex in the rat. J. Comp. Neurol. 300:593-606. Van Groen, Th., and J.M. Wyss (1991) Projections from the laterodorsal nucleus of the thalamus to the limbic and visual cortices. SOC. Neurosci. Abs. I7:1583. Van Hoesen, 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 Research 95: 1-24. Vastola, E.F. (1982) Electrical signs of an oligo-synaptic visual projection to rat hippocampus. Brain Behav. Evol. 20:1-18.

T. VAN GROEN AND J.M. WYSS Vogt, B.A. (1974) A reduced silver stain for normal axons in the central nervous system. Physiol. Behav. 139337-840. Vogt, B.A. (1985) Cingulate Cortex. In A. Peters and E.G. Jones (eds): Cerebral Cortex, Vol. 4. NewYork: Plenum Press, pp. 89-149. Vogt, B.A., and A. Peters (1981) Form and distribution of neurons in rat cingulate cortex: areas 32,24, and 29. J. Comp. Neurol. 195r603-625. Vogt, B.A., and M.W. Miller (1983) Cortical connections between rat cingulate cortex and visual, motor, and postsubicular cortices. J. Comp. Neurol. 216:192-210. Vogt, B.A., R.W. Sikes, H.A. Swadlow, and T.G. Weyand (1986) Rabbit cingulate cortex: cytoarchitecture, physiological border with visual cortex, and afferent cortical connections of visual, motor, postsubicular, and intracingulate origin. J. Comp. Neurol. 248t74-94. Witter, M.P., R.H. Ostendorf, and H.J. Groenewegen (1990) Heterogeneity in the dorsal subiculum of the rat: Distinct neuronal zones project to different cortical and subcortical targets. Eur. J. Neurosci. 2718-725. Wouterlood, F.G., E . Saldana, and M.P. Witter (1990) Projection from the nucleus reuniens thalami to the hippocampal region: light and electron microscopic tracing study in the rat with the anterograde tracer Phuseolus vulgaris-leucoagglutinin.J. Comp. Neurol. 296: 179-203. Wyss, J.M., and K. Sripanidkulchai (1984)The topography of the mesencephalic and pontine projections from the cingulate cortex of the rat. Brain Res. 293:l-15. Wyss, J.M., Th. Van Groen, and C. Rodenburg (1988) Topography of anterior thalamic afferents from the posterior limbic cortex and the contralateral anteroventral nucleus. SOC.Neurosci. Abs. 14:921. Wyss, J.M., Th. Van Groen, and K. Sripanidkulchai (1990) Dendritic bundling in layer I of granular retrosplenial cortex: Intracellular labeling and selectivity of innervation. J. Comp. Neurol. 295:33-42.