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Organization of retrosplenial cortical projections to the anterior cingulate, motor, and prefrontal cortices in the rat
Neuroscience Research 49 (2004) 1–11
Organization of retrosplenial cortical projections to the anterior cingulate, motor, and prefrontal cortices in the rat Hideshi Shibata a,∗ , Shiori Kondo a , Jumpei Naito b a b
Department of Veterinary Anatomy, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Department of Animal Science, School of Science and Engineering, Teikyo University of Science and Technology, Yamanashi 409-0913, Japan Received 19 September 2003; accepted 9 January 2004
Abstract The retrosplenial cortex (areas 29a–29d) has been implicated in spatial memory, which is essential for performing spatial behavior. Despite this link with behavior, neural connections between areas 29a–29d and frontal association and motor cortices—areas also essential for spatial behavior—have been analyzed only to a limited extent. Here, we report an analysis of the anatomical organization of projections from areas 29a–29d to area 24 and motor and prefrontal cortices in the rat, using the axonal transport of biotinylated dextran amine (BDA) and cholera toxin B subunit (CTb). Area 29a projects to rostral area 24a, whereas area 29b projects to caudodorsal area 24a and ventral area 24b. Caudal area 29c projects to mid-rostrocaudal area 24b, whereas rostral area 29c projects to caudal areas 24a and 24b and caudal parts of primary and secondary motor areas. Caudal area 29d projects to mid-rostrocaudal areas 24a and 24b, whereas rostral area 29d projects to the caudalmost parts of areas 24a and 24b and the secondary motor area and to the mid-rostrocaudal part of the primary motor area. Area 29d also projects weakly to the prefrontal cortex. These differential corticocortical projections may constitute important pathways that transmit spatial information to particular frontal cortical regions, enabling an animal to accomplish spatial behavior. © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. Keywords: Area 29; Retrosplenial cortex; Area 24; Secondary motor area; Primary motor area; Corticocortical projections
1. Introduction The retrosplenial cortex, which occupies the caudal half of the cingulate cortex in the rat, consists of four cytoarchitectonically distinct areas: 29a, 29b, 29c and 29d (Vogt and Peters, 1981; Vogt, 1993). The relatively small areas 29a and 29b are located at levels caudal to the splenium of the corpus callosum, whereas the relatively larger areas 29c and 29d extend almost throughout the rostrocaudal axis of the retrosplenial cortex (see Fig. 1 for reference). Recent studies show that either cytotoxic lesions involving the entire retrosplenial cortex, or even those restricted to its caudal parts, cause spatial memory impairment (Vann and Aggleton, 2002; Vann et al., 2003). Electrophysiological evidence also supports the idea that this region is involved some way in spatial memory or associated functions. Cells in the retrosplenial cortex have been implicated in encoding ∗ Corresponding author. Tel.: +81-42-367-5766; fax: +81-42-367-5766. E-mail address: [email protected] (H. Shibata).
the head direction (Chen et al., 1994a,b) or the combinations of direction, location, and movement of an animal (Cho and Sharp, 2001); retrosplenial cells have also been implicated in path integration associated with spatial navigation (Cooper and Mizumori, 1999; Cooper et al., 2001). Thus, the retrosplenial cortex may play critical roles in spatial memory and navigation. It is assumed that the retrosplenial cortex transmits such spatial information via corticocortical pathways to the frontal association and/or motor cortical regions, which are intimately involved in spatial and motor behaviors (e.g. Hall and Lindholm, 1974; Becker et al., 1980). The corticocortical projections from the retrosplenial cortex to the frontal cortical regions thus far reported in the literature indicate that the region including areas 29a and 29b projects to areas 24a and 32 (Van Groen and Wyss, 1990), that area 29c projects to areas 24a and 24b (Vogt and Miller, 1983; Van Groen and Wyss, 2003), and that area 29d projects to areas 24a and 24b, the orbital cortex, and the medial part of the secondary motor area (Vogt and Miller, 1983; Van Groen and Wyss, 1992). However, the projections from the caudal part of the retrosplenial cortex, a region that is
0168-0102/$ – see front matter © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.01.005
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Fig. 1. Schematic drawings of the distribution of anterogradely labeled terminals in frontal cortical regions following injections of BDA into area 29a (A–E), area 29b (F–J), caudal area 29c (K–O), and rostral area 29c (P–T). The injection site of each case is depicted in A, F, K, and P, and the resulting labeled fibers and terminals are depicted in the frontal sections (B–D, G–I, L–N, and Q–S), arranged in the caudorostral order. Arrowheads indicate the boundaries between different cortical areas. Medial views of locations of injection sites and the relative density of labeled terminals were reconstructed in sagittal planes to produce panels E, J, O, and T; arrows indicate rostrocaudal levels of frontal sections. Abbreviations used in this and the following figures: ac, anterior commissure; CA, cornu ammonis; cc, corpus callosum; c29c, caudal part of area 29c; c29d, caudal part of area 29d; DG, dentate gyrus; II–IV, layers II–IV of cortex; LO, lateral orbital area; LV, lateral ventricle; MO, medial orbital area; M1, primary motor area; M2, secondary motor area; PL, prelimbic area; Pre, presubiculum; r29c, rostral part of area 29c; r29d, rostral part of area 29d; Sep, septum; Sub, subiculum; V, layer V of the cortex; VI, layer VI of the cortex; and VO, ventral orbital area.
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particularly important for spatial memory, are still unclear. For example, it is as yet undetermined whether projections from area 29a differ from those originating from area 29b. Furthermore, to date, projections to motor areas have not been studied in detail (Vogt and Miller, 1983; Van Groen and Wyss, 1992). Thus, the present study was carried out to clarify in more detail the organization of projections from each area of the retrosplenial cortex to the anterior cingulate, motor, and prefrontal cortices in the rat. First, using the anterograde neuronal tracer biotinylated dextran amine (BDA), we determined the terminal distribution of projections originating from each area of the retrosplenial cortex. Second, using the retrograde neuronal tracer cholera toxin B subunit (CTb), we determined the distribution of cells of origin of these projections. The results show that each area of the retrosplenial cortex projects to distinct parts of the anterior cingulate, motor, and prefrontal cortices.
2. Materials and methods A total of 45 Wistar rats of both sexes, weighing 210–460 g, were used in the present study. All surgical procedures were done under deep anesthesia consisting of intraperitoneal injections of sodium pentobarbital (60 mg/kg body weight) or a combination of xylazine (10 mg/kg body weight) and ketamine (50 mg/kg body weight). Before and after surgery, the rats were allowed free access to food and water. All the experimental procedures complied with the guidelines of the National Institute of Health (USA). BDA injections were done first and their terminal fields were analyzed to determine the placement of our subsequent retrograde injections in different animals. For BDA injections, a glass micropipette (10–30 m i.d.) was filled with 10% BDA (Molecular Probes, Eugene, OR, USA) solution; lyophilized BDA was dissolved in saline (Brandt and Apkarian, 1992). Each area of the retrosplenial cortex was sampled with discrete BDA injections in different animals, using coordinates derived from the stereotaxic atlas of Paxinos and Watson (1997). BDA was deposited iontophoretically by passing a 3–5 A positive current pulse for a total on-time of 5–15 min (Brandt and Apkarian, 1992). After 10 or 14 days, the rats were anesthetized with intraperitoneal injections of sodium pentobarbital (60 mg/kg body weight) and perfused transcardially with saline, followed by 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After saturation in 30% sucrose in 0.1 M phosphate buffer at 4◦ C, the brains were frozen, and frontal sections were cut at 40 or 50 m thickness on a freezing microtome. To visualize BDA-labeled terminals, every other section was placed overnight in Tris–HCl buffer (TB) containing 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), rinsed briefly in TB, and incubated for 1 h at room temperature in TB containing either streptavidin-conjugated horseradish
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peroxidase (1:500; Dakopatts, Glostrup, Denmark) or Elite avidin-biotinylated horseradish peroxidase complex (ABC) (1:100 or 1:200; Vector Laboratories, Burlingame, CA, USA). After three 5 min rinses in TB, the sections were incubated in TB containing 0.04% 3,3 -diaminobenzidine (DAB) and 0.01% H2 O2 for 15–20 min. Some sections were incubated in Ni–DAB solution (TB containing 0.02% DAB, 0.6% nickel ammonium sulfate and 0.003% H2 O2 ) for 5–10 min. Finally, all the sections were thoroughly rinsed in TB and mounted onto gelatin-coated slides. For CTb injections, 0.5 mg of lyophilized CTb (List Biological Laboratories, Campbell, CA, USA) was reconstituted with 0.5 ml of 0.1 M phosphate buffer (pH 6.0) and concentrated to 1% by ultrafiltration, according to the protocol of Luppi et al. (1990). In some cases, 1% “low salt” CTb (List Biological Laboratories) dissolved in distilled water was used without prior ultrafiltration. Glass micropipettes (10–30 m i.d.) were filled with CTb, which was iontophoretically injected into parts of the anterior cingulate or motor cortices that were previously shown in our BDA experiments to receive dense terminal projections from the retrosplenial cortex. CTb was deposited by passing a 2–4 A positive current pulse for 5–10 min in total. After 3, 5 or 7 days, the rats were deeply anesthetized with intraperitoneal injections of sodium pentobarbital (60 mg/kg body weight) and perfused according to the same protocol used for the BDA-injected cases. The brains were removed, saturated with 30% sucrose in 0.1 M phosphate buffer, and cut into frontal sections at 40 or 50 m thickness on a freezing microtome. To visualize CTb-labeled cells immunohistochemically, every other section was first incubated overnight in Tris–HCl-buffered saline (TBS) containing 2% normal rabbit serum and 0.5% Triton X-100, and then in a primary antibody solution containing goat anti-CTb (1:40,000; List Biological Laboratories), 1% normal rabbit serum, 0.5% Triton X-100 and TBS for 48 h at 4 ◦ C. After three 5 min rinses in TBS, the sections were treated with a Vectastain ABC kit (Vector Laboratories), and CTb-labeled elements were visualized by incubating the sections in the Ni–DAB solution as described above. The sections processed for either BDA histochemistry or CTb immunohistochemistry were dehydrated in ethanol, cleared in xylene, and coverslipped. Some sections were counterstained with thionin. The sections were examined with bright- and darkfield illuminations. Locations of injection sites and the distribution of labeled cells, fibers, and terminals were first plotted onto representative drawings of frontal sections with the aid of a camera lucida apparatus, then reconstructed onto drawings of sagittal sections so that their distributions could be appreciated from a medial view of the cerebral hemispheres. For demarcating retrosplenial and anterior cingulate cortical areas, we adopted the nomenclature described by Vogt and Peters (1981), and for other cortical areas, we followed the nomenclature outlined by Paxinos and Watson (1997).
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3. Results In the anterograde tracing experiments, BDA was injected into each area of the retrosplenial cortex at various rostrocaudal levels in order to delineate terminal fields of retrosplenial cortical projection fibers to frontal cortical regions, such as the anterior cingulate, motor, and prefrontal cortices. The injections involved all cortical layers and were successfully made into area 29a (2 cases), area 29b (1 case), areas 29b and 29c (2 cases), area 29c (11 cases), and area 29d (7 cases). The results from six representative cases will be presented to illustrate the projection patterns. Data from the remaining cases were confirmatory. Although labeled terminals in all cases were organized homotopically in the frontal cortical regions in both hemispheres, contralateral labeling was sparser than ipsilateral labeling. Thus, we describe only labeling ipsilateral to the injection sites. The distribution of labeled terminals within the retrohippocampal region and the anterior and laterodorsal thalamic nuclei in some cases has been reported previously (Shibata, 1994, 1998, 2000). BDA injections into areas 29a or 29b anterogradely labeled terminals mainly in the rostral part of the anterior cingulate cortex (Fig. 1A–J). An injection restricted to area 29a labeled terminals in the rostral part of area 24a (Figs. 1A and C–E, and 2A and B). Terminal labeling in layers I–III was denser than that in layers V and VI (Figs. 1C and D, and Fig. 2B). A few labeled terminals were also observed within the ventral part of rostral area 24b (Fig. 1D). An injection restricted to area 29b labeled terminals somewhat more caudodorsally within the anterior cingulate cortex (FFig. 1F–J). This area 29b injection also labeled many terminals within all layers of area 24a, with the densest labeling in layers I and III (Fig. 1G–I). Labeled terminals were also observed in the adjoining part of area 24b (Fig. 1G–I). Few labeled terminals were found in the rostralmost and caudal parts of area 24a (Fig. 1J). Injections into area 29c labeled terminals in parts of area 24 and the primary and secondary motor areas (Fig. 1K–T). An injection into the caudal part of area 29c labeled terminals in areas 24a and 24b and the secondary motor area, only at the level of the anterior commissure (Fig. 1K–O). As one examines labeling within this level in the caudorostral direction, labeling gradually shifts ventrodorsally such that at caudal levels, labeling is found in the dorsal part of area 24a, then at mid-rostrocaudal levels in area 24b, and finally at rostral levels in the secondary motor area (Fig. 1L–O). Terminal labeling was particularly dense in layers I and III of these cortical areas. An injection into the rostral part of area 29c labeled massive terminal fields in caudal parts of areas 24a and 24b and in caudal parts of the primary and secondary motor areas, particularly in layers I and III (Figs. 1P, Q and T, and 2C and D). In areas 24a and 24b, labeling was dense within dorsal parts (Fig. 1Q). At more rostral levels (Fig. 1R–T), some labeled terminals were noted mainly in layers I and III of the secondary motor area; only a few, scattered terminals were observed in the primary motor area.
Injections into area 29d labeled terminals in the anterior cingulate, motor, and prefrontal cortices (Fig. 3A–L). An injection into the caudalmost part of area 29d labeled relatively few terminals in areas 24a and 24b at caudal levels and in the ventral orbital area near the frontal pole (Fig. 3A–E). Labeling in these areas was distributed mainly in layer I. An injection into the rostral part of area 29d labeled massive terminal fields in areas 24a and 24b and in the primary and secondary motor areas (Fig. 3F–J and L). In the caudal parts of areas 24a and 24b, labeled terminals were distributed throughout all layers, with the densest labeling in layers I–III of the caudalmost part of area 24b (Fig. 3G and H). At caudal levels of the secondary motor area, diffuse terminal labeling was observed in all layers (Fig. 3G, H and L), whereas at more rostral levels, labeled terminals were concentrated in layers I and III (Fig. 3I). At mid-rostrocaudal levels of the primary motor area, terminal labeling was densest in layers I and III (Fig. 3J and L). At rostral levels of areas 24a and 24b and the motor areas, few terminals were labeled (Fig. 3L). Minute, scattered labeling was also observed in the prefrontal cortex, mainly in layers I–III of the ventral, medial, and lateral orbital areas and prelimbic area (Fig. 3K and L). In the retrograde tracing experiments, CTb was injected into the parts of the anterior cingulate (13 cases) and motor cortices (9 cases) where dense labeling of terminals was observed in the anterograde tracing experiments; CTb injections involved all cortical layers. The results of the CTb experiments confirmed those of the BDA experiments, and delineated the laminar distribution of retrosplenial cells that projected to the anterior cingulate and motor cortices. The results of seven representative cases will be described below. As in the anterograde tracing experiments, we shall only describe the distribution of retrogradely labeled cells ipsilateral to each injection site. CTb was injected into various rostrocaudal levels of area 24a (Fig. 4A–O). An injection into area 24a at the level of the caudal septum retrogradely labeled cells mainly in areas 29b and 29c, at levels just caudal to the splenium of the corpus callosum (Figs. 2E and F, and 4A–C and E). In these areas, most of the labeled cells were located in layer V, and a few labeled cells were observed in layers II–IV and VI (Fig. 4C). A few labeled cells were also observed in layers V and VI of the caudalmost part of area 29a (Fig. 4B and E), in layers V and VI of rostral area 29c (Fig. 4D and E), and in layer V of area 29d throughout its rostrocaudal extent (Fig. 4B–E). A CTb injection into area 24a at the level of the rostral septum labeled cells more caudally within the retrosplenial cortex (Fig. 4F–J). Many labeled cells were observed in layer V of areas 29a and 29b, with fewer cells in layers II–IV (Fig. 4H and I). The labeled cells in area 29b were located mainly in the part adjoining area 29a (Fig. 4H and I). Some labeled cells were also found in layers II–IV and V of area 29d at levels caudal to the splenium of the corpus callosum (Fig. 4G–J). An injection confined to the most rostral part of area 24a labeled cells mainly in layers
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Fig. 2. Photomicrographs of tracer injection sites (A, C and E) and distribution of resulting labeled terminals or cells (B, D and F). (A) Thionin-counterstained section showing a BDA injection site localized within area 29a. (B) Darkfield photomicrograph of anterogradely labeled terminals in area 24a of the anterior cingulate cortex, resulting from area 29a injection in panel A. (C) Thionin-counterstained section showing a BDA injection site localized within area 29c. (D) Darkfield photomicrograph of anterogradely labeled terminals in primary (M1) and secondary motor areas (M2), resulting from area 29c injection shown in panel C. (E) CTb injection site localized within area 24a. (F) Brightfield photomicrograph of retrogradely labeled cells in areas 29b and 29c, just caudal to the splenium of the corpus callosum, resulting from area 24a injection shown in panel E. Arrowheads indicate the boundaries between different cortical areas.
II–IV and V of area 29a and the adjoining part of area 29b (Fig. 4K–O). A CTb injection into the caudalmost part of area 24b labeled many cells in layers II–IV and V and in the superficial portion of layer VI of areas 29b, 29c, and 29d at a level caudal to the splenium of the corpus callosum (Fig. 4P–R
and T). In area 29b, labeled cells were located in the part near area 29c, whereas in area 29d more labeled cells were located in the part near the visual association cortex (Fig. 4Q and R). At more rostral levels of the retrosplenial cortex, only a few labeled cells were found in areas 29c and 29d, mainly in layers II–IV and V (Fig. 4S and T).
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Fig. 3. Schematic drawings of the distribution of anterogradely labeled terminals in frontal cortical regions following injections of BDA into caudal (A–E) or rostral parts of area 29d (F–L). The injection site of each case is depicted in A and F, and the resulting labeled fibers and terminals are depicted in the frontal sections (B–D and G–K) arranged in the caudorostral order. Medial views of locations of injection sites and the relative density of labeled terminals were reconstructed in sagittal planes to produce panels E and L. The same diagram conventions as those described for Fig. 1.
An injection into the caudal part of the secondary motor area at the level of the caudal septum labeled many cells in all the cellular layers of areas 29b, 29c, and 29d in the mid-rostrocaudal third of the retrosplenial cortex (Fig. 5A, C, D and F). In areas 29b and 29c, labeled cells were most
numerous in layer V. Within rostral and caudal thirds of the retrosplenial cortex, only a few labeled cells were scattered in areas 29c and 29d (Fig. 5B, E and F). An injection into the mid-rostrocaudal part of the secondary motor area at the level of the rostral septum labeled only a few, scat-
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Fig. 4. Schematic drawings of the distribution of retrogradely labeled cells in the retrosplenial cortex following injections of CTb into caudal (A–E), mid-rostrocaudal (F–J), and rostral parts of area 24a (K–O), and into area 24b (P–T). The injection site of each case is depicted in A, F, K, and P, and the resulting labeled cells are depicted in frontal sections (B–D, G–I, L–N or Q–S) arranged in the caudorostral order. Large dots represent 5–10 labeled cells, whereas small dots represent 1–4 labeled cells. Medial views of locations of injection sites and the relative density of labeled cells were reconstructed in sagittal planes to produce panels E, J, O, and T. The same diagram conventions as those described for Fig. 1.
tered cells in rostral areas 29c and 29d, mainly in layer V (Fig. 5G–J). A CTb injection into the primary motor area at the level of the mid-rostrocaudal septum labeled a group of cells distributed throughout dorsal area 29c and ventromedial area 29d at rostralmost levels (Fig. 5K, M and N). Most of the labeled cells were located in layer V and a few were observed in layers II–IV and the superficial part of layer VI
(Fig. 5M). A few labeled cells were also observed in the deep part of layer V of caudal area 29d (Fig. 5L and N).
4. Discussion The present study demonstrated the anatomical organization of projections originating from different
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Fig. 5. Schematic drawings of the distribution of retrogradely labeled cells in the retrosplenial cortex following injections of CTb into caudal (A–F) or mid-rostrocaudal parts of M2 (G–J), and in M1 (K–N). The injection site of each case is depicted in A, G, and K, and the resulting labeled cells are depicted in frontal sections (B–E, H and I, and L and M) arranged in the caudorostral order. Large dots represent 5–10 labeled cells, whereas small dots represent 1–4 labeled cells. Medial views of locations of injection sites and the relative density of labeled cells were reconstructed in sagittal planes to produce panels F, J, and N. The same diagram conventions as those described for Fig. 1. Although the injection appears to have spared layer I in the drawings of each case shown, the injected tracer did involve layer I in nearby sections (not illustrated).
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4.2. Projections from areas 29c and 29d
Fig. 6. Schematic drawings of the medial aspect of the rat cerebral cortex, summarizing the organization of projections from retrosplenial area 29 to the anterior cingulate, motor, and prefrontal cortices. The contralateral projections, which are homotopical but relatively sparse, are not illustrated. Arrow thickness represents the strength of the projections.
cytoarchitectonic areas of the retrosplenial cortex and terminating in frontal cortical regions, specifically the anterior cingulate, motor, and prefrontal cortices. Using sensitive anterograde and retrograde neuronal tracers, we showed that distinct areas of the retrosplenial cortex provide predominantly ipsilateral projections to distinct parts of these frontal cortical regions (Fig. 6). 4.1. Projections from areas 29a and 29b The projections from areas 29a and 29b to the frontal cortical regions have not been studied in detail. Van Groen and Wyss (1990) demonstrated only weak projections from the retrosplenial granular a cortex (i.e., areas 29a and 29b) to dorsal area 24a and the infralimbic area. In the present study, BDA injections restricted to either area 24a or area 24b, and CTb injections confined to the terminal fields of each of these areas enabled us to demonstrate that areas 29a and 29b provide massive projections to distinct parts of area 24. Area 29a projects mainly to the rostral part of area 24a, whereas area 29b projects mainly to the caudodorsal part of area 24a and the ventral part of area 24b (Fig. 6). Both of these projections originate mainly from layer V and terminate mainly in layers I and III. Neither area 29a nor 29b projects to motor areas (see below).
The present study showed that area 29c provides massive projections to area 24 in a topographic manner. The caudal part of area 29c projects to area 24b and dorsal area 24a at the level of the anterior commissure, whereas the rostral part of area 29c projects to areas 24a and 24b at more caudal levels (Fig. 6). The projections originate from layer V, and to a lesser extent layers II–IV and VI, and terminate mainly in layers I and III of area 24. These findings confirm the results of a previous retrograde and anterograde tracing study (Van Groen and Wyss, 2003). The present study clearly showed that the rostral part of area 29c provides massive projections to the caudal parts of primary and secondary motor areas (Fig. 6). These projections originate from layer V, and to some extent layers II–IV, and terminate mainly in layers I and III of both motor areas. The caudal part of area 29c also provides a weak projection to layers I and III of the secondary motor area. Only some of these projections from area 29c to the secondary motor area have been demonstrated previously by retrograde tracing (Reep et al., 1990). Our tracer experiments also demonstrated that projections from area 29d to area 24 are organized topographically. The caudal part of area 29d projects to dorsal area 24a and ventral area 24b at the level of the anterior commissure, whereas the rostral part of area 29d projects to areas 24a and 24b at more caudal levels (Fig. 6). The projections from rostral area 29d are more massive than those from caudal area 29d. The projections of area 29d originate from layer V and terminate mainly in layers I and V. These findings also extend the results of previous retrograde and anterograde tracing studies (Vogt and Miller, 1983; Reep et al., 1990; Van Groen and Wyss, 2003) that demonstrated only part of projections from area 29d to area 24. Furthermore, the present study demonstrated that the projections from area 29d to the primary and secondary motor areas are more substantial than previously believed (Donoghue and Parham, 1983; Reep et al., 1990; Van Groen and Wyss, 1992). These projections originate from layer V of rostral area 29d and terminate mainly in layers I and V of the caudal parts of the primary and secondary motor areas. We also demonstrated weak projections from area 29d to the most rostral part of the prefrontal cortex, particularly to layer I of the ventral orbital area, confirming the findings of Van Groen and Wyss (1992). 4.3. Functional implications The present study demonstrated that the retrosplenial cortex provides massive projections to area 24a and the caudal part of area 24b. Since previous physiological studies have suggested that the retrosplenial cortex, particularly its caudal part, plays critical roles in spatial memory and navigation (Sutherland and Hoesing, 1993; Chen et al., 1994a,b; Cooper and Mizumori, 1999; Cho and Sharp, 2001; Cooper et al., 2001; Vann and Aggleton, 2002; Vann et al., 2003), projections originating predominantly from
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areas 29a, 29b, and caudal area 29c may provide spatial information, or some aspect or transformation of it, to area 24. This is significant, since area 24 was found to be involved in head movement (Sinnamon and Galer, 1984) and spatial behavior (Becker et al., 1980). Areas 29 and 24 were also found to play complementary roles in various discriminative behavioral learning tasks (Meunier et al., 1991; Gabriel, 1993; Bussey et al., 1996). Thus, projections from areas 29 to 24, as well as their reciprocal projections (Beckstead, 1979; Vogt and Miller, 1983; Finch et al., 1984; Van Groen and Wyss, 1990, 1992; Fisk and Wyss, 1999), may contribute to interactions between these cortical areas during discriminative behavioral learning. The present study also demonstrated that the rostral parts of areas 29c and 29d provide massive projections to the caudal part of the primary and secondary motor areas, suggesting a more direct involvement in motor behavior. Electrical stimulation of the caudal part of the primary motor area evokes trunk and limb muscle contractions (Hall and Lindholm, 1974; Donoghue and Wise, 1982; Gioanni and Lamarche, 1985; Neafsey et al., 1986; Wise and Donoghue, 1986). Stimulation of the caudal part of the secondary motor area evokes eye movement (Hall and Lindholm, 1974; Guandalini, 1998) and pupillary constriction (Gioanni and Lamarche, 1985; Guandalini, 2003). This cortical area also projects to brainstem visuomotor nuclei (Leichnetz and Gonzalo-Ruiz, 1987; Stuesse and Newman, 1990; Zeng and Stuesse, 1993; Guandalini, 2001, 2003; Tsumori et al., 2001). Thus, the projections from the retrosplenial cortex, particularly those originating from rostral parts, may provide spatial information to primary and secondary motor areas that are involved in an animal’s movements, including eye movement and pupillary contraction. Because the control of coordinated eye and head movements is essential for attentional processes (Crowne and Pathria, 1982; Neafsey et al., 1993), these projections may also contribute to an animal’s ability to attend to important stimuli. In conclusion, the present study demonstrated that each retrosplenial area provides predominantly ipsilateral projections to distinct parts of the anterior cingulate, motor, and prefrontal cortices. These projections constitute important neural pathways that may transmit spatial information to frontal cortical regions.
Acknowledgements The present study was, in part, supported by a grant (No. 07660400) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Van Groen, T., Wyss, J.M., 1990. Connections of the retrosplenial granular a cortex in the rat. J. Comp. Neurol. 300, 593–606. Van Groen, T., Wyss, J.M., 1992. Connections of the retrosplenial dysgranular cortex in the rat. J. Comp. Neurol. 315, 200–216. Van Groen, T., Wyss, J.M., 2003. Connections of the retrosplenial granular b cortex in the rat. J. Comp. Neurol. 463, 249–263. Vann, S.D., Aggleton, J.P., 2002. Extensive cytotoxic lesions of the rat retrosplenial cortex reveal consistent deficits on tasks that tax allocentric spatial memory. Behav. Neurosci. 116, 85–94. Vann, S.D., Wilton, L.A.K., Muir, J.L., Aggleton, J.P., 2003. Testing the importance of the caudal retrosplenial cortex for spatial memory in rats. Behav. Brain Res. 140, 107–118. Vogt, B.A., 1993. Structural organization of cingulate cortex: areas, neurons, and somatodendritic transmitter receptors. In: Vogt, B.A., Gabriel, M. (Eds.) Neurobiology of Cingulate Cortex and Limbic Thalamus. Birkhäuser, Boston, pp. 19–70. Vogt, B.A., Miller, M.W., 1983. Cortical connections between rat cingulate cortex and visual, motor, and postsubicular cortices. J. Comp. Neurol. 216, 192–210. Vogt, B.A., Peters, A., 1981. Form and distribution of neurons in rat cingulate cortex: areas 32, 24, and 29s. J. Comp. Neurol. 195, 603– 625. Wise, S.P., Donoghue, J.P., 1986. Motor cortex of rodents. In: Jones, E.G., Peters, A. (Eds.) Cerebral Cortex, vol. 5. Sensory-Motor Areas and Aspects of Cortical Connectivity. Plenum Press, New York, pp. 243–270. Zeng, D., Stuesse, S.L., 1993. Topographic organization of efferent projections of medial frontal cortex. Brain Res. Bull. 32, 195–200.
Organization of retrosplenial cortical projections to the anterior cingulate, motor, and prefrontal cortices in the rat Hideshi Shibata a,∗ , Shiori Kondo a , Jumpei Naito b a b
Department of Veterinary Anatomy, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Department of Animal Science, School of Science and Engineering, Teikyo University of Science and Technology, Yamanashi 409-0913, Japan Received 19 September 2003; accepted 9 January 2004
Abstract The retrosplenial cortex (areas 29a–29d) has been implicated in spatial memory, which is essential for performing spatial behavior. Despite this link with behavior, neural connections between areas 29a–29d and frontal association and motor cortices—areas also essential for spatial behavior—have been analyzed only to a limited extent. Here, we report an analysis of the anatomical organization of projections from areas 29a–29d to area 24 and motor and prefrontal cortices in the rat, using the axonal transport of biotinylated dextran amine (BDA) and cholera toxin B subunit (CTb). Area 29a projects to rostral area 24a, whereas area 29b projects to caudodorsal area 24a and ventral area 24b. Caudal area 29c projects to mid-rostrocaudal area 24b, whereas rostral area 29c projects to caudal areas 24a and 24b and caudal parts of primary and secondary motor areas. Caudal area 29d projects to mid-rostrocaudal areas 24a and 24b, whereas rostral area 29d projects to the caudalmost parts of areas 24a and 24b and the secondary motor area and to the mid-rostrocaudal part of the primary motor area. Area 29d also projects weakly to the prefrontal cortex. These differential corticocortical projections may constitute important pathways that transmit spatial information to particular frontal cortical regions, enabling an animal to accomplish spatial behavior. © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. Keywords: Area 29; Retrosplenial cortex; Area 24; Secondary motor area; Primary motor area; Corticocortical projections
1. Introduction The retrosplenial cortex, which occupies the caudal half of the cingulate cortex in the rat, consists of four cytoarchitectonically distinct areas: 29a, 29b, 29c and 29d (Vogt and Peters, 1981; Vogt, 1993). The relatively small areas 29a and 29b are located at levels caudal to the splenium of the corpus callosum, whereas the relatively larger areas 29c and 29d extend almost throughout the rostrocaudal axis of the retrosplenial cortex (see Fig. 1 for reference). Recent studies show that either cytotoxic lesions involving the entire retrosplenial cortex, or even those restricted to its caudal parts, cause spatial memory impairment (Vann and Aggleton, 2002; Vann et al., 2003). Electrophysiological evidence also supports the idea that this region is involved some way in spatial memory or associated functions. Cells in the retrosplenial cortex have been implicated in encoding ∗ Corresponding author. Tel.: +81-42-367-5766; fax: +81-42-367-5766. E-mail address: [email protected] (H. Shibata).
the head direction (Chen et al., 1994a,b) or the combinations of direction, location, and movement of an animal (Cho and Sharp, 2001); retrosplenial cells have also been implicated in path integration associated with spatial navigation (Cooper and Mizumori, 1999; Cooper et al., 2001). Thus, the retrosplenial cortex may play critical roles in spatial memory and navigation. It is assumed that the retrosplenial cortex transmits such spatial information via corticocortical pathways to the frontal association and/or motor cortical regions, which are intimately involved in spatial and motor behaviors (e.g. Hall and Lindholm, 1974; Becker et al., 1980). The corticocortical projections from the retrosplenial cortex to the frontal cortical regions thus far reported in the literature indicate that the region including areas 29a and 29b projects to areas 24a and 32 (Van Groen and Wyss, 1990), that area 29c projects to areas 24a and 24b (Vogt and Miller, 1983; Van Groen and Wyss, 2003), and that area 29d projects to areas 24a and 24b, the orbital cortex, and the medial part of the secondary motor area (Vogt and Miller, 1983; Van Groen and Wyss, 1992). However, the projections from the caudal part of the retrosplenial cortex, a region that is
0168-0102/$ – see front matter © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.01.005
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Fig. 1. Schematic drawings of the distribution of anterogradely labeled terminals in frontal cortical regions following injections of BDA into area 29a (A–E), area 29b (F–J), caudal area 29c (K–O), and rostral area 29c (P–T). The injection site of each case is depicted in A, F, K, and P, and the resulting labeled fibers and terminals are depicted in the frontal sections (B–D, G–I, L–N, and Q–S), arranged in the caudorostral order. Arrowheads indicate the boundaries between different cortical areas. Medial views of locations of injection sites and the relative density of labeled terminals were reconstructed in sagittal planes to produce panels E, J, O, and T; arrows indicate rostrocaudal levels of frontal sections. Abbreviations used in this and the following figures: ac, anterior commissure; CA, cornu ammonis; cc, corpus callosum; c29c, caudal part of area 29c; c29d, caudal part of area 29d; DG, dentate gyrus; II–IV, layers II–IV of cortex; LO, lateral orbital area; LV, lateral ventricle; MO, medial orbital area; M1, primary motor area; M2, secondary motor area; PL, prelimbic area; Pre, presubiculum; r29c, rostral part of area 29c; r29d, rostral part of area 29d; Sep, septum; Sub, subiculum; V, layer V of the cortex; VI, layer VI of the cortex; and VO, ventral orbital area.
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particularly important for spatial memory, are still unclear. For example, it is as yet undetermined whether projections from area 29a differ from those originating from area 29b. Furthermore, to date, projections to motor areas have not been studied in detail (Vogt and Miller, 1983; Van Groen and Wyss, 1992). Thus, the present study was carried out to clarify in more detail the organization of projections from each area of the retrosplenial cortex to the anterior cingulate, motor, and prefrontal cortices in the rat. First, using the anterograde neuronal tracer biotinylated dextran amine (BDA), we determined the terminal distribution of projections originating from each area of the retrosplenial cortex. Second, using the retrograde neuronal tracer cholera toxin B subunit (CTb), we determined the distribution of cells of origin of these projections. The results show that each area of the retrosplenial cortex projects to distinct parts of the anterior cingulate, motor, and prefrontal cortices.
2. Materials and methods A total of 45 Wistar rats of both sexes, weighing 210–460 g, were used in the present study. All surgical procedures were done under deep anesthesia consisting of intraperitoneal injections of sodium pentobarbital (60 mg/kg body weight) or a combination of xylazine (10 mg/kg body weight) and ketamine (50 mg/kg body weight). Before and after surgery, the rats were allowed free access to food and water. All the experimental procedures complied with the guidelines of the National Institute of Health (USA). BDA injections were done first and their terminal fields were analyzed to determine the placement of our subsequent retrograde injections in different animals. For BDA injections, a glass micropipette (10–30 m i.d.) was filled with 10% BDA (Molecular Probes, Eugene, OR, USA) solution; lyophilized BDA was dissolved in saline (Brandt and Apkarian, 1992). Each area of the retrosplenial cortex was sampled with discrete BDA injections in different animals, using coordinates derived from the stereotaxic atlas of Paxinos and Watson (1997). BDA was deposited iontophoretically by passing a 3–5 A positive current pulse for a total on-time of 5–15 min (Brandt and Apkarian, 1992). After 10 or 14 days, the rats were anesthetized with intraperitoneal injections of sodium pentobarbital (60 mg/kg body weight) and perfused transcardially with saline, followed by 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After saturation in 30% sucrose in 0.1 M phosphate buffer at 4◦ C, the brains were frozen, and frontal sections were cut at 40 or 50 m thickness on a freezing microtome. To visualize BDA-labeled terminals, every other section was placed overnight in Tris–HCl buffer (TB) containing 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), rinsed briefly in TB, and incubated for 1 h at room temperature in TB containing either streptavidin-conjugated horseradish
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peroxidase (1:500; Dakopatts, Glostrup, Denmark) or Elite avidin-biotinylated horseradish peroxidase complex (ABC) (1:100 or 1:200; Vector Laboratories, Burlingame, CA, USA). After three 5 min rinses in TB, the sections were incubated in TB containing 0.04% 3,3 -diaminobenzidine (DAB) and 0.01% H2 O2 for 15–20 min. Some sections were incubated in Ni–DAB solution (TB containing 0.02% DAB, 0.6% nickel ammonium sulfate and 0.003% H2 O2 ) for 5–10 min. Finally, all the sections were thoroughly rinsed in TB and mounted onto gelatin-coated slides. For CTb injections, 0.5 mg of lyophilized CTb (List Biological Laboratories, Campbell, CA, USA) was reconstituted with 0.5 ml of 0.1 M phosphate buffer (pH 6.0) and concentrated to 1% by ultrafiltration, according to the protocol of Luppi et al. (1990). In some cases, 1% “low salt” CTb (List Biological Laboratories) dissolved in distilled water was used without prior ultrafiltration. Glass micropipettes (10–30 m i.d.) were filled with CTb, which was iontophoretically injected into parts of the anterior cingulate or motor cortices that were previously shown in our BDA experiments to receive dense terminal projections from the retrosplenial cortex. CTb was deposited by passing a 2–4 A positive current pulse for 5–10 min in total. After 3, 5 or 7 days, the rats were deeply anesthetized with intraperitoneal injections of sodium pentobarbital (60 mg/kg body weight) and perfused according to the same protocol used for the BDA-injected cases. The brains were removed, saturated with 30% sucrose in 0.1 M phosphate buffer, and cut into frontal sections at 40 or 50 m thickness on a freezing microtome. To visualize CTb-labeled cells immunohistochemically, every other section was first incubated overnight in Tris–HCl-buffered saline (TBS) containing 2% normal rabbit serum and 0.5% Triton X-100, and then in a primary antibody solution containing goat anti-CTb (1:40,000; List Biological Laboratories), 1% normal rabbit serum, 0.5% Triton X-100 and TBS for 48 h at 4 ◦ C. After three 5 min rinses in TBS, the sections were treated with a Vectastain ABC kit (Vector Laboratories), and CTb-labeled elements were visualized by incubating the sections in the Ni–DAB solution as described above. The sections processed for either BDA histochemistry or CTb immunohistochemistry were dehydrated in ethanol, cleared in xylene, and coverslipped. Some sections were counterstained with thionin. The sections were examined with bright- and darkfield illuminations. Locations of injection sites and the distribution of labeled cells, fibers, and terminals were first plotted onto representative drawings of frontal sections with the aid of a camera lucida apparatus, then reconstructed onto drawings of sagittal sections so that their distributions could be appreciated from a medial view of the cerebral hemispheres. For demarcating retrosplenial and anterior cingulate cortical areas, we adopted the nomenclature described by Vogt and Peters (1981), and for other cortical areas, we followed the nomenclature outlined by Paxinos and Watson (1997).
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3. Results In the anterograde tracing experiments, BDA was injected into each area of the retrosplenial cortex at various rostrocaudal levels in order to delineate terminal fields of retrosplenial cortical projection fibers to frontal cortical regions, such as the anterior cingulate, motor, and prefrontal cortices. The injections involved all cortical layers and were successfully made into area 29a (2 cases), area 29b (1 case), areas 29b and 29c (2 cases), area 29c (11 cases), and area 29d (7 cases). The results from six representative cases will be presented to illustrate the projection patterns. Data from the remaining cases were confirmatory. Although labeled terminals in all cases were organized homotopically in the frontal cortical regions in both hemispheres, contralateral labeling was sparser than ipsilateral labeling. Thus, we describe only labeling ipsilateral to the injection sites. The distribution of labeled terminals within the retrohippocampal region and the anterior and laterodorsal thalamic nuclei in some cases has been reported previously (Shibata, 1994, 1998, 2000). BDA injections into areas 29a or 29b anterogradely labeled terminals mainly in the rostral part of the anterior cingulate cortex (Fig. 1A–J). An injection restricted to area 29a labeled terminals in the rostral part of area 24a (Figs. 1A and C–E, and 2A and B). Terminal labeling in layers I–III was denser than that in layers V and VI (Figs. 1C and D, and Fig. 2B). A few labeled terminals were also observed within the ventral part of rostral area 24b (Fig. 1D). An injection restricted to area 29b labeled terminals somewhat more caudodorsally within the anterior cingulate cortex (FFig. 1F–J). This area 29b injection also labeled many terminals within all layers of area 24a, with the densest labeling in layers I and III (Fig. 1G–I). Labeled terminals were also observed in the adjoining part of area 24b (Fig. 1G–I). Few labeled terminals were found in the rostralmost and caudal parts of area 24a (Fig. 1J). Injections into area 29c labeled terminals in parts of area 24 and the primary and secondary motor areas (Fig. 1K–T). An injection into the caudal part of area 29c labeled terminals in areas 24a and 24b and the secondary motor area, only at the level of the anterior commissure (Fig. 1K–O). As one examines labeling within this level in the caudorostral direction, labeling gradually shifts ventrodorsally such that at caudal levels, labeling is found in the dorsal part of area 24a, then at mid-rostrocaudal levels in area 24b, and finally at rostral levels in the secondary motor area (Fig. 1L–O). Terminal labeling was particularly dense in layers I and III of these cortical areas. An injection into the rostral part of area 29c labeled massive terminal fields in caudal parts of areas 24a and 24b and in caudal parts of the primary and secondary motor areas, particularly in layers I and III (Figs. 1P, Q and T, and 2C and D). In areas 24a and 24b, labeling was dense within dorsal parts (Fig. 1Q). At more rostral levels (Fig. 1R–T), some labeled terminals were noted mainly in layers I and III of the secondary motor area; only a few, scattered terminals were observed in the primary motor area.
Injections into area 29d labeled terminals in the anterior cingulate, motor, and prefrontal cortices (Fig. 3A–L). An injection into the caudalmost part of area 29d labeled relatively few terminals in areas 24a and 24b at caudal levels and in the ventral orbital area near the frontal pole (Fig. 3A–E). Labeling in these areas was distributed mainly in layer I. An injection into the rostral part of area 29d labeled massive terminal fields in areas 24a and 24b and in the primary and secondary motor areas (Fig. 3F–J and L). In the caudal parts of areas 24a and 24b, labeled terminals were distributed throughout all layers, with the densest labeling in layers I–III of the caudalmost part of area 24b (Fig. 3G and H). At caudal levels of the secondary motor area, diffuse terminal labeling was observed in all layers (Fig. 3G, H and L), whereas at more rostral levels, labeled terminals were concentrated in layers I and III (Fig. 3I). At mid-rostrocaudal levels of the primary motor area, terminal labeling was densest in layers I and III (Fig. 3J and L). At rostral levels of areas 24a and 24b and the motor areas, few terminals were labeled (Fig. 3L). Minute, scattered labeling was also observed in the prefrontal cortex, mainly in layers I–III of the ventral, medial, and lateral orbital areas and prelimbic area (Fig. 3K and L). In the retrograde tracing experiments, CTb was injected into the parts of the anterior cingulate (13 cases) and motor cortices (9 cases) where dense labeling of terminals was observed in the anterograde tracing experiments; CTb injections involved all cortical layers. The results of the CTb experiments confirmed those of the BDA experiments, and delineated the laminar distribution of retrosplenial cells that projected to the anterior cingulate and motor cortices. The results of seven representative cases will be described below. As in the anterograde tracing experiments, we shall only describe the distribution of retrogradely labeled cells ipsilateral to each injection site. CTb was injected into various rostrocaudal levels of area 24a (Fig. 4A–O). An injection into area 24a at the level of the caudal septum retrogradely labeled cells mainly in areas 29b and 29c, at levels just caudal to the splenium of the corpus callosum (Figs. 2E and F, and 4A–C and E). In these areas, most of the labeled cells were located in layer V, and a few labeled cells were observed in layers II–IV and VI (Fig. 4C). A few labeled cells were also observed in layers V and VI of the caudalmost part of area 29a (Fig. 4B and E), in layers V and VI of rostral area 29c (Fig. 4D and E), and in layer V of area 29d throughout its rostrocaudal extent (Fig. 4B–E). A CTb injection into area 24a at the level of the rostral septum labeled cells more caudally within the retrosplenial cortex (Fig. 4F–J). Many labeled cells were observed in layer V of areas 29a and 29b, with fewer cells in layers II–IV (Fig. 4H and I). The labeled cells in area 29b were located mainly in the part adjoining area 29a (Fig. 4H and I). Some labeled cells were also found in layers II–IV and V of area 29d at levels caudal to the splenium of the corpus callosum (Fig. 4G–J). An injection confined to the most rostral part of area 24a labeled cells mainly in layers
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Fig. 2. Photomicrographs of tracer injection sites (A, C and E) and distribution of resulting labeled terminals or cells (B, D and F). (A) Thionin-counterstained section showing a BDA injection site localized within area 29a. (B) Darkfield photomicrograph of anterogradely labeled terminals in area 24a of the anterior cingulate cortex, resulting from area 29a injection in panel A. (C) Thionin-counterstained section showing a BDA injection site localized within area 29c. (D) Darkfield photomicrograph of anterogradely labeled terminals in primary (M1) and secondary motor areas (M2), resulting from area 29c injection shown in panel C. (E) CTb injection site localized within area 24a. (F) Brightfield photomicrograph of retrogradely labeled cells in areas 29b and 29c, just caudal to the splenium of the corpus callosum, resulting from area 24a injection shown in panel E. Arrowheads indicate the boundaries between different cortical areas.
II–IV and V of area 29a and the adjoining part of area 29b (Fig. 4K–O). A CTb injection into the caudalmost part of area 24b labeled many cells in layers II–IV and V and in the superficial portion of layer VI of areas 29b, 29c, and 29d at a level caudal to the splenium of the corpus callosum (Fig. 4P–R
and T). In area 29b, labeled cells were located in the part near area 29c, whereas in area 29d more labeled cells were located in the part near the visual association cortex (Fig. 4Q and R). At more rostral levels of the retrosplenial cortex, only a few labeled cells were found in areas 29c and 29d, mainly in layers II–IV and V (Fig. 4S and T).
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Fig. 3. Schematic drawings of the distribution of anterogradely labeled terminals in frontal cortical regions following injections of BDA into caudal (A–E) or rostral parts of area 29d (F–L). The injection site of each case is depicted in A and F, and the resulting labeled fibers and terminals are depicted in the frontal sections (B–D and G–K) arranged in the caudorostral order. Medial views of locations of injection sites and the relative density of labeled terminals were reconstructed in sagittal planes to produce panels E and L. The same diagram conventions as those described for Fig. 1.
An injection into the caudal part of the secondary motor area at the level of the caudal septum labeled many cells in all the cellular layers of areas 29b, 29c, and 29d in the mid-rostrocaudal third of the retrosplenial cortex (Fig. 5A, C, D and F). In areas 29b and 29c, labeled cells were most
numerous in layer V. Within rostral and caudal thirds of the retrosplenial cortex, only a few labeled cells were scattered in areas 29c and 29d (Fig. 5B, E and F). An injection into the mid-rostrocaudal part of the secondary motor area at the level of the rostral septum labeled only a few, scat-
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Fig. 4. Schematic drawings of the distribution of retrogradely labeled cells in the retrosplenial cortex following injections of CTb into caudal (A–E), mid-rostrocaudal (F–J), and rostral parts of area 24a (K–O), and into area 24b (P–T). The injection site of each case is depicted in A, F, K, and P, and the resulting labeled cells are depicted in frontal sections (B–D, G–I, L–N or Q–S) arranged in the caudorostral order. Large dots represent 5–10 labeled cells, whereas small dots represent 1–4 labeled cells. Medial views of locations of injection sites and the relative density of labeled cells were reconstructed in sagittal planes to produce panels E, J, O, and T. The same diagram conventions as those described for Fig. 1.
tered cells in rostral areas 29c and 29d, mainly in layer V (Fig. 5G–J). A CTb injection into the primary motor area at the level of the mid-rostrocaudal septum labeled a group of cells distributed throughout dorsal area 29c and ventromedial area 29d at rostralmost levels (Fig. 5K, M and N). Most of the labeled cells were located in layer V and a few were observed in layers II–IV and the superficial part of layer VI
(Fig. 5M). A few labeled cells were also observed in the deep part of layer V of caudal area 29d (Fig. 5L and N).
4. Discussion The present study demonstrated the anatomical organization of projections originating from different
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Fig. 5. Schematic drawings of the distribution of retrogradely labeled cells in the retrosplenial cortex following injections of CTb into caudal (A–F) or mid-rostrocaudal parts of M2 (G–J), and in M1 (K–N). The injection site of each case is depicted in A, G, and K, and the resulting labeled cells are depicted in frontal sections (B–E, H and I, and L and M) arranged in the caudorostral order. Large dots represent 5–10 labeled cells, whereas small dots represent 1–4 labeled cells. Medial views of locations of injection sites and the relative density of labeled cells were reconstructed in sagittal planes to produce panels F, J, and N. The same diagram conventions as those described for Fig. 1. Although the injection appears to have spared layer I in the drawings of each case shown, the injected tracer did involve layer I in nearby sections (not illustrated).
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4.2. Projections from areas 29c and 29d
Fig. 6. Schematic drawings of the medial aspect of the rat cerebral cortex, summarizing the organization of projections from retrosplenial area 29 to the anterior cingulate, motor, and prefrontal cortices. The contralateral projections, which are homotopical but relatively sparse, are not illustrated. Arrow thickness represents the strength of the projections.
cytoarchitectonic areas of the retrosplenial cortex and terminating in frontal cortical regions, specifically the anterior cingulate, motor, and prefrontal cortices. Using sensitive anterograde and retrograde neuronal tracers, we showed that distinct areas of the retrosplenial cortex provide predominantly ipsilateral projections to distinct parts of these frontal cortical regions (Fig. 6). 4.1. Projections from areas 29a and 29b The projections from areas 29a and 29b to the frontal cortical regions have not been studied in detail. Van Groen and Wyss (1990) demonstrated only weak projections from the retrosplenial granular a cortex (i.e., areas 29a and 29b) to dorsal area 24a and the infralimbic area. In the present study, BDA injections restricted to either area 24a or area 24b, and CTb injections confined to the terminal fields of each of these areas enabled us to demonstrate that areas 29a and 29b provide massive projections to distinct parts of area 24. Area 29a projects mainly to the rostral part of area 24a, whereas area 29b projects mainly to the caudodorsal part of area 24a and the ventral part of area 24b (Fig. 6). Both of these projections originate mainly from layer V and terminate mainly in layers I and III. Neither area 29a nor 29b projects to motor areas (see below).
The present study showed that area 29c provides massive projections to area 24 in a topographic manner. The caudal part of area 29c projects to area 24b and dorsal area 24a at the level of the anterior commissure, whereas the rostral part of area 29c projects to areas 24a and 24b at more caudal levels (Fig. 6). The projections originate from layer V, and to a lesser extent layers II–IV and VI, and terminate mainly in layers I and III of area 24. These findings confirm the results of a previous retrograde and anterograde tracing study (Van Groen and Wyss, 2003). The present study clearly showed that the rostral part of area 29c provides massive projections to the caudal parts of primary and secondary motor areas (Fig. 6). These projections originate from layer V, and to some extent layers II–IV, and terminate mainly in layers I and III of both motor areas. The caudal part of area 29c also provides a weak projection to layers I and III of the secondary motor area. Only some of these projections from area 29c to the secondary motor area have been demonstrated previously by retrograde tracing (Reep et al., 1990). Our tracer experiments also demonstrated that projections from area 29d to area 24 are organized topographically. The caudal part of area 29d projects to dorsal area 24a and ventral area 24b at the level of the anterior commissure, whereas the rostral part of area 29d projects to areas 24a and 24b at more caudal levels (Fig. 6). The projections from rostral area 29d are more massive than those from caudal area 29d. The projections of area 29d originate from layer V and terminate mainly in layers I and V. These findings also extend the results of previous retrograde and anterograde tracing studies (Vogt and Miller, 1983; Reep et al., 1990; Van Groen and Wyss, 2003) that demonstrated only part of projections from area 29d to area 24. Furthermore, the present study demonstrated that the projections from area 29d to the primary and secondary motor areas are more substantial than previously believed (Donoghue and Parham, 1983; Reep et al., 1990; Van Groen and Wyss, 1992). These projections originate from layer V of rostral area 29d and terminate mainly in layers I and V of the caudal parts of the primary and secondary motor areas. We also demonstrated weak projections from area 29d to the most rostral part of the prefrontal cortex, particularly to layer I of the ventral orbital area, confirming the findings of Van Groen and Wyss (1992). 4.3. Functional implications The present study demonstrated that the retrosplenial cortex provides massive projections to area 24a and the caudal part of area 24b. Since previous physiological studies have suggested that the retrosplenial cortex, particularly its caudal part, plays critical roles in spatial memory and navigation (Sutherland and Hoesing, 1993; Chen et al., 1994a,b; Cooper and Mizumori, 1999; Cho and Sharp, 2001; Cooper et al., 2001; Vann and Aggleton, 2002; Vann et al., 2003), projections originating predominantly from
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areas 29a, 29b, and caudal area 29c may provide spatial information, or some aspect or transformation of it, to area 24. This is significant, since area 24 was found to be involved in head movement (Sinnamon and Galer, 1984) and spatial behavior (Becker et al., 1980). Areas 29 and 24 were also found to play complementary roles in various discriminative behavioral learning tasks (Meunier et al., 1991; Gabriel, 1993; Bussey et al., 1996). Thus, projections from areas 29 to 24, as well as their reciprocal projections (Beckstead, 1979; Vogt and Miller, 1983; Finch et al., 1984; Van Groen and Wyss, 1990, 1992; Fisk and Wyss, 1999), may contribute to interactions between these cortical areas during discriminative behavioral learning. The present study also demonstrated that the rostral parts of areas 29c and 29d provide massive projections to the caudal part of the primary and secondary motor areas, suggesting a more direct involvement in motor behavior. Electrical stimulation of the caudal part of the primary motor area evokes trunk and limb muscle contractions (Hall and Lindholm, 1974; Donoghue and Wise, 1982; Gioanni and Lamarche, 1985; Neafsey et al., 1986; Wise and Donoghue, 1986). Stimulation of the caudal part of the secondary motor area evokes eye movement (Hall and Lindholm, 1974; Guandalini, 1998) and pupillary constriction (Gioanni and Lamarche, 1985; Guandalini, 2003). This cortical area also projects to brainstem visuomotor nuclei (Leichnetz and Gonzalo-Ruiz, 1987; Stuesse and Newman, 1990; Zeng and Stuesse, 1993; Guandalini, 2001, 2003; Tsumori et al., 2001). Thus, the projections from the retrosplenial cortex, particularly those originating from rostral parts, may provide spatial information to primary and secondary motor areas that are involved in an animal’s movements, including eye movement and pupillary contraction. Because the control of coordinated eye and head movements is essential for attentional processes (Crowne and Pathria, 1982; Neafsey et al., 1993), these projections may also contribute to an animal’s ability to attend to important stimuli. In conclusion, the present study demonstrated that each retrosplenial area provides predominantly ipsilateral projections to distinct parts of the anterior cingulate, motor, and prefrontal cortices. These projections constitute important neural pathways that may transmit spatial information to frontal cortical regions.
Acknowledgements The present study was, in part, supported by a grant (No. 07660400) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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