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Heterogeneity in the Dorsal Subiculum of the Rat. Distinct Neuronal Zones Proiect to Different Cortical and *

Subcortical Targets

Menno P. Witter, Robert H. Ostendorf and Henk J. Groenewegen Department of Anatomy and Embryology, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Key words: hippocampus, efferent connections, tracing study, limbic system

Abstract

The aim of the present study was to relate the distribution of efferents of the dorsal subiculum to their origin along the proximG distal axis of the subiculum. The distribution of subicular projections was studied in detail by means of the sensitive anterograde tracer fhaseolus vulgaris-leucoagglutinin (PHA-L), and the precise origin of these projections analysed with retrogradely transported fluorescent tracers, using double- and triple-labelling protocols. Injections of PHA-L in the proximal part of the dorsal subiculum, i.e. that part which borders field CA1, result in labelling of the infralimbic, entorhinal and perirhinal cortices, the nucleus accumbens and the lateral septal region, the interanteromedial nucleus of the thalamus, the core of the nucleus gelatinosus, and the mammillary nuclei, in particular in the rostra1 parts of the medial nucleus. In contrast, injections in the distal part of the dorsal subiculum, i.e. that part which borders the presubiculurn, give rise to labelling in the retrosplenial and postrhinal cortices, the presubiculum, the anterior thalamic complex, the shell of the nucleus gelatinosus, and the mammillary nuclei, preferentially in the caudal part of the medial nucleus. The results of injections of different retrograde tracers, simultaneously placed in two or three targets of the subicular efferents, confirm the results of the anterograde tracing experiments. Moreover, they clearly demonstrate that the population of subicular neurons which, for example, projects to the nucleus accumbens and the interanterornedial nucleus of the thalamus is almost completely segregated from the population that projects to the retrosplenial cortex and the anterior complex of the thalamus. Thus within the dorsal subiculum, populations of neurons can be differentiated so that each population projects to a unique set of target structures. These cell populatioris are differentially positioned along the proximdistal axis. In view of additional evidence indicating that some of the major afferents to the subiculurn are organized along the same axis, we suggest that the heterogeneity of the dorsal subiculum along the proximedistal axis reflects a general organizational characteristic of this hippocampal field.

Introduction The subiculum constitutes a major output structure of the hippocampal formation. Fibres that leave the subiculum travel preferentially by way of two pathways, the fornix and the angular bundle. The first tract is known to distribute its fibres to the septal region, the anterior complex of the thalamus, the nucleus accumbens, the ventral hypothalamic area, and the mammillary nuclei. Fibres that follow the angular bundle reach the para- or retrohippocampal cortical areas, such as the entorhinal and perirhinal cortices, the presubiculum and parasubiculum, as well as the retrosplenial cortex. The amygdala is reached by fibres that traverse the amygdalahippocampal area (Swanson and Cowan, 1977; see also Swan- son et al., 1987; Lopes da Silva et al., 1990).

It is well established that the dorsal and ventral parts of the subiculum differ with respect to the structures that are reached by their efferents. For example, the projections to the mammillary nuclei and the anterior complex of the thalamus arise mainly from the dorsal subiculum. In con-

trast, those to the ventromedial hypothalamic area and the amygdala originate predominantly in the ventral subiculum (Swanson and Cowan, 1977; see also Witter, 1986). The projections to the septal region and the nucleus accumbens arise from the entire dorso-ventral extent of the subiculum, but are topographically organized; the distribution of subicular fibres in these areas is related to the origin along the dorso-ventral or longitudinal axis of the subiculum (Groenewegen et a / . , 1982, 1987; Swanson and Cowan, 1979). A similar topography has been described with respect to the reciprocal connections between the subiculum and the entorhinal cortex (Van Groen et al . , 1986; Witter et al., 1989).

Apart from this dorsoventral differentiation within the subiculum, we recently noted, in the rat, that the projections from the subiculum to the nucleus accumbens do not arise from the entire proximo-distal or transverse extent of the dorsal subiculum. By means of anterograde tracing experiments with the sensitive tracer Phaseolus vulgaris-

Correspondence to: M . P. Witter, as above

Received 12 January 1990, revised 21 March 1990, accepted 22 March 1990

Efferents of the dorsal subiculum 719

leucoagglutinin (PHA-L), we observed that only injections in the proximal part of the dorsal subiculum, i.e. the part that borders field CAI of the Ammon's horn, resulted in labelling in the nucleus accumbens. Con- versely, injections of retrograde tracers into the nucleus accumbens resulted in labelled cells that were confined to the proximal part of the dorsal subiculum (Groenewegen er al., 1987). These latter observations suggest that the origin of the projections from the subiculum is organized not only along its dorso-ventral axis, but also along its proximodistal axis. In agreement with this are the observations of Meibach and Siege1 (1977a, b). They reported in the rat that the projections to the septal region predominantly originate in the proximal part of the subiculum. In contrast, the projections to the anterior nuclei of the thalamus mainly originate in the distal part of the subiculum and in the adjacent presubiculum. These authors also described that the projections to the mammillary nuclei show a topographical organization related to the proximodistal axis of the subiculum. As yet, no data are available on the exact origin of other projections from the dorsal subiculum. Therefore, the aim of the present study was to explore whether the origin of all the major efferent projections from the dorsal subiculum respect its differentiation into proximal and distal parts.

Materials and methods For the analysis of the subicular efferents we used a series of nine rats in which PHA-L had been injected into the dorsal subiculum. This material has previously been used for a description of the projections from the subiculum to the striatum (Groenewegen et al., 1987). This paper also provides the methods for delivering the tracer and the immuno- cytochemical procedure to visualize the PHA-L (cf. Gerfen and Sawchenko, 1984).

To determine the precise location of the populations of subicular neurons that project to the various targets, in a second series of experiments we injected each animal with two or three different retrograde fluorescent tracers into different targets of the subiculum. A total of 24 female Wistar rats (body weight 180-220 g) were used. Animals were anaesthetized by an intramuscular injection of a mixture of ketamine and xylazine (4:3, 1 mllkg). Subsequently, stereotaxic injections (coordinates derived from the atlas by Paxinos and Watson, 1986) were made using either fast blue or diamidino yellow (FB or DY; Kuypers et al., 1977) or fluoro-gold (FG; Schmued and Fallon, 1986). Varying volumes (30- 100 nl) of an aqueous solution of FB or DY (2.5% in distilled water) were pressure-injected using a 1 or 5 pl Hamilton syringe. Injections with FG (2% FG in 0.05 M Na-acetate buffer, pH 5.0) were made iontophoretically, for 5-10 min with 7.5 pA, using a glass micropipette with a tip diameter of approximately 13 pm. Follow- ing survival times of 4 - 12 days, the animals were deeply anaesthetized and transcardidly perfused with 0.9% saline, followed by 4% para- formaldehyde or 10% formaldehyde in phosphate buffer (pH 7.4). After removal from the skull, the brains were postfixed for approximately 1.5 h in the same fixative that was used for the perfusion. Following over- night storage in a solution of 20% glycerol and 2% dimethyl sulphoxide (DMSO), the brains were sectioned on a freezing microtome (30 pm) and one out of two series of sections were mounted immediately after sectioning. One series was stored desiccated by -2O"C, and the other series was examined uncovered with the aid of a Zeiss IV F fluorescence microscope with filter-mirror system 01 (365 nm). This allowed us to distinguish all three retrograde tracers. The distribution of retrogradely labelled cells was charted with the aid of an X-Y recorder connected to the microscope stage. Subsequently, the sections were counterstained

with cresyl violet, coverslipped, and re-examined in order to relate the distribution of the labelled cells to the cytoarchitectonic borders of the subiculum.

Results Anterograde tracing with PHA-L Nine cases with injections of PHA-L in the dorsal part of the subiculum were analysed. Two of the injections were located in the proximal part of the subiculum and labelled neurons in the part of the subiculum that borders field CA1 of the Ammon's horn. Five injections were confined to the distal part of the subiculum and filled cells close to the border of the subiculum with the pmbiculum. The other two injections labelled cells in the region of the subiculum, intermediate between the proximal and distal injections. Following all injections, clear fibre labelling was present throughout the fornix and in various parts of the white matter underlying the cortex of the telencephalon, predominantly in the angular bundle and in the internal and external capsules. We will subsequently describe the distribution of labelled fibres and terminals following two representative injections in the proximal or distal part of the dorsal subiculum at a comparable dorso-ventral level (Fig. 1).

In case 85392, the injection had an approximate diameter of 450 pm, and PHA-L positive cells were located in the proximal part of the subiculum, directly adjacent to the border with CA1 (Fig. 1A). Terminal labelling in the cortex was mainly present in the deep layers of the infralimbic (area 25; Figs. 2A, 4A), and entorhinal (area 28; Fig. 2D) cortices. In the entorhinal cortex, the labelling was confined to its lateral part, adjacent to the rhinal sulcus, and was continuous with labelling in the deep layers of the perirhinal cortex. Weak terminal labelling was present in the dorsal part of the presubiculum (Fig. 2D) and in parts of the medial prefrontal cortex (Fig. 2A). In the presubiculum, labelling was mainly observed in the molecular layer, whereas in the medial prefrontal cortex the labelled fibres were rather diffusely distributed over the various cell layers. In subcortical structures of the telencephalon, terminal-like labelling was observed in the rostro-lateral part of the nucleus accumbens and in the lateral septal nucleus, mainly in the dorsal part of its intermediate subdivision (Fig. 2B, C; nomenclature of the septal region is according to Swanson and Cowan, 1979). In the diencephalon, labelled varicose fibres were present in the thalamus, predominantly in the interanteromedial nucleus and weak terminal labelling occupied the core of the nucleus gelatinosus (Figs. 2G, H, 4B). Fibres reaching these thalamic nuclei followed two pathways. A few fibres coursed in the fornix and, after leaving this bundle, traversed the nucleus reuniens. Other fibres travelled in the internal capsule and entered the thalamus through the anterior complex (Fig. 2G). The labelled fibres in these two trajectories did not show varicosities, and thus most probably represent passing fibres. In the hypothalamus, strong labelling was observed bilaterally in the mammillary nuclei, predominantly in the rostral part of the medial nucleus (Figs. U, 4C). At this level, there were also a few labelled varicose fibres in the supramammillary region and in the ventro-lateral part of the hypothalamus, directly ventral to the supramamrmll ' ary region and lateral to the lateral mammillary nucleus (Fig. U).

Following an injection with an approximate diameter of 450 pm in the distal part of the subiculum (case 86292; Fig. 1B) labelling was absent in the infralimbic and entorhinal cortices, the nucleus accumbens, the lateral septal nucleus, and the interanteromedial thalamic nucleus (Fig. 3A-E, G). In contrast, dense labelling was found in the retrosplenial and postrhinal cortices, whereas the dorsal part of the

720 Efferents of the dorsal subiculum

FIG. 1. Photomicrographs of transverse sections through the dorsal subiculum illustrating the location and extent along the transverse axis of representative injections of PHA-L in its proximal (A) and distal (B) parts (calibration bars represent 200 pm).

FIG. 2. Camera lucida drawings of a series of transverse sections through the telencephalon (A-E) and diencephalon/mesencephalon (F-K), illustrating the distribution of anterogradely transported PHA-L after an injection in the proximal part of the dorsal subiculum (case. 85392). Sections are organized from rostra1 to caudal.


FIG. 3. Camera lucida drawings of a series of transverse sections through the telencephalon (A-E) and diencephalonhnesencephalon (F-K), illustrating the distribution of anterogradely transported PHA-L after an injection in the distal part of the dorsal subiculum (case 86292). Sections are organized from rostra1 to caudal.

presubiculum showed moderately dense labelling (Figs. 3D, E, 4D, E). In the retrosplenial cortex, labelling is particularly dense in layer XI. The deeper layers displayed weaker, difisely organized labelling, with the highest density in layer IV. In the presubiculum and the postrhinal cortex, labelling was most prominent in the superficial layers II and III (Fig. 4D, E). Labelling was also observed in the thalamus, particularly in the anterior complex. The antemventral nucleus displayed the densest labelling, but also the anterodorsal nucleus contained a few labelled varicose fibres (Fig. 3F, G). In the nucleus gelatinosus, moderately dense labelling was confined to the dorsal part of the shell of the nucleus (Figs. 3H, 4F). The fibres that distribute to the thalamus travel almost exclusively by way of the fornix. In the hypothalamus, both the lateral and medial mammillary nuclei displayed terminal-like labelling, predominantly in the posterior part of the medial nucleus (Figs. 3K, 4G). As in the previous experiment, a few varicose fibres were labelled in the supramammillary region and the narrow area directly lateral to the lateral mammillary nucleus.

Injections that involve a more intermediately located population of cells in the dorsal subiculum resulted in a pattern of labelling that resembles a combination of the two patterns described above.

Retrograde tracing with various fluorescent substances

The results of the anterograde tracing experiments indicate that the subiculum contains different populations of neurons each projecting to a specific set of target structures. In order to study the origin of these projections in more detail, we injected rats with different fluorescent retrograde tracers. In each animal, injections were placed in at least one of the targets of the proximally located subicular cells and in one of the targets of the distally located subicular neurons. Samples from 24 animals successfully injected have been analysed. Injections were placed in the infralimbic cortex (n = l), the septal region (n = l), the infralimbic cortex/septal region (n = 3), the mammillary nuclei (n = 3, the anterior complex (n = 2) and the interanteromedialhidline region (n = 4) of the thalamus, the retrosplenial (n = 7) and the entorhinal (n = 6) cortices, and the nucleus accumbens (n = 3). Five representative cases will be described and illustrated. As all experiments were meant to determine only the presumed proximo-distal origin of the subicular efferents, the following account will not deal with questions of possible collateralization of subicular projections (cf. Swanson et al., 1981; Donovan and Wyss, 1983).


FIG. 4. Photomicrographs of labelling in various brain areas following injections of PHA-L in the proximal (A-C) and distal (D-G) parts of the dorsal subiculum (calibration bars represent 200 Fm). (A) Labelling in the deep layers of the infralimbic cortex and in the rostral nucleus accumbens. (B) Labelling in the interanteromedial nucleus of the thalamus. (C) Labelling in the rostral part of the medial mammillary nucleus. (D) Labelling in the retrosplenial cortex. Note the clear terminal fields in layer II and at the transition from layer IV to layer V. (E) Labelling in layers I1 and I11 of the postrhinal cortex. (F) Labelling in the outer dorsal zone of the nucleus gelatinosus thalami (arrowhead). (G) Labelling at caudal levels in the medial mammillary nucleus.


88389

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ANTERIOR THALAMUS A MIDLINE THALAMUS

INFRALIMBIC CTXlSEPTAL COMPLEX RETROSPLENIAL CTX.

MAMM.BODY.CAUDOMEDIAL ENTORHINAL CTX.

MAMM.BODY,ROSTROLATERAL o NC. ACCUMBENS

FIG. 5. Schematic drawings of transverse sections through the dorsal subiculum of five representative cases, illustrating the distribution of neurons filled with FG, FB or DY injected in various targets of the subiculum. Arrowheads indicate the borders of the subiculum with the adjacent presubiculum and field CAI. (A) Case 88536 with injections of FG in the anteroventral nucleus of the thalamus and FB in the infralimbic cortex and septal region. (B) Case 88535 with injections of FB in the dorsal subdivision of the lateral septal nucleus and FG in the caudomedial part of the medial mammillary nucleus. (C) Case 88494 with injections of FG in the rostro-lateral part of the medial mammillaly nucleus, DY in the midline of the thalamus involving the interanteromedial nucleus, and FB in the retrosplenial cortex. (D) Case 88522 with injections of FB in the retrosplenial cortex and DY laterally in the entorhinal cortex. (E) Case 88389 with injections of DY in the retrosplenial cortex and FB in the rostro-lateral part of the nucleus accumbens.


The results of the anterograde experiments and of two retrograde cases, in which the septal region and the infralimbic cortex were injected separately (not illustrated), indicate that both structures receive input from proximally located subicular neurons. Based on these observations, we placed large injections of FB in the infralimbic cortex and adjacent septal region in some other animals. In these cases, FB-labelled cells were observed almost exclusively in the proximal part of the dorsal subiculum and in the adjacent part of CAI (Fig. 5A, B). It is clear from these experiments that the proximo-distal position of labelled neurons does not change along the dorso-ventral axis of the subiculum. This conclusion also holds for all other experiments described below. Two of these animals were also successfully injected with FG in one of the target structures of the distal part of the dorsal subiculum. In the first rat, the injection was placed in the anterior thalamic complex, mainly centred in the antero-ventral and antero-dorsal nuclei (exp. 88536). This resulted in a large number of fluorescent cells in the presubiculum, whereas only a few cells were labelled in the adjacent, most distal part of the dorsal subiculum (Fig. 5A). However, as is illustrated in Figure 5A, the largest portion of the distal part of the dorsal subiculum did not contain labelled cells. The other animal received the injection of FG in the caudo-medial part of the mammillary nuclei (exp. 88535; Fig. 5B). In this case, labelled cells were confiied to the distal part of the dorsal subiculum. The distribution of these FG-labelled neurons showed only minor overlap with that of the FB-labelled cells in the proximal part of the dorsal subiculum.

In a third animal an injection of FG involved the rostro-lateral part of the mammillary nuclei, and resulted in retrogradely filled neurons in the proximal part of the dorsal subiculum. No cells were labelled in the distal part of the subiculum nor in CAI (exp. 88494; Fig. 5C). This animal was also successfully injected with DY in the midline thalamus and with FB in the retrosplenial cortex. The distribution of DY-labelled cells showed almost complete overlap with that of the FG- labelled cells. In contrast, FB-labelled cells were confined to the distal part of the dorsal subiculum. A similar distribution of retrogradely labelled cells was observed in two other cases with injections of FB (exp. 88522) and DY (exp. 88393) in the retrosplenial cortex. In case 88522 (Fig. 5D), a second injection of DY was placed laterally in the entorhinal cortex, whereas animal 88393 (Fig. 5E) received an additional injection of FG into the nucleus accumbens. In both cases, the labelled cells in the dorsal subiculum resulting from the second injection were confined to its proximal part. These labelled cells showed a clear spatial separation from the labelled cells in the distal part of the dorsal subiculum labelled after the injections in the retrosplenial cortex.

Discussion The results of the present study confirm previously published observations (Swanson and Cowan, 1977; Meibach and Siegel, 1977a; Kohler, 1985) that the dorsal subiculum of the rat gives rise to projections to the infralimbic, entorhinal, perirhinal, and retrosplenial cortices, the presubiculum, the lateral septal region, the nucleus accumbens, and the mammillary nuclei. Although the anterograde tracing experiments examined by us indicate that the subiculum projects to the anterior complex of the thalamus, the results of our retrograde experiments show that the fierents to the antero-ventral/anterodorsal nuclei predominantly arise from the presubiculum. Only a few cells in the part of the subiculum that directly borders the presubiculum appear to contribute to the projections to the anterior thalamus. As various authors have reported similar observations (Meibach and Siegel, 1977a, b; Swanson and Cowan, 1977;

Donovan and Wyss, 1983), we conclude that the major projections to the anterior complex of the thalamus arise from the presubiculum.

The projections of the dorsal subiculum to the interanteromedial nucleus of the thalamus have been described previously by Meibach and Siegel (1977b), although they termed this cell group the anteromedial nucleus. Interestingly, these authors noted that this termination area is reached by subicular fibres following not only the fornix, but also the internal capsule. The latter fibres enter the thalamus through its dorsal peduncle. Similar routes to the interanteromedial nucleus were found in the present study.

To the best of our knowledge, the presently observed bilateral projections from the subiculum to the nucleus gelatinosus of the thalamus have not been described before. This also holds true for the projection from the dorsal subiculum to the postrhinal cortex, which is an extremely caudo-dorsally located cortical area, at the level where the perirhinal cortex abuts the parasubiculum. Interestingly, in the cat, a cortical area has been described which, among others, has strong connections with the subiculum, and has a similar location between the perirhinal cortex and the parasubiculum as the postrhinal cortex of the rat (see Deacon et al., 1983; Witter et al., 1986; Witter et al., 1989). Regarding the presently observed weak projections to the lateral mammillary nucleus, there appears to be consensus that the source is the presubiculum rather than the subiculum (cf. Allen and Hopkins, 1989; Shibata, 1989).

Without doubt, the most important finding of the present study is that the dorsal subiculum is not homogeneous with respect to the origin of its efferents. The results of our anterograde and retrograde tracing experiments both show that within the dorsal subiculum at least two populations of neurons exist that differ in the distribution of their efferents. Although the efferents of the subiculum and their sites of origin have been described in several reports, only a few indications have been given of the presently observed heterogeneity of the subiculum. Meibach and Siegel (1977a, b) described, in the rat, projections to the septal region originating predominantly from the more proximal portions of the subiculum. They also noted that the fibres to the interanteromedial nucleus of the thalamus (the anteromedial nucleus according to their terminology) arise almost exclusively from the proximal part of the subiculum, whereas the distal part of the subiculum, and, even more prominently, the presubiculum project densely to the antero-ventral nucleus.

With respect to projections from the subiculum to the mammillary nuclei, Meibach and Siegel (1977a) observed that fibres from cells in the proximal part of the subiculum preferentially reach medial parts of the posterior subdivision of the medial mammillary nucleus. More distally located cells project medially also and, in addition, distribute their fibres to lateral parts of the posterior subdivision. Such a topography has not been observed by us. By contrast, our findings indicate that a proximal- to-distal origin of subiculo-mammillary fibres is related to a rostral-to- caudal termination in the medial mammillary nucleus. This is in accordance with the results of a recent study on the organization of afferents to the mammillary nuclei in the rat (Shibata, 1989).

The present findings indicate that within the subiculum different populations of cells exist, each projecting to a different set of targets or to different regions within the target structures of the subiculum. However, it is not yet clear whether the origin of all these projections respect the same organizational principle and thus characterize the same subdivisions of the subiculum. For example, it was presently found that the projections to the entorhinal cortex and the septal region originate in restricted populations of cells that overlap each other along the proximodistal axis of the subiculum (cf. Swanson et al., 1981). Furthermore, both these populations are clearly separated from the cells that give origin to the


projections to the retrosplenial cortex. In contrast, the origins of the subicular projections to the mammillary nuclei are arranged in a gradient- like pattern.

Do the presently observed hodological differences within the subiculum reflect the presence of functional subunits comparable, for example, to a columnar organization known for the n m r t e x ? Although this question cannot yet be answered, it is of interest to note that the projections to the subiculum from the entorhinal cortex and field CA 1 support a parhtion of the subiculum along its proximodistal axis (Tamamaki et af., 1987; Witter and Jorritsma-Byham, in preparation). In conclusion, we suggest that the presently observed heterogeneity in the efferents of the dorsal subiculum along its proximodistal axis represents a general organizational feature of this hippocampal field. It should be kept in mind, however, that this hodologically based heterogeneity is not reflected in any known way in cytoarchitectonic or chemoarchitectonic differences within the dorsal subiculum.

Acknowledgements We would like to thank Ms Barbara Jomtsma-Byham for her excellent assistance with the preparation of many of the retrograde cases, Mr Dirk de Jong for help with the illustrations, and Dr Tony Lohman for critically reading the manuscript and for his helpful suggestions.

Abbreviations ac Acb AD AM AV CAI DG DMD DMSO DY EC FB f FG G hf IAM IL LSD LSI LSV MD MM MP mP Pas PHA-L PRC hS PVA Rh Re RSpl S sm SUM TT VMH

anterior commissure nucleus accumbens anterodorsal nucleus of the thalamus antero-medial nucleus of the thalamus antero-ventral nucleus of the thalamus subfield of the Ammon’s horn dentate gyrus dorso-medial nucleus of the hypothalamus dimethyl sulphoxide diamidino yellow entorhinal cortex fast blue fornix fluorogold nucleus gelatinosus hippocampal fissure interanteromedial nucleus of the thalamus infralimbic cortex dorsal subdivision of the lateral septum intermediate subdivision of the lateral septum ventral subdivision of the lateral septum medio-dorsal nucleus of the thalamus medial mammillary nucleus posterior subdivision of the medial mammillary nucleus mammillary peduncle parasubiculum Phaseolus vulgaris-leucoagglutinin postrhinal cortex presubiculum paraventricular nucleus of the thalamus rhomboid nucleus nucleus reuniens retrosplenial cortex subiculum stria medullaris supramammillary nucleus tenia tecta ventro-medial nucleus of the hypothalamus

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