A cytoarchitectonic study of the hippocampal formation of the tree shrew (Tupaia belangeri)

Journal of Chemical Neuroanatomy 26 (2003) 1 /15 www.elsevier.com/locate/jchemneu

A cytoarchitectonic study of the hippocampal formation of the tree shrew (Tupaia belangeri) Jeanine I.H. Keuker a,*, Christian D.P. Rochford a,1, Menno P. Witter b, Eberhard Fuchs a a

Clinical Neurobiology Laboratory, German Primate Center, Kellnerweg 4, Go¨ttingen 37077, Germany Department of Anatomy, Vrije Universiteit Medical Center, 1081 BT Amsterdam, The Netherlands

b

Received 26 November 2002; received in revised form 19 March 2003; accepted 19 March 2003

Abstract Tree shrews constitute an interesting animal model to study the impact of stress or aging on the hippocampal formation, a brain structure known to be affected under such environmental or internal influences. To perform detailed investigations of the hippocampal formation, adequate knowledge of its anatomy should be present. Until now, the hippocampal formation of the tree shrew has not yet been studied extensively. The main objective of this study, therefore, was to describe the subfield boundaries in various levels of the dorsoventral hippocampal axis of the tree shrew (Tupaia belangeri ) in detail. The secondary aim was to clarify whether a separate CA2 field can actually be distinguished in the tree shrew hippocampus, a fact that was denied in former reports. In addition, we aimed at investigating whether or not a CA4 subfield can be identified in the tree shrew’s hippocampus. The immunocytochemical distribution of microtubule-associated protein 2 and the calcium-binding proteins, parvalbumin and calbindin, and the characteristics of Nissl staining in adjacent sections were compared. Because of the rather dorsoventral orientation of the long hippocampal axis in tree shrews, staining patterns were analyzed mainly in horizontal sections. The subiculum and the hippocampal CA1 and CA3 areas were easily identified. Moreover, we were able to demonstrate the existence of a distinct CA2 subfield in the tree shrew’s Ammon’s horn, contrary to previous reports. However, our results indicate that a CA4 field in the tree shrew hippocampal formation cannot be identified with the methods that we used. Therefore, supposed CA4 pyramidal neurons should be included into the CA3 field. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Hippocampus; Neuroanatomy; Immunocytochemistry; Calcium-binding proteins; MAP2; Scandentia

1. Introduction The hippocampal formation2 is a brain structure that shows pronounced structural and morphological changes in response to environmental changes or altered internal states such as stress exposure or aging (Kerr et al., 1991; Fuchs et al., 1995; McEwen, 1999; McKittrick et al., 2000). To study the neurobiological effects of stress or aging on the hippocampal formation, it is

* Corresponding author. Tel.:
/49-551-3851134; fax:
/49-5513851307. E-mail address: [email protected] (J.H. Keuker). 1 Present address: Department of Neuroimmunology, European Neuroscience Institute, Go¨ttingen 37073, Germany. 2 The term hippocampal formation includes the subiculum, the hippocampus (or hippocampus proper), and the dentate gyrus.

necessary to have adequate knowledge of its anatomy. In the past, a great number of reports have described the apparent macro- and microscopical structure of the hippocampal formation. Such studies focused mainly on the rat (Seress, 1988, 1992; Amaral and Witter, 1989, 1995; Woodhams et al., 1993; Witter, 1993), the nonhuman primate (Rosene and Van Hoesen, 1987; Seress, 1988, 1992; Frahm and Zilles, 1994; Alonso and Amaral, 1995), and human hippocampal formation (Braak, 1972; Seress, 1988; Amaral and Insausti, 1990; Insausti, 1993; Lim et al., 1997). The ultimate goal of experimental neurobiological research is to understand the mechanisms of neurological disease in humans. Thus, a particular species as an experimental model should closely mimic pathological processes in man. Among others, the tree shrew may be

0891-0618/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0891-0618(03)00030-9

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an interesting species to study the effects of stress or aging on the hippocampal formation. Originally regarded as primitive primates (Le Gross-Clark, 1956), tree shrews are nowadays considered as an intermediate between insectivores and primates and are placed in the separate order Scandentia (Starck, 1978; Martin, 1990). In many characteristics, tree shrews are closer to primates than are rodents. The high degree of genetic homology between tree shrews and primates found for several receptor proteins of neuromodulators and the amyloid-b precursor protein (Meyer et al., 1998, 2000; Palchaudhuri et al., 1998, 1999; Pawlik et al., 1999) raises the possibility for tree shrews to become an alternative model for studying stress- or age-related brain changes in socially homogenous and stable cohorts (Michaelis et al., 2001). The lifespan of the day-active tree shrews in captivity is in the range of 10 years (Fuchs, 1999). In the wild, adult male tree shrews display an intense territoriality that can be used to establish a naturally occurring stressful situation under experimental control in the laboratory (Fuchs and Flu¨gge, 2002; Van Kampen et al., 2002). Animals exposed to stress show an atrophy of hippocampal CA3 pyramidal neurons (Magarin˜os et al., 1996), reduced neurogenesis in the dentate gyrus (Gould et al., 1997; Czeh et al., 2001), and lowered hippocampal apoptosis (Lucassen et al., 2001). Although total hippocampal volume was found to be decreased, the volume of the dentate gyrus was stable (Czeh et al., 2001). To be able to study which hippocampal subfield causes stressinduced hippocampal shrinkage, detailed anatomical knowledge is required. A few studies deal in detail with the hippocampal formation and compare volumetric aspects of the hippocampal formation between species like the rat, hedgehog, and various primate species, including the marmoset monkey and man (West and Schwerdtfeger, 1985; West, 1990; Frahm and Zilles, 1994). Among mammalian species investigated, the hippocampal formation of the tree shrew is less well studied. Although a stereotaxic brain atlas of the tree shrew (Tupaia glis ) provides the anatomy of the complete brain (Tigges and Shantha, 1969), the hippocampal formation is not described in great detail. These studies, therefore, are most probably not sufficient to enable a clear definition of the hippocampal subfields throughout the entire hippocampal formation of the tree shrew. The first and main objective of the present study was to perform a detailed cytoarchitectonic analysis of the anatomy of the tree shrew (Tupaia belangeri ) hippocampal formation by comparing immunocytochemical distributions of the calcium-binding proteins parvalbumin (PV) and calbindin (CB), as well as microtubuleassociated protein 2 (MAP2) in coronal and horizontal sections. We aimed at describing the anatomy in such detail, that the results from this study may well be used

for volumetric or stereological studies, in which it is important to use constant and reproducible hippocampal subfield borders (Keuker et al., 2001). The second objective was to use the combined immunocytochemical stainings to clarify whether a separate CA2 subfield can actually be distinguished in the tree shrew hippocampus, a fact that was denied in former reports (Schwerdtfeger, 1984; West and Schwerdtfeger, 1985). Finally, we were interested in whether or not a CA4 subfield can be defined, which is true for primates, including humans (Lorente de No´, 1934; Rosene and Van Hoesen, 1987; Amaral and Insausti, 1990), but less certain in rodents (Amaral and Witter, 1995). In the present study, we used the term hippocampal formation for the subiculum, hippocampus (or hippocampus proper, Cornu Ammonis, or Ammon’s horn), and dentate gyrus. Nevertheless, we want to point out that different authors include varying components in the term hippocampal formation. Amaral and Insausti (1990) and Amaral and Witter (1995), for example, also included the presubiculum, parasubiculum, and the entorhinal cortex, because all these components are linked by unique and largely unidirectional projections. We excluded these fields from the term hippocampal formation in the current study. A major justification for the exclusion is the controversy whether the pre- and parasubiculum have more than three layers and the fact of six layers in the entorhinal cortex. Therefore, in accordance with Rosene and Van Hoesen (1987), our definition of the hippocampal formation comprises only the three-layered regions (a single neuronal layer with fiber or plexiform layers above and below the cell layer).

2. Materials and methods 2.1. Experimental animals Three adult female tree shrews (T. belangeri ) with a body weight of 9/200 g were used from the breeding colony at the German Primate Center (Go¨ttingen, Germany; for details about housing, see Fuchs, 1999). All animal experimentations were conducted in accordance with ‘‘Principles of Laboratory Animal Care’’ (NIH publication No. 86-23, revised 1985) and the European Communities Council Directive of November 24, 1986 (86/EEC), and were approved by the Government of Lower Saxony, Germany. 2.2. Perfusion and tissue preparation The animals were terminally anaesthetized by intraperitoneal administration of an overdose of ketamine (Ketavet† , Pharmacia and UpJohn, Erlangen, Germany), xylazine (Rompun† , Bayer, Leverkusen, Germany), and atropine (WDT, Hannover, Germany) in the

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ratio 5:1:0.01. The descending aorta was clamped and the animals were perfused transcardially with cold 0.9% NaCl for 5 min, followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2, for 22 min. To prevent the development of post-perfusion artifacts (Cammermeyer, 1978), the heads were post-fixed in fresh fixative at 4 8C. On the following day, the brains were gently removed and stored overnight in 0.1 M PB at 4 8C. To cryoprotect the brains, they were immersed in 2% DMSO and 20% glycerol in 0.125 M phosphatebuffered saline (PBS) at 4 8C. The hemispheres were then dissected into blocks that contained the entire hippocampal formation, frozen on dry ice, and stored at /80 8C before serial cryosectioning at a thickness of 50 mm. A stereotaxic brain atlas of the tree shrew (Tigges and Shantha, 1969) was used for reference during the dissecting and cryosectioning procedures. A total of 10 horizontal or 8 coronal series were collected per hemisphere and stored in 0.1 M PBS until staining. 2.3. Staining procedures For the immunocytochemical procedures, the freefloating sections were washed in 0.1 M PBS and then treated with 0.5% H2O2 for 30 min. After washing, nonspecific binding of antibodies, resulting in high background staining, was prevented by incubating the sections for 1 h with 5% normal goat serum (NGS; DAKO, Glostrup, Denmark) in 0.1 M PBS containing 0.5% triton-X-100. The sections were subsequently incubated with the primary antibodies. In the case of the mouse monoclonal primary antibody against PV (Sigma-Aldrich), the sections were incubated for 40 h at 4 8C in 0.1 M PBS containing 0.5% triton-X-100 and 3% NGS. The anti-PV antibody was used at a working dilution of 1:2000. The incubation period used for the mouse monoclonal primary antibodies against the calcium-binding protein CB (Sigma-Aldrich) and the cytoskeletal protein MAP2 (Sigma-Aldrich) was 18 h at 4 8C, with working dilutions of 1:500 and 1:1000, respectively. After incubation with the primary antibodies, the sections were washed thoroughly with 0.1 M PBS and incubated with biotinylated goat anti-mouse antibody (DAKO), 1:200 in 0.1 M PBS with 3% NGS and 0.5% triton-X-100, for 1.5 h, followed by washing in 0.1 M PBS. Subsequently, the sections were incubated with 1:200 horseradish peroxidase-conjugated streptavidin (DAKO) in 0.1 M PBS with 3% NGS and 0.5% triton-X-100 for 1.5 h. After washing, the sections were stained with a DAB kit (Vector Laboratories, Burlingame, CA), which employs 3,3?-diaminobenzidine (DAB) as a chromogen. The exposure to DAB was 5 min for all sections. The reaction was stopped by washing the sections in 0.1 M PBS. Sections were mounted on glass slides in a 0.1% gelatin solution and dried overnight at 37 8C, after which they were cleared

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in xylene for 30 min and finally coverslipped with Eukitt. Series of adjacent horizontal and coronal sections were also mounted on glass slides, dried overnight, and stained with cresyl violet for a clear comparison with the immunocytochemical images. Staining patterns in the dentate gyrus, the hippocampus, and in the subiculum were analyzed and photographed using a Zeiss Axiophot II photomicroscope (Carl Zeiss). The original negative films were scanned at original size with 1200 dpi resolution. Adobe Photoshop software was used for minor darkness/brightness adjustments. The sharpness of the images was slightly increased. The pictures did not require any modification, for e.g. artifacts.

3. Results Macroscopically, the tree shrew hippocampal formation is a C-shaped structure, which is situated underneath the neocortex (Fig. 1). The long axis of the tree shrew hippocampal formation is tilted ventrorostrally at an angle of approximately 708 when compared to the rat (Schwerdtfeger, 1984). This difference brings about that, in coronal sections from rostral to caudal, the rat hippocampal formation first appears in the dorsal part (Schwerdtfeger, 1984), whereas in tree shrews the hippocampal formation starts to come into view ventrally. It is not our objective to elaborate the phylogenetic characteristics of the hippocampal formation, because other authors have done so extensively (see Schwerdtfeger, 1984; Rosene and Van Hoesen, 1987). However, it is noteworthy to mention that rostral coronal sections of the ventrotemporal pole of the tree shrew hippocampal formation display a similar appearance as such sections of the marmoset hippocampal formation (see Figs. 12 and 13 in Schwerdtfeger, 1984). Because the angle of the long axis of the tree shrew hippocampal formation is almost vertical, not all subfields are encountered in every coronal section. For example, the CA2 subfield is actually encountered only in levels A 4.5 through A 2.5 in the tree shrew atlas (Tigges and Shantha, 1969; not shown), whereas the complete hippocampal formation spans levels A 5.0 /A 1.0. For better and more precise analyses of a hippocampal formation with a rather vertical orientation, it is recommended that the brain is sectioned horizontally. For this reason, we will show images from horizontal sections. In the tree shrew, the dorsal two-thirds of serial horizontal sections show a classical representation of the hippocampal formation. The axis of the very rostroventral part of the tree shrew hippocampal formation is close to horizontal (see Fig. 1), so that the arrangement of the hippocampal subfields in the ventral third of the

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Fig. 1. Line drawing of the tree shrew brain showing the general orientation of the hippocampal formation. (A) In the lateral view, the hippocampal formation (gray) is shown for the left hemisphere. In the ventral view, the outline of the ventral appearance of the hippocampal formation (gray) is shown for both hemispheres. The dashed lines indicate that the hippocampal formation is covered by the cortex. (B) The horizontal levels of Figs. 5 / 9 are drawn in the left hippocampus. Scale bar/1 cm. Abbreviations: MAP2, microtubule-associated protein 2; PV, parvalbumin; CB, calbindin; C, caudal direction; L, lateral direction; M, medial direction; R, rostral direction; sub, subiculum; CA1, CA1 subfield of the hippocampus; CA2, CA2 subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; DG, dentate gyrus; hil, hilus of the dentate gyrus; gcl, granule cell layer of the dentate gyrus; mol, molecular layer of the dentate gyrus; h.f., hippocampal fissure; str or, stratum oriens; str pyr, stratum pyramidale; str rad, stratum radiatum; str luc, stratum lucidum; str l-m, stratum lacunosum-moleculare.

horizontal sections may be confusing at first sight. However, if one is to follow a horizontal series of sections from dorsal to ventral, the subfields can be traced with more ease. Fig. 1 illustrates the position of the tree shrew hippocampal formation within the brain and shows the levels of the horizontal sections in Figs. 5/9. We chose to demonstrate more levels through the ventral than the dorsal and middle hippocampal formation, since the subfield positions in the ventral hippocampal formation are subjected to a relatively strong shifting when compared to the middle and dorsal parts of the hippocampal formation. The denominations of all neuron- and dendritecontaining layers in the hippocampal formation are depicted once in Fig. 6B. The ‘‘stratum pyramidale’’ contains the cell layer with the principal neurons of the hippocampus. Deep to this layer, that is distant from the hippocampal fissure, is the ‘‘stratum oriens’’ that con-

tains the basal dendrites of the pyramidal neurons. The apical dendrites are located on the other side of the pyramidal cells in the ‘‘stratum radiatum’’. Bordering the hippocampal fissure is the ‘‘stratum lacunosummoleculare’’ that contains the most distal parts of the apical dendrites. The ‘‘granule cell layer’’ of the dentate gyrus contains the somata of the principal cells, i.e. the granule cells. Their dendrites extend into the ‘‘molecular layer’’ of the dentate gyrus, facing the hippocampal fissure and the ventricle of the temporal lobe. 3.1. Detailed description of subfield boundaries in the tree shrew hippocampal formation 3.1.1. Subiculum/CA1 border As seen in a Nissl stain, the subiculum consists of a broad cell band of pyramidal neurons, whereas the CA1 pyramidal layer appears narrow (Fig. 2A). In the CA1 region, the deeper edge of the cell layer is more diffuse

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Fig. 2. Detailed images of the border between subiculum and CA1 in horizontal sections from the tree shrew hippocampus (arrows, ¡/). (A) The Nissl staining shows dispersed neurons in the subiculum, whereas the CA1 cells are arranged in a narrow band. Some neurons in the CA1 seem to have migrated into the stratum oriens (arrowhead, ). (B) MAP2 immunocytochemistry reveals the dendritic structure. The main dendritic shafts in the subiculum are larger than in CA1 (arrowhead, ). (C) PV immunocytochemistry shows dispersed interneurons and diffuse fibers in the subiculum, but a dense fiber plexus around the pyramidal cell layer in CA1 (arrowhead, ). Interneurons in the CA1 are observed mainly in the stratum oriens and stratum radiatum, closely to the pyramidal cell layer. (D) CB immunocytochemistry reveals CA1 pyramidal neurons to be positive, whereas only at the subiculum/CA1 border, principal subicular neurons are positive. The remaining subiculum bears some CB-positive interneurons (arrowhead, ) and leaves the cell bodies of the other subicular pyramidal neurons blank. Scale bar/200 mm. For abbreviations see Fig. 1.

than the superficial edge (Fig. 2A). This feature of a less strictly bordered CA1 layer is not so clear in the rat. In nonhuman primates, however, CA1 neurons do have a tendency of a looser appearance toward the stratum oriens. This widening of the CA1 cell layer is present to such an extent that the CA1 region in nonhuman primates almost resembles the subiculum in nonprimates such as rats and tree shrews. The stratum oriens in CA1 is no longer present in the subiculum. As seen in rats and primates (Rosene and Van Hoesen, 1987; Amaral and Witter, 1995), the CA1 stratum radiatum disappears in the tree shrew subiculum. Instead, it is replaced by the wide molecular layer of the subiculum. When observing the cytoskeletal protein MAP2 (Fig. 2B), the main dendritic shafts of subicular neurons are longer than in CA1 and they are not as uniformly arranged as in CA1. In the subiculum, PV-positive interneurons are present scattered throughout the pyramidal layer in equal overall density as in the CA1 subfield. However, in the CA1, in contrast to the subiculum, the PV-positive interneur-

ons are mostly located just above and below the pyramidal cell layer, and in the stratum oriens. The CA1 pyramidal layer is characterized by a quite compact PV-immunoreactive fiber plexus, with long dendrites extending into the strata oriens and radiatum. In the latter hippocampal layer, the orientation of most dendrites is perpendicular to the pyramidal cell layer and some of these dendrites have a recognizable beaded appearance. The dendrites sometimes extend into the deeper layer of the stratum lacunosum-moleculare, whereas the superficial part of the stratum lacunosummoleculare does not contain any PV-positive dendrites. The pattern of PV-immunopositive fibers in the pyramidal cell layer of the subiculum is rather diffuse, yet relatively intense. In the molecular layer of the subiculum, the density of PV-positive fibers is lower (Fig. 2C). The CA1 pyramidal neurons are rather strongly immunoreactive for CB, whereas the subicular principal cells are CB-negative. Cells at the border of the subiculum and the CA1 are strongly CB-immunoreac-

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tive and the fibers of such neurons stain more intensively for CB than in the rest of CA1 (Fig. 2D). Furthermore, CB is expressed in a subset of interneurons, which are stained with diverging intensity, and may be encountered in small numbers throughout the subiculum, all hippocampal subfields, and the dentate gyrus. 3.1.2. CA1/CA2/CA3 borders The CA1 subfield in the tree shrew, compared to the CA2 and CA3 regions, is characterized by a very thin, tight layer of pyramidal neurons. As seen in rodents and primates, CA1 neurons in tree shrews tend to be smaller than CA3 neurons. When following the CA1 region towards the CA3 area, most horizontal sections show an apparent ‘‘drop’’ of pyramidal cells towards the hippocampal fissure (Fig. 3A). This point is the beginning of the CA2 region. In addition, this change in position of the neurons is also recognized in immunostainings for MAP2, PV, and to a lesser extent for CB. The main dendritic shafts in the CA1 and CA3 regions, as seen in the MAP2 staining, are neatly aligned. In the CA2

region, however, a lesser degree of organized orientation is encountered (Fig. 3B). By manipulating the focus in MAP2-stained sections at high magnification, thick thorns that are typical for CA3 neuronal shafts, but not for CA2 and CA1 neurons, can be observed as fine lines parallel to the pyramidal cell layer (not shown). Especially remarkable for the CA2 region is the lack of the well-organized layered PV fiber plexus around the pyramidal neurons in CA1 and CA3 (Fig. 3C). Yet the CA2 region contains, compared to CA1 and CA3 subfields, a high number of PV-positive interneurons and accompanying fibers and fiber terminals in the strata pyramidale and radiatum. The density of PVpositive fibers that run through the stratum radiatum and that extend into the stratum lacunosum-moleculare is markedly higher in CA2 and CA3 than in CA1. This feature is also observed in macaque monkeys, but not in rats. The CB antibody visualizes pyramidal cells in CA1, but not CA3 pyramidal cells. As in the rat and macaque

Fig. 3. Characteristic features of the hippocampal CA2 boundaries to the CA1 and CA3 subfields in horizontal sections from the tree shrew (arrows, ¡/). (A) Nissl-stained pyramidal neurons of the CA2 region seem to drop toward the stratum radiatum. The area of the mossy fibers is occupied with small-scattered glia cells (arrowheads, ). (B) MAP2 reveals the dendritic structure, but spares the immunonegative somata and confirms the ‘‘drop’’ seen in the Nissl staining. (C) The stratum lucidum is readily observed in PV immunocytochemistry (arrowheads, ). The pyramidal cells in CA2 are missing the characteristically organized plexus as seen in the pyramidal layer in CA1 and CA3. Moreover, the CA2 region contains more PV interneurons and associated fibers (between open arrows, ). (D) The CA2 region stained for CB shows a mixture of mossy fiber endings (arrowheads, ) and light CB-positive pyramidal neurons (open arrows, ). Scale bar/200 mm. For abbreviations see Fig. 1.

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monkey CA2 region, however, some lightly CB-immunoreactive principal neurons are spread throughout the entire portion of the CA2 region (Fig. 3D). The mossy fibers, existing of axons coming from the granule cells of the dentate gyrus, are also immunoreactive for CB and they extend halfway into the CA2 subfield. The area where these mossy fibers run can be recognized in a Nissl stain by the presence of a high number of small, scattered glia cells (Fig. 3A) and in the PV staining by a lighter band (Fig. 3C); this zone in the CA3 area is also called ‘‘stratum lucidum’’. 3.1.3. CA3/dentate gyrus border The CA3 field of the pyramidal cell layer ends between the blades of the dentate gyrus, opposing the hilus (Fig. 4A). At the very tip of CA3, the principal cells seem slightly dispersed. It may be argued that these

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neurons belong to CA4, as Lorente de No´ (1934) first described. Also, Schwerdtfeger (1984) shows a picture of the ending of the pyramidal cell layer and dentate gyrus area, and classifies a separate CA4 in the tree shrew, although his description is not very thorough. In primates, including man, a CA4 region can be discerned by the fact that, relative to the CA3 pyramidal layer, the CA4 cell layer makes a bend to turn back on the CA3 area (Rosene and Van Hoesen, 1987; Amaral and Insausti, 1990). However, because of the lack of strong anatomical features in the tree shrew, and in concert with Amaral and Witter (1995) and Amaral and Insausti (1990), we would rather avoid the term ‘‘CA4’’ and instead include these neurons in the CA3 region. Also, the immunostainings for MAP2, PV, and CB do not provide unique attributes to discriminate a separate CA4 subfield (Figs. 4B /D).

Fig. 4. Detailed images of the end of CA3, the hilus of the dentate gyrus, and the dentate gyrus granule cell layer in horizontal sections. (A) The CA3 pyramidal layer ends in between the blades of the dentate gyrus. At the extremity (arrowheads, ) the pyramidal neurons seem diffuse. The neurons in the hilus (bordered by the black line) are rather large, and scattered, without the organized orientation as in the CA3 region. The granule cells of the dentate gyrus are small and densely packed. (B) MAP2 immunostaining exposes identical parallel arrangements of the main dendritic shafts of CA3 neurons, whereas the hilus is devoid of such. The dendrites of the granule cells are thick and straight within the granule cell layer, and divide and spread in the molecular layer. (C) The layered arrangement of the PV-positive plexus in CA3 is vanishing toward the end of the pyramidal layer. The hilus contains several PV-reactive interneurons that seem to concentrate at the border between the hilus and the granule cell layer of the dentate gyrus. The granule cells of the dentate gyrus are heavily innervated from such interneurons. The plexus is consolidated especially at the upper and lower borders of the cell layer. (D) The granule cells of the dentate gyrus are completely immunopositive for CB. The somata are intensely stained, whereas their dendrites are moderately marked. The axons of the granule cells are stained as intense as the dendrites, and run through the hilus toward the CA3 pyramidal cells as the mossy fiber bundle. Scale bar/200 mm. For abbreviations see Fig. 1.

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Fig. 5. Horizontal sections (top view) through the left tree shrew hippocampal formation at the most dorsal level. Quite obvious in dorsal levels of the hippocampal formation is the CA2 subfield, which shows an apparent ‘‘drop’’ of pyramidal neurons in the Nissl staining (A), and a heavy PVpositive plexus (C). (A) Nissl staining. (B) Schematic drawing of hippocampal subfields, exactly matching the Nissl section in (A), with indication of coordinates. (C) PV immunocytochemistry. (D) CB immunocytochemistry. Scale bar/1 mm. For abbreviations see Fig. 1.

The hilus belongs to the dentate gyrus, and holds scattered, large polymorphic neurons (Fig. 4A). In the hilus, the parallel orientation of the dendrites, as seen in the CA3 region, is entirely missing (Fig. 4B). Apart from polymorphic neurons, the hilus is also characterized by a moderate number of PV-positive interneurons (Fig. 4C), which are distributed throughout the hilus. Some of these interneurons are aligned at the border of the granule cell layer and the hilus. Furthermore, the hilus is occupied by the mossy fibers that arise from the dentate gyrus granule cells (Fig. 4D). The granule cell layer of the dentate gyrus contains the principal neurons, which are smaller in size than pyramidal neurons of the hippocampus. The densely packed cell layer of the dentate gyrus comprises a stack of approximately 10 granule cells, in contrast to the pyramidal layer of the

hippocampus, which is roughly 3/6 neurons thick. As seen in the MAP2 staining, the granule cells have a main dendritic shaft within the granule cell layer, before the dendrites start to branch and widen into the molecular layer (Fig. 4B). The cell bodies of the granule cells are heavily innervated by PV-positive interneurons (Fig. 4C). Especially, the inner and outer edges of the granule cell layer have many PV-reactive fiber terminals. Besides, some dendrites of PV-positive interneurons spread into the molecular layer (Fig. 4C). Granule cells are immunopositive for CB in their entirety. As mentioned before, their axons can be observed as the mossy fiber bundle, their somata are intensely stained, and their dendrites are stained to a lesser extent (Fig. 4D). All the features of stainings described above in detail can be used to differentiate the various subfields of the

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Fig. 6. Horizontal sections (top view) through the dorsoventral middle of the left tree shrew hippocampal formation. (A, C, and D) Arrows ( ¡/) point to the stratum lucidum, which can be seen throughout the entire hippocampal formation. In the PV staining (C), the stratum lucidum actually appears as a lighter band, which the Nissl staining (A) reveals as a glia-rich area, and which accommodates the CB-positive mossy fibers (D). (B) The hippocampal nomenclature of neuron- and fiber-containing layers. For description of (A) /(D) and scale bar, see legend of Fig. 5. For abbreviations see Fig. 1.

hippocampal formation in all horizontal (and coronal; not shown) levels. 3.2. The dorsoventral axis of the tree shrew hippocampal formation All subfields of the tree shrew hippocampal formation are encountered over a large extent of the dorsoventral axis (Figs. 5 /7). The typical sequence of the subiculum, subfields CA1 /CA3, hilus, and dentate gyrus (Figs. 5 and 6) appears more complex in the ventral part of the hippocampal axis (Fig. 7), but that is the result of its three-dimensional structure. The hippocampal formation may be thought of as a ‘‘tube’’ in which, perpendicular to the tube, the typical subfield sequence is present over the entire extent. The largest part of the tree shrew hippocampus has a dorsoventral orientation, such that horizontal sections through the dorsal two-thirds of the long axis produce a representative orientation of the

subfields. In the ventral part, the hippocampal tube bends horizontally towards the medial temporal pole and ultimately slightly dorsally. This brings about that, when sectioning horizontally, the ventrorostral tip of the curved part of the hippocampal formation appears additional to the transected ‘‘main’’ tube. For this reason, a ‘‘double’’ dentate gyrus is encountered (Fig. 7), that in ventral direction fuses to a circular appearance (Fig. 8) and finally disappears (Fig. 9). The CA3 region, that is partially enclosed between the blades of the dentate gyrus (Fig. 6), becomes discontinuous in horizontal sections (Fig. 7). The CA3 segment that is captured between the ‘‘double’’ dentate gyrus then decreases in size and may appear as a compact circular area of pyramidal neurons (level between Figs. 7 and 8; not shown) surrounded by hilar neurons. Eventually, this CA3 part ceases to appear and only hilar neurons can be observed (Fig. 8). The other part of the CA3 in Fig. 7 is not encountered anymore in Fig. 8. In the level

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Fig. 7. Horizontal sections (top view) through the left tree shrew hippocampal formation at a ventral level, where a ‘‘double’’ dentate gyrus can be observed. Arrows ( ¡/) in (D) point to CB-positive mossy fibers. At the corresponding site in the PV-stained section (C), the stratum lucidum is observed just underneath, i.e. superficial to, the pyramidal cell layer. The inlay in (D) shows the elongated CA2 region at the level between Figs. 7 and 8. For description of (A) /(D) and scale bar, see legend of Fig. 5. Scale bar of inlay in (D) is 200 mm. For abbreviations see Fig. 1.

between Figs. 7 and 8, this particular cell band shows strong characteristics of CA2 (inlay Fig. 7D). Then, in Fig. 8, only the CA1 is transected at this position. The subfields at these transverse ventral levels appear so elongated, because the hippocampal ‘‘tube’’, and therefore the pyramidal cell layer, is curved at this point. Especially helpful for demonstrating this subfield transition is the CB immunocytochemistry. Fig. 7D shows the CB-positive CA1 pyramidal neurons and the mossy fibers in the CA3 region. Further ventrally, between Fig. 7D and Fig. 8D, the part of the pyramidal layer, that is CA3 in Fig. 7D, is designated as CA2, since over the complete extent of this area, moderate mossy fiber input and lightly stained pyramidal neurons are seen (inlay Fig. 7D). Then, in Figs. 8 and 9, no mossy fibers can be recognized anymore, but instead, the region is completely occupied by CB-positive CA1 pyramidal neurons. Over the entire dorsoventral extent of the tree shrew hippocampal formation, we only observed minor differences within certain stainings. The invasion by pyramidal cells in the CA2 into the stratum radiatum became more obvious from ventral to dorsal. This could be noticed not only in the Nissl staining but in PVimmunocytochemistry as well. As for the CB stainings,

we noticed that in any horizontal level, including the ventral level with a relatively long CA2, the mossy fibers partially reached into the lightly stained CA2-type pyramidal neurons, but they never reached towards the beginning of the CA1 region.

4. Discussion In the present study, we used immunocytochemistry for calcium-binding proteins and for MAP2 to describe the boundaries in horizontal sections of the tree shrew hippocampal formation. The calcium-binding proteins analyzed in this study were chosen as they show expression in specific classes of hippocampal neurons. PV is known to be localized in a subpopulation of nonpyramidal neurons in a wide variety of brain regions (Celio, 1990); studies of PV-immunoreactive neurons in the rat and monkey hippocampal formation show its colocalization with markers for GABA-ergic neurotransmission (Nitsch et al., 1990; Pitka¨nen and Amaral, 1993). CB differs from PV in that it is present in glutamatergic neurons, namely granule cells of the dentate gyrus and pyramidal cells of the CA1 and CA2 subfields. Furthermore, CB is present in GABA-

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Fig. 8. Horizontal sections (top view) through the left tree shrew hippocampal formation at a ventral level, where the dentate gyrus appears ringshaped. In this particular level, the dentate gyrus encloses only hilar neurons. Slightly more dorsally, CA3 neurons would come into view in the midst of the hilus. For description of (A) /(D) and scale bar, see legend of Fig. 5. For abbreviations see Fig. 1.

ergic nonpyramidal cells throughout the hippocampus proper (Seress et al., 1991). One of the most striking aspects of CB immunostaining are the strongly immunopositive mossy fibers of the granule cells (Seress et al., 1991, 1993, 1994; Woodhams et al., 1993; Hof et al., 1996; Lim et al., 1997). For MAP2, a cytoskeletal protein, immunostaining of the dentate granule cells and the somatodendritic compartment of pyramidal neurons has been described previously in the tree shrew (Wolter et al., 1999). In addition to immunocytochemical analyses, a Nissl stain was performed to allow for a clear analysis of the pyramidal cell-containing layers. One of the main findings of the current study is the presence of field CA2 in the tree shrew hippocampus. From our data, it appears that the stratum lucidum, i.e. the area occupied by the mossy fibers, extends into the CA2 subfield and does not stop at the CA3/CA2 border. This result is in agreement with previously reported findings in the rat (Woodhams et al., 1993). Therefore, our data are not in line with the general description of CA2 given by Lorente de No´ (1934), who reported one of the characteristics of this region to be the absence of innervation by mossy fibers. Another main finding is the

lack of a clearly definable CA4 region in the tree shrew hippocampal formation. Although a separate CA4 region can be distinguished in the nonhuman primate and human hippocampal formation (Lorente de No´, 1934; Rosene and Van Hoesen, 1987; Amaral and Insausti, 1990), our results from the tree shrew hippocampal formation did not show any region-specific features that would allow a clear definition of subfield CA4 on an anatomical basis. Conflicting or confusing anatomical findings probably rely on falsely used nomenclature. The most commonly used nomenclature for the hippocampal formation of mammals is that introduced by Lorente de No´ (1934) based on Nissl and Golgi stainings of the hippocampal formation in mouse, rhesus monkey, and man. Although Lorente de No´ (1934) divided Ammon’s horn into four fields, CA1/CA4, with fields CA2 and CA3 corresponding to the regio inferior and the CA1 subfield to regio superior, as described earlier by Ramo´n y Cajal (1893), most anatomical studies refer only to the CA1 and CA3 subfields with little or no mention of the CA2 and CA4 subfields. Although not very apparent from histological sections, the subiculum shares the three layers of the

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Fig. 9. Horizontal sections (top view) through the left tree shrew hippocampal formation at most ventral level, where the granule cell layer of the dentate gyrus is no longer present. Dashed line in (B) delineates the molecular layer, which can be observed in the horizontal level of (A), but not (C) and (D). In the ventral tree shrew hippocampal formation, shifting of hippocampal subfields occurs rapidly over a relatively short vertical extent. For this reason, the outline of the molecular layer in the Nissl staining (A) can be observed, but no CB-positive dendrites of granule cells can be seen (D). However, at this level, large blood vessels that run horizontally can be encountered. For description of (A) /(D) and scale bar, see legend of Fig. 5. For abbreviations see Fig. 1.

allocortex that are seen in the hippocampus proper. According to Lorente de No´ (1934), rodents, hedgehogs, cats, dogs, monkeys, and humans have a wide prosubiculum situated between CA1 and subiculum, of which the superficial part of the stratum pyramidale is composed of small, modified ammonic pyramids. Especially in rhesus monkeys and humans, a separate prosubiculum may be distinguished (Rosene and Van Hoesen, 1987). However, Amaral and Witter (1995) recommend not to use the term prosubiculum, at least in the rat hippocampal formation. Many researchers agree in regarding it as an oblique border where the CA1 gradually replaces the subiculum. In an anatomical study on the flying fox hippocampal formation, a separate prosubiculum could neither be detected (Buhl and Dann, 1991). Also, Amaral and Insausti (1990) do not mention a prosubiculum in the human hippocampal formation. In none of the hippocampal sections from the tree shrews, a particular group of small neurons at the end of the stratum radiatum, pointing to a possible prosubiculum, could be observed.

4.1. Tree shrews possess an anatomically defined CA2 subfield Following the description by Lorente de No´ (1934) in mice and macaques, the CA2 subfield consists of large pyramidal cells, similar to those found in the CA3 subfield, but with shafts that are devoid of thorns. Therefore, the dendritic shaft of CA2 pyramidal neurons is thicker than that of CA1 pyramidal neurons. These findings are confirmed for CA2 neurons in Golgistained hippocampal sections of the guinea pig (Bartesaghi and Ravasi, 1999). In rodents and tree shrews, neurons in the CA2 subfield appear to extend superficially from the pyramidal layer. It should be mentioned, however, that this typical feature of the CA2 in a Nissl stain may not easily be observed in rather thin sections. The slight spreading of CA2 neurons superficially from the pyramidal layer is not as easily recognized in primates as in rodents, because the CA2 pyramidal layer is more compact and narrower than the CA1 and CA3 regions (Amaral and Insausti, 1990). A

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further feature of CA2 neurons described by Lorente de No´ (1934) was the lacking of mossy fiber input, and many studies agree with this definition (Schwerdtfeger, 1984; Rosene and Van Hoesen, 1987; Amaral and Insausti, 1990; Seress et al., 1991; Amaral and Witter, 1995; Lim et al., 1997). However, there is accumulating evidence that mossy fibers reach into the CA2 field. This has been demonstrated in the temporal rat hippocampus (Woodhams et al., 1993) and the dorsal canine hippocampus (Amayasu et al., 1999). The fact that mossy fibers taper into the CA2 region could have changed a statement of a former investigation on the tree shrew hippocampal formation (West and Schwerdtfeger, 1985). This study employed both Timm’s and Nissl staining techniques in different subsets of sections and reported the inability to define a separate CA2 subfield in the tree shrew hippocampal formation (West and Schwerdtfeger, 1985). Pyramidal neurons of the CA2 region have been demonstrated to have unique features that clearly distinguish it from the CA1 and CA3 fields. These include dense PV-positive terminals, high levels of acetylcholinesterase, the presence of pericellular nets of matrix, and basic fibroblast growth factor immunoreactivity (Woodhams et al., 1993). Because it is quite difficult to inject tracers into the small CA2 region, not much is known about the efferent projections. Also, retrograde tracing studies have not given much information about CA2 afferents. However, it has been shown that CA2 pyramidal neurons receive input from the hypothalamus and tubero- and supramammilary nuclei (Woodhams et al., 1993), indicating a role in processing information of the physiological state of the animal, in cortical activation and in behavioral arousal. Although the CA2 function is still not clear, this subfield deserves more attention, since CA2 pyramidal neurons are spared in pathologies such as Alzheimer’s disease and temporal lobe epilepsy (Leranth and Ribak, 1991; Woodhams et al., 1993). The resistance of CA2 to epileptic damage has been linked to the presence of high levels of calcium-binding proteins in this field (Leranth and Ribak, 1991). However, the CA2 region of schizophrenic and manic depressive patients may be more vulnerable, since a decrease in density of nonpyramidal neurons in CA2 was reported (Benes et al., 1998). Altogether, the CA2 region should not merely be considered as a transition zone between the CA1 and CA3 regions, but should be regarded as a separate cytoarchitectonic field that deserves more investigation.

part of the pyramidal cell layer that reaches the hilus, bends on itself, first upwards and then downwards, constituting two blades which more or less run parallel to the granule cell layer of the dentate gyrus. However, in the present study, in sections that were perpendicular to the hippocampal axis, this bending was not observed in the tree shrew. Lorente de No´ (1934) yet stated that the development of the field CA4 varies along the hippocampal axis, but even more so in different mammals. In the primate hippocampal formation, a distinct hippocampal CA4 subfield can be recognized with relative certainty (Lorente de No´, 1934; Rosene and Van Hoesen, 1987; Amaral and Insausti, 1990). In lower mammalian species, it is harder to do so. For example, Rosene and Van Hoesen (1987) defined in the rat hippocampus a cluster of neurons at the tip of the CA3 region as the CA4 area. However, such a cluster may not always be clearly observed. In fact, as Amaral and Witter (1995) describe for the rat hippocampal formation, the polymorphic neurons in the hilus of the dentate gyrus may easily be confused with neurons of the pyramidal cell layer of the hippocampus. Amaral and Insausti (1990) indicate that Lorente de No´ (1934) may have designated in his drawings polymorphic hilar neurons as comprising the CA4 region. Information about the CA4 region in tree shrews is very limited. In a study with shrews, tree shrews, and primates, Schwerdtfeger (1984) suggests that the CA4 region has evolved from a zone of scattered cells in shrews to a moderate dense cell band in primates. Schwerdtfeger (1984) is, however, unclear about the tree shrew CA4 region. Unfortunately, the figures from Schwerdtfeger’s publication do not provide sufficient information to understand whether or not the author correctly used the term CA4 for the corresponding pyramidal cells. CA4 pyramidal cells and polymorphic hilar neurons are shown to have dissimilar connections. Only polymorphic neurons give rise to dentate gyrus association and commissural projections, and CA4 neurons project out of the hippocampal formation to the septal area (Rosene and Van Hoesen, 1987; Amaral and Insausti, 1990; Amaral and Witter, 1995). To avoid confusion, yet depending on the type of study, it is better to discard the term CA4 and include the pyramidal cells between the dentate gyrus blades in the CA3 region.

4.2. Tree shrews do not possess an anatomically definable CA4 subfield

The hippocampal formation is known to play an important role in learning and memory processes (Squire and Zola-Morgan, 1991; Eichenbaum and Otto, 1992). To improve the knowledge of causal mechanisms of memory decline in humans, e.g. after stress exposure or during aging, we need adequate

According to Lorente de No´ (1934), the CA4 region can be recognized in mouse, rabbit, monkey, and human sections perpendicular to the hippocampal axis, as that

5. Conclusions

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animal models that mimic the stress-derived or agerelated neuropathophysiological mechanisms in humans. Tree shrews have been shown to constitute a valuable animal model to investigate neurobiological changes after stressful experiences (Fuchs and Flu¨gge, 2002; Van Kampen et al., 2002). Experiments with the chronic psychosocial stress paradigm showed, for example, that there was a tendency of hippocampal volume loss (Ohl et al., 2000; Czeh et al., 2001). Localized proton magnetic resonance spectroscopy revealed stress-related decreased concentrations of N -actetyl-aspartate (NAA), creatine and phosphocreatine, and choline-containing compounds in the tree shrew forebrain, which included the hippocampal formation (Czeh et al., 2001). Furthermore, exposure to acute and chronic psychosocial stress resulted in a decreased number of BrdU-labeled cells in the dentate gyrus (Gould et al., 1997; Czeh et al., 2001). Also, chronic psychosocial stress specifically increased the number of apoptotic cells in the hilus, whereas a decreased number of apoptotic cells was demonstrated in the CA1 stratum radiatum (Lucassen et al., 2001). In addition, apical dendrites of CA3 pyramidal neurons from chronically stressed tree shrews had a decreased number of branch points and reduced total dendritic length as compared with controls (Magarin˜os et al., 1996). However, stereological evaluations gave evidence of preserved neuronal numbers in hippocampal subfields CA1 and CA3 of stressed tree shrews (VollmannHonsdorf et al., 1997; Keuker et al., 2001). Most of the above-mentioned stress-related changes in the hippocampal formation are shown to be subfieldspecific. To understand what mechanisms in which subfields contribute to the observed general changes in the hippocampal formation, it is most important that adequate knowledge about the hippocampal cytoarchitecture exists. The present description yields clear boundary definitions to conduct subfield-specific neuroanatomical studies on the hippocampal formation in the tree shrew.

Acknowledgements This work was partially supported by the Graduate School ‘‘Perspectives of Primatology’’ of the German Science Foundation (DFG).

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