New boundaries and dissociation of the mouse hippocampus along the dorsal‐ventral axis based on glutamatergic, GABAergic and catecholaminergic receptor densities

In rodents, gene‐expression, neuronal tuning, connectivity and neurogenesis studies have postulated that the dorsal, the intermediate and the ventral hippocampal formation (HF) are distinct entities. These findings are underpinned by behavioral studies showing a dissociable role of dorsal and ventral HF in learning, memory, stress and emotional processing. However, up to now, the molecular basis of such differences in relation to discrete boundaries is largely unknown. Therefore, we analyzed binding site densities for glutamatergic AMPA, NMDA, kainate and mGluR2/3, GABAergic GABAA (including benzodiazepine binding sites), GABAB, dopaminergic D1/5 and noradrenergic α1 and α2 receptors as key modulators for signal transmission in hippocampal functions, using quantitative in vitro receptor autoradiography along the dorsal‐ventral axis of the mouse HF. Beside general different receptor profiles of the dentate gyrus (DG) and Cornu Ammonis fields (CA1, CA2, CA3, CA4/hilus), we detected substantial differences between dorsal, intermediate and ventral subdivisions and individual layers for all investigated receptor types, except GABAB. For example, striking higher densities of α2 receptors were detected in the ventral DG, while the dorsal DG possesses higher numbers of kainate, NMDA, GABAA and D1/5 receptors. CA1 dorsal and intermediate subdivisions showed higher AMPA, NMDA, mGluR2/3, GABAA, D1/5 receptors, while kainate receptors are higher expressed in ventral CA1, and noradrenergic α1 and α2 receptors in the intermediate region of CA1. CA2 dorsal was distinguished by higher kainate, α1 and α2 receptors in the intermediate region, while CA3 showed a more complex dissociation. Our findings resulted not only in a clear segmentation of the mouse hippocampus along the dorsal‐ventral axis, but also provides insights into the neurochemical basis and likely associated physiological processes in hippocampal functions. Therein, the presented data has a high impact for future studies modeling and investigating dorsal, intermediate and ventral hippocampal dysfunction in relation to neurodegenerative diseases or psychiatric disorders.


| INTRODUCTION
Since the first introduction of the term hippocampus and its anatomical description in the 16th century of Arantius (1564), the hippocampus became one of the most studied brain regions for neuroscientists of all fields (Andersen, 2007). Cytoarchitectonically, the hippocampus consists of the dentate gyrus (DG) and the Cornu Ammonis fields (CA1-4) (Amaral, 1978;Nó, 1934;Ramon y Cajal, 1893;Ramón y Cajal, 1911). Together with the subiculum and the entorhinal cortex, all of these structures belong to the hippocampal formation (HF) (Andersen, 2007;Witter & Amaral, 2004).
The HF is extensively connected to other cortical and subcortical areas that are involved in processing and controlling cognitive functions and plays a prominent role in memory processing, learning, spatial navigation and anxiety and fear (Bannerman et al., 2002;Bannerman et al., 2004;Moser & Moser, 1998;Saxe et al., 2006;van Strien, Cappaert, & Witter, 2009). With increasing evidence for a different distribution of several functions along the dorsal-ventral (septo-temporal) axis of the HF, researchers suggested further separations, that is, that the dorsal hippocampus (posterior in primates) is involved in spatial navigation and memory, whereas the ventral hippocampus (anterior in primates) mediates more anxiety and fear-related responses (Anacker & Hen, 2017;Bannerman et al., 2004;H. W. Dong, Swanson, Chen, Fanselow, & Toga, 2009;Fanselow & Dong, 2010;Kheirbek et al., 2013;Moser & Moser, 1998;Muzzio et al., 2009;Plachti et al., 2019;Strange, Witter, Lein, & Moser, 2014). Up to now, it seems to be that there is no clear cut and a common functional specialization scheme along the dorsal-ventral axis of the mammalian HF is still a matter of debate, very likely depending on different parameters like intrinsic and extrinsic connectivity patterns, neurochemical systems, cell types, adult neurogenesis, vulnerability to ischemia, and discrete genetic domains (Anacker & Hen, 2017;Bienkowski et al., 2018;Cembrowski, Wang, Sugino, Shields, & Spruston, 2016;Fanselow & Dong, 2010).
Due to the increasing amount of functional studies that take dorsal-ventral differences with more or less precise anatomical borders in the hippocampus into account, we set out to study the receptor densities of glutamatergic, GABAergic, dopaminergic and noradrenergic receptors along the dorsal-ventral axis with autoradiography in combination with a detailed cyto-and myelo-architecture analysis in serial coronal sections of the mouse hippocampus. In addition, we analyzed the detailed receptor architecture in the different layers of dorsal, intermediate and ventral subregions, which has further functional implications to understand the effects of neurotransmitter receptor modulation on synaptic activity depending on different cell types and the position at cellular structures. Thus, we mapped different neurochemical functional domains in the mouse hippocampus, integrated the findings in current maps of dorsal, intermediate and ventral subregions and included these borders and domains into an atlas scheme to provide a robust parcellation for future functional studies.

| Animals and tissue preparation
We used 10, adult male C57BL/6 mice that were obtained from CERJ (Janvier Labs, Germany). The number of animals was chosen due to consideration of statistical power as well as previous anatomical/ future functional studies conducting similar analysis. After arrival, animals were housed in groups (five/cage) in an enriched environment, under constant room temperature and humidity control in a 12-hour light/dark cycle for 8 weeks. Food pellets and water were ad libitum. All procedures were in accordance with the German law to protect animals and the guidelines of the "Landesamt für Natur, Umwelt und Verbraucherschutz NRW, Germany (LANUV)" as well as with the guidelines of the European Council Directive 2010/63/EU. For the histological procedures and the receptor autoradiography, mice were decapitated, brains were removed from the skull and frozen in isopentane at −40 C. At this timepoint all mice were at the age of 27 weeks (5 month) and weighed 28-33 g. Unfixed frozen tissue was stored at −80 C until sectioning.
For the receptor binding studies the following binding sites were labelled: α-amino-3-hydroxy-5-methyl-4-isoxalone propionic 2. During the main incubation step binding sites were labeled with the respective tritiated ligand (total binding), or co-incubated with the tritiated ligand and a 1,000-10,000-fold excess of specific nonlabeled ligand (displacer) determined non-displaceable, and thus, non-specific binding. Specific binding is the difference between total and non-specific binding. It was less than 5% in all cases. 4. Radioactively labeled sections were air-dried overnight and coexposed with plastic [ 3 H]-standards (Microscales, Amersham, UK) of known radioactivity concentrations against tritium-sensitive films (Hyperfilm, Amersham, UK) for 4-18 weeks.

| Image analysis
The resulting autoradiographs were processed using densitometry with a video-based image analyzing technique . Autoradiographs were digitized by means of a KS-400 image analyzing system (Kontron, Germany) connected to a CCD camera (Sony, Japan) equipped with a S-Orthoplanar 60-mm macro lens (Zeiss, Germany). The images were stored as binary files with a resolution of 512 x 512 pixels and 8-bit gray value. The gray value images of the co-exposed microscales were used to compute a calibration curve by nonlinear, least-squares fitting, which defined the relation- where K D is the equilibrium dissociation constant of ligand-binding kinetics, L is the incubation concentration of ligand, and A S the specific activity of the ligand. The results of these calculations were used for binding site density measurements. The digitized autoradiographic images were color-coded to facilitate the detection of regional differences in binding site densities by visual inspection.
Thereby a color bar encodes the receptor density in fmol/mg protein.

| Anatomical identification
The borders and the subregions of the HF were anatomically identified based on our cyto-, myelo-and receptorarchitectonic data (Figures 1-3) in series of sections in the mouse brain and previous cytoarchitectural, connectional and genetic maps from the Allen Brain Atlas (Hong Wei Dong, 2008;Lein et al., 2007), the Franklin and Paxinos atlas (Franklin & Paxinos, 2008), the comparative cytoarchitectonic atlas of the C57BL/6 and 120/Sv mouse brains (Hof, Young, Bloom, & Belichenko, 2000), Thompson and colleagues T A B L E 1 Neurotransmitter receptor densities (fmol/mg protein) in different subregions of the mouse hippocampus (Mean ± SEM) and results of the Friedman ANOVA displaying regional differences for all regions for each receptor type (all N = 10, df = 4)  Dong et al., 2009) and the HGEA of Bienkowski and colleagues (Bienkowski et al., 2018).  differences in receptor densities due to adjusting optimal density color coding for each area and each receptor type in series of sections for each mouse brain, differences in the myeloarchitecture and according to a set of individual references of stereotactic coordinates including different parameters in terms of measurements (Bienkowski et al., 2018;H. W. Dong et al., 2009;Fanselow & Dong, 2010;Lein et al., 2007;Strange et al., 2014;Thompson et al., 2008  into a receptor concentration per unit protein, that is, fmol/mg protein . The receptor density values of each subregion of the HF was then calculated over the sampled brain levels from each animal, averaged across the 10 animals, and is reported as the overall receptor concentration (mean ± SEM in fmol/mg protein). Quantitative, multi-receptor data is provided in Tables 1,2 and separately presented in the color-coded autoradiographs (Figures 4, 6, 8), histograms (Figures 5,7,9) as well as in the regional-and laminar specific receptor fingerprints that are prepared as polar plots ( Figure 10).
Thereby, the shape of the fingerprint defines the individual receptor density profiles for each hippocampal subregion and provides a detailed and comparable overview of specific outlines in receptor densities for each layer in dorsal, ventral and intermediate subregions.
Additionally, heat maps of receptor denseties for all subregions are provided in the supplements (Figures S1-S3). Further (but see also discussion), we could not confirm an iDG based on our cyto-, myelo-and receptor analysis in the coronal sections of our study, although we carefully inspected and analyzed the most caudal DG. This may have methodological reasons, but based on current published data in mice (for example Bienkowski et al., 2018: partly no distinction between iDG and iCA3, few concrete data on differences between dDG, iDG and vDG), we think that future studies have to clarify if an iDG in mice can be repeatedly and separately identified with a greater set of variables including more precise functional (for example, lesions or inactivation and behavioral tests) and/or electrophysiological data.
Additionally, we may speculate that on the basis of the development, evolution, position, connectivity and size of the hippocampus, species-specific differences may also play a role, including differences between mice and rats but also other mammals that may have led to a different functionally relevant compartmentalization of the hippocampus, that is, dentate gyrus.

| Statistics
To investigate the chemoarchitectural differences between the different main areas of the HF and between dorsal, ventral and intermediate areas, first a Friedman ANOVA across all subregions for each ligand was applied. If significant, this was followed by Wilcoxon-rank tests for pair-wise comparisons between subdivisions. For the general statistical analyses, Statistica 10 (StatSoft, Tulsa, RRID:SCR_015627) was used. The significance level was set at .05.

| RESULTS
We analysed the receptor-architecture of the different subdivisions of the HF in the mouse brain with a focus on dorsal-ventral differences The results of the Friedman ANOVA display the regional differences for all subregions for each receptor type (all N = 10, df = 9). ***p < .001. The DG is a three-layered C-shaped structure within the HF consisting of the principle granule cell layer (sg), containing densely packed granule cells, nicely resolved in Nissl-stained sections, an overlaying molecular layer (mo) and a subjacent polymorph layer of cells (po; Figures 1-3, first column). The surrounding structure of the HF is formed by the alveus that is particularly visible in myelin-stained sections reflecting the outermost subependymal fiber layer of the HF (Figures 1-3, second column). The hippocampal fields are formed by a characteristic cellular layer, the stratum pyramidale (sp), clearly visible in Nissl-stained tissues (Figures 1-3 Strange et al., 2014;Thompson et al., 2008), and our new determined borders in the dorsal-ventral axis based on a highly significant data set for the analyzed receptor classes are described below. Therefore, first, we will briefly outline the general differences in receptor densities in the HF and most important, along the dorsal-ventral axis. Second, we will present the detailed analysis of specific layers in this context. The combined analysis of all data was integrated into the presented atlas scheme (Figures 1-3, fourth column).

Glutamatergic receptors
The glutamatergic receptors were individually expressed at different density levels in specific subdivisions (Tables 1 and 2 -p, 5b and S2).

GABAergic receptors
While the two types of GABA A receptor bindings sites tested separated the subdivisions of the HF, GABA B receptors showed an equal distribution in all subdivisions (Tables 1 and 2). The highest amounts of GABA A receptors were detected in CA1 followed by decreasing amounts in CA2, CA4/hilus, DG and CA3 (Figures 6a-d, 7a and S1).
Benzodiazepine binding sites showed only a slightly different picture.
Here, the highest densities were detected in CA1 and CA2 that were distinguished from CA3 and DG, with the lowest amounts in CA4/hilus (Figures 6e-h, 7a and S1). Along the dorsal-ventral axis, the density of GABA A receptors was three times higher in DGd compared to DGv (Figures 6a-d, 7b and S2). A stepwise decrease in densities was observed from CA1d to CA1i to CA1v, while both, CA3d and CA3i showed higher densities compared to CA3v (Figures 6a-d, 7b and S2). GABA A(BZ) densities differed between CA1d, CA1i and CA1v and showed a 1.5-fold higher density in CA1d compared to CA1v. In addition, GABA A(BZ) receptors were higher expressed in CA3i compared to CA3v (Figures 6e-h, 7b and S2). Further, none of the GABA A receptors showed detectable differences between CA2d and CA2i.

Catecholaminergic receptors
In comparison to the densities of glutamatergic and GABAergic receptors, catecholaminergic receptors were relatively low expressed in the HF (Tables 1 and 2). Thereby, densities of noradrenergic α 1 and dopaminergic D 1/5 showed similar values, while for example, noradrenergic α 2 receptor densities were five times higher expressed in CA1 if compared to α 1 and D 1/5 levels. Further, the highest densities of α 1 receptors were detected in CA1, while the lowest were found in CA4/hilus (Figures 8a-d, 9a and S1). The α 2 receptors were also high expressed in CA1 and showed a stepwise decrease to CA2 to CA3 to CA4/hilus.  from previous studies (Bienkowski et al., 2018;Dong et al., 2009;Lein, Zhao, & Gage, 2004;Schultz & Engelhardt, 2014;Thompson et al., 2008;Zhao et al., 2001), our results provide a high-resolution scheme of neurochemically discrete structures that can be used for future hippocampal structural and functional studies. Here, our analyses showed a specific regional receptor distribution along the dorsal-ventral axis of the hippocampus that adds to the multiscale nature of the different subdivisions.
Up to now, the Hilus/CA4 region of the mouse hippocampus is often omitted in anatomical studies, and if presented, sometimes referred to as hilar region of the DG or included into the CA3 region, while functional studies differentiate hilar neurons from the rest of the DG (Danielson et al., 2017;Scharfman & Myers, 2012;Swaminathan, Wichert, Schmitz, & Maier, 2018;Tong et al., 2015;S. Zhao, Chai, Forster, & Frotscher, 2004). Here we detected overall substantial differences in the receptor architecture of the Hilus/CA4 region compared to the neighbouring CA3 and DG. Therefore, the combined analysis of receptor densities and the inspection of the cellular and fiber architecture resulted into a delineation of the Hilus/CA4 region and the inclusion of a small Hilus/CA4 subdivision as an individual region in the presented atlas. Based on our analysis we would argue that CA4/hilus is a transitional region between DG and CA3, with an individual chemoarchitecture that reflects its functional diversification. This would be further in line with the high amount of different cell types in this region (Amaral, 1978;Nó, 1934;Ramón y Cajal, 1911;Scharfman & Myers, 2012) and again matches the multi-receptor profile of human CA4 (Palomero-Gallagher et al., 2020). Besides, recent evidence further supports the idea to think of the hippocampus as a bilateral structure with functional specializations of subfields in either the right or left hemisphere that are accompanied by the asymmetrical presence of neurotransmitter receptor subunit compositions in these subfields and strata (Jordan, 2020;Kawakami et al., 2003;Shinohara et al., 2008;Shipton et al., 2014). Therefore, we cannot exclude that an additional lateralization of the chemoarchitecture of hippocampal subfields per se and in the dorsal-ventral axis exists, which was not in the focus of this study, but should be considered for future studies.

| Neurochemical differences in dorsal, ventral and intermediate hippocampus
Beyond the more multi-receptor differences in the mouse hippocampus per se, the analysis of receptor densities provided a comprehensive view of neurotransmitter targets at the protein level in order to clarify subdivision-or sublayer specific density patterns in dorsal, intermediate and ventral hippocampus. Here, our data not only complements gene expression studies (Bienkowski et al., 2018;Cembrowski et al., 2016;H. W. Dong et al., 2009;Lein et al., 2007; F I G U R E 1 0 Legend on next coloumn.  Thompson et al., 2008), but shows neurochemical heterogeneity in line with different functional connectivity along the dorsal-ventral axis (Anacker & Hen, 2017;Bannerman et al., 2014;Bast, Wilson, Witter, & Morris, 2009;Kheirbek et al., 2013;Lee, Kim, Cho, Kim, & Park, 2017;Moser & Moser, 1998;Strange et al., 2014). Further, our findings of different as well as indifferent (co-) distributions of specific neurotransmitter receptors along the dorsal-ventral axis may offer a link between the dichotomy of functionally segregated subfields with precise borders on the one hand and a more gradual organization of processing functions on the other and show that both views are not exclusive (Bast et al., 2009;Brun et al., 2008;Lee, Rao, & Knierim, 2004;Leutgeb, Leutgeb, Moser, & Moser, 2005;Leutgeb, Leutgeb, Moser, & Moser, 2007;McHugh et al., 2007;Neunuebel & Knierim, 2014;Strange et al., 2014;Vogel et al., 2020). Despite ubiquitous glutamatergic and GABAergic inputs to all hippocampal subdivisions Freund & Buzsáki, 1996;Klausberger, 2009;Klausberger & Somogyi, 2008 (Pandis et al., 2006). However, we detected no differences for AMPA receptor densities between DGd and DGv, and higher amounts in CA3i/v, which is in line with an earlier receptor binding study in rats (Martens et al., 1998). In addition, we detected lower levels of kainate receptors in DGv and higher levels in CA1v, CA2i and CA3v, while Martens et al. (1998) observed no differences along the septotemporal axis. Beside advanced methods and more precise maps used in this study, it is possible that species differences between mice and rats had led to these discrepancies. Further, mGlu 2/3 receptors, which serve as autoreceptors at glutamatergic terminals (Shigemoto et al., 1997), showed lower levels in CA1v and higher levels in CA3v, which we think has not been reported yet but is in line with different treatment effects in these areas due to differ- and CA3 of rats (Sotiriou, Papatheodoropoulos, & Angelatou, 2005).
Additionally, we found a stepwise decrease from CA1d to CA1i to CA1v of both, GABA A receptor and GABA A receptor benzodiazepine binding sites.
Further, noradrenergic α 1 and α 2 , as well as dopaminergic D 1/5 receptors showed different densities along the dorsal-ventral axis along with the reported source inputs implying a stronger input to ventral hippocampal areas (Edelmann & Lessmann, 2018;Gasbarri et al., 1997;Gasbarri, Packard, Campana, & Pacitti, 1994;Haring & Davis, 1985;Verney et al., 1985). Here, in line with higher noradrena-  in Fremeau Jr. et al. (1991) but showed no overall differences if compared to ventral (Fremeau Jr. et al., 1991), while other studies reported equal or higher levels in the ventral CA regions via in situ hybridization of D 1 or D 5 receptor genes or binding to D 1/5 (Edelmann & Lessmann, 2018;Fremeau Jr. et al., 1991;Gangarossa et al., 2012;Khan et al., 2000;Lazarov, Schmidt, Wanner, & Pilgrim, 1998;Wei et al., 2018). Despite these controversies, stimulation of D 1/5 receptors in dorsal hippocampus has been shown to promote spatial learning and memory (Kempadoo et al., 2016), and recently different functional aspects of D 1/5 receptors in the dorsal hippocampus with respect to novelty and memory consolidation in line with the different inputs from the VTA and the LC have been discussed (Duszkiewicz et al., 2019).
Overall, we could not establish a border between DGd, DGi, and DGv based on our receptor data in the mouse hippocampus compared to the recent HGEA atlas (Bienkowski et al., 2018) and therefore divided the DG only in a DGd and DGv. Although we inspected the most posterior hippocampal slices at the point the DG blades merge, based on our analysis of the receptor architecture in serial frontal sections, no differences were observed, and the DGi of the study from Bienkowski and colleagues is mostly included into the dorsal DG. Nevertheless, herein, our data is in line with respect to a septotemporal organization of inputs to the DG, without considering differences in the internal fiber network organization of the DG mentioned in the HGEA atlas or a strict dissociation based on stereotaxic coordinates. This may also imply that the DG is functionally more gradually organized, which future studies may investigate in more detail.

| Specific receptor profiles in layers of dorsal, intermediate and ventral hippocampus
The different multi-receptor maps for the dorsal, intermediate and ventral CA subdivisions were accompanied by specific receptor density peaks in the hippocampal layers and co-distributions of receptors, which is in line with the heterogeneity of pyramidal cell types and associated functions, for example in place field modulation, along the dorsal-ventral axis in CA3 and CA1 found by comprehensive mapping of genomic-wide in situ hybridization data and electrophysiological recordings (Cembrowski et al., 2016;Cembrowski & Spruston, 2019;Danielson et al., 2017;Dong et al., 2009;Hunt, Linaro, Si, Romani, & Spruston, 2018;Komorowski et al., 2013;Thompson et al., 2008).
Here, the different glutamatergic receptors revealed an individual and specific pattern for each receptor type, with high levels of AMPA receptors in spCA1d/I and spCA3i/v, Kainate receptors in slmCA1i, soCA1v, slmCA1v, slmCA2i and sluCA3 with overall higher levels in CA3i/v in all layers, NMDA receptors in srCA1d and srCA3i and mGlu 2/3 receptors in slmCA1d/i, slmCA3d but overall higher levels in all layers of CA3v, which is in line with the observed selectivity for neuronal targets (Buhl & Whittington, 2007;Nilssen et al., 2019).
GABAergic GABA A receptors showed a more homogenously distribution pattern in all layers of CA1d and peaked only in srCA3i/v. Noradrenergic α 1 -receptors were enhanced in all layers of CA1v/i and CA2i and peaked in srCA3i/v, while α 2 -receptors specifically peaked in slmCA1i and slmCA2i and soCA3v. Dopaminergic D 1/5 receptors were highly distributed in all layers of CA1d that differed from the observed peak in slmCA3d. Co-distribution of high levels were detected in srCA3i/v for α 1 , GABA A and NMDA receptors, in slmCA3d for D 1/5 and mGlu 2/3 , in slmCA1i for α 2 , kainate and mGlu 2/3 receptors and in slmCA2i for α 2 and kainate receptors. Therefore, we would argue, that the discriminative pattern of individual layers can be further used to define different hippocampal components in line with specific cellular properties for future functional studies.
In DG, noradrenergic α 2 -receptors showed the highest density in all layers of DGv, while the DGd exhibited the highest receptor concentrations in the moDGd and in particular in kainate, NMDA, GABA A and D 1/5 receptors if compared to ventral layers. The moDG is a relative cell-free layer occupied by dendrites of dentate granule cells, fibres of the perforant path originating in the entorhinal cortex and a small number of interneurons, while sgDG mainly contains granule cells and poDG includes different cell types, but the most prominent is the mossy cell . Here we can assume that α 2 -receptors play a pivotal role in modulating the functions of the ventral DG, particularly with respect to the noradrenergic modulation of stress-response and depression related behavioral measures that are further linked to adult neurogenesis and disturbed pattern separation in the ventral DG (Anacker et al., 2018;Anacker & Hen, 2017;Hu et al., 2007). Vice versa, the co-occurrence of high NMDA, GABA A and D 1/5 receptor levels in moDGd may point to a more complex dissociation. Both, NMDA and GABA A receptors can interact with D 1/5 receptor, and this interaction can shape the signal transfer along dendrites by potentiation as well as depression of currents via different mechanisms (Duszkiewicz et al., 2019;Lee et al., 2002;Liu et al., 2000;Varela, Hirsch, Chapman, Leverich, & Greene, 2009;Yang, 2000). Future functional and electrophysiological studies as well as detailed cell type analysis and further co-expression analysis may target this complex dissociation.

| Conclusion
The present study provides a detailed neurochemical receptor map of the mouse hippocampus in the dorsal-ventral axis. Therefore, the combination of the neurochemical profile and cytoarchitectural analysis resulted in a refined parcelling of the mouse HF based on quantitative molecular measurements. This allowed us not only to specify borders between different subdivisions along the dorsal-ventral axis but also to show that CA2 in mice exists as an independent area with a dorsal and an intermediate component.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest in the manuscript.

DATA AVAILABILITY STATEMENT
Data availability statement: The data that support the findings of this study is available in the main text, tables and figures.