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. 2019 Oct 10;528(4):523–541. doi: 10.1002/cne.24773

Differential expression of secretagogin immunostaining in the hippocampal formation and the entorhinal and perirhinal cortices of humans, rats, and mice

Silvia Tapia‐González 1,2,3, Ricardo Insausti 4, Javier DeFelipe 1,2,3,✉
PMCID: PMC6972606  PMID: 31512254

Abstract

Secretagogin (SCGN) is a recently discovered calcium‐binding protein belonging to the group of EF‐hand calcium‐binding proteins. SCGN immunostaining has been described in various regions of the human, rat and mouse brain. In these studies, it has been reported that, in general, the patterns of SCGN staining differ between rodents and human brains. These differences have been interpreted as uncovering phylogenetic differences in SCGN expression. Nevertheless, an important aspect that is not usually taken into account is that different methods are used for obtaining and processing brain tissue coming from humans and experimental animals. This is a critical issue since it has been shown that post‐mortem time delay and the method of fixation (i.e., perfused vs. nonperfused brains) may influence the results of the immunostaining. Thus, it is not clear whether differences found in comparative studies with the human brain are simply due to technical factors or species‐specific differences. In the present study, we analyzed the pattern of SCGN immunostaining in the adult human hippocampal formation (DG, CA1, CA2, CA3, subiculum, presubiculum, and parasubiculum) as well as in the entorhinal and perirhinal cortices. This pattern of immunostaining was compared with rat and mouse that were fixed either by perfusion or immersion and with different post‐mortem time delays (up to 5 hr) to mimic the way the human brain tissue is usually processed. We found a number of clear similarities and differences in the pattern of labeling among the human, rat, and mouse in these brain regions as well as between the different brain regions examined within each species. These differences were not due to the fixation.

Keywords: comparative studies; entorhinal cortex; hippocampal formation; human, rat and mouse brains; immunostaining; perirhinal cortex; post‐mortem; RRID:AB_1079874; RRID:AB_10807945; secretagogin

Graphical abstract showing secretagogin immunostaining pattern in various regions of the human, rat, and mouse brain. Clear similarities and differences are found in the pattern of labeling among the human, rat, and mouse in the hippocampal formation and entorhinal and perirhinal cortices, as well as between the different brain regions examined within each species.

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1. INTRODUCTION

Secretagogin (SCGN) is a recently discovered calcium‐binding protein belonging to the group of EF‐hand Ca2+‐binding proteins (Archer & Wagner, 2000). As reported by Wagner et al. (2000), sequence analysis shows that SCGN has six Ca2+‐binding loops with typical EF‐hand tandem repeats and has marked homology to the Ca2+‐binding proteins calbindin D‐28k and calretinin, which also have six EF‐hand motifs. However, the distribution in the brain of calbindin D‐28k‐, calretinin‐, and SCGN‐immunoreactive (‐ir) neurons and neuropil immunostaining is different (e.g., Arellano, Munoz, Ballesteros‐Yanez, Sola, & DeFelipe, 2004; Barinka et al., 2012; Cho et al., 2011; Miettinen, Pitkanen, & Miettinen, 1997; Seress et al., 1993; Wouterlood, van Denderen, van Haeften, & Witter, 2000). SCGN has been less intensively studied (particularly in the human brain) than the other calcium‐binding proteins, but it has been described in various regions of the human, rat, and mouse brain (e.g., Gartner et al., 2001; Kosaka & Kosaka, 2013; Mulder, Bjorling, et al., 2009; Mulder et al., 2010; Mulder, Zilberter, et al., 2009; Wagner et al., 2000).

These studies reported that, in general, the patterns of SCGN‐immunoreactivity differ between rodents and human brains, although few detailed comparative studies have been carried out. It seems that SCGN labeling identifies certain neuronal subtypes and might be used as a marker to delineate particular brain structures, as well as to identify hierarchical organizing principles. Therefore, the study of SCGN‐immunoreactivity in the brain is of great interest. For example, in a recent study in the mouse brainstem, few SCGN‐ir cells were found, whereas numerous SCGN‐ir neurons were observed in the rat brainstem (Kosaka & Kosaka, 2018). Another study reported that a subpopulation of parvalbumin immunoreactive interneurons coexpressed SCGN in the dorsal striatum of rats, but not in mice (Garas et al., 2016). Furthermore, it has been shown that SCGN is expressed by cholinergic neurons in embryonic basal forebrain and neuroblasts in the rostral migratory stream in gray mouse lemurs, but not in mice. There are also differences in SCGN immunostaining during development in the granule cell layer of the dentate gyrus (DG) and pyramidal cell layer of CA1–CA3 fields between gray mouse lemurs and mice (Mulder et al., 2010; Mulder, Zilberter, et al., 2009). These differences have been interpreted by Mulder et al. as uncovering phylogenetic differences in SCGN expression. More recently, it has also been reported that SCGN is expressed by developing neocortical GABAergic neurons in humans but not in mice (Raju, Spatazza, Stanco, Sorrells, & Kelley, 2017). In the human brain, SCGN immunostaining has been described in both cerebellar basket and stellate cells and in neurons of temporal, frontal, hippocampal, and hypothalamic regions (Gartner et al., 2001). In addition, Attems et al. (2007) reported that SCGN‐immunoreactivity in the human hippocampus was restricted to a subset of pyramidal neurons in subiculum, CA1, CA2, CA3, and CA4 and that these cells were mostly unaffected by tau pathology in patients with Alzheimer's disease (Attems et al., 2008).

Nevertheless, an important aspect that is not usually taken into account is that different methods are used for obtaining and processing brain tissue coming from humans and experimental animals. Indeed, it has been shown that post‐mortem time (PT) delay and the method of fixation (i.e., perfused vs. nonperfused brains) may influence the results of the immunostaining (e.g., see Gonzalez‐Riano et al., 2017; Lavenex, Lavenex, Bennett, & Amaral, 2009). Thus, it is not clear whether differences found in comparative studies with the human brain are simply due to technical factors or species‐specific differences. In the present study, we analyzed the pattern of SCGN immunostaining in the adult human hippocampal formation compared with rat and mouse that were fixed either by perfusion or immersion and with different PT delays (up to 5 hr) to mimic the way that the human brain tissue is usually processed. We focused on the characterization of the pattern of SCGN‐immunoreativity in different hippocampal fields (DG, CA1, CA2, CA3, subiculum, presubiculum, and parasubiculum) as well as in the entorhinal (EC) and perirhinal (PRC) cortices of the three species. We found a number of clear similarities and differences in the pattern of labeling among the human, rat, and mouse in these brain regions as well as between the different brain regions examined within each species. These differences were not due to the fixation.

2. MATERIALS AND METHODS

2.1. Humans

Human brain tissue was obtained at autopsy from the Unidad Asociada Neuromax—Laboratorio de Neuroanatomía Humana, Facultad de Medicina, Universidad de Castilla‐La Mancha, Albacete, and the Laboratorio Cajal de Circuitos Corticales UPM‐CSIC, Madrid, Spain. The tissue was obtained following national laws and international ethical and technical guidelines on the use of human samples for biomedical research purposes. In this study, we used samples of human brain tissue from 3 control human brains (subjects with no recorded neurological or psychiatric alterations): 2 males, aged 45 (AB1) and 53 (AB3) and 1 female, aged 53 (AB2). The PT between death and brain fixation varied between 1.5 and 4 hr (AB1: 1.5 hr; AB3: 3.5 hr; and AB2: 4 hr PT). Tissue from human brain AB1 and AB2 has been used in a previous study (Anton‐Fernandez, Aparicio‐Torres, Tapia, DeFelipe, & Munoz, 2017). The cause of death was pleural mesothelioma (case AB1), septic shock of pulmonary origin (case AB2), and metastatic bladder carcinoma (case AB3). Upon removal, the brains (AB2, AB3) were immediately fixed in cold 4% paraformaldehyde (PFA) in 0.1 mol/L, pH 7.4 phosphate buffer (PB), and sectioned into 1.5 cm‐thick coronal slabs. The hippocampus was cut into 1 cm blocks and post‐fixed in the same fixative for 24–48 hr at 4°C. Case AB1 was perfused through both internal carotid arteries <1 hr after death with a saline solution followed by 4% PFA in PB. The brain was then removed and post‐fixed as described above. The tissue blocks were then cryoprotected in 25% sucrose in PB and stored at −20°C in a solution of glycerol, ethylene glycol and PB. Serial hippocampal sections (50 μm) of each case were obtained using a vibratome (Leica VT2100S St. Louis, MO), and processed for histochemical and immunohistochemical experiments. The sections immediately adjacent to those stained immunohistochemically were Nissl‐stained in order to identify the hippocampal fields and the laminar boundaries.

2.2. Animals

This study was performed in 2‐month‐old male C57BL/6 J mice and 3‐month‐old male Wistar albino male rats (Charles River Laboratories, Wilmington, MA). Animals were kept in a 12:12‐hr light/dark cycle and received food and water ad libitum. All experimental protocols involving the use of animals were performed in accordance with recommendations for the proper care and use of laboratory animals, and under the authorization of the regulations and policies governing the care and use of laboratory animals (EU directive no. 86/609 and Council of Europe Convention ETS1 23, EU decree 2001‐486 and Statement of Compliance with Standards for Use of Laboratory Animals by Foreign Institutions no. A5388‐01, National Institutes of Health [USA]). Special care was taken to minimize animal suffering and to reduce the number of animals used to the minimum required for this study.

In order to perform comparative studies between human and rodent species in the present study, we analyzed SCGN‐ir in two groups: one group of mice (n = 4) and another of rats (n = 6). All animals were anesthetized with a pentobarbital lethal injection (40 mg/kg BW, Vetoquinol, Madrid, Spain) and transcardially perfused with a saline solution followed by 4% PFA in PB. The brains were removed from the skull and post‐fixed by immersion in the same fixative for 20 hr at 4°C.

Furthermore, we tested the possible effects of 2 and 5 hr PT on SCGN immunostaining in two groups of rats and two of mice. Rats and mice were sacrificed with the same pentobarbital lethal injection as described above. Thereafter, their brains were removed at 2 and 5 hr PT (n = 2 rats and n = 2 mice, for each time point) as described in our previous study (Gonzalez‐Riano et al., 2017), and fixed in PB‐buffered 4% PFA overnight (20 hr) at 4°C.

After rinsing in PB, the brain tissue from the six groups of animals was cut into 50‐μm‐thick, coronal slices using a vibratome (Leica VT2100S, St. Louis, MO). The brain tissue was processed as described above and then processed for immunohistochemistry.

2.3. Immunohistochemistry

The rabbit anti‐SCGN antibody (RRID:AB_1079874) used in the present study was obtained from Sigma‐Aldrich (see below). This antibody was raised against recombinant human SCGN and has been described, characterized and tested in detail for its specificity by Mulder et al. (Mulder, Zilberter, et al., 2009, Mulder, Bjorling, et al., 2009, Mulder et al., 2010, see also Ellis et al., 2019). Using immunohistochemical techniques with this antibody, SCGN has been detected in the brain of various mammalian species including rat (Mulder, Bjorling, et al., 2009), mouse and mouse lemur (Mulder et al., 2010; Mulder, Zilberter, et al., 2009), ferret (Ellis et al., 2019), and human (Raju et al., 2017). This anti‐SCGN antibody has also been characterized by performing western blot analysis, yielding a single protein band of ~32 kDa in BRIN‐BD11 insulinoma cell lysates (Sanagavarapu, Weiffert, Ni Mhurchu, O'Connell, & Linse, 2016), rat brain homogenates (Mulder, Bjorling, et al., 2009), extracts of different brain areas from mouse brain (Mulder et al., 2010; Mulder, Zilberter, et al., 2009), and ferret whole brain lysates (Ellis et al., 2019). The rabbit anti‐NeuN antibody (RRID:AB_10807945) employed in the present study was obtained from Millipore (see below). It was characterized by the manufacturer by performing immunohistochemistry techniques in human (cerebellum and cerebral cortex) and mouse (hippocampus) brain tissue sections, as well as western blot analyses in mouse and rat brain tissue lysates, observing 2–3 bands in the 46–48 kDa range. Immunohistochemical experiments were carried out in free‐floating sections under moderate shaking. The endogenous peroxidase activity was quenched in a solution of 1.66% hydrogen peroxide in 50% ethanol in PB for 30 min at room temperature. After several washes in 0.1 M phosphate buffer (pH 7.4) containing 0.3% TritonX‐100 (washing buffer), sections were incubated overnight at 4°C with one of the following primary antibodies: anti‐NeuN (ABN78, rabbit polyclonal, Millipore, Billerica, MA; diluted 1:2000) or anti‐SCGN (rabbit polyclonal Sigma‐Aldrich Cat# HPA [Human Protein Atlas Number] 006641, RRID:AB_1079874, St Louis, MO; diluted 1:1000). Primary antibodies were diluted in washing buffer containing 3% normal goat serum. After incubation with the primary antibody, sections were then rinsed in buffer and incubated for 2 hr at room temperature with biotinylated goat anti‐rabbit immunoglobulin G (BA1000, Vector laboratories, Burlingame, CA) diluted 1:250 in washing buffer. After several washes in buffer, sections were incubated for 1 hr at room temperature with avidin–biotin peroxidase complex (ImmunoPure ABC, Pierce, Rockford, IL; diluted 1:125). Peroxidase activity was revealed with 0.01% hydrogen peroxide, using 3,3′‐diaminobenzidine (Sigma, St Louis. MO; 0.05%). Finally, sections were mounted, dehydrated and coverslipped with DEPEX (VWR, Rannor, PA). In addition, other sections were chosen for counterstaining with toluidine blue (Merck, Darmstadt, Germany) with the purpose of identifying hippocampal fields and layers, as well as other brain areas. Observations were made with a digital microscope (BX51, Olympus). Immunostaining was absent when the primary antibody was omitted. All the experimental groups were assayed in parallel.

3. RESULTS

Since small changes visualized using immunohistochemical techniques are typically found from experiment to experiment, even using sections from the same brains, subtle changes are difficult to interpret. Thus, we were only looking for large, obvious differences between species or in different experimental conditions. Therefore, we analyzed qualitatively the pattern of immunostaining for SCGN in the different subdivisions of the head and body portions of the human hippocampus (Figures 1, 2, 3, 4, 5) (DG, CA1, CA2, CA3, subiculum, presubiculum, and parasubiculum) as well as in the EC and PRC cortices, according to the indications of the atlas of Mai, Majtanik, and Paxinos (2016). In rats and mice, we examined these regions from Bregma −2.04 to −6.60 (rat atlas; Paxinos & Watson, 2007) and from Bregma −0.94 to −3.88 (mouse atlas, Paxinos & Franklin, 2001), respectively, (Figures 6, 7, 8, 9, 10). In order to separate the different fields of the hippocampal formation and the boundaries between them, we used an antibody for NeuN, which specifically recognizes a soluble, nuclear, neural vertebrate DNA‐binding protein that is present in the vast majority of mature neurons in both the central and peripheral nervous systems of several vertebrate species, including humans (Mullen, Buck, & Smith, 1992; Sarnat, Nochlin, & Born, 1998; Wolf et al., 1996).

Figure 1.

Figure 1

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Coronal sections of the human hippocampus at the level of the hippocampal body (a–d) and the hippocampal head (e, f). Low‐power photographs from sections immunostained for anti‐NeuN (a, c, e) and anti‐SCGN (b, d, f) from cases AB1 (a, b, e, f) and AB3 (c, d). Note the similar pattern of SCGN immunostaining in the human cases as well as in the different levels of the hippocampus (body and head). The labeled neuropil showed two types of staining (see also Figure 2): a dense brownish diffuse staining (Type I neuropil staining) (solid arrows ), and a light diffuse staining (Type II neuropil staining) (open arrows). The area indicated by a rectangle in (d) is shown at a higher magnification in Figure (3d). alv, alveus; CA1–CA3, hippocampal CA fields; DG, dentate gyrus; EC, entorhinal cortex; f, fimbria; gl, granular layer; ml, molecular layer; Pas, parasubiculum; pl, polymorphic layer; PRC, perirhinal cortex; PrS, presubiculum; slm, stratum lacunosum moleculare; sp, stratum piramidale; sr, stratum radiatum; Sub, subiculum; so, stratum oriens. Scale bar shown in (f) indicates 1,460 μm in (a–d), 1,600 μm in (e), and 1,720 μm in (f) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2.

Figure 2

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Photomicrographs from case AB3 illustrating the different patterns of SCGN immunostaining in the neuropil and neurons of the human hippocampus (a–c). (a) Shows Type I neuropil staining (dense brownish diffuse staining: no individual neuronal processes are distinguished. (b) Shows the three possible staining patterns of the neuropil; Type I neuropil staining in the inner third of the molecular layer (ml); Type II immunostaining (light diffuse staining in which scattered stained neuronal processes are observed) in the polymorphic layer (pl); and no labeling of the neuropil in the granular layer (gl) and outer upper molecular layer (ml). Some Type I (N I) and II (N II) SCGN‐ir neurons are indicated (arrows). (c) Illustrates a lack of staining of the neuropil while several Type I (N I) SCGN‐ir neurons are observed (arrows). Note the nonpyramidal cell morphology of the labeled cells in the subiculum (sub). CA1, CA1 field of the hippocampus; DG, dentate gyrus; gl, granular layer; ml, molecular layer; pl, polymorphic layer; Sub, subiculum. Scale bar shown in (c) indicates 90 μm in all panels [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3.

Figure 3

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Photomicrographs from case AB3 illustrating the differences in the pattern of SCGN staining in the DG, CA1–CA3 (a–c), subiculum and presubiculum (d). Rectangles in (a) indicate the areas of magnification in (b) and (c). Note a clear decrease in SCGN immunostaining of neuropil in the alveus (alv) and in the stratum lacunosum moleculare of CA1 and granular layer (gl) of the DG. By contrast, there is an intense neuropil staining in the pyramidal cell layer in CA1, CA2, and CA3 (a–c), and in the inner third of molecular layer (ml) and polymorphic layer of the DG (c). CA1 shows very few immunostained neurons, whereas some stained neurons (arrows) are present in CA2, CA3, and DG (a–c). (d) Higher magnification of the area indicated by a rectangle in Figure 1d to show the sharp decrease in staining of the neuropil of CA1 close to the subiculum. There is also a virtual lack of immunostaining in the neuropil of the subiculum and presubiculum. alv, alveus; CA1, CA1 field of the hippocampus; CA2, CA2 field of the hippocampus; CA3, CA3 field of the hippocampus; DG, dentate gyrus; gl, granular layer; ml, molecular layer; pl, polymorphic layer; PrS, presubiculum; slm, stratum lacunosum moleculare; sp, stratum piramidale; sr, stratum radiatum; Sub, subiculum; so, stratum oriens. Scale bar shown in (d) indicates 240 μm in (a), 70 μm in (b) and (c), and 340 μm in (d) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4.

Figure 4

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(a, b) Low‐power photographs of sections stained for SCGN (a) and NeuN (b) from case AB3 and higher magnification of SCGN immunostaining of cells in the subiculum, presubiculum, and parasubiculum. Rectangles in (a) indicate the areas of magnification in (c, d) and in Figure 5. (c, d) Higher magnification photomicrographs showing Type II SCGN cells in Layers II–III of the presubiculum (c) and in layer V of the parasubiculum (d). Note the differences in the distribution of Type II SCGN‐ir cells between the layers, with them being more abundant in Layers II–III in presubiculum (c) and in Layer V in parasubiculum (d). A35, area 35; A36, area 36; EC, entorhinal cortex; Pas, parasubiculum; PRC, perirhinal cortex; PrS, presubiculum; Sub, subiculum. Scale bar shown in (d) indicates 2,300 μm in (a) and (b), 100 μm in (c) and (d) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5.

Figure 5

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Higher magnification of the areas indicated by a rectangle in Figure 4a. (a–c) SCGN‐ir cells in the upper layers of the entorhinal cortex (EC) (a); perirhinal cortex‐area 35 (PRC‐A35) (b); and perirhinal cortex‐area 36 (PRC‐A36) (c). (d–f) Labeled neurons in deep layers of EC, PRC‐A35, and PRC‐A36. Note the nonpyramidal cell morphology of the labeled cells. Scale bar shown in (f) indicates 90 μm in all panels [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6.

Figure 6

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Coronal sections of the rat and mouse brain (from perfused brains). Low‐magnification photomicrographs showing differences in the general pattern of SCGN immunostaining of sections from the rat (a, c, e) and mouse (b, d, f) brains. alv, alveus; AHi, amygdalohippocampal area, posteromedial part; APir, amygdalopiriform transition area; BLP, basolateral amygdaloid nucleus, posterior part; CA1, CA1 field of the hippocampus; CA2, CA2 field of the hippocampus; CA3, CA3 field of the hippocampus; DG, dentate gyrus; DS, dorsal subiculum; EC, entorhinal cortex; FC, fasciola cinerea; gl, granular layer; ml, molecular layer; Pas, parasubiculum; pl, polymorphic layer; PP, peripeduncular nucleus; PRC, perirhinal cortex; PrS, presubiculum; SC, superior colliculus; SG, suprageniculate thalamic nucleus; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum; VS, ventral subiculum. Scale bar shown in (f) indicates 490 μm in (a–d) and 670 μm in (e) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 7.

Figure 7

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Differential pattern of SCGN immunostaining in rat (a, c) and mouse (b, d) hippocampal field (from perfused brains). Note that in the rat there are SCGN‐ir cells located in the stratum pyramidale in CA1 (a) and CA2 (c) (arrows). In the mouse (b, d), there are no immunostained neurons in the stratum pyramidale of CA1 (b) but these are present in CA2 (d) (arrow). Furthermore, there is a virtual lack of immunostaining in the DG of the rat (a) in contrast to the mouse (b), where the neuropil is stained as well as the granule cells (arrows). alv, alveus; CA1, CA1 field of the hippocampus; CA2, CA2 field of the hippocampus; CA3, CA3 field of the hippocampus; DG, dentate gyrus; gl, granular layer; ml, molecular layer; pl, polymorphic layer; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum. Scale bar shown in (d) indicates 100 μm in (a, c, and d) and 70 μm in (b) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 8.

Figure 8

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Differential distribution of SCGN immunostaining in the rat and mouse subicular complex (from perfused brains). (a, b) Low‐magnification photomicrographs showing SCGN immunostaining of sections from subicular complex and neocortex of the perfused brains of rat (a) and mouse (b). The areas indicated by rectangles in (a) and (b) indicate the areas of magnification in (c–h). In (c), note the presence of scattered Types I and II SCGN‐ir cells in the deep and superficial pyramidal layers of the dorsal subiculum, whereas—in the ventral subiculum—mostly SCGN‐ir cells are detected, especially close to the boundary with the alveus (d). As shown in (e), the parasubiculum displays Types I and II SCGN‐ir cells, whereas—in the presubiculum—there is labeling of the neuropil with few or no Type II SCGN‐ir cells. Note that in the brain of mouse (b), there is an intense staining of neuropil in the subiculum, presubiculum, and parasubiculum (f–h) and few Type II SCGN‐ir cells located in subiculum, especially at the ventral portion of the hippocampus (f). alv, alveus; CA1, CA1 field of the hippocampus; DS, dorsal subiculum; EC, entorhinal cortex; ml, molecular layer of the dentate gyrus; mlS, molecular layer of the subiculum; Pas, parasubiculum; pl, polymorphic layer; PRC, perirhinal cortex; PrS, presubiculum; SC, superior colliculus; Sub, subiculum; VS, ventral subiculum. Scale bar shown in (h) indicates 580 μm in (a), 460 μm in (b), 110 μm in (c–h) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 9.

Figure 9

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Differential distribution of SCGN immunostaining in rat and mouse brain (from perfused brains). (a, b) Low‐magnification photomicrographs showing SCGN immunostaining of sections to illustrate the differential pattern of immunostaining at the level of the entorhinal and perirhinal cortices in the rat (a) and mouse (b). The areas indicated by rectangles in (a) and (b) indicate the areas of magnification in (c–g). Note that in the rat EC (c, d), SCGN‐ir cells are present in all layers but they are mainly located in Layers V–VI. However, in the EC of the mouse (e), SCGN‐ir cells are virtually absent. In PRC A35‐A36 of the rat (f), there are SCGN‐ir cells located in all layers. However, in the PRC A35‐A36 of the mouse (g), very few SCGN‐ir cells were present. BLP, basolateral amygdaloid nucleus, posterior part; EC, entorhinal cortex; LaDL, lateral amygdaloid nucleus, dorsolateral part; LaVM, lateral amygdaloid nucleus, ventromedial part; PRC, perirhinal cortex. Scale bar shown in (g) indicates 720 μm in (a), 570 μm in (b), and 90 μm in (c–g) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 10.

Figure 10

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Comparison of the patterns of SCGN‐immunoreactivity in the hippocampus of the rat and mouse from brains fixed by perfusion or by immersion. (a–d) Low‐magnification photomicrographs showing the distribution patterns of SCGN‐immunoreactivity in sections through the CA1 and DG of rat brain fixed by perfusion (a and c) or by immersion after 5 hr PT (b, d). (e–h) Low‐magnification photomicrographs showing the distribution patterns of SCGN immunostaining in sections through CA1 and DG of mouse brain fixed by perfusion (e, g) or by immersion after 5 hr PT (f, h). Note the similar pattern of immunostaining obtained for the two experimental conditions (perfused vs. fixed by immersion after 5 hr PT). alv, alveus; CA1, CA1 field of the hippocampus; CA2, CA2 field of the hippocampus; CA3, CA3 field of the hippocampus; DG, dentate gyrus; gl, granular layer; ml, molecular layer; pl, polymorphic layer; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum. Scale bar shown in (h) indicates 90 μm in all panels [Color figure can be viewed at http://wileyonlinelibrary.com]

3.1. SCGN immunostaining patterns in the hippocampal formation

The pattern of SCGN immunostaining in the human, rat, and mouse hippocampus showed numerous similarities and also clear differences between each of these species. Interestingly, some patterns of immunostaining showed a higher degree of similarity between humans and rats, whereas others had a greater degree of similarity between humans and mice.

In general, the neuropil was either labeled or not labeled. The labeled neuropil showed two types of staining: a dense brownish diffuse staining (Type I neuropil staining), in which no individual neuronal processes are distinguished (Figures 1b,d and 2b), and a light diffuse staining (Type II neuropil staining), in which scattered stained neuronal processes are observed (Figures 1b,d and 2b). We also found two main types of labeling of neurons and their proximal processes: a dark brown (Golgi‐like) staining (Type I neuronal staining) (Figure 2b,c) and a light brownish staining (Type II neuronal staining) (Figure 2b). Regarding the morphology of the SCGN‐ir cells, when the dendrites were clearly labeled (Type I neuronal staining), it was observed that the vast majority showed a nonpyramidal morphology (interneurons) in all species. Light labeled cells were difficult to classify as pyramidal or nonpyramidal cells, but they were considered pyramidal when their soma was a triangular shape with a vertically oriented apical dendrite emerging from it. In regions with pattern I neuropil staining, labeled neurons were absent or only occasionally found (Figure 2a). Unless otherwise specified, we will use “SCGN‐ir cells” to refer to both Types I and II neurons. What follows is a description of the SCGN staining in the human, rat, and mouse.

3.1.1. Pattern of SCGN immunostaining in the human hippocampal formation

The hippocampus, together with the EC and PRC, parahippocampal cortex, and HC, forms the medial temporal lobe system, considered pivotal to memory (Eichenbaum & Lipton, 2008; Squire & Zola‐Morgan, 1991). In the head and body of the human hippocampus, the fields with SCGN immunostaining were observed in the neuropil of the pyramidal cell layer and strata oriens and radiatum of CA1, CA2, and CA3, although these three fields exhibited differences in SCGN immunostaining intensity. As shown in Figures 1, 2, 3, 4, CA1 and CA2 show Type I neuropil staining, whereas CA3 and DG display Type II neuropil staining. There is a clear decrease in SCGN immunostaining in stratum lacunosum moleculare and alveus of CA1–CA3 and in the outer two thirds of the molecular layer of the DG. Furthermore, we observed a clear decrease in the staining of the neuropil in strata pyramidale and radiatum of CA, close to the subiculum.

The subiculum is the major output structure of the hippocampus proper, which receives a large and robust projection from CA1 and in turn sends its major projection to EC (Amaral, Dolorfo, & Alvarez‐Royo, 1991; O'Mara, 2006; O'Mara, Commins, Anderson, & Gigg, 2001). The subiculum has three layers with a deep, polymorphic layer; a pyramidal cell layer containing the principal cells; and a molecular layer, which is continuous with the stratum lacunosum moleculare of field CA1 (Ding, 2013). As shown in Figures 1, 2, 3, 4, the subiculum showed a clear decrease in staining of the neuropil, and an increase in the number of SCGN‐ir cells (Figure 3d). Furthermore, we also observed this pattern of staining in presubiculum and parasubiculum. The CA1/subicular border is marked by a rather abrupt narrowing of the neuropil immunostaining of the CA1 pyramidal cell layer. In the subiculum, SCGN‐ir cells were found in all layers but they were more abundant in the deep polymorphic layer. In the presubiculum, SCGN‐ir cells were more numerous in Layers II–III, whereas—in the parasubiculum—SCGN‐ir cells were localized in deep layers, mostly Layer V (Figure 4c,d).

The EC is the main interface between the hippocampus and neocortex (Insausti, Amaral, & Cowan, 1987), which projects to the DG and hippocampal fields CA3 and CA1 via the perforant pathway (Kerr, Agster, Furtak, & Burwell, 2007). In the EC, most SCGN‐ir cells were located in Layer II, while in Layer V these neurons were scarce (Figure 5a,d). In the PRC, which includes Brodmann's areas 35 (A35) and 36 (A36), SCGN‐ir cells were located mainly in Layers II–III of A35 (Figure 5b,e) as well as in deep layers, whereas A36 showed scattered cells throughout all layers, although fewer immunoreactive cells were found in deep layers (Figure 5c,f).

3.1.2. Pattern of SCGN immunostaining in the rat hippocampal formation

This study was carried out in the brain of animals that were either perfused or fixed by immersion after different PT delays. In hippocampus, the pattern of immunostaining in the rat hippocampal formation showed notable differences in comparison to humans. Furthermore, we found clear differences in the pattern of immunostaining between dorsal and ventral hippocampal regions. In the dorsal zone, the labeling of neurons and neuropil was found mainly in the strata lacunosum moleculare, radiatum, pyramidale, and oriens of CA1, which extended to the fasciola cinerea (Figures 6a,c,d and 7a). We observed—especially in the border of CA1 and the fasciola cinerea—a relatively large number of cell bodies and fibers in the neuropil (Figure 6a), and a decrease in labeling of the neuropil and somata, in stratum pyramidale of CA2 and CA3 (Figure 7c), and in the granular and polymorphic layers of the DG (Figure 7a). In the granular layer of DG, there were few or no Type II SCGN‐ir cells in the subgranular zone (Figures 6a,c and 7a). Curiously, in the temporal extreme, the DG had relativity more SCGN‐ir cells in the inner third of the molecular layer, similar to human brain (Figure 6e). The hilus exhibited intense labeling in the neuropil and a relatively large number of SCGN‐ir cells in the polymorphic layer of the DG. Moreover, there were fewer labeled somata in the stratum pyramidale of CA1, relative to the dorsal zone (Figure 6e). Another difference that could be observed along the dorso‐ventral axis was neuropil staining of strata pyramidale, radiatum, and lacunosum moleculare of CA1, which waned gradually and faded out in the ventral part of the hippocampus (Figure 6e).

In the subiculum, pre‐ and parasubiculum, the SCGN immunostaining was observed mainly in cell bodies. The pyramidal cell layer of the subiculum contains deep aspects (adjacent to the alveus) and superficial aspects (adjacent to the molecular layer of the subiculum) (Drexel, Preidt, Kirchmair, & Sperk, 2011). We observed that the dorsal and ventral zones of the subiculum had different distribution patterns (Figures 6e and 8a,c,d). At the ventral subiculum (VS), a relatively large number of SCGN‐ir cells were observed next to the border with the alveus (deep layer). However in the dorsal zone, the stained cells were scattered through the subiculum, and the superficial pyramidal layer had mostly Type‐I SCGN‐ir somata. The presubiculum only had SCGN immunostaining in the neuropil and the parasubiculum in somata (Figure 8e).

In rodents, the EC has two main classical subdivisions, namely the lateral and medial entorhinal area (LEA and MEA, respectively), with three subdivisions having been described for LEA and two for MEA (Insausti, Herrero, & Witter, 1997; van Groen, 2001). In the present work, the observations have been focused on the LEA. In this subdicision of the EC, SCGN‐ir cells were mainly located in deep Layers V–VI (Figure 9c,d). In the PRC (comprising area 35 and area 36) exhibited SCGN‐ir somata mainly in Layers III and V, although they were observed in other layers (Figure 9f).

Although in the present study we have focused on the hippocampal formation, as well as the EC and PRC, we observed in the rat and mouse an intense immunostaining for SCGN in other brain areas such as the superior colliculus (SC), the peripeduncular nucleus (PP) (Figure 6e), and the basolateral amygdaloid nucleus (BLP) (Figure 9a). These other brain regions were not examined in the human brain.

3.1.3. Pattern of SCGN immunostaining in the mouse hippocampal formation

The pattern of SCGN located in the mouse hippocampus was also different compared to rat and human, although some similarities were shared between mouse and each of the other two species. Apparently, the mouse hippocampus does not show differences between the dorsal and ventral zones as pronounced as in the rat hippocampus, although our observations revealed a decrease in the intensity of labeling in all the layers of ventral hippocampus (Figure 6b,d,f).

In the dorsal hippocampus, the fasciola cinerea exhibited an intense labeling in neuropil and somata (Figure 6b,d). Furthermore, it could be observed clearly that—as was the case in human hippocampus—the pyramidal cell layer of CA1 did not display cellular immunostaining, or it was weak in this layer. A similar pattern was seen in CA3, where the labeling was more intense in the neuropil, especially in mossy fibers. On the other hand, intense staining was observed in somata located in the pyramidal cell layer of CA2 and also in the neuropil consistently immunostained for SCGN (Figure 6b,d), which allowed the boundaries of CA2 to be displayed clearly (Figures 6b,d and 7d). Interestingly, this staining faded out in the ventral zone of the hippocampus. By contrast, this pattern of staining in CA2 was not present in the rat hippocampus (Figures 6a,c and 7c,d). The SCGN labeling in the neuropil was also detected in the stratum oriens of CA1 that exhibited strong SCGN staining, as well as in the strata radiatum and lacunosum moleculare, and it decreased along the dorso‐ventral axis, eventually disappearing (Figure 6f). The DG displayed a darkly stained granular cell layer, particularly close to the boundary with the molecular layer. This pattern of immunostaining was different from that observed in the rat and human hippocampi (Figure 6b). The molecular cell layer was intensely immunostained in the neuropil and some scattered SCGN‐ir somata were found (Figure 7b).

At ventral levels of the hippocampus, the pattern of labeling showed a decrease in the immunoreactivity—although the distribution was quite similar to the dorsal hippocampus (Figure 6f), especially in the molecular and polymorphic layers and stratum oriens. The granular layer exhibited a decrease of SCGN‐ir cells compared to the septal pole. At this level, the pattern of labeling was the opposite to that found in rat ventral hippocampus (Figure 6e,f).

The subiculum, unlike the human hippocampus, displayed very few labeled cell bodies at dorsal and ventral levels (Figures 6f and 8b,f–h), although some SCGN‐ir cells were found in the subiculum, and these cells were more numerous at the ventral portion of the hippocampus (Figures 6f and 8b,f). The presubiculum and parasubiculum had no Type I SCGN‐ir cells, although in the presubiculum a few weakly immunostained Type II SCGN‐ir cells were observed and the neuropil was also stained (Figure 8b,g–h).

Likewise, in the EC and PRC, scarce and very weakly immunostained SCGN‐ir cells were detected throughout the cortical layers, showing a pronounced difference with respect to the human and rat cortex (Figure 9b,e,g). It should be noted that some SCGN‐ir somata were labeled intensely, chiefly in Layers V and VI of the somatosensorial and auditory cortices.

In other brain regions, strong immunoreactivity against SCGN was visualized; for example in the SC, the PP, the BLP, and the posteromedial part of the amygdalohippocampal area (AHi) (Figure 6f), where, curiously, an intense neuropil staining was observed, especially in the border between VS and the posteromedial part of the amygdalohippocampal area (Figure 6f). This was not found in rat brain (Figure 6e).

3.1.4. Pattern of SCGN immunostaining in rodent brains fixed by immersion after different post‐mortem delays

We tested the possible effects of fixation by immersion after 2 and 5 hr PT delays on the immunostaining for SCGN in rat and mouse brains, as described in our previous study (Gonzalez‐Riano et al., 2017). As shown in Figure 10, in general, the pattern of immunostaining was similar in brains fixed by perfusion or by immersion after up to 5 hr PT delay, in both rats (Figure 10a–d) and mice (Figure 10 e–h). The only apparent difference was a general decrease in the intensity of the labeling of Type II cells.

4. DISCUSSION

In the present study, we have demonstrated clear differences and similarities in the pattern of SCGN immunostaining among the human, rat, and mouse hippocampal formation (DG, CA1, CA2, CA3, subiculum, presubiculum, and parasubiculum) as well as in the EC and PRC. We also found obvious differences and similarities between the different brain regions examined within each species (Tables 1, 2, 3 and Figure 11). None of these features were due to methodological factors, since the general pattern of immunostaining was similar in brains fixed by perfusion or by immersion after up to 5 hr PT delay, that is, similar to the way in which human brain tissue is obtained and processed. Importantly, the present results also indicate that PT delays of up to 5 hr do not affect the general pattern of SCGN labeling. Furthermore, some patterns were different between the human and either rat or mouse as well as between the two nonhuman species themselves. For example, the pyramidal cell layer of human CA1 (both at the level of the hippocampal body and the hippocampal head) shows an intense neuropil staining but very few SCGN‐ir cells. However, regarding rat and mouse, the pyramidal cell layer of CA1 in the dorsal hippocampus showed immunostaining of both neuropil and neurons in the rat, while in the mouse the neuropil was stained but very few SCGN‐ir cells were found. Furthermore, in rodents, there was a clear difference in the staining between the dorsal and ventral hippocampal formation, with a notable decrease in immunostaining in the ventral region. Another interesting example is regarding the human PrS and Pas which showed numerous SCGN‐ir cells, whereas no or rarely labeled cells were found in the PrS of rat and mouse. However, SCGN‐ir cells were observed in Pas of rat, but not in mouse. Finally, in the human and rat EC and PRC, there were intensely stained SCGN‐ir cells (Type I cells), although the distribution patterns differed throughout the cortical layers. In the mouse EC and PRC, only occasional and weakly labeled SCGN‐ir cells were observed. Regarding the morphology of the SCGN‐ir cells, when the dendrites were well labeled, it was observed that the vast majority showed a nonpyramidal morphology (indicating interneurons) in all species. Light labeled cells were hard to classify as pyramidal or nonpyramidal cells, but they were considered pyramidal when their soma had a triangular shape and a vertically oriented apical dendrite emerging from it (e.g., in CA2 of the mouse and CA1 of the rat). The pattern of immunostaining that we observed in the human hippocampus is different to that observed by Attems et al. (2007). These authors examined the human hippocampus and EC and reported that both cellular and neuropil immunoreactivity were restricted to the subiculum and hippocampus and that, at the cellular level, only pyramidal cells in CA1–CA4 or the hilar region of the DG were stained. However, there are important methodological differences between our study and that of Attems and colleagues. They used human brain tissue with a PT interval that ranged from 6 to 18 hr; the brain tissue was fixed in 7.5% formaldehyde; they used paraffin‐embedded tissue; and before immunostaining, they incubated the sections in citrate‐buffer in a microwave oven (3 min, 630 W and 30 min, 240 W) (antigen retrieval). However, in our study we used human brain tissue with a PT interval ranging from 1.5 to 4 hr; the brain tissue was fixed in 4% PFA; and the brain tissue was directly cut using a vibratome and processed for immunohistochemical experiments without antigen retrieval; that is, similar to the way in which tissue is usually processed in experimental animals.

Table 1.

Summary of the pattern of SCGN‐immunoreactivity in the human hippocampus, subiculum, pre‐ and parasubiculum, EC, and PRC

Human brain area Labeling type
Neuropil Neurons
Hippocampus
CA1

Type I: so, sp, sr

Type II: alv

 No or occasional labeling
CA2

Type I: so, sp, sr, slm

Type II: alv

 Type II: so, sp
CA3

Type I: so, sp, sr, slm

Type II: alv

 Types I and II: so, sp
DG Type II: ml (inner third), pl Type II: pl
Subiculum No labeling Types I and II: ml, sp, pl
Presubiculum No labeling Types I and II: Layers II–III
Parasubiculum No labeling Types I and II: Layer V
EC No labeling Types I and II: Layers II, V
PRC‐A35 No labeling Types I and II: Layers II, V
PRC‐A36 No labeling Types I and II: Layers II, III, V
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Abbreviations: alv, alveus; CA1, CA2 and CA3 fields of the hippocampus; DG, dentate gyrus; EC, entorhinal cortex; ml, molecular layer; pl,polymorphic layer; PRC‐A35, perirhinal cortex‐area 35; PRC‐A36, perirhinal cortex‐area 36; slm, stratum lacunosum moleculare; so, stratum oriens; sp,stratum piramidale; sr, stratum radiatum.

Table 2.

Summary of the pattern of SCGN‐immunoreactivity in the rat hippocampus, subiculum, pre‐ and parasubiculum, EC, PRC, and other brain areas

Rat brain area Labeling type
Neuropil Neurons
Dorsal hippocampus
CA1 Type II: so, sp, sr, slm, fc Types I and II: so, sp
CA2 No labeling Types I and II: so, sp
CA3 No labeling No or occasional labeling: so, sp
DG No labeling Types I and II: gl, pl
Dorsal subiculum Type II Types I and II: deep/superficial pcl
Ventral hippocampus
CA1 No labeling Types I and II: so, sp
DG Type II: ml (inner third), pl Types I and II: pl
Ventral subiculum Type II Types I and II: deep pcl
Presubiculum Type II No or occasional labeling
Parasubiculum Type II Type II
EC Type II Types I and II: Layers V, VI
PRC‐A35 Type II Types I and II: Layers III, V
PRC‐A36 Type II Types I and II: Layers III, V
LaDL/ LaVM Type I Types I and II
BLP Type II No or occasional labeling
APir Type II No or occasional labeling
AHi No labeling No labeling
SC Type II Types I and II
SG Type II Types I and II
PP Type II Types I and II
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Abbreviations: AHi, amygdalohippocampal area,posteromedial part; APir, amygdalopiriform transition area; BLP, basolateral amygdaloid nucleus, posterior part; CA1, CA2 and CA3 fields of the hippocampus; DG, dentate gyrus; EC, entorhinal cortex; fc, fasciola cinerea; LaDL, lateral amygdaloid nucleus, dorsolateral part; LaVM, lateral amygdaloid nucleus; gl, granular layer; ml, molecular layer; pcl, pyramidal cell layer; pl, polymorphic layer; PRC‐A35, perirhinal cortex‐area 35; PRC‐A36, perirhinal cortex‐area 36; PP, peripeduncular nucleus; SC, superior colliculus; SG, suprageniculate thalamic nucleus; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum.

Table 3.

Summary of the pattern of SCGN‐immunoreactivity in the mouse hippocampus, subiculum, pre‐ and parasubiculum, EC, PRC, and other brain areas

Mouse brain area Labeling type
Neuropil Neurons
Dorsal hippocampus
CA1 Type II: fc, so, sr, slm No or occasional labeling
CA2 Type II: fc, so, sr, slm Type II: sp
CA3 Type II: fc, so, sr, slm No or occasional labeling
DG Type I: pl, ml Types I and II: gl
Dorsal subiculum Type II Type II: deep/superficial pcl
Ventral hippocampus
CA1 Type II No or occasional labeling
DG Type I: pl, ml No labeling
Ventral subiculum Type II Types I and II
Presubiculum Type I Type II
Parasubiculum Type I No or occasional labeling
EC Type I No labeling
PRC‐A35 Type I No labeling
PRC‐A36 Type I No labeling
BLP Type I No labeling
APir Type I No labeling
AHi Type I No or occasional labeling
SC Type II Types I and II
SG Type II No labeling
PP Type II No labeling
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Abbreviations: AHi, amygdalohippocampal area,posteromedial part; APir, amygdalopiriform transition area; BLP, basolateral amygdaloid nucleus, posterior part; CA1, CA2 and CA3 fields of the hippocampus; DG, dentate gyrus; EC, entorhinal cortex; fc, fasciola cinerea; gl, granular layer; ml, molecular layer; pcl, pyramidal cell layer; pl: polymorphic layer; PRC‐A35, perirhinal cortex‐area 35; PRC‐A36, perirhinal cortex‐area 36; PP:peripeduncular nucleus; SC, superior colliculus; SG, suprageniculate thalamic nucleus; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum.

Figure 11.

Figure 11

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Graphical representations derived from human, rat, and mouse coronal sections, illustrating the pattern of SCGN immunostaining in neuropil and somata, through hippocampal formation (CA1, CA2, CA3, DG, subiculum, presubiculum, and parasubiculum), entorhinal (EC), and perirhinal (PRC) cortices. The areas labeled in dark gray, light gray, or white indicate Types I, II neuropil staining or no staining in neuropil, respectively. The circles in dark and light gray indicate Types I and II neuronal staining, respectively. The presence of occasional immunostained Type I or II somata are not represented in the schemes. alv, alveus; CA1, CA2, and CA3 fields of the hippocampus; DG, dentate gyrus; DHc, dorsal hippocampus; DS, dorsal subiculum; EC, entorhinal cortex; gl, granular layer; Hc, hippocampus; ml, molecular layer; Pas, parasubiculum; Pcl, pyramidal cell layer; pl, polymorphic layer; PRC‐A35, perirhinal cortex‐area 35; PRC‐A36, perirhinal cortex‐area 36; PrS, presubiculum; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum; Sub, subiculum; VHc, ventral hippocampus; VS, entral subiculum. Asterisks in VHc indicate that only CA1 is represented in the ventral hippocampus, since virtually no labeling or occasional stained cells were found in CA2, CA3, and DG. See text for further details [Color figure can be viewed at http://wileyonlinelibrary.com]

In the human hippocampus, evidence of a possible functional segregation into the anterior and posterior zones has been reported (Strange, Witter, Lein, & Moser, 2014). Briefly, in humans, memory systems present a double dissociation between semantic processing in the anterior hippocampus and nonsemantic processing in the posterior hippocampus (Chua, Schacter, Rand‐Giovannetti, & Sperling, 2007; Giovanello, Schnyer, & Verfaellie, 2009; Strange et al., 2014). Also, in episodic memory, detailed spatial or autobiographical memory engages the posterior hippocampus (Addis, Moscovitch, Crawley, & McAndrews, 2004; Hirshhorn, Grady, Rosenbaum, Winocur, & Moscovitch, 2012). In spatial cognition, some studies have demonstrated a double dissociation in anterior versus posterior human hippocampal responses (Nadel, Hoscheidt, & Ryan, 2013; Strange, Fletcher, Henson, Friston, & Dolan, 1999). In the present study, a similar pattern of SCGN labeling was found at the level of the head and body of the human hippocampus. Therefore, the similar pattern of immunostaining along the anterior–posterior axis of the hippocampus seems to be unrelated to the functional differences between the head and body of the hippocampus.

This is in sharp contrast to the SCGN labeling in rodents, which is strong in the dorsal hippocampus and gradually decreases to the point of almost disappearing more ventrally. In the rodent brain, it has been reported that there is a functional segregation of the hippocampus into dorsal, intermediate, and ventral zones, where the dorsal hippocampus mediates cognitive functions, especially spatial memory, whereas the ventral hippocampus is involved in emotional responses (Fanselow & Dong, 2010). The ventral hippocampus seems to have a preferential role in the regulation of emotional behavior in rodents (LeDoux, 2000), and behavioral deficits correlate with the loss of hippocampal hilar neurons after kainate treatment mainly in the ventral pole of the DG (Maia et al., 2014). Since SCGN labeling is intense in the dorsal hippocampus and almost disappears ventrally in rodents, it is tempting to speculate that this differential pattern is related to the functional segregation of the hippocampal formation. However, there are also other studies showing similarities and differences in neuronal composition between the dorsal and ventral hippocampus. For example, parvalbumin‐ir neurons show no dorso‐ventral difference in any subdivisions of the hippocampus, whereas the density of neurons immunostained for calretinin, nitric oxide synthase, and somatostatin is significantly larger in ventral levels than at dorsal levels of the hippocampus (Nomura et al., 1997a, 1997b).

In general, Ca2+‐binding proteins in the brain play important roles in the initiation and maintenance of long‐term potentiation—and hence in learning and memory (Molinari et al., 1996; Schurmans et al., 1997). Although the Ca2+‐binding protein content does not define the main functional role of the cells, it seems that SCGN could play different roles in the modulation of cellular firing properties and the timing of the neurotransmitter released by eliminating intracellular Ca2+ from the cytosol (Rogstam et al., 2007). SCGN has also been related to an increase in neurite length and complexity by promoting calcium‐dependent exocytosis of plasmalemmal precursor vesicles in growth cones in diverse neuronal subtypes (Raju et al., 2017). Finally, SCGN may also play a role in the regulation of the release factors related to the survival or development of new neurons in the olfactory bulb and DG (Mulder, Zilberter, et al., 2009). However, the possible significance of the dorso‐ventral differences in SCGN in rodents but not in the anterior–posterior axis in humans is difficult to determine. First, there are multiple neural systems involved in the different functional attributes of the hippocampal formation and second, these systems may differ between the three species regarding cytoarchitectonic and neurochemical characteristics as well as in terms of the patterns of connectivity.

4.1. Evaluation of post‐mortem delay and fixation in rodents

The PT delay and the method of fixation are important factors to be taken into account when interpreting immunocytochemical staining. For example, previous studies in the mouse hippocampus in our laboratory described changes in immunoreactivity depending on both the PT delay and on whether parvalbumin or calbindin D‐28k was used (Gonzalez‐Riano et al., 2017). Since the goal in the present study was to compare between human, rat, and mouse, we investigated the possible effects of these technical factors in the mouse and rat to determine whether the possible differences in immunostaining between these species were simply methodological. We have not found apparent differences in the general pattern of SCGN staining in the mouse or rat, up to 5 hr of PT. Nevertheless, we observed a decreased intensity of the SCGN immunostaining at 2 hr PT, with this decrease becoming more evident at 5 hr PT in both species. In rat hippocampus, there was a decrease in the type II SCGN‐somata (lightly labeled cells) in CA1 and CA2 and in the subgranular zone of the DG. In mouse brain there was a decrease in the labeling of type II SCGN‐somata in the granular cell layer of DG. Therefore, the differences between the pattern of SCGN immunostaining in the hippocampal formation found in the present study between the human and rodents are not due to the fixation.

ACKNOWLEDGMENTS

We would like to thank Lorena Valdes and Carmen Alvarez for technical assistance, and Nick Guthrie for his excellent text editing. This work was supported by grants from the following entities: Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain); the Spanish “Ministerio de Ciencia, Innovación y Universidades” (grant PGC2018‐094307‐B‐I00); and the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 785907 (Human Brain Project, second specific grant agreement [SGA2]).

Tapia‐González S, Insausti R, DeFelipe J. Differential expression of secretagogin immunostaining in the hippocampal formation and the entorhinal and perirhinal cortices of humans, rats, and mice. J Comp Neurol. 2020;528:523–541. 10.1002/cne.24773

Peer Review The peer review history for this article is available at https://publons.com/publon/10.1002/cne.24773.

Funding information Centro de Investigación en Red sobre Enfermedades Neurodegenerativas, Grant/Award Number: CB06/05/0066; European Union's Horizon 2020 Research and Innovation Programme (Human Brain Project), Grant/Award Number: 785907; Ministerio de Ciencia, Innovación y Universidades, Grant/Award Number: PGC2018‐094307‐B‐I00

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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