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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Brain Struct Funct. 2020 Sep 9;225(8):2521–2531. doi: 10.1007/s00429-020-02139-x

Quantification of neurons in the hippocampal formation of chimpanzees: Comparison to rhesus monkeys and humans

Christina N Rogers Flattery 1,*,₸, Rebecca F Rosen 2,₸, Aaron S Farberg 3, Jeromy M Dooyema 3, Patrick R Hof 4, Chet C Sherwood 5, Lary C Walker 3,6, Todd M Preuss 3,7
PMCID: PMC7775633  NIHMSID: NIHMS1656309  PMID: 32909100

Abstract

The hippocampal formation is important for higher brain functions such as spatial navigation and the consolidation of memory, and it contributes to abilities thought to be uniquely human, yet little is known about how the human hippocampal formation compares to that of our closest living relatives, the chimpanzees. To gain insight into the comparative organization of the hippocampal formation in catarrhine primates, we quantified neurons stereologically in its major subdivisions - the granular layer of the dentate gyrus, CA4, CA2–3, CA1, and the subiculum – in archival brain tissue from six chimpanzees ranging from 29 to 43 years of age. We also sought evidence of Aβ deposition and hyperphosphorylated tau in the hippocampus and adjacent neocortex. A 42-year-old animal had moderate cerebral Aβ-amyloid angiopathy and tauopathy, but Aβ was absent and tauopathy was minimal in the others. Quantitatively, granule cells of the dentate gyrus were most numerous, followed by CA1, subiculum, CA4, and CA2–3. In the context of prior investigations of rhesus monkeys and humans, our findings indicate that, in the hippocampal formation as a whole, the proportions of neurons in CA1 and the subiculum progressively increase, and the proportion of dentate granule cells decreases, from rhesus monkeys to chimpanzees to humans. Because CA1 and the subiculum engender key hippocampal projection pathways to the neocortex, and because the neocortex varies in volume and anatomical organization among these species, these findings suggest that differences in the proportions of neurons in hippocampal subregions of catarrhine primates may be linked to neocortical evolution.

Keywords: Stereology, CA1 region, dentate gyrus, Alzheimer’s disease, Abeta, tauopathy

1. Introduction

The hippocampal formation (here defined as the hippocampus proper, dentate gyrus and subiculum) is critical for cognitive functions such as spatial navigation (Ekstrom et al. 2003; Hartley et al. 2014) and the establishment and retrieval of memories (Squire 1992; Eichenbaum 2017), and it contributes to abilities that are highly developed in humans, such as imagining the future (Schacter et al. 2017). However, relatively little is known about structural features of the human hippocampal formation that set it apart from other primate species. Moreover, certain subdivisions of the human hippocampal formation experience neuron loss with normal aging (West 1993), whereas this has not been reported in rats (Rasmussen et al. 1996) or rhesus monkeys (Macaca mulatta) (Keuker et al. 2002). In addition, neurons of the hippocampal formation, particularly region CA1, are highly susceptible to loss in Alzheimer’s disease (Price et al. 2001), a neurodegenerative disorder to which senescent nonhuman primates appear to be resistant to the full extent of pathology (Rapoport 1990; Rapoport and Nelson 2011; Heuer et al. 2012; Finch and Austad 2015; Rosen et al. 2016; Walker and Jucker 2017; Edler et al. 2018; Munger et al. 2019). Therefore, a comparative analysis of neuronal populations in the hippocampal formation of chimpanzees (Pan troglodytes) – our closest living biological relatives (Waterson et al. 2005) – might provide insights into both phylogenetic differences in its organization and to characteristics of neurobiological aging that may be unique to humans.

In this study, we used stereologic methods to estimate the total number of neurons in the major hippocampal subdivisions - the dentate granule cell layer, the hilus of the dentate gyrus (hereafter CA4, which includes all neurons within the granule cell pocket except those of the proximal part of CA3), CA2–3, CA1, and the subiculum - in six adult chimpanzees (Figure 1). This parcellation and nomenclature were chosen to enable a direct comparison with earlier stereologic analyses in adult humans (West and Gundersen 1990) and rhesus macaques (Keuker et al. 2003). We found that the general proportions of neurons in these regions are similar in chimpanzees to those previously reported in rhesus monkeys (Keuker et al. 2003) and humans (West and Gundersen 1990; West 1993; Šimić et al. 1997), but that, relative to the total number of hippocampal neurons, the proportions of neurons in CA1 and the subiculum increase markedly from rhesus monkeys to chimpanzees to humans along with a decrease in the proportion of dentate granule cells. Our findings indicate that the overall arrangement of neuronal somata in the hippocampal formation places chimpanzees in an intermediate position between rhesus monkeys and humans. In light of differences among these species in neocortical size (Van Essen et al. 2018), we suggest that the subregional differences in the hippocampal formation may be linked to the degree of expansion and/or reorganization of the neocortex in catarrhine primates.

Fig. 1.

Fig. 1.

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A representative systematic-random series of tissue sections (every 60th section throughout the entire hippocampal formation) from one chimpanzee (Pt06), with the quantified subdivisions schematically depicted in color beneath each panel.

To assess the presence of potentially confounding neurodegenerative changes in these subjects, we also performed immunohistochemical analyses of amyloid beta (Aβ) and hyperphosphorylated tau proteins in the hippocampal formation and adjacent temporal neocortex. Aβ and tau misfold, multimerize, and profusely deposit in the brains of Alzheimer’s disease (AD) patients as Aβ plaques and neurofibrillary tangles, respectively, and these two lesions have been identified (albeit in smaller quantities) in aging chimpanzees (e.g., Rosen et al. 2008; Edler et al. 2017). In the present cohort, Aβ deposits were present only in the 42-year-old chimpanzee (Pt05), primarily in the form of cerebral amyloid angiopathy (CAA). Variable and mostly sparse hyperphosphorylated tau-immunoreactive neurites and neuronal somata were detected in all chimpanzees, but neither these nor the pattern of Aβ deposition resembled the changes seen in humans with AD.

2. Materials and Methods

2.1. Subjects

Complete temporal lobe tissue samples were collected opportunistically at necropsy from six chimpanzees (Pan troglodytes), two females and four males, aged 29–43 years (Table 1), all of whom died before 2007 of natural causes or were euthanized due to intractable clinical conditions by experienced veterinarians at the New Iberia Research Center or the Yerkes National Primate Research Center. All tissues were collected in accordance with federal and institutional guidelines for the humane care and use of experimental animals. The New Iberia and Yerkes Centers are fully accredited by AAALAC International.

Table 1:

Subjects.

Chimpanzee # Age (years) Sex PMI (hours)† Brain weight (g) Cause of death
Pt01 29 F <12 n/a heart failure
Pt02 34 M 1.5 376 pharyngeal mass
Pt03 37 M 1.0 461 intractable infection
Pt04 40 M 2.0 423 myocardial fibrosis
Pt05 42 F n/a 363 indeterminate
Pt06 43 M 2.5 420 myocardial fibrosis
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g: grams; PMI: Postmortem interval; n/a: not available.

2.2. Tissue preparation

Whole brains were immersion-fixed in 4% depolymerized paraformaldehyde in 0.01 M phosphate-buffered saline (PBS; pH 7.4) for at least 14 days on an orbital shaker at 4°C. Left temporal lobe tissue blocks, extending posteriorly to the level of the splenium of the corpus callosum, were dissected from the brain in 5 cases; in 1 case (Pt04) the right temporal lobe was taken. Temporal lobe blocks were bisected in the coronal plane so that they could be stably mounted on the microtome stage (below). They were then cryoprotected for 2–3 days each in a progressive series of 10%, 20%, 30%, and 40% sucrose/PBS (pH 7.4), and stored at −20°C until sectioning.

To ensure that the anterior and posterior blocks comprising each temporal lobe were cut in the same plane, each frozen block was mounted on a leveled microtome stage with the cut face down. They were sectioned coronally at 50 μm thickness on a Microm HM440E sliding microtome equipped with a freezing stage (Microm International GmbH, Walldorf, Germany). Care was taken to collect all sections containing the hippocampal formation, including those at the interface of the bisected blocks, so that little or no tissue was lost. Cut sections were collected in a cryopreservative solution of 30% ethylene glycol, 30% sucrose, and 1% polyvinyl pyrrolidone in PBS overnight at 4°C (Hoffman and Le 2004), and then stored at −20°C until staining.

2.3. Tissue staining

Nissl stain.

In preparation for stereological counts of hippocampal neurons, every 12th tissue section (600 μm between sections) from each animal was mounted on a 2 × 3-inch gelatin-coated glass slide and dried at room temperature for one week. Mounted sections were immersed for 3 minutes each in distilled water, 50% ethanol, 70% ethanol, 95% ethanol, and 100% ethanol, then a mixture of 50% chloroform/50% ethanol for 30 minutes, followed by stepwise rehydration in 100%, 95%, 70%, and 50% ethanol and then distilled water. They were then immersed in a solution of 0.05% thionin (dissolved in 0.2 M acetic acid/0.2 M sodium acetate, pH 3.0; filtered before use) for 5 minutes. After 3-minute immersions in distilled water, 50% ethanol, and 70% ethanol, the stained sections were differentiated in 95% ethanol/1% acetic acid, dehydrated in 100% ethanol and 2 immersions in xylene (5 minutes each), and then coverslipped with dibutylphthalate polystyrene xylene (DPX) mountant (Sigma-Aldrich, St. Louis, MO).

Immunohistochemistry and antibody characterization.

Floating tissue sections from each subject (a series of every 96th section throughout each temporal lobe block) were rinsed in 0.01 M PBS to remove cryopreservative and then immersed for 10 minutes in 3% H2O2 in methanol to block endogenous peroxidases. Nonspecific reagent binding was then blocked by a 60-minute shaking immersion in 2% normal horse serum/0.2% Tween-20 (MilliporeSigma, St. Louis, MO)/PBS (blocking solution). Sections next were immersed in primary antibody diluted in blocking solution on an orbital shaker overnight at 4°C. For alternating sections within each series, the primary monoclonal antibodies used were 6E10 against an N-terminal epitope at residues 3–8 of the Aβ peptide (Kim et al. 1988) (Covance, Princeton, NJ, Cat# SIG-39320, RRID:AB_662798) at 1:25,000 dilution, or CP13 against an epitope around phosphorylated serine 202 of the tau protein (Jicha et al. 1999; antibody provided by Dr. Peter Davies, Feinstein Institute for Medical Research, Manhasset, NY, USA Cat# CP13, RRID:AB_2314223) at 1:10,000 dilution. Antibody specificity was determined by Western blot for 6E10 (Covance) and CP13 (Jicha et al. 1997). For Aβ immunohistochemistry, sections were pretreated with 100% formic acid for 10 minutes to expose antigenic sites (Kitamoto et al. 1987). Primary antibodies bound to tissue sections were detected using an ABC kit (Vector Laboratories, Burlingame, CA). Specifically, sections were rinsed in 0.01 M PBS, incubated for one hour in biotinylated horse anti-mouse antibody (1:200 in blocking solution), rinsed again, immersed in avidin-biotin complex for 30 minutes, and then reacted with 3,3’-diaminobenzidine, all at room temperature. A Nissl counterstain was applied to some sections prior to coverslipping. Alzheimer’s disease brain tissue sections served as positive controls for immunostaining, and primary antibodies were omitted for negative controls.

2.4. Stereological determination of neuronal numbers and hippocampal subregional volumes

We quantified neuronal somata and regional volumes in five hippocampal subdivisions: the granule cell layer of the dentate gyrus, regions CA4, CA2–3, CA1, and the subiculum, using stereological methods comparable to those employed in humans by West and Gunderson (1990) and Šimić et al. (1997), and in rhesus monkeys by Keuker et al. (2003). Note that the hilar region, deep to the polymorphic cell layer of the dentate gyrus, is referred to as CA4, following Lorente de No (1934), although some researchers consider this to be a part of CA3 (e.g., Amaral 1978; Jabès et al. 2011).

First, the Nissl-stained sections throughout the temporal lobe were examined on a Leica DM RXA2 microscope (Leica Microsystems, Wetzlar, Germany) at low magnification to identify the anterior and posterior limits of the hippocampal formation according to the appearance of CA1 pyramidal cells rostrally and their disappearance caudally. Because every 12th section was Nissl-stained, the systematic-random series began with the random selection of one of the first 5 sections that included the rostral CA1. Thereafter, every 60th section was quantified, at a distance of 3 mm (60 sections × 50 μm) between sections. Using this technique, each series contained 8–10 sections. If the hippocampal tissue was not fully intact in a section within the original stereology subseries, the section was replaced with an adjacent, intact section for stereological analysis. The new block-face distance was then recorded in the Serial Section Manager of Stereo Investigator 7 (MBF Biosciences, Williston, VT).

Using a 1.6X objective (16X total magnification) and Stereo Investigator 7 software, contours were tightly drawn around the collective Nissl-stained cells within each subdivision (hereafter ROI, region of interest) using cytoarchitectural criteria (West and Gundersen 1990) (Figure 1). Neurons were identified by the presence of Nissl-stained cytoplasm and, particularly in larger neurons, the presence of a prominent nucleolus, whereas cytoplasmic staining was consistently absent in glial cells (Figure 2).

Fig. 2.

Fig. 2.

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High-magnification images of Nissl-stained cells in the five regions of the hippocampal formation in which neurons were quantified (chimpanzee Pt03). CA1 (a), CA3 (b), CA4 (c), granule cell layer of the dentate gyrus (d); subiculum (e). Scale bar = 20 μm for all panels.

Although all tissues were sectioned at a thickness of 50 μm, there was expected shrinkage of the sections during processing and some variation in their measured thickness due mainly to variable temperature of the block during cutting. Final section thickness was preliminarily measured at approximately 5 distinct sites within the hippocampal formation, and the mean of these values (which tended to be relatively uniform) was used to set the height of the counting boxes in that section. The investigator recorded the tissue depth in every fifth counting box throughout each ROI in order to double-check the thickness of the section. Due to the variation in thickness, the actual height of the disector boxes across the six cases ranged from 8.8 μm to 10.9 μm. In all counting boxes, a guard zone of 1 μm was applied at the top of the section.

The optical fractionator probe was used to count neuronal nuclei in the hippocampal subdivisions. The planar dimensions of the horizontal surfaces (squares) of the counting boxes were 75 × 75 μm for CA4, CA2–3, CA1, and subiculum, and 50 × 50 μm for the dentate granule cell layer. These values were set to obtain 2–3 neurons per counting box when using a 63X oil-immersion objective (Leica Microsystems, Wetzlar, Germany) for the CA4, CA2–3, CA1, and subiculum, and a 100X oil-immersion objective for the dentate granule cells. Sampling grids were individually determined for ROIs in each brain to accommodate approximately 15 counting boxes per contour in each section.

The total number of neurons in each ROI was estimated with Stereo Investigator by extrapolating the number of neurons in the collective volume of counting boxes to the volume of the entire region as estimated according to Cavalieri’s principle (Mouton 2011). Schmitz-Hof coefficients of error (standard deviation divided by the mean; Schmitz and Hof, 2000; 2005) were below 0.09 for all calculated neuronal counts (Table 2). Neuronal density was calculated as the number of neurons per unit of volume (Table 2). For statistical analysis, Pearson’s correlation coefficients (Pearson’s r) were calculated between the subjects’ ages and subregional volumes, neuronal numbers, and neuronal densities.

Table 2.

Neuronal numbers, coefficients of error, regional volumes, and neuronal densities in 5 subregions of the hippocampal formation in 6 chimpanzees.

Chimpanzee # → Pt01 Pt02 Pt03 Pt04 Pt05 Pt06 MEAN SD
Age/Sex → 29 / F 34 / M 37 / M 40 / M 42 / F 43 / M
GCL NN† 8.27 × 106 1.21 × 107 1.33 × 107 9.88 × 106 7.65 × 106 1.60 × 107 1.12 × 107 3.21 × 106
CE‡ 0.083 0.066 0.055 0.055 0.075 0.046
Vol§ 23.34 20.68 22.37 19.99 19.35 27.36 22.18 2.94
N/V 3.54 × 105 5.84 × 105 5.96 × 105 4.94 × 105 3.95 × 105 5.85 × 105 5.02 × 105 1.06 × 105
CA4 NN† 1.17 × 106 9.23 × 105 1.02 × 106 2.62 × 106 1.02 × 106 1.42 × 106 1.36 × 106 6.40 × 105
CE‡ 0.056 0.062 0.049 0.052 0.056 0.050
Vol§ 81.45 57.83 61.78 121.99 70.91 76.22 78.36 23.11
N/V 1.44 × 104 1.60 × 104 1.65 × 104 2.15 × 104 1.43 × 104 1.86 × 104 1.69 × 104 2.75 × 103
CA2–3 NN† 9.77 × 105 9.39 × 105 9.67 × 105 9.55 × 105 1.13 × 106 9.52 × 105 9.87 × 105
CE‡ 0.046 0.046 0.039 0.047 0.048 0.052 7.19 × 104
Vol§ 44.67 32.34 35.83 50.48 49.12 44.95 42.90 7.28
N/V 2.19 × 104 2.90 × 104 2.70 × 104 1.89 × 104 2.30 × 104 2.12 × 104 2.35 × 104 3.79 × 103
CA1 NN† 3.20 × 106 4.11 × 106 3.25 × 106 3.32 × 106 3.27 × 106 5.23 × 106 3.73 × 106 8.1 × 105
CE‡ 0.052 0.048 0.043 0.055 0.059 0.056
Vol§ 165.79 273.32 139.77 259.05 209.23 321.12 228.05 68.84
N/V 1.93 × 104 1.50 × 104 2.33 × 104 1.28 × 104 1.56 × 104 1.63 × 104 1.71 × 104 3.7 × 103
SUB NN† 1.68 × 106 1.05 × 106 2.17 × 106 2.04 × 106 1.04 × 106 2.33 × 106 1.71 × 106 5.6 × 105
CE‡ 0.054 0.068 0.052 0.065 0.066 0.045
Vol§ 108.84 104.57 123.79 160.36 103.47 121.55 120.43 21.36
N/V 1.55 × 104 1.00 × 104 1.76 × 104 1.27 × 104 1.01 × 104 1.91 × 104 1.42 × 104 3.8 × 103
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†

NN: Estimated number of neurons

‡

CE: Coefficient of error (Schmitz-Hof) for estimated neuronal numbers

§

Vol: Volume of hippocampal subregion in mm3

N/V: Neuronal density (neuronal number/mm3)

GCL: Granule cell layer; CA: Cornu ammonis; SUB: Subiculum

SD: Standard deviation

2.5. Assessment of Aβ- and hyperphosphorylated tau-immunoreactivity

To optimize detection of Aβ and hyperphosphorylated tau (both of which can be infrequent in chimpanzees in this age range), we included both the hippocampal formation and the adjacent neocortex (parahippocampal gyrus, inferior, middle and superior temporal gyri) to determine the prevalence of the lesions in each subject.

Aβ plaques and cerebral Aβ-amyloid angiopathy (CAA).

From the series of temporal lobe sections immunostained for Aβ, a subset of every fourth tissue section (3 sections spaced approximately 19.2 mm apart) was analyzed for the presence of Aβ deposition. Using Stereo Investigator 7 software, a single closed contour was drawn around the perimeter of the tissue section using a 1.6X objective. The entire tissue section was scanned using the Meander Scan function with a 5X objective, and Aβ-immunoreactive lesions were point-counted with two separate markers indicating either parenchymal Aβ plaques or CAA. In the latter case, each independent vessel profile was denoted with a single marker, regardless of its orientation. Thus, in many instances, separate profiles may represent segments of a common vessel that was rendered discontinuous by sectioning of the tissue. Areal densities were calculated for each section as the number of lesions per mm2 using MBF NeuroExplorer software.

Tauopathy.

Because most hyperphosphorylated tau-immunoreactive profiles in the temporal lobe sections were scattered neurites rather than neuronal somata, tauopathy in each case was scored as follows: From the series of temporal lobe sections immunostained with the CP13 antibody, a subseries of every fourth tissue section (3 sections spaced approximately 19.2 mm apart) was analyzed for somatal and neuritic tau immunoreactivity using a qualitative rating scale (0: no signal; I: some scattered tau-positive neurites, few or no tau-immunoreactive somata; II: profuse tau-positive neurites and some tau-containing somata; and III: profuse tau-positive neurites and numerous tau-positive somata). Using a 10X objective, each section was examined in its entirety by two independent investigators, and a rating score was assigned. Scores were averaged to determine final case ratings.

3. Results

3.1. Subregional volumes, neuronal numbers and neuronal densities in the hippocampal formation

The mean volume of each hippocampal subregion in a single hemisphere, uncorrected for fixation shrinkage, along with the estimated total number of neurons and the density of neurons per mm3, are shown in Table 2 and Figure 3. We calculated a mean total of approximately 1.90 × 107 neurons in the combined hippocampal subregions, of which the densely packed neurons of the granule cell layer were the most numerous, followed by CA1, subiculum, CA4, and CA2–3. When expressed per unit volume, the density of neurons was highest in the granule cell layer, whereas neurons were much sparser in the other four regions analyzed (Figures 2 and 3). In the limited age-range examined (29–43 years), no significant correlations were found between age and total hippocampal neuron number, (r(4) = 0.461, p = 0.357) total hippocampal volume (r(4) = 0.575, p = 0.232), or overall neuron density (r(4) = −0.032, p = 0.952), nor for these variables in any of the hippocampal subdivisions.

Fig. 3.

Fig. 3.

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Schematic representation of the average proportion of neurons, volume of each subregion, and density of neurons in 5 subdivisions of the hippocampal formation in 6 adult chimpanzees.

Within the framework of prior investigations of adult humans (West and Gundersen 1990; West 1993; Šimić et al. 1997) and rhesus monkeys (Keuker et al. 2002), the absolute number of neurons in all hippocampal subregions increased from rhesus monkeys to chimpanzees, and from chimpanzees to humans. However, this increase was not uniform in magnitude across all subdivisions. Compared to rhesus monkeys, the smallest increase in total neuronal number in chimpanzees was in the granule cell layer, followed by CA2–3, CA1, subiculum, and CA4; in contrast, compared to chimpanzees, the smallest increase in total neuronal number in humans was in CA4, followed by the granule cell layer, CA2–3, subiculum, and CA1 (Table 3).

Table 3.

Hippocampal neuronal numbers in adult rhesus monkeys, chimpanzees and humans.

Study Species N Age range Mean NN GCL Mean NN CA4 Mean NN CA2–3 Mean NN CA1 Mean NN SUB
Keuker et al., 2003 Rhesus monkeys 5 18–31 7,799,000 394,000 622,000 1,381,000 612,000
Present study Chimpan-zees 6 29–43 11,200,000 1,360,000 987,000 3,730,000 1,720,000
Šimić et al., 1997 Humans 10 71–99 16,600,000 1,400,000 2,500,000 11,800,000 4,200,000
West, 1993 Humans 19 50–85 18,310,000 1,650,000 2,760,000 14,110,000 5,550,000
West & Gundersen, 1990 Humans 5 47–85 15,400,000 1,980,000 2,700,000 16,400,000 4,510,000
Mean values from human studies above 16,770,000 1,676,667 2,653,333 14,103,333 4,753,333
Increase in total neurons, rhesus to chimpanzee 1.4 x 3.5 x 1.6 x 2.7 x 2.8 x
Increase in total neurons, chimpanzee to human 1.5 x 1.2 x 2.7 x 3.8 x 2.8 x
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NN: Neuronal number; GCL: Granule cell layer; CA: Cornu ammonis; SUB: Subiculum

3.2. Aβ deposition

Immunohistochemistry with antibody 6E10 revealed Aβ deposition in the temporal lobe only in the 42-year-old female (Pt05; Figure 5a, Table 4). Most Aβ aggregation was in the walls of cerebral blood vessels as CAA, whereas parenchymal Aβ plaques were sparse. CAA was less abundant in the hippocampal formation than in the adjacent temporal lobe cortex.

Fig. 5.

Fig. 5.

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Cerebral amyloid angiopathy (a) and hyperphosphorylated tau (b and c) in the temporal lobe of chimpanzees. a and b are from the 42-year-old female (Pt05), and c is from the 37-year-old male (Pt03). Parenchymal Aβ plaques were rare in Pt05 and absent in the other animals. b depicts a hyperphosphorylated tau-immunoreactive neuronal soma and neurites, and c depicts a neuritic cluster, possibly arising from an out-of-plane soma. Aβ was immunostained using antibody 6E10, and tau was immunostained using antibody CP13. Nissl counterstain. Bar = 50 μm in b and c, and 200 μm in a.

Table 4.

Aβ plaques, CAA, and hyperphosphorylated tau in the hippocampal formation and temporal neocortex of 6 chimpanzees.

Chimpanzee # Age (years) Sex Aβ plaques (#/mm2) CAA (#/mm2) Tau lesion score
Pt01 29 F 0 0 I
Pt02 34 M 0 0 I
Pt03 37 M 0 0 I
Pt04 40 M 0 0 I
Pt05 42 F 0.02 5.19 II
Pt06 43 M 0 0 I
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3.3. Tauopathy

Immunohistochemistry with antibody CP13 revealed mostly sparse and variable accumulations of hyperphosphorylated tau-positive structures in the temporal cortex of all six animals examined (Figure 5b, 5c; Table 4). Most tau-immunoreactive profiles were cellular processes, most likely neurites (axons or dendrites), whereas immunoreactive somata were very rare, possibly reflecting sampling bias due to the greater volume occupied by the neuritic tree than by somata. Tau-positive elements - mostly neurites - were most abundant in Pt05, the 42-year-old female with significant CAA (above), and in this animal, they were seen throughout much of the temporal cortex. There was no consistent spatial overlap of hyperphosphorylated tau and CAA/Aβ. In no subject was an AD-like pathologic phenotype evident (i.e., profuse Aβ plaques and bona fide neurofibrillary tangles in the hippocampal formation and adjacent cortex), and there was no histologic evidence of other acute or chronic neurodegenerative brain conditions.

4. Discussion

The structure and function of the hippocampal formation are generally similar in many extant mammalian species (Manns and Eichenbaum 2006; Clark and Squire 2013). In stereological analyses of primates including humans (West and Gundersen 1990; West 1993; Šimić et al. 1997), rhesus monkeys (Keuker et al. 2003; Jabès et al. 2011), and marmosets (van Dijk et al. 2016), along with several non-primate species (van Dijk et al. 2016), a common theme is that the neuronal population in CA1 is expanded in primates, particularly in humans (Stephan and Manolescu 1980). Our findings in chimpanzees support this view, and also suggest a smaller relative increase in the subicular neurons; when comparing the overall neuronal composition in three species of catarrhine primates - rhesus monkeys, chimpanzees and humans - the proportions of neurons in CA1 and the subiculum increase from rhesus monkeys to chimpanzees to humans (Figure 4 and Table 3). At the same time, the proportion of dentate granule cells progressively decreases (Figure 4 and Table 3), an important factor in defining the overall neuronal composition of the hippocampal formation. While our analysis focused on adult animals, it is worth noting that a stereological analysis of the hippocampal formation in pre-adult rhesus monkeys (Jabès et al. 2011) corroborates the regional proportions of neurons in adult monkeys as determined by Keuker et al. (2003). Note that in the present study, the weights of the blocks before the histological procedure were not available, preventing us from applying a correction reliably to the other dimensions than the vertical plane. Whereas stereological quantification yields a reasonably accurate estimate of the total number of neurons in a defined volume regardless of shrinkage, we did not account for shrinkage in the calculation of subregional volumes, and therefore our results should be compared cautiously to neuronal densities and regional volumes reported in other studies.

Fig. 4.

Fig. 4.

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Schematic representation of the proportions of neurons in five hippocampal subregions of adult rhesus monkeys (Keuker et al. 2003), chimpanzees (present study) and humans (mean values of West 1993; West and Gundersen 1990; and Šimić et al. 1997) relative to total hippocampal number. The proportion of neurons in CA1 and subiculum increases, whereas the proportion of GCL (granule cell layer of the dentate gyrus) neurons decreases, from rhesus monkeys to chimpanzees to humans.

The collective evidence indicates that key information flow through the hippocampal formation passes sequentially through the dentate gyrus, CA3, CA1, and the subiculum, which then is the major source of hippocampal output to various cortical and subcortical sites (Rosene and Van Hoesen 1977; Witter and Amaral 1995; Manns and Eichenbaum 2006). CA1 also innervates the entorhinal cortex, and it is thought to be particularly important for cognitive skills such as mental time-travel and autobiographical memory (Bartsch et al. 2011). CA1 is also involved in spatial memory (Pittenger et al. 2002; Place et al. 2012), and this region is particularly vulnerable to neuronal loss caused by such insults as hypoxia/ischemia (Schmidt-Kastner and Freund 1991) and AD (West et al. 2000). Interestingly, social and ecological variables correlate with volumetric variation in different hippocampal subregions in primates. Specifically, the entire cornu ammonis appears to co-evolve with group size, whereas the volumes of CA1 and the subiculum are associated with animals’ home-range size, a feature that is linked to spatial cognition (Todorov et al. 2019).

As key mediators of hippocampal output, CA1 and the subiculum are essential for effective communication between the hippocampal formation and the rest of the brain. Given their connectivity with periallocortical, proisocortical, and neocortical regions, along with the enlargement of the neocortex in great apes and especially humans (Sherwood et al. 2008; Van Essen et al. 2018), it is possible that the progressive increase in neuron number in CA1 and the subiculum in rhesus monkeys, chimpanzees, and humans is linked to the relative neocortical volume in these species. Furthermore, while the evolutionary relationship between hippocampal neuron number and neocortical expansion is still speculative, a comparative volumetric analysis of primates indicates that humans have a unique combination of neocortical and hippocampal organization that involves a parallel expansion of the rhinal cortex, the CA3, and the subiculum (Vanier et al. 2019).

The subiculum, in addition to receiving crucial input from the CA1, also intercommunicates with several neocortical regions, and it sends projections to the nucleus accumbens, septum, hypothalamus, and amygdala (Manns and Eichenbaum, 2006). The subicular region is one of the earliest brain areas affected by tauopathy in AD (Braak and Braak, 1995; Carlesimo et al. 2015), and experimental studies in mice have shown that disrupting hippocampal connections with other brain regions impedes the progression of Aβ deposition (George et al. 2014; Lazarov et al. 2002). Because Aβ deposition in humans first becomes evident in the neocortex (Thal et al. 2002), the expansion of hippocampal communication with neocortical regions in humans (Insausti 1993) could be a factor that renders CA1 and the subiculum especially susceptible to neuron loss and tauopathy in AD.

In summary, stereologic quantification of neurons in the hippocampal formation indicates that, in relation to the total number of hippocampal neurons, CA1 and the subiculum contain relatively fewer neurons, and the granule cell layer contains relatively more neurons, in chimpanzees than in humans. This pattern places the overall neuronal organization of the chimpanzee hippocampal formation in an intermediate position between rhesus monkeys and humans. These findings suggest a reorganization of the hippocampal formation that may be related to the differential expansion of the neocortex (Van Essen et al. 2018) following the evolutionary divergence of these three species of catarrhine primates.

Funding:

This research was supported by National Institutes of Health (NIH) grants P01 AG026423 (to James G. Herndon), P50 AG025688 (to Allan I. Levey), and P50 AG005138 (to Patrick R. Hof), and by the James S. McDonnell Foundation (JSMF 21002093) to Todd M. Preuss.

Footnotes

These data were in part collected while Rebecca Rosen was employed at Emory University (2004–2010). The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States Government.

Conflicts of Interest: The authors have no conflicts of interest to report.

Availability of data and material: Data are accessible in a public repository at https://github.com/anthrochristina/ChimpHippocampus

Code availability: Not applicable.

Ethics approval: Research was conducted on post-mortem/archival chimpanzee brain tissue that had been opportunistically collected at necropsy, thus IACUC approval was not required. All tissues were collected in accordance with federal and institutional guidelines for the humane care and use of experimental animals. The New Iberia and Yerkes Centers are fully accredited by AAALAC International.

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