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. 2025 Feb 6;533(2):e70025. doi: 10.1002/cne.70025

Characterization of the Porcine Cingulate Sulcus Cytoarchitecture

Brendan Hoffe 1,✉, Lisa Hebert 1,2, Oren E Petel 3, Matthew R Holahan 1
PMCID: PMC11800179  PMID: 39912370

ABSTRACT

Cortical folding (gyrification) is a unique process by which the brain can expand and increase surface area while confined by the boundaries of the inner wall of the skull. Although there is still much debate about the exact mechanisms concerning the genetic and cellular factors involved in this process, gyrification results in a heterogenous organization of neuronal layering and cell types not seen in the smooth, lissencephalic brain of rodents. In this article, we describe differences in neuronal density and supporting cells within the depths (fundus) and adjacent walls of the cingulate sulcus of the porcine brain. We also measured the distance between pyramidal neurons within Layers III and V to investigate if the observed increase in density of neurons within the cingulate fundus is associated with a decrease in distance between neurons in these layers. We also identify the presence of the gigantopyramidal neuron within the fundus of the porcine cingulate sulcus, a pyramidal neuron subtype seen in nonhuman primates and human brains. Taken together, this article provides evidence that further supports the heterogeneous composition of the gyrified brain by describing the cellular organization of the porcine cingulate sulcus.

Keywords: cingulate sulcus, cytoarchitecture, cortical layering, fundus, porcine brain, research resource identifiers (RRIDs)

Analysis of the porcine cingulate sulcus was conducted using immunohistochemical techniques. Results showed a higher neuronal density within the depths of the cingulate sulcus (fundus), as well as decreased distance between neuronal cell bodies within the different cortical layers of the fundus.

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1. Introduction

The folding of the brain, a process referred to as gyrification, occurs largely during development and results in changes to the morphology of the brain surface resulting in gyri and sulci. The gyrification of the brain takes place during the third trimester in humans, where brain surface area increases while maintaining the same volume within the cranial cavity (White et al. 2010; Bayly, Taber, and Kroenke 2014). The degree of gyrification, correlating to age, body weight, and brain volume, is described as the gyrification index (Zilles et al. 1988). Brains with a high gyrification index, such as the nonhuman primate and porcine brain, have distinct neural connectivity patterns and structure that closely resemble the organization of the human brain (Larsen et al. 2004).

The mechanisms by which the brain becomes folded are still largely unknown; however, there are several hypotheses proposed to explain how this process occurs from a material standpoint. The buckling hypothesis states that the outer, more elastic, regions of the brain grow faster than that of the inner, stiffer, regions (Richman et al. 1975). This difference in growth rates results in buckling of tissue into folds as the outer regions expand creating a compressive stress on the inner regions (Richman et al. 1975). The theory of multiplicative growth (Rodriguez et al. 1994) states that as the outer cortical layer of the brain experiences rapid growth, it is limited by the size of the skull. The rapid growth due to genetic factors and differing rates of cell proliferation is constrained by the boundary of the skull, resulting in the tissue folding inward to allow for further development in the limited space provided (Rodriguez et al. 1994; Bayly, Taber, and Kroenke 2014).

There are several hypotheses that aim to explain the development of brain gyrification at the cellular level (for full review see Fernández and Borrell 2023). These explanations focus on the migration of intermediate progenitor cells (IPCs) from the ventricular and subventricular zones (divided into inner and outer subventricular zones in some species) to the outer cortical plate. This is achieved by radial glial cells providing scaffolding structures that allow IPCs to expand outward, increasing the size of the brain before developing into cortical neurons (Borrell and Götz 2014). A specialized type of radial glial cell, known as basal radial glial cell, has been identified in gyrencephalic brains linked to cortical expansion and cortical folding (Hansen et al. 2010; Pilz et al. 2013). These cells do exist in the smooth, lissencephalic rodent brain, but their rate of cellular division and differentiation is significantly less compared to that seen in gyrencephalic brains (Hansen et al. 2010; Betizeau et al. 2013).

The cytoarchitecture of the mammalian brain is a complex arrangement of cortical layers, comprised of different cell types and degrees of myelination. The organization of the six cortical layers is highly conserved across evolution, with the same cell types observed in rodents, ferrets, pigs, and humans (Palomero‐Gallagher and Zilles 2019). A distinction between gyrified and lissencephalic brains is the thickness of the cortical gray matter at different parts and the variation in cytoarchitecture of the folded brain. The depths of the sulci (fundus) are roughly half the thickness compared to the adjacent walls of the sulci and top of the gyri (Chance et al. 2004). Moreover, Chance et al. (2004) found that as the gyri move into the sulcal depths, there is an increase in overall cell density (measured both neurons and glial cells) at the fundus in Layers II and V compared to the cell layers in adjacent regions of the sulcus.

The porcine cortex shares similar characteristics to the human brain and makes it a suitable candidate for studying properties of the gyrified brain. These similarities include gyrification index, white and gray matter densities, overall growth, and myelination (Tallinen et al. 2014; Cullen et al. 2016; Villadsen et al. 2018). To further understand the cytoarchitecture of the gyrified brain, we examined distances between neurons within Layers III and V of the porcine cingulate cortex, among other characteristics. By describing the characteristics of the porcine cortex, we hope to further the basic understanding of the particular properties of the folded brain that may predispose it to certain pathological outcomes not seen in smooth cortices.

2. Methods

2.1. Brain Removal

Whole porcine heads (10–12‐month‐old Yorkshire pigs) were sourced from a local abattoir. The pigs were slaughtered by electrocution to the back of the neck and bled, a common slaughter procedure used in the agriculture industry (Terlouw, Bourguet, and Deiss 2016). Whole pig heads were collected approximately 15 min after slaughter and transported back to Carleton University campus for removal. Total time for transportation was approximately 50 min. The procedure for brain removal has been previously described (Bjarkam, Orlowski et al. 2017; Hoffe et al. 2021). Slices for immunohistological preparations were taken 3 cm from the anterior tip of the porcine brain (Figure 1), in what would be considered between the primary somatosensory cortex and motor cortex (Sauleau et al. 2009).

FIGURE 1.

FIGURE 1

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Horizontal and sagittal view of the porcine brain removed from the skull. Dashed lines represent where sectioning commenced for immunohistological preparations. Scale bar = 3 cm.

2.2. Immunohistochemistry

After incubation in 4% PFA was completed, brain slabs were placed into 30% sucrose/0.1 M phosphate buffer saline (PBS) for storage. Coronal slabs were hemi‐sectioned, and the right hemisphere was sectioned on a cryostat at 60 µm and placed into 0.1% sodium azide/0.1 M PBS solution for storage.

Brain sections underwent three 5‐min washes in 0.2% PBS‐Triton X (PBS‐TX), followed by a 30‐min incubation in 3.0% hydrogen peroxide. After this incubation, sections were then placed into a peroxidase blocking solution (BLOXALL, Vector Laboratories) for 10 min, followed by a single, 5‐min wash in 0.1 M PBS. Sections were placed into primary antibody solution (rabbit anti‐NeuN, 1:5000; Abcam, Lot. Ab177487; RRID: AB_2532109) and left to incubate on a shaker table overnight at room temperature.

The following day, the sections were washed in three 10‐min washes of 0.2% PBS‐TX and incubated in secondary antibody solution (biotinylated goat anti‐rabbit, 1:1000; Vector Laboratories, Lot: ZJ0418; RRID: AB_2313606) for 2 h at room temperature on a shaker table. Following incubation, slices were washed in three 10‐min washes of 0.2% PBS‐TX before being placed into Avidin/Biotin Complex Solution (ABC; Vector Laboratories). Sections were washed in three 5‐min washes of 0.1 M PBS. A diaminobenzene kit (DAB; Vector Laboratories) was used to visualize stain, and company protocol was followed. Sections were then mounted onto slides and counterstained using 0.1% Cresyl‐Violet solution and differentiated with glacial acetic acid.

2.3. Image Acquisition and Neuron Distance Measurement

Images were taken using an MBF CX9000 camera (MBF Bioscience, Williston, VT) mounted onto an Olympus Bx51 microscope with a Prior motorized stage. Images for measuring neuronal distance were taken at 40× magnification within the porcine cingulate sulcus fundus and arm region of interests (ROI). A Z‐stack image acquisition was taken with a depth of 20 µm, with each image taken at 1 µm intervals, producing twenty images per stack. Z‐stack image acquisition allowed for precise definition of cell boundaries.

Distances were measured between NeuN‐labeled pyramidal neurons within Layers III and V of the arm and fundus of the porcine cingulate sulcus in ImageJ. Briefly, a single neuron was highlighted as the primary reference point. Individual lines were then traced from the center of the highlighted neuron to the center of adjacent neurons using the measure tool (e.g., see Figure 2). The distance for that measurement was recorded in a separate file. This process of measuring to adjacent neurons was repeated 8–10 times for that primary reference neuron. From this, 8–10 primary reference neurons were selected, resulting in a total of 80–100 measurements per Z‐stack. Measurements were run in triplicate for each ROI per brain, with a total group size of three per condition. All together, a total of roughly 720–900 measurements were taken for both the fundus and arm of the cingulate sulcus.

FIGURE 2.

FIGURE 2

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Representative image of distance measurement. Reference neuron (blue) has 8–10 radial measurements (yellow lines) to adjacent neurons within ROI. Measurements were taken from center of reference neuron to the center of adjacent neuron.

2.4. Quantification of the Cellular Composition Within the Sulcus

Unbiased stereological quantification investigating the cellular composition of the porcine cingulate sulcus was performed using Stereo Investigator (MBF Bioscience, Williston, VT). ROI containing the neuronal layers was traced for both the sulcus fundus and adjacent arms. ROI tracing was performed in such a way that both the sulcus fundus and arms contained approximately the same size for tracing area. Separate markers were used to identify neurons and Cresyl‐Violet‐stained cells. Cresyl‐Violet‐stained cells were considered any cells that did not overlap with a NeuN‐labeled neuron.

2.5. Statistical Analysis

The data are presented as mean ± standard error of the mean (SEM). For the cellular composition of the sulcus, data were analyzed using two‐tailed t‐tests to compare mean NeuN density, Cresyl‐Violet density, total cellular density (NeuN and Cresyl‐Violet combined), and the ratio of Cresyl‐Violet to NeuN‐labeled cells within the fundus of the sulcus and the adjacent arm (Prism Graph Pad 8.0). For distance measurements between NeuN‐labeled cells within Layers III and V of the sulcus, mean distance measurements within the fundus of the sulcus and adjacent arm were compared using two‐tailed t‐tests (Prism Graph Pad 8.0). An alpha level of p < 0.05 was considered statistically significant.

3. Results

3.1. Cellular Composition of the Cingulate Sulcus

The cingulate sulcus is located laterally to the corpus callosum in the gyrified brain and is one of the first folds coming from the central midline. The cingulate sulcus was chosen as a distinct landmark to provide reliable measurements across the samples. Although this area in pigs has been previously anatomically described (Félix et al. 1999), the cytoarchitecture and cellular composition of this region have yet to be quantified in detail.

Here, we demonstrate that there are unique cellular compositions that distinguish neuronal and cellular populations in the fundus and adjacent arm of the cingulate sulcus (Figure 3). Along the sulcal walls, there is clear delineation between the neuronal layers of the cortex, with each layer having distinct boundaries and clearly identifiable neuronal subtypes. Within Layer I of the cortex, there are sparse NeuN‐labeled cells. This has been previously described in the rodent brain where the Cajal‐Retzius cells occupy this layer but lack NeuN‐labeling (Duan et al. 2016). Layer II of the porcine cortex is clearly defined by the clustering of the granular‐type neurons (Figure 4). These neurons are characterized by small cell bodies and projections leading into Layer I of the cortex. It is important to note that within Layer II of the porcine cortex, there is the presence of small‐bodied pyramidal cells. Layer III of the porcine cortex contains medium‐sized pyramidal neurons with projections leading into the ascending layers of the cortex (Figure 4). Layer V of the porcine cortex contains the large pyramidal neurons with large gaps to accommodate their size (Figure 4). Layer V can be divided into two sub‐layers, with Layer Va containing medium‐large pyramidal neurons and Layer Vb containing the large pyramidal neurons. Layer VI of the porcine cortex contains oddly shaped neurons that run along the white matter tract. There are some large pyramidal neurons within this layer; however, there also are more stellate‐shaped neurons as well as bipolar‐shaped neurons (Figure 4). Due to the location where brain tissue was sampled from, Layer IV does not appear to be present. This is in accordance with previous reports that describe the presence of Layer IV to be located in the caudal regions of the porcine neocortex (Bjarkam, Glud et al. 2017; Graïc et al. 2022).

FIGURE 3.

FIGURE 3

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Whole coronal slice representation of the porcine brain and cortical layering. (A) Coronal slice of the porcine brain (2.5× magnification) indicating cingulate sulcus fundus and arm (dashed boxes). Scale bar = 5 mm. (B) Cortical Layers I–VI of the porcine cingulate sulcal fundus and arm (10× magnification). Dashed lines indicate rough delineation between cortical layers based on neuron type. Scale bar = 150 µm.

FIGURE 4.

FIGURE 4

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Representative image of the different neuron types within the cortical layers. (II) Layer II of the porcine cortex consists primarily of granular type neurons, as well as small‐bodied pyramidal neurons. (III) Layer III consists of small to medium sized pyramidal neurons that are close in proximity to one another (photo taken in fundus). (V) Layer V consists of the medium to large sized pyramidal neurons with apical dendrite “trunks” reaching far into the superior cortical layers. (VI) Layer VI consists of numerous different neuronal subtypes such as stellate, large‐bodied pyramidal neurons and bipolar neurons. All images were taken at 40× magnification, scale bar = 25 µm.

3.2. Measurements of Cytoarchitecture Within the Porcine Cingulate Sulcus

As the cortex moves from the sulcal wall to the fundus, the neuronal layers condense, making the distinctive gaps between layers seemingly disappear. This change in cytoarchitecture around the fundus was confirmed by a two‐tailed t‐test which revealed a difference in neuronal density within these two regions, with the sulcus fundus, showing higher neuronal density (4.693 cells/µm3 ± 0.300) compared to the neuronal density in the adjacent arm (2.791 cells/µm3 ± 0.247) of the cingulate sulcus (t = 4.889, p = 0.0081; Figure 5A,B,G).

FIGURE 5.

FIGURE 5

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Analysis of the cytoarchitecture within the porcine cingulate sulcus. (A) Representative image of cingulate sulcus fundus (60× magnification, scale bar = 25 µm). (B) Representative image of cingulate sulcus arm (60× magnification, scale bar = 25 µm). (C) Representative image of Cresyl‐Violet cells and NeuN‐labeled neurons within the fundus of the cingulate sulcus (100× magnification, scale bar = 10 µm). (D) Representative image of Cresyl‐Violet cells and NeuN‐labeled neurons within the arm of the cingulate sulcus (100× magnification, scale bar = 10 µm). (E) Representative image of overall cell density within the porcine cingulate sulcus fundus (20× magnification, scale bar = 50 µm). (F) Representative image of overall cell density within the porcine cingulate sulcus arm (20× magnification, scale bar = 50 µm). (G) and (H) NeuN and Cresyl‐Violet density data represented as mean ± SEM. (I) Cresyl‐Violet/NeuN ration data represented as mean ± SEM. (J) Overall cell density data represented as mean ± SEM. Star = p < 0.05.

To determine if this change in cytoarchitecture was due to neurons, we analyzed Cresyl‐Violet‐stained cells in the same region to investigate if there were changes to other cell types. A two‐tailed t‐test revealed a significant difference between Cresyl‐Violet‐labeled cell density, with the arm density (7.952 cells/µm3 ± 0.431) being higher than the fundus Cresyl‐Violet density (6.150 cells/µm3 ± 0.148) within the cingulate sulcus (t = 6.854, p = 0.0024; Figure 5H). Further investigation into these cellular composition characteristics revealed about a 3:1 Cresyl‐Violet/NeuN‐labeled ratio in the sulcus arm to roughly 1:1 Cresyl‐Violet/NeuN‐labeled ratio in the fundus (t = 6.571, p = 0.0028; Figure 5C,D,I).

Despite the differences in neuronal and Cresyl‐Violet‐labeled cell densities, when analyzing total cell density (NeuN‐labeled cells + Cresyl‐Violet‐labeled cells), a two‐tailed t‐test revealed no difference in total cell densities between the arm (10.74 cells/µm3 ± 0.447) and fundus (10.84 cells/µm3 ± 0.377; t = 0.171, p = 0.8723; Figure 5E,F,J).

The higher neuronal density of the cingulate sulcus fundus prompted the question: If there is a higher density of neurons within this region, does this mean these cells are physically closer together? Taking into consideration the differences in cell composition within the cortical layers, we investigated the differences between the pyramidal neurons, the primary neuronal type in the cerebral cortex, within cortical Layers III and V of the cingulate sulcus arm and fundus. A two‐tailed t‐test revealed a significant difference in distance between pyramidal cells within Layer III the fundus (36.52 µm ± 1.558) and arm (48.16 µm ± 2.805; t = 3.626, p = 0.022; Figure 6). This difference in distance between cells was not observed in Layer V of the cingulate cortex (t = 0.1937, p = 0.856; Figure 6).

FIGURE 6.

FIGURE 6

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Measurement of distance between neurons in cortical Layers III and V of the porcine cingulate sulcus. (A) Representative image of Layer III of the porcine cingulate sulcus fundus. (B) Representative image of Layer III of the porcine cingulate sulcus arm. (C) Representative image of Layer V of the porcine cingulate sulcus fundus. (D) Representative image of Layer V of the porcine cingulate sulcus arm. Dashed boxes in all images represent the delineation of the cortical layer. All images were taken at 40× magnification, scale bar = 25 µm. Distance between neurons data represented as mean ± SEM. Star = p < 0.05.

Along with these differences in cytoarchitecture, and the physical space of the different cortical layers of the arm and fundus, there were unique pyramidal cell types within the fundus of the sulcus. The gigantopyramidal cells were observed within the fundus of the porcine cingulate sulcus (Figure 7). These unique pyramidal neurons have been previously described in other mammals, including humans, with projections reaching to deep brain structures as well as into the peripheral nervous system (Nolan et al. 2024). While the arm of the porcine sulcus does contain a subpopulation of large pyramidal neurons, these gigantopyramidal neurons were found along the ventral portions of the sulcus arm that extend into the fundus of the sulcus. Surrounding these neurons was a higher presence of Cresyl‐Violet‐stained cells, with some gigantopyramidal neurons having two to three Cresyl‐Violet‐stained cells adjacent to the neuronal membrane.

FIGURE 7.

FIGURE 7

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Representative image of gigantopyramidal neurons within Layer V of the porcine cingulate sulcus fundus. These neurons were characterized by their large pyramidal shape, with some gigantopyramidal neurons exhibiting a more rotund base (left), whereas other gigantopyramidal neurons had a more distinct triangular shape. Several Cresyl‐Violet‐stained cells can be seen surrounding these neurons. Images taken at 100× magnification. Scale bar = 10 µm.

4. Discussion

In this article, we described the cytoarchitecture of the cingulate sulcus wall and fundus in the porcine brain. The porcine brain, as well as other mammals such as nonhuman primates, canines, and bovines, exhibits striking similarities in the development and organization of the brain (Lind et al. 2007; Nolan et al. 2024). Using porcine brains can be beneficial to understanding how the gyrified brain develops, how cellular organization occurs and potential explanations for neurological conditions that are unique to the folded brain.

4.1. Neuronal Characteristics of the Gyrified Brain

The coronal layering and composition of neuronal subtypes within the cerebral cortex is fairly conserved across species, including the porcine brain, with a few exceptions found in gyrified brains. Here we describe the porcine brain as having six cortical layers, each with their own neuronal subtype that makes each layer distinct from one another. Moreover, the porcine brain also exhibits layer‐specific protein characteristics, with the near complete lack of NeuN‐labeling of the Cajal‐Retzius cells found within Layer I. This characteristic has been observed in other species, but the reasoning as to why these neurons within Layer I do not express NeuN remains elusive (Duan et al. 2016). Layer II of the cortex, also referred to as the extra‐granular layer, contains a densely packed region of granular cells that shows a clear delineation from the absent‐stained Layer I (Figure 3). Within Layer II, there are also sparsely scattered small, pyramidal neurons, but the primary neuron within this layer appears to be granular neurons. This intermingling of granular neurons and small‐sized pyramidal neurons is a characteristic found in both human and nonhuman primate brains (Rosabal 1967; Bludau et al. 2014). Cortical Layer III is primarily made up of small‐ to medium‐sized pyramidal neurons. With the NeuN stain, the apical dendrite “trunks” are visible and reach into the superior layers. It is within Layer III of the porcine cortex that we first observe a major change in the neuronal organization of the gyrified brain. The internal granular Layer IV was not observed within the region described, which is consistent with previous studies (Jelsing et al. 2006; Pirone et al. 2019; Desantis et al. 2021).

To our knowledge, we are the first to quantify the physical distance between neurons within the porcine cingulate sulcus arm and fundus. There have been several instances of qualitative observations that the neurons within this region are closer together, given the curvature of the sulcal depths (Welker 1990; Chance et al. 2004; Vogt et al. 2005). We describe here a statistically significant decrease in distance between neurons within Layer III of the porcine cingulate sulcus fundus, with the Layer III pyramidal neurons being, on average, 10 µm closer compared to the adjacent sulcal wall. This could be due to a variety of reasons, such as the soma of pyramidal neurons within the fundus being larger in size compared to those found in adjacent layers (Vogt et al. 2005), or cortical thinning that occurs around the base of the fundus (Chance et al. 2004; Borrell 2018). Interestingly, we did not observe a difference in the distance between the neurons that make up Layer V of the cingulate sulcus (Figure 6). This could be due to the increased size of pyramidal neurons basal dendritic field within this region (Rosabal 1967; Vogt et al. 2005), and the visual presence of the largest pyramidal neuron subtype, the gigantopyramidal neuron or Betz cell (Figure 7; for an in‐depth review of Betz cells see Nolan et al. 2024). The presence of these unique pyramidal neurons also supports the similarities between the porcine brain and the human brain, as these unique pyramidal neurons are exclusively found in mammals with gyrified cortices (Nolan et al. 2024). Larger pyramidal neurons have also been described in the motor cortex fundus of similar species within the Cetartiodactyl order, such as sheep, dolphin, and wild boar, with connections to thalamic regions and the spinal cord (John et al. 2017; van Kann et al. 2017). These deep brain connections are believed to be responsible for the cortical involvement in encoding complex movements (Nolan et al. 2024).

An interesting observation made when characterizing these neurons was their location within the cingulate sulcus. Similar to humans and some nonhuman primates (Szocsics et al. 2021; Nolan et al. 2024), the gigantopyramidal neurons observed in this study were mainly found within the fundus and the early curvature into the adjacent walls of the sulcus. Classification of this specific type of pyramidal neuron was done so by visual morphological criteria established in previous literature (Nolan et al. 2024). Along with this, these gigantopyramidal neurons were accompanied by several Cresyl‐Violet‐stained supporting cells, possibly glial cells, around the soma of the gigantopyramidal neurons. Future studies using glial markers should be done to confirm the cell type that supports the gigantopyramidal neurons within this cortical layer, as well as confirming the presence of gigantopyramidal neurons in the porcine brain based on quantifiable criteria (i.e., somal volume).

Von Economo Neurons have been characterized in the gyrified brain of numerous animal species, including humans for nearly a century, with primary location of the neurons observed in Layers III and V of the anterior cingulate cortex, anterior insular cortex, and the frontal pole region (von Economo 1929; Triarhou 2006; Raghanti et al. 2015). These neurons are characterized by thick apical and basal dendrites stemming from a spindle‐like soma, with the apical and basal dendrites spanning multiple cortical layers. Von Economo Neurons have been studied in their involvement with social awareness, cognition, and interoception (Allman et al. 2010; Seeley et al. 2012). Interestingly, within the neocortex, Von Economo Neurons have the highest density at the tip of the gyral crown, and lower density within the sulcal depths (Raghanti et al. 2015). The porcine brain has reported to have high numbers of Von Economo Neurons when compared to the human brain, which might account for the robust social hierarchy and cognition that have been previously described in porcine behavior (Lind et al. 2007; Raghanti et al. 2015). Similar to Betz cells, future studies should investigate the in‐depth characterization of Von Economo Neurons within the porcine brain, such as distribution to normal, pyramidal neurons as well as somal volumes and presence of glial cells surrounding these neurons.

4.2. Explanations for Heterogeneous Neuronal and Cell Densities of the Cingulate Sulcus

Having established that there is a decrease in distance between the neurons within specific layers of the porcine cingulate cortex, we next investigated the neuronal density of the fundus and adjacent sulcal walls. Here we describe that there is a statistically higher density of neurons within the fundus compared to the adjacent arm (Figure 4). This analysis looked at all the cortical layers that were NeuN‐labeled (Layers II–VI), with the upper threshold of the region of interest consisting of the boundary between Layers I and II and the lower threshold consisting of the boundary between Layer VI and the white matter. We found that overall, there was a higher density of neurons within the fundus of the cingulate sulcus compared to the adjacent arm when analyzing similar gray matter volumes. Upon visual inspection, the upper cortical layers (Layers II and III) appear to be where the higher density of neurons is located, which supports the decreased cell distance within Layer III. Previous work examining NeuN‐labeled neurons in Layer V of the porcine cortex found that there was no difference in the density of neurons between the two regions of interest (Hoffe et al. 2021) which has also been observed in Layer V of the human brain (Bludau et al. 2014).

When measuring the other cell types within the fundus and adjacent arm, we discovered that there was a decrease in the number of Cresyl‐Violet‐stained cells within the fundus. These other types of cells are believed to be different types of glial and supporting cells, given their cell size and lack of NeuN (Pilati et al. 2008; Favorito et al. 2017). Moreover, the ratio of Cresyl‐Violet‐stained cells to NeuN‐labeled neurons shifts between the sulcal arm and fundus, with the sulcal arm having roughly three Cresyl‐Violet‐stained cells to one neuron, whereas the fundus has roughly a 1‐to‐1 ratio. Upon further inspection, a large portion of the Cresyl‐Violet stained cells appear to occupy Layer V, with supporting cells surrounding the larger pyramidal neurons and gigantopyramidal neurons. In Layer III of the cingulate sulcus fundus, the number of Cresyl‐Violet‐stained cells is lower possibly due to limited physical space between the neurons. This could have implications for possible mechanisms of neuropathology as the reduced amount of supporting cells within the fundus might prolong homeostatic restoration and maintenance of the extracellular space.

Despite the differences in neuronal density and the ratio of neurons to Cresyl‐Violet‐stained cells, we found that the overall cell density (both neurons and supporting cells) remained constant between the sulcal walls and the fundus of the cingulate sulcus. This is rather interesting as it shows a preferential shift towards neurons within the depths of the cingulate sulcus. There are several explanations for this occurrence based on previous literature investigating the cytoarchitecture of larger, gyrified brains. First, gray matter thinning has been established to occur in fundal areas compared to the gyrus tip and sulcal walls (Chance et al. 2004). Considering how the cortex maintains the six primary layers, the space between these neurons and the density of the supporting cells must be reduced to compensate for the reduction in gray matter (Chance et al. 2004). Another explanation can be derived from the dendritic arborization and clustering of neurons within the fundus. It has been demonstrated that in marmosets and humans, the fundal basal dendritic field is more extensive than those in the surrounding sulcal and gyral areas (Welker 1990; Elston, Rosa, and Calord 1996; Kroenke and Bayly 2018). At the gyral tip and descending into the sulcal wall, the neuronal cytoarchitecture represents a column‐like structure, with pyramidal neurons characterized as having long apical dendrites and clear distinction among the different cortical layers (Welker 1990). As the neuronal density increases, the space between the neurons decreases (especially in Layer III), and the neurons within these layers display shorter apical dendrites, and the basal dendritic fields spread out in a parallel fashion to the underlying white matter (Welker 1990; Kroenke and Bayly 2018). Moreover, the presence of possible gigantopyramidal neurons within Layer V of the cingulate fundus may require higher levels of support given their vulnerability to damage and degeneration, leaving less available supporting cells for the superior cortical layers (Mattson and Magnus 2006; Tsuchiya et al. 2006; Genç et al. 2017). A third explanation is that there are variations in genetic expression similar to those observed in humans, ferrets, and experimental models of mouse brain gyrification. Del Toro et al. (2017) demonstrated in a Flrt1/3 DKO brain that reduced levels of cell adhesion promoted nonuniform neuronal migration profiles and clustering of neurons that formed sulci. Wild‐type mice that had high levels of Flrt1/3 exhibited typical lissencephalic structures and homogenous cortical columns (Del Toro et al. 2017). Flrt1/3 DKO mice showed faster proliferation of pyramidal neurons in the brain, possibly leading to the folding of the cortex (Del Toro et al. 2017). The faster proliferation and clustering of the neurons around the sulcus could potentially explain the reduced levels of Cresyl‐Violet‐stained cells neurons within the sulcus, but similar overall cell densities when comparing the sulcal wall and the fundus. While beyond the scope of the present study, genetic sequencing of factors involved in neuronal clustering and cell proliferation could further exemplify the similarities between the porcine brain and other gyrified brains (Liko et al. 2016).

This unique process of cortical folding can lead to higher susceptibility of damage due to strain and biomechanical forces applied to the brain. Simulations of brain impact using finite element and three‐dimensional brain representation have shown some of the highest levels of strain occur within the fundus as well as along white matter tracts such as the corpus callosum (Ghajari, Hellyer, and Sharp 2017; Laksari et al. 2018). Data gathered from computer simulations (e.g., Cloots et al. 2008) showing the sensitivity of the sulci fundus regions have implications for certain pathological conditions. Chronic traumatic encephalopathy (CTE) and Alzheimer's disease both show significant neurodegeneration within the fundus regions of the sulci (McKee et al. 2023, 2013; Stern et al. 2013). Postmortem analysis of athletes who received numerous repetitive head impacts shows higher deposits of hyper‐phosphorylated tau, neurofibrillary tangles, and gliosis within the depths of the sulcus (McKee et al. 2023, 2013). The reason as to why this area is particularly vulnerable to neurodegeneration due to brain trauma remains unclear. Because the porcine brain has a high gyrification index, similar to that seen in humans (Nielsen et al. 2010; McBride and Morton 2018), one can model particular aspects of the folded brain, such as development and material dynamics, and compare these characteristics to the gyrified human brain.

4.3. Limitations and Future Directions

To the best of our knowledge, this is the first description of the neuronal cytoarchitecture of the porcine cingulate sulcus by quantifiably measuring the physical distance between the neurons as well as the neuronal, supporting, and overall cell density of the porcine cingulate sulcus. However, there are a few limitations that should be discussed. First, although Cresyl‐Violet‐stained cells and differentiation using NeuN staining have shown to be useful in the current study, we cannot definitively say what types of glial cells are observed. Previous studies have described different subtypes of glial cells based on physical size (Pilati et al. 2008; Favorito et al. 2017); however, we did not measure the size of the Cresyl‐Violet‐stained cells for subtype categorization. For the purpose of establishing a base knowledge of describing the porcine cingulate cortex, we negated measuring the size of the Cresyl‐Violet‐stained cell and simply classified them as a whole group against NeuN differentiation. Further studies investigating the different subtypes of glial cells, through double‐ or triple‐labeling immunofluorescence, could provide further insight into the cytoarchitecture and spatial distribution of the supporting cells found within the sulcal walls and fundus of the cingulate cortex. Additionally, the categorization of the gigantopyramidal neurons within Layer V was done on visual inspection of the neurons based on morphological characteristics previously described (Nolan et al. 2024). Further confirmation of these neurons as genuine Betz cells using quantifiable measurements should be conducted. Lastly, cortical thinning based on gray matter thickness was claimed through visual inspection of the porcine cortex. Further studies measuring the variability of the porcine cortex as it transitions from gyrus tip to sulcal depths could aid this claim. Future studies should also investigate if this pattern of neuronal clustering is found in other sulci throughout the cortex, or if this pattern is unique to the cingulate sulcus.

Although there are well‐documented similarities between the porcine brain and the human brain as previously mentioned, there remain some key differences in brain cytoarchitecture and brain development that should be addressed. First, van Kann et al. (2017) described the human brain as having thick, well‐defined Layer III within the primary sensory projection areas, whereas the wild boar and other terrestrial artiodactyls have a narrow, less densely packed Layer III in the same region. Another important discrepancy that is important to take into consideration is the presence, or lack thereof, of the cortical Layer IV in the porcine brain. In the human brain, as well as in other hominoids (chimpanzees, gorillas, bonobos), there exists a well‐defined internal granular Layer IV within regions of the frontal cortex (Vogt et al. 1995, 2005; Semendeferi et al. 2001). The prefrontal cortex of terrestrial artiodactyls lacks the presence of Layer IV within the cortical layering (Jelsing et al. 2006; Pirone et al. 2019; Desantis et al. 2021); however, the presence of an internal granular Layer IV does appear to exist in the caudal regions of the neocortex (Bjarkam, Glud et al. 2017; Graïc et al. 2022).

With this novel insight into the cytoarchitecture of the porcine cingulate sulcus, future research can investigate the unique feature of neuronal clustering and cellular distribution within this area. Recent studies analyzing the brains of individuals with CTE have found neurodegenerative markers, such as hyperphosphorylated tau, axonal disruption, and activated microglia, particularly within the depths of the cortical sulci (Holleran et al. 2017; Arena et al. 2020; McKee et al. 2023). This particular pathological development highlights the selective vulnerability of the fundus, as evident by the increased clustering within this region, particularly of highly vulnerable pyramidal neurons (Scheibel, Tomiyasu, and Scheibel 1977; Mattson and Magnus 2006; Nolan et al. 2024). Another direction can be the further justification for using porcine brains as an experimental model of the human brain. Macroscopic characteristics have been previously established (Lind et al. 2007; Hoffe and Holahan 2019), with this study adding the microscopic details showing similarities between the human brain and the porcine brain. Some differences must be taken into account when comparing the porcine brain to the human brain, such as gray matter thickness, differences in cell density populations, and structural composition (van Kann et al. 2017).

In conclusion, this study demonstrated the heterogenous cytoarchitecture within the porcine cingulate sulcus by measuring the neuronal, supporting, and overall cell densities, as well as the physical distance between the neurons that make up various layers of the porcine cingulate sulcus. We found an increase in neuronal density within the fundus, as well as a decrease in physical distance between the neurons in Layer III of the cortex when compared to the adjacent sulcal wall. Moreover, there was an increase in Cresyl‐Violet‐stained supporting cells within the adjacent sulcal wall, with a Cresyl‐Violet/NeuN ratio being roughly 3:1, whereas in the fundus this ratio was roughly 1:1. This novel insight into the cellular composition of the porcine cingulate sulcus can provide further insight into various human conditions by modeling the gyrified, human‐like cortex.

Author Contributions

Brendan Hoffe and Matthew R. Holahan performed the brain removals and immunohistochemical staining for the experiments. Brendan Hoffe and Lisa Hebert performed data analysis for the figures reported in the current manuscript. Brendan Hoffe drafted most of the manuscript, with writing contributions from Lisa Hebert on the cell distance and interpretation of results. Matthew R. Holahan and Lisa Hebert provided edits to the final draft of the manuscript. Experimental conceptualization was from Brendan Hoffe, Oren E. Petel, and Matthew R. Holahan, and scientific contributions were from Brendan Hoffe, Matthew R. Holahan, and Lisa Hebert. All authors have read and approved the final version of the manuscript.

Ethics Statement

No animal ethics were needed for the current study. Biosafety and Biohazards was approved by Carleton University Biohazards Committee (Biohazards Certification 109314).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/cne.70025.

Acknowledgments

The authors would like to thank Molly Mullan and Aida Attar for their assistance in data analysis and immunohistochemical preparations during the experimental procedure. The authors would like to thank Rohan Banton and Thuvan Piehler for the financial support from ARL for the materials used in these experiments.

Funding: Financial support was provided by the Department of the Army, U.S. Army Research Office under contract WP911F‐17‐2‐0222.

Data Availability Statement

The dataset used in the current 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 dataset used in the current study are available from the corresponding author upon reasonable request.

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