Ganglion cells and displaced amacrine cells density in the retina of the collared peccary (Pecari tajacu)

In the present study, we investigated the topographical distribution of ganglion cells and displaced amacrine cells in the retina of the collared peccary (Pecari tajacu), a diurnal neotropical mammal of the suborder Suina (Order Artiodactyla) widely distributed across central and mainly South America. Retinas were prepared and processed following the Nissl staining method. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from three animals. The average density of ganglion cells was 351.822 ± 31.434 GC/mm2. The peccary shows a well-developed visual streak. The average peak density was 6,767 GC/mm2 and located within the visual range and displaced temporally as an area temporalis. Displaced amacrine cells have an average density of 300 DAC/mm2, but the density was not homogeneous along the retina, closer to the center of the retina the number of cells decreases and when approaching the periphery the density increases, in addition, amacrine cells do not form retinal specialization like ganglion cells. Outside the area temporalis, amacrine cells reach up to 80% in the ganglion cell layer. However, in the region of the area temporalis, the proportion of amacrine cells drops to 32%. Thus, three retinal specializations were found in peccary’s retina by ganglion cells: visual streak, area temporalis and dorsotemporal extension. The topography of the ganglion cells layer in the retina of the peccary resembles other species of Order Artiodactyla already described and is directly related to its evolutionary history and ecology of the species.


Introduction
The Amazon rainforest is the most biodiverse biome of the planet. It is the home of many animal species, including mammals, thus being a significant source of data for comparative anatomy and physiology of tropical wildlife. Throughout the years, several studies have focused on the visual system morphophysiological organization in rodents and primates . two hours at 60 ºC. Next, the retina was washed in distilled water and stained with 0.5% cresyl violet for 10 min and dehydrated in a series of graded ethanol concentrations, cleared in xylene and coverslipped with Permount.

Imaging and analysis
The ganglion and amacrine cells were viewed and digitally documented using a microscope Axion Scope A1 (Carl Zeiss) with camera Axiocam HRc. The cell counting was made directly in the microscope using square fields and an objective A-plan 100x/1.25 Oil (Carl Zeiss). The cells were counting at 1 mm intervals along two meridians: (1) horizontal, in the naso-temporal axis along the visual streak; (2) vertical, dorsal-ventral axis, which crosses the horizontal meridian and the optic disc perpendicularly. Counts were also made along the vertical meridian from temporal to nasal border. For some special-target areas, as the visual streak or area temporalis, the interval between the counting fields was 0.5 mm and 0.25 mm, respectively. The ganglion and amacrine cell counts were converted to density in cells/mm 2 , and the estimation of the isodensity lines was described in earlier studies of our group [15,21]. Briefly, the isodensity contours were plotted linking the points over an isodensity contour and the points located between densities higher and lower than that corresponding to the isodensity contours. The total number of ganglion cells (GC) and displaced amacrine cells were then obtained by a simple measure of the area between two isodensity contours multiplying the area times the mean density value of the two isodensity contours. The total number was then the sum of all the resulting isodensity figures. To color the topographic maps the isodensity contours were drawn and converted into color-coded isodensity lines using SigmaPlot 1 for Windows™ Version 12.5 software (Systat Software, Inc., Richmond, CA).

Gross anatomy, retinal area and identification of ganglion cells and displaced amacrine cells
The peccary's retina had a typical vascular pattern called holangiotic as early described [39], the optic disc (OD) has an oval appearance. It is located just below the center of the retina and temporally displaced (Fig 1). The retinal area comprised 837.8 ± 56.5 mm 2 (N = 6) before the histological procedure and 828.8 ± 52.3 mm 2 after the histological procedure. The shrinkage due to histological procedures was estimated and ranged from 0.40% to 1.87%, a compilation of retinal area measurements performed before and after histology is showed on Table 1. The shrunken area was restricted to the periphery. Thus, ganglion cell counting was completed with no corrections for shrinkage.
We used morphological criteria to differentiate ganglion cells from neuroglial cells and displaced amacrine cells according to Hughes [41] The same criteria was used in other studies for artiodactyls species [32,42,43] and other species [17,40,44]. Briefly, ganglion cells have a large soma with an oval pale nucleus and a prominent nucleolus with abundant Nissl substance visible in the cytoplasm. On the other hand, displaced amacrine cells have small soma and cytoplasm containing less Nissl (Fig 2).

Total number and topography of ganglion cells
The total of the ganglion and displaced amacrine cells was estimated by the integration of isodensity contours from the isodensity maps (Fig 3). The total number of ganglion cells for all retinas was 1,029,498 (±121,060) ( Table 1). Table 2 shows the descriptive statistic for the density of ganglion cells; the mean density for all retinas analyzed was 2254 GC/mm 2 (± 346.7). The animal 03 showed the lower values, around 1,990 GC/mm 2 for both retinas. The density peak also varied strongly, the highest value was 9,900 GC/mm 2 , and the lower value was 5,100 GC/mm 2 , with peak mean at 6,767 GC/mm 2 (±1,914).
When we analyzed the ganglion cell isodensity maps (Fig 3), it can be seen that the lower cellular density occurred at the retinal periphery and was around 500 GC/mm 2 . The density increases concentrically towards the visual streak with density contours varying from around 2000 to 4000 GC/mm 2 . Besides, in visual streak, there was a temporarily dislocated circular region with densities between 4000 and 5000 GC/mm 2 , inside this area it´s located the peak density ( � ), this area is called area temporalis [36]. Fig 4 shows the "average" isodensity map for all retinas used in this study. For the ''average" map, the isodensity contours were drawn from mean density values of six retinas and plotted on the map of Animal 03 (left retina). Here we analyzed the number of ganglion cells by region. Each region was defined for lines isodensity contours. Thus, region A corresponded to the area between the wholemount border and the 500 GC/mm 2 contour. The B region is the area between 500 and 1000 GC /mm 2 ; C region

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was the area between 1000 and 2000 GC/mm 2 ; D region was the area between 2000 and 3000 GC/mm 2 ; E region was the area between 3000 and 4000 GC/mm 2 ; F region was the area between 4000 GC/mm 2 and density peak area represent by ( � ). In Fig 5 we presented a column graphic compared to the number of ganglion cells by region identify in the "average" isodensity map showed in Fig 4. Here we do not consider the density peak for this comparison. We observed that the region with most ganglion cells was C, followed for the B and D region.
The Figs 6 and 7 show the ganglion cells density profile in the dorsal-ventral and temporalnasal axes, respectively. For the dorsal-ventral axis ( Fig 6) the peak density was temporarily located at a mean distance of 3.13 mm (± 0.38) from the dorsal region to the optic nerve. The peak density varied among retinas with values ranging from 5100 to 9900 GC/mm 2 ( Table 2). From the optic nerve, there is an abrupt decrease in cell density to values close to 1000 GC/ mm 2 in the dorsal and ventral. Similarly, in the dorsal and ventral periphery, especially between the retinal border and first-line contour, the density falls to about 500 GC/mm 2 .
For the temporal-nasal axis, along the visual streak (Fig 7), the density was around 4000 GC/mm 2 from nasal to central direction. The peak density was temporarily located at approximately 6.77 mm (± 0.60) from the optic nerve.

Displaced amacrine cells
The topographical distribution of displaced amacrine cells (DAC) differs significantly from the ganglion cells distribution in two critical aspects: First, there was no spatial arrangement such as a visual streak; second, there was a significant decrement of displaced amacrine cells in the area temporalis (Fig 8). The distribution of displaced amacrine cells is homogeneous in the majority of the retina (Fig 8B) with a density of around 3000 DAC/mm 2 , and in the area temporalis region (white stippled circle), there was a considerable decrease in the density of displaced amacrine cells.  From the neurons in the ganglion cell layer the percentage of amacrine cells was always larger (reaching up to 70%) than the percentage of ganglion cells except for the area temporalis ( Fig 9A). Notably, in the area temporalis, there was an inversion of the percentage of amacrine and ganglion cells where the amacrine cells decreased to 30.52% ± 3.91 on average against 69.48% ±3.09 of ganglion cells (Fig 9B).

Discussion
In the present study, we have investigated the density distribution of ganglion and displaced amacrine cells using six retinas from the collared peccary, a diurnal mammal found

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throughout all Amazon Rainforest. The ganglion cell layer showed three characteristic regions with high cellular density: visual streak; area temporalis and a dorsotemporal extension named anakatabatic area, the later also found in other animals such as giraffe [32]. We located the visual streak above the optic nerve and the area temporalis superposed on the visual streak and the dorso temporal extension forming a dorsal arch of increased number of ganglion cells.
These results suggested that despite the difference in mean density values as well as peak density values, collared peccary and other species of the order Artiodactyla seem to share essential characteristics like retinal organization in ganglion cells layer. For example, in peccary, the mean cell density of ganglion cells in all retinas analyzed was 2253.5 GC/mm 2 , this value is higher than was reported for the wild pig (Sus scrofa), whose mean density was about 1133 GC/mm 2 [31]. On the other hand, if we consider just the peak density value for collared peccary, the average was 6767±1.94 GC/mm 2 , which is very close to what was found for wild pig's retina, that was 5735 ± 1066 GC/mm 2 [31]. Besides, the density peaks in both animals are displaced temporally relative to the optic nerve inside the visual streak. When our results for the collared peccary are compared to the retina topography analysis carried out in other species from the Artiodactyla order, some similarities are also noticed. For the goat, Gonzales-Soriano et al., reported a peak density of 3592 GC/mm 2 restricted to a circular area temporal to the optic disc, besides the existence of a visual streak [34]. In another study, this time in adult sheep retinas, the authors also found a visual streak formed by ganglion cells and area temporalis with a peak density of 18,000 GC/mm 2 [35]. These visual specializations (visual streak and area temporalis) were described in many artiodactyls such as giraffes [32], goats [41], cattle, pigs, and sheep [32,33], animals that inhabit a variety of environments, from the savannah to dense rainforests. Whereas the dorsal extension was found in artiodactyls such as sheep [35], giraffes [32], and the Nubian ibex [42].
Coimbra et al., [32] argued based on the theory of phylogenetic ancestry of Stone [45] that, as the horizontal streak and area temporalis are frequent in retinas of artiodactyls, these The A region corresponded to the area between the whole mount border and the 500 GC/mm 2 contour. The B region was the area between 500 and 1000 GC/mm 2 ; C region was the area between 1000 and 2000 GC/mm 2 ; D region was the area between 2000 and 3000 GC/mm 2 ; E region was the area between 3000 and 4000 GC/mm 2 ; F was the area between 4000 and 5000 GC/mm 2 and G region was the area on the peak density.
https://doi.org/10.1371/journal.pone.0239719.g005 specializations are most likely plesiomorphic characters inherited from a common ancestor. Thus, regardless of habitat or lifestyle, the visual streak and area temporalis are conserved traits in the retina of the order [26,31,32,34,46]. Based on Hughes [47], Coimbra et al., [32] also claim that the joint presence of these two conspicuous retinal characteristics certainly indicates their relation with important behavioral marks like foraging and avoiding predators. The dorsotemporal extension forms a dorsal arch of ganglion cells increase Some authors reported that this specialization is closely related with demands required by the inferior visual field such as spotting predators, orientation during foraging or ambulation [32]. It was also found in other artiodactyl living in mountains and valleys [42], which points to a shared ecological niche.
Undoubtedly, independent of the topographical arrangement of retinal cells, the ubiquitous presence of the visual streak, dorsotemporal extension and area temporalis provide a good adaptation despite the variety of natural environments inhabited for these animals. In this respect, we can conclude that the distribution of ganglion cells in the retina of the collared peccary is not different from the distribution of ganglion cells observed in the retinas of other diurnal artiodactyls. Interestingly, the collared peccary is found in a wide range of  environments such as desertic regions and tropical forests, suggesting again that the topographical map described for ganglion cells serves well for different habitats being important for foraging and avoiding predators.

Displaced amacrine cells
The displaced amacrine cells have been reported in other species but with little emphasis on their topographical distribution [48][49][50][51][52][53][54]. The topography of amacrine cells in the collared peccary retina differs from ganglion cell distribution in some critical aspects related to the retina specializations, visual steak, dorsotemporal extension and area temporalis.
The topography of the amacrine cells displaced in the peccary's retina is almost homogeneous throughout its extension and does not form a retinal specialization. Except in the area centralis, the proportion of displaced amacrine cells in the ganglion cell layer of the peccary retina is higher than the proportion of ganglion cells. Likewise, displaced amacrine cells are absent from the central area in the retina of the teleost thraira [52]. In addition, studies of a variety of primate retinas have shown that the proportion of displaced amacrine cells in the peripheral retina is significantly higher than that in central retina [14,55,56].
In the howler monkey (alouatta caraya), a diurnal primate, the proportion of amacrine cells versus ganglion cells is like that found in the collared peccary. In the alouatta on the periphery of the retina, amacrine cells represent about 67% to 84% of the total cells in the ganglion cell layer. In contrast, in the central retina (fovea) there is an inversion in the proportion of amacrine and ganglion cells, and amacrine cells represent about 6% of the cells in that region [14]. In aotus, a nocturnal primate, the proportion of amacrine and ganglion cells is

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Ganglion cells and displaced amacrine cells density in the retina of the collared peccary similar in most of the retina, except for the central region where displaced amacrine cells also decrease their percentage concerning ganglion cells [56]. The peculiarities and similarities found in the peccary' retina concerning other species, represent essential features of the order Artiodactyla to which the P. tajacu is inserted. The presence of retinal specializations in the peccary's retina, such as the visual streak, dorsotemporal extension and the area temporalis, which are directly related to its evolutionary history and ecology of the species, allow initiating morphophysiological comparisons of the retina of the collared peccary with that of other animal species.