Neuropil Distribution in the Anterior Cingulate and Primary Visual Cortex of Cetartiodactyla, Primates, and Afrotheria 2 1 2 3 Amy Nguyen, Nina Patzke, Kathleen Bitterman, Paul Manger, and Muhammad Spocter 2,3 (1) College of Pharmacy and Health Sciences, Drake University (2) School of Anatomical Sciences, The University of Witwatersrand (3) Deparment of Anatomy, Des Moines University ABSTRACT The Ceratriodactyla contain a great diversity in brain morphology and behavior as exemplified by the elaborate behavioral repertoire of the bottlenose dolphin. Despite this, little is known about species-specific variation in neuronal packing, connectivity, 1 and variation in neuropil space for this clade. Using image analysis techniques, the neuropil fraction (NF) in the anterior cingulate cortex (ACC), and the primary visual cortex (OCC) in layers I-VI of the cerebral cortex was quantified to identify species and regional differences in connectivity. It is2 believed that qualitative differences in connectivity underlie behavioral differences between species. Given the role of the ACC as a component of the limbic system involved in the emotional aspects of motivating and 3 initiating goal-directed behaviors, it is argued that modifications in the neuropil are responsible for some of the complex behaviors exhibited by the members of this group. The study examines the NF in the ACC and OCC across 18 mammalian species. MATERIALS & METHODS Specimens and histological preparation The brains of eighteen adult mammalian species of unknown sex were used in this current study (Fig. 1). All animals were euthanized and brain specimens harvested during necropsy, with permission and guidelines from the University of the Witwatersrand Animal Ethics Committee and in accordance with comparable provisions from the NIH. After harvesting, whole brains were submerged in 4% until further processing. formaldehyde solution Chlorocebus aethiops (vervet mo n k e y ) Primates Catarrhini Vm H o m o sapiens ( h u ma n s) *not u s e d in study Platyrrhini Saimiri sciureus (squirrel m o n k e y ) Sqm Cere b White Matter Olf 4 0.9 OCC Artiodactyls 0.8 Image analysis The neuropil fraction (NF) was sampled in Nissl sections in layers I to VI by quantifying the relationship between the proportion of unstained and stained cellular elements. Using the StereoInvestigator program, contours were drawn around the region of interest at 2X magnification and fractionator sampling was used to randomly sample digital images at 20X magnification (Fig. 3). Image stacks consisting of approximately 20 images per section were collected and converted into a series of 8 bit grayscale image frames. The following parameters describe the sampling design: grid spacing 1000 μm X 1000 μm; an average target intensity of 71%; image size 1600 x 1200 pixels, resolution 0.37 pixels per μm. 4 0.7 Hippopotamus 0.6 4 0.5 Primates 0.4 4 0.3 Whales 0.2 0.1 0.0 Fig. 3- An example of the image sampling method used for estimating the neuropil fraction. Section through the ACC in Trichechus senagalensis. C a p r a ibex (alpine ibex)- Ci 0.9 AM CD Ci Ct HA Ss Ba M3 Pt Sqm Tm VM Gs Sc TA TS Dp Cg B a l a e n o p t e r a a c u t o r o s t r a t a (m i n k e w h a l e ) Ba Suidae Camelidae Macroscelididae Afrotheria H e x a p r o t o d o n l ib e r ie n s is ( p y g m y hi p p o ) - H a ACC 0.8 0.7 0.6 0.5 0.4 C a m e l u s d r o m e d a r iu s (camel)- C d S u s s c r o f a (wild boar ) - S s Trichechus manatus (manatee)- Trichechidae Fig. 4 - Example of Petrodromus tetradactylus (shrew)-Pt TT m r i ,c M h e3 c h u s s e n e g a l e n s i s ( m a n a t e e ) - T s pixels NF = + the image conversion pixels from grey scale to binary and NF equation. 0.3 0.2 0.1 0.0 AM CD Ci Ct HA Ss Ba M3 Pt Sqm Tm VM Gs Sc TA TS Dp Cg Blocks of the occipital and frontal lobes, containing the OCC and the ACC respectively, were removed with a coronal cut at the level of the precentral gyrus rostrally and a further cut at the level of the angular gyrus caudally (Fig. 2). Tissue blocks were cryoprotected by immersion with increasing concentrations of sucrose solutions up to 30%. Blocks were covered with sucrose, frozen on a slur of dry ice, and sectioned at 50μm using a sliding microtome. A 1:10 series of sections was stained for Nissl substance with a solution of 0.5% cresyl violet. Sig. 0.010 0.018 0.014 0.018 0.010 0.016 0.012 0.016 0.018 0.016 0.019 0.022 0.014 0.012 0.019 0.019 1.00E+00 5.81E-05 1.90E-17 2.89E-09 1.00E+00 1.71E-07 2.20E-19 4.63E-09 5.81E-05 1.71E-07 2.33E-21 5.54E-15 1.90E-17 2.20E-19 2.33E-21 1.00E+00 0.018 0.016 0.022 0.019 2.89E-09 4.63E-09 5.54E-16 1.00E+00 DISCUSSION CONCLUSION Fig. 5 - Regional variation in measurements of neuropil fraction (NF) in the 18 species according to the methods used in this study. Bar graphs of the mean neuropil fraction by cortical area. Error bars indicate standard error. In conclusion, all eighteen species display a higher neuropil fraction in the anterior cingulate cortex in comparison to that observed in the primary visual cortex. Species with complex socio-cognitive behaviors may have evolved different mechanisms to support these functions. Further studies are required to identify specific adaptations in the ACC, especially in the much understudied hippopotamus due to its surprisingly high NF values. Betweengroups df Acknowledgments This work was funded by a National Research Foundation (NRF) grant to Dr. Paul Manger and an IOER-FAC startup grant awarded to Dr. Muhammad Spocter. . Fig. 1 - Phylogenetic relationships of the species used in the current study. Statistical analysis All analyses were undertaken using the NF calculated for each section from the series of six sections sampled by region and species. The data was placed into an Excel worksheet and imported into SPSS Statistical Software package Version 22.0 for statistical comparison of significant differences in mean values. A Repeated Measures Analysis of Variance (ANOVA) with species as the between-subjects factor and brain region as the within-subjects factor was conducted. All graphs were plotted using the graphing function in PAST (version 1.18; PAST © Hammer et al., 2001). 0.014 -0.086* 0.152* 0.126* -0.014 -0.100* 0.139* 0.112* 0.086* 0.100* 0.238* 0.211* 0.211* -0.152* -0.139* -0.238* -0.027 -0.112* -0.211* 0.027 b Std. Error The increased connectivity observed in the ACC relative to the OCC supports our understanding of the ACC and its role in the limbic system. We would hypothesize that regional variation in neuropil space is indicative of behavioral differences supporting socio-cognitive functions relevant for each species. The contradiction within primates and cetaceans can be explained by modifications in neural circuitry. The study by Peters 6 and Yilmaz identified smaller minicolumn widths in primates, and we argue that the reduction of minicolumn widths, and in effect, smaller NF, allowed for specialization in the cortical microcircuitry. Identification of specialized modules in the primary visual cortex of primates7 supports the idea of modifications. The hippopotamus has on average, the largest neuropil space sampled to date; however, its aggressiveness makes studying its behavior and sociality difficult. In the absence of a full chemoarchitectural analysis and reconstruction of neuronal complexity, the behavioral complexity of the hippopotamus is relatively unknown. The use of the pygmy hippopotamus in the study is not representative of H. amphibius, and comparative studies such as these are limited by tissue availability and the “endangered status” of some of these species. Gazella subgutturosa (goitered gazelle)- G s Image frame acquisition was monitored during fractionator sampling, and all frames that fell outside of the region of interest boundaries were omitted. Each image series was imported into Image J (v.1.32j). Each series was subjected to background subtraction and then converted to binary by an automated threshold routine (Fig. 4).5 The percentage of the measuring frame occupied by pixels representing unstained elements was calculated and used to determine the NF using the equation below (Fig. 4). Artiodactyls Hippopotamus Primates Whales Afrotheria Hippopotamus Primates Whales Afrotheria Artiodactyls Primates Whales Afrotheria Artiodactyls Hippopotamus Whales Afrotheria Artiodactyls Hippopotamus Primates Difference (I-J) Table 2. Pairwise Comparisons. Highlighted areas indicate a significant difference. 1.0 Antidorcas marsupialis (springbok)- A m Tragelaphus angasii (nyala)- T a 4 Afrotheria Fig. 2 - Schematic illustrating the cortical areas where the neuropil was sampled. Damaliscus pygargus (blesbok)- D p S y n c e r u s caffer (African buffalo)- S c Comparison Groups 1.0 C o n n o c h a e t e s taurinus (blue wildebest)- C t Cetartiodactyla Our results indicate a consistent pattern of greater NF in the ACC in comparison to that observed in the OCC. The two primate species (vervet monkey and squirrel monkey) included in this study expressed the lowest mean NF across all species studied for both the ACC ( .697) and OCC ( .506). By contrast the hippopotamus ( H. liberiensis ), displayed the largest mean NF in both regions (ACC=.860 OCC=.819) and appeared to be an outlier in comparison to the broader Artiodactyla and Cetacea groups. Mean NF values in the artiodactyls were ACC= .763 and OCC =.717 while for the minke whale, a NF of .724 and .532 for the ACC and OCC were collected respectively. A significant difference in mean NF between the ACC and the OCC (F=775.684 P<0.05) was observed. In addition, a significant difference was observed between species (F=59.682 P<0.05) and the interaction between brain region and species (F=153.204 P<0.05) (Table 1). Pairwise comparisons of the mean difference in NF reveal that the hippopotamus differed significantly from all other groups included in this study while primates and cetaceans showed significant similarities in neuropil space. Significant similarities in neuropil space were also exhibited between Artiodactyls and Afrotheria (Table 2). Mean Caudat e C o n n o c h a e t e s g n o u (black wildebeest)- C g Bovidae RESULTS AC C Corte x Neuropil Fraction BACKGROUND INFORMATION OC C Neuropil Fraction Previous studies of the cerebral cortex have utilized the neuropil space as a proxy for connectivity, highlighting structural differences between cortical areas. The following study aims to investigate the distribution of neuropil space across 18 mammalian Orders in two cortical areas, sampled from the frontal and occipital lobes. Results indicate a significant difference in neuropil space between cortical areas and species. The anterior cingulate cortex maintained a higher neuropil fraction than the primary visual cortex among all species studied. Brain Area Species Brain Area * Species 1 F P 4 775.684 9.435E-50 1 59.862 4.986E-26 153.204 1.963E-42 Table 1. - Results of repeated measures of ANOVA of neuropil fraction across all 18 species References 1. Vislobokova I. 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