Neuron Types in the Presumptive Primary Somatosensory Cortex of the Florida Manatee (Trichechus manatus latirostris).

Original Paper Brain Behav Evol 2015;86:210–231 DOI: 10.1159/000441964

Received: April 27, 2015 Returned for revision: May 11, 2015 Accepted after revision: October 25, 2015 Published online: November 28, 2015

Neuron Types in the Presumptive Primary Somatosensory Cortex of the Florida Manatee (Trichechus manatus latirostris) Laura D. Reyes a Cheryl D. Stimpson a Kanika Gupta a Mary Ann Raghanti b Patrick R. Hof c Roger L. Reep d Chet C. Sherwood a a

Department of Anthropology and Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington, D.C., b Department of Anthropology and School of Biomedical Sciences, Kent State University, Kent, Ohio, c Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, N.Y., and d Department of Physiological Sciences, University of Florida, Gainesville, Fla., USA

Abstract Within afrotherians, sirenians are unusual due to their aquatic lifestyle, large body size and relatively large lissencephalic brain. However, little is known about the neuron type distributions of the cerebral cortex in sirenians within the context of other afrotherians and aquatic mammals. The present study investigated two cortical regions, dorsolateral cortex area 1 (DL1) and cluster cortex area 2 (CL2), in the presumptive primary somatosensory cortex (S1) in Florida manatees (Trichechus manatus latirostris) to characterize cyto- and chemoarchitecture. The mean neuron density for both cortical regions was 35,617 neurons/mm3 and fell within the 95% prediction intervals relative to brain mass based on a reference group of afrotherians and xenarthrans. Densities of inhibitory interneuron subtypes labeled against calcium-binding proteins and neuropeptide Y were relatively low compared to afrotherians and xenarthrans and also formed a

© 2015 S. Karger AG, Basel 0006–8977/15/0864–0210$39.50/0 E-Mail [email protected] www.karger.com/bbe

small percentage of the overall population of inhibitory interneurons as revealed by GAD67 immunoreactivity. Nonphosphorylated neurofilament protein-immunoreactive (NPNFP-ir) neurons comprised a mean of 60% of neurons in layer V across DL1 and CL2. DL1 contained a higher percentage of NPNFP-ir neurons than CL2, although CL2 had a higher variety of morphological types. The mean percentage of NPNFP-ir neurons in the two regions of the presumptive S1 were low compared to other afrotherians and xenarthrans but were within the 95% prediction intervals relative to brain mass, and their morphologies were comparable to those found in other afrotherians and xenarthrans. Although this specific pattern of neuron types and densities sets the manatee apart from other afrotherians and xenarthrans, the manatee isocortex does not appear to be explicitly adapted for an aquatic habitat. Many of the features that are shared between manatees and cetaceans are also shared with a diverse array of terrestrial mammals and likely represent highly conserved neural features. A comparative study across manatees and dugongs is necessary to determine whether these traits are specific to one or more of the manatee species, or can be generalized to all sirenians. © 2015 S. Karger AG, Basel

Laura D. Reyes Center for the Advanced Study of Human Paleobiology The George Washington University, Science and Engineering Hall 800 22nd St. NW, Suite 6000, Washington, DC 20052 (USA) E-Mail ldreyes @ gwu.edu

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Key Words Manatee · Interneurons · Cerebral cortex · Mammal · Pyramidal cell · Cytoarchitecture · Afrotheria · Brain evolution · Immunohistochemistry

Neuron Types in the Florida Manatee Presumptive Somatosensory Cortex

Brain Behav Evol 2015;86:210–231 DOI: 10.1159/000441964

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Examining the architecture of the cerebral cortex from a wide range of mammals contributes to a more comprehensive view of brain evolution. The evolutionary history of mammals spans more than 200 million years, with the divergence of placental mammals having occurred over 100 million years ago [Pyron, 2010]. Consequently, mammals display considerable diversity in terms of brain size and isocortical organization [Krubitzer and Kaas, 2005; Boddy et al., 2012]. One well-supported phylogeny divides placental mammals into four major groups based on molecular genetic analyses: Euarchontoglires, Laurasiatheria, Xenarthra and Afrotheria [Murphy et al., 2001a, b; Springer et al., 2004; Hallström et al., 2007; Murphy et al., 2007; Nikolaev et al., 2007; Wildman et al., 2007; Prasad et al., 2008; Asher et al., 2009; Meredith et al., 2011; Song et al., 2012; but see Kriegs et al., 2006] (fig. 1). Although these four major groups are generally accepted, certain aspects of the eutherian tree, such as its root and relationships among orders within Euarchontoglires and Laurasiatheria, remain unresolved [Asher et al., 2009; Song et al., 2012]. Euarchontoglires and Laurasiatheria are a part of the magnorder Boreoeutheria, which arose on the Northern Hemisphere subcontinent of Laurasia, while Xenarthra and Afrotheria are part of the magnorder Atlantogenata that appeared on the Southern Hemisphere supercontinent Gondwana [Wible et al., 2007]. Xenarthrans (e.g. sloths, tamanduas, anteaters, armadillos) and afrotherians (e.g. elephant shrews, tenrecs, golden moles, aardvarks, hyraxes, elephants, manatees, dugongs) radiated in the South American and African continents, respectively [Archibald, 2003]. Neuroanatomical variation among extant members of atlantogenatans is of particular interest due to their divergence at the stem of the placental mammal lineage [Murphy et al., 2007]. The scaling of neurons and glial cells in the afrotherian cerebral cortex and cerebellum relative to brain mass was likely shared with the last common eutherian ancestor [Neves et al., 2014]. Afrotherians and xenarthrans also retain many plesiomorphic traits in terms of their cortical cell types based on their divergence near the root of the eutherian clade [Sherwood et al., 2007]. Afrotherians and xenarthrans closely resemble monotremes and marsupials in the morphology and laminar distribution of nonphosphorylated neurofilament protein-immunoreactive (NPNFP-ir) neurons, the proportion of calcium-binding protein-ir interneurons, and the typology of glial fibrillary acidic protein-ir astrocytes in the somatosensory cortex [Sherwood et al., 2009]. Previ-

ous investigations of chemoarchitecture in atlantogenatans, however, have not included sirenians (e.g. manatee, dugong). Sirenians are unusual within the afrotherian clade, since they have adapted to an entirely aquatic lifestyle [Hartman, 1979; O’Shea and Reep, 1990]. The sirenian brain is also distinctive compared with other afrotherians. Its external morphology is characterized by lissencephaly, or a smooth surface lacking in convolutions [Edinger, 1933, 1939; Reep et al., 1989; Reep and O’Shea, 1990] (fig. 2). Mammalian brains with a mass of over 300 g are usually gyrencephalic, and it is thus unusual that manatees and other sirenians, with an average brain mass of 350 g, are lissencephalic [Pillay and Manger, 2007]. Cytoarchitectural parcellation of the manatee cerebral cortex has revealed a total of 29 different regions [Reep et al., 1989; Marshall and Reep, 1995]. Cellular layers are distinct, and some regions have a granular layer IV while others are characterized by neuronal clusters, or Rindenkerne, in layer VI [Dexler, 1912; Reep et al., 1989; Marshall and Reep, 1995; Sarko et al., 2007] that are similar to clusters of neurons identified in layer V in cetaceans [Hof et al., 2005]. The Rindenkerne also resemble the barrel cortex in S1 of rodents and closely related species, in which input from each whisker terminates in a distinct cytoarchitectural area in layer IV [Woolsey et al., 1970, 1975; Rice, 1995]. In the barrel cortex, each individual barrel encodes information about the movement of a corresponding whisker [Swadlow et al., 1989]. However, barrels are present in the afferent layer IV while Rindenkerne are present in the efferent layer VI. Sarko et al. [2007] suggest that there may be a difference in how these two complexes are organized within the sensorimotor system, with Rindenkerne playing a larger role in descending pathways. Further investigation of the manatee cerebral cortex has revealed seven areas that comprise the presumptive primary somatosensory cortex (S1), including areas DM3 (dorsomedial cortex), DL1 (dorsolateral cortex), CL2 (cluster cortex), CL1, and parts of dorsal cortex, DL2 and DM2. CL areas contain Rindenkerne and are likely the site of vibrissal input. Areas DL, DM and dorsal cortex appear to process information from cutaneous afferents [Sarko and Reep, 2007]. The current study explored the neuronal organization of the Florida manatee (Trichechus manatus latirostris) cerebral cortex, with a focus on the presumptive primary somatosensory cortex, facilitating comparison with other afrotherians and their closest living relatives, the xenarthrans. We first investigated total neuron density based on Nissl-stained cytoarchitecture. We then examined the proportion, typology and morphology of inhibitory in-

Introduction

Boreoeutheria

Laurasiatheria Euarchontoglires

Eutheria

Xenarthra

Folivora (sloths) - two-toed sloth Vermilingua (anteaters) - giant anteater, lesser anteater Cingulata (armadillos)

Atlantogenata

Tenrecidae (tenrecs) Chrysochloridea (golden moles) Macroscelidea (elephant shrews) - giant elephant shrew Afrotheria

Tubulidentata (aardvarks) Sirenia (manatees, dugongs) - Florida manatee Hyracoidea (hyraxes) - rock hyrax

Fig. 1. Mammalian phylogenetic tree indi-

cating the position of manatees within the order Sirenia. The other species listed in bold italics include members of Xenarthra (two-toed sloth, giant anteater, lesser anteater) and Afrotheria (giant elephant shrew, rock hyrax, African elephant) that were compared with manatees. Adapted from Sherwood et al. [2009].

Proboscidea (elephants) - African elephant Marsupialia Monotremata

dorsal DD2 DM3 DD

CL3

DL4

DL3

DM2

rostral

CL2

CP

FR

CL4 cb

CL1

DL1 CL2

CL5 RH ENT

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bs

CL1

Reyes/Stimpson/Gupta/Raghanti/Hof/ Reep/Sherwood


brain. Dotted lines indicate the location of sulci. The areas examined in this study are shown in gray: dorsolateral somatosensory cortex (DL1) and cluster somatosensory cortex (CL2). Areas adapted from Reep et al. [1989] and Marshall et al. [1995].

OLF DL2

Fig. 2. Right lateral view of the manatee

Materials and Methods Sample The brains from two adult female Florida manatees (T. manatus latirostris) were obtained from the Lowry Park Zoo in Tampa Bay, Florida. Both manatees were wild and were euthanized after being struck by boats. Following euthanasia, pathology examination was performed by the Florida Fish and Wildlife Conservation Commission Marine Mammal Pathobiology Laboratory in St. Petersburg, Fla., USA. Both brains were gravity perfused with paraformaldehyde (one manatee at 2%, the other at 4%) within 24 h of death. Staining quality was comparable in both specimens despite different paraformaldehyde concentrations. All procedures were carried out under the approval of the University of Florida IACUC protocol No. C233. Histological Preparation and Immunohistochemistry In both manatees, the dorsolateral cortex (DL1) and the cluster cortex (CL2) were processed from tissue blocks dissected from whole brain specimens. Both regions were identified based on cytoarchitecture described previously. DL1 exhibited an indistinct layer II that was combined with layer III. Layer IV had small round cells and also contained small pyramidal cells from layers III and V. Layer V was thin with no sublayers, and layer VI was thick with tangential striations [Reep et al., 1989]. CL2 is distinguished by a relatively thick layer V especially compared to DL1, and a large layer VI with clusters of cells known as Rindenkerne [Reep et al., 1989; Loerzel and Reep, 1991; Marshall and Reep, 1995]. DL1 was selected for study because it is the S1 region with the most pronounced granular layer IV, while CL2 was selected because it is one of the regions with Rindenkerne, or clusters of neurons in layer VI. The tissue blocks from one manatee individual had a mass of 1.60 g for CL2 and 1.63 g for DL1, while the tissue blocks for the other manatee had a mass of 2.62 g for CL2 and 2.30 g for DL1. These two regions have previously been identified as part of S1 of the manatee by cytochrome oxidase staining [Sarko et al., 2007]. The S1 region in the manatee is considered presumptive as it has not been investigated with electrophysiological or tracing studies. These blocks were fixed for 10 days and were then transferred to a solution of phosphate-buffered saline (PBS) with 0.1% sodium azide. In preparation for sectioning, the blocks were cryoprotected by immersion in increasing concentrations of sucrose solutions up to 30%. The samples were subsequently frozen in dry ice and isopentane, embedded in tissue-freezing medium, and sections were cut from the blocks at a 40-μm thickness on a sliding microtome


(Leica SM2000 R, Nussloch, Germany). For each specimen, a 1:10 series of sections was Nissl-stained with a solution of 0.5% cresyl violet. Immunohistochemistry was performed for each antigen on adjacent 1: 20 series of sections. Free-floating sections were stained with monoclonal antibodies against nonphosphorylated epitopes on the neurofilament protein triplet (NPNFP; dilution 1: 3,000, SMI-32 antibody; Covance International, Princeton, N.J., USA), neuropeptide Y (NPY; dilution 1: 1,500; Peninsula Laboratories, San Carlos, Calif., USA), parvalbumin (PV; dilution 1: 10,000; Swant, Bellinzona, Switzerland), calbindin D-28k (CB; dilution 1:8,000; Swant) and GAD67 (dilution 1:2,000; Millipore, Billerica, Mass., USA), and with a polyclonal antibody against calretinin (CR; dilution 1: 10,000; Swant). Sections stained against NPNFP, PV, GAD67 and CB were processed using horse serum (H0146; Sigma-Aldrich, St. Louis, Mo., USA) and anti-mouse secondary antibody (BA-2000; Vector Laboratories, Burlingame, Calif., USA). Sections stained against CR and NPY were incubated using goat serum (Sigma, G9023) and anti-rabbit secondary antibody (BA-1000; Vector Laboratories). Prior to immunostaining, the sections were rinsed thoroughly in PBS, pretreated for antigen retrieval by incubation in 10 mM sodium citrate buffer (pH 3.5) at 37 ° C in an oven for 30 min, then immersed in a solution of 0.75% hydrogen peroxide in 75% methanol to eliminate endogenous peroxidase activity. For the SMI-32 antibody against NPNFP, antigen retrieval used the same buffer at pH 8.5 and at 85 ° C in a water bath to achieve optimal staining. Primary antibodies were diluted in a solution containing PBS with 2% normal serum (4% normal serum for SMI-32) and 0.1% Triton X-100 and incubated for approximately 48 h on a rotating table at 4 ° C. After rinsing in PBS, sections were incubated in the biotinylated secondary IgG antibody (dilution 1: 200, Vector Laboratories) and processed with the avidinbiotin-peroxidase method using a Vectastain Elite ABC kit (Vector Laboratories). Nickel enhancement of the chromogen was used in some cases. Positive control sections of mouse brain obtained from adult male C57Bl6 mice perfused with 4% paraformaldehyde were also used as an additional control in all experiments. Calcium-binding protein and NPY stains were selected to provide a comparison with previously published studies on other mammals [Glezer et al., 1993; Hof et al., 1999; Sherwood et al., 2009] and to indicate specific subtypes of GABAergic interneurons [Celio, 1986; Hendry et al., 1987, 1989; Celio, 1990; Blümke et al., 1990, 1991; Hashikawa et al., 1991; Hendry and Jones, 1991]. GAD67 is an enzyme that catalyzes the conversion of L-glutamic acid to GABA [Erlander et al., 1991]; immunohistochemical staining against GAD67 was therefore used to label all GABAergic interneurons and was selected to show the total density of interneurons regardless of type in the manatee S1. Staining for NPNFP was selected for this study to form a comparison with results from other mammals [Sherwood et al., 2009] and to assess a link between NPNFP expression and brain size in the manatee S1.

Regional and Laminar Analysis The regional and laminar analyses of stained sections were performed using a Zeiss Axioplan 2 photomicroscope equipped with a Ludl XY motorized stage (Ludl Electronics, Hawthorne, N.Y., USA), Heidenhain z-axis encoder (Heidenhain, Schaumburg, Ill., USA), an Optronics MicroFire color videocamera (Optronics, Goleta, Calif., USA) and a Dell PC workstation running StereoInvestigator software, version 10 (MBF Bioscience, Willis-


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terneurons identified by calcium-binding proteins (calbindin, calretinin and parvalbumin), neuropeptide Y and glutamic decarboxylase 67 (GAD67), as well as cells expressing NPNFP that likely represent excitatory projection neurons. These specific calcium-binding proteins were selected because they are present in subpopulations of neurons across a large number of vertebrate species including mammals [Hof et al., 1999] and would allow for comparisons with previous findings in afrotherians and xenarthrans [Sherwood et al., 2009].

CB

CR

PV

NPY

GAD67

I II/III

CL2

IV

V/VI

a

b

c

d

e

f

g

h

i

j

I II/III

DL1

IV

Small round multipolar

Large multipolar

Pyramidal

Round bipolar

Fusiform bipolar

Bitufted

Fig. 3. Schematic representation of inhibitory interneurons in manatee S1 areas CL2 (a–e) and DL1 (f–j). CB (a, f; CB-ir), CR (b, g; CR-ir), PV (c, h; PV-ir), NPY (d, i; NPY-ir) and GAD67 (e, j) stains are shown. Neuron types were comparable across stains, with round multipolar cells as the most common type. CR immunoreactivity was most distinctive, with a majority of bipolar neu-

rons. CB, PV, and NPY immunostaining also showed multipolar neurons in infragranular layers. Stained pyramidal neurons are also shown in the schematic, with the gray shades indicating the intensity of the stain. Staining for pyramidal neurons was visible in layers III and V and occasionally in layer VI, and was darkest for CR and GAD67 immunoreactivity in both cortical areas.

ton, Vt., USA). We identified cortical regions for examination based on cytoarchitecture observed in Nissl-stained sections and in reference to previous parcellations [Reep et al., 1989; Marshall and Reep, 1995].

of 50 sections (∼2 mm). Disector frames were set at 50 × 50 μm for determining total neuron density with Nissl-stained sections and at 100 × 100 μm for assessing density for CB-, CR-, PV-, NPY-, GAD67- and NPNFP-ir neurons. Grid spacing varied depending on the size of the reference area. On average, 109.04 ± 8.04 (mean ± standard deviation) counting frames per layer for each cell type were investigated. The optical disector analysis was performed under Koehler illumination using a ×63 objective lens (Zeiss Plan-Apochromat, NA 1.4). The thickness of optical disectors was set to 6 μm to allow for a sufficient guard zone at the top and bottom of the sections, and an optical measurement of mounted section thickness was collected at every eighth sampling site. Neuronal densities were derived from these stereological

Quantification Estimates of neuronal densities were obtained using the optical disector with fractionator sampling implemented in three equidistantly spaced brain sections for cell type in each S1 area. For the Nissl stain, three sections were selected at a regular interval to obtain a representative sample from the entire block. These regular intervals varied based on the size of the block. For the immunohistochemistry stains, three sections were selected at an interval

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V/VI

a

b

c

e

d

Fig. 4. CB-ir neurons in areas CL2 (a–c) and DL1 (d–g). A variety of CB-ir inter-

f

g

counts and were corrected for z-axis shrinkage from histological processing by the number-weighted mean measured section thickness as described previously [Sherwood et al., 2007]. For NPNFP-ir neurons, neuron density and neuron type in layer V were analyzed. The percentages of pyramidal, extraverted, inverted, fusiform and multipolar neuron types of all NPNFP-ir stained

neurons were also calculated according to the descriptions of these different morphological types found in Sherwood et al. [2009]. To obtain densities and proportions for both regions combined as S1, the mean value was taken for the two regions. Combined S1 neuron density was calculated by taking the mean of all layer measurements for both areas.



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neuron shapes were identified in the manatee S1. Shown here from CL2 are a fusiform bipolar neuron from layer III (a) and round multipolar neurons from layers III (b) and V (c). Shown from DL1 are a bitufted neuron (e) and round multipolar neurons (f, g) from layer III. Pyramidal neurons were also lightly stained in layers III and V of both areas (DL1 layer III shown in d). Scale bars = 25 μm.

Results

Description of Immunohistochemical Staining Calbindin Overall, few neurons expressed CB immunoreactivity in either cortical region (CL2 and DL1; fig. 3a, f). In both regions, layer I did not include any CB-ir neurons. The neuropil in layers II and IV was more darkly stained, particularly in area DL1, and only contained very sparse round stellate interneurons. Layer III had the highest density of darkly stained interneurons across the cortex, containing mostly round multipolar types (fig. 4b, f, g). In area DL1, layer III contained bitufted neurons (fig. 4d). Layer III also had few, very lightly stained pyramidal neurons (fig. 4d). Layers IV, V and VI had fewer darkly stained CB-ir interneurons, though layers V and VI also had a small number of lightly stained pyramidal neurons. Neurons in layers IV and V were primarily round multipolar types (fig. 4c), although there were a few fusiform bipolar neurons (fig. 4a). Neurons in layer VI included these types along with a few triangular multipolar neurons. No specific staining pattern was identified in area CL2 in relation to the distribution of Rindenkerne, or clusters of cells in layer VI that are associated with vibrissal input. Calretinin There were very few CR-ir interneurons in either cortical region (fig. 3b, g), and light neuropil staining was observed in layers II and IV. Layer I did not contain many apparent interneurons. Layer II had bipolar neurons, while layer III contained lightly stained pyramidal neurons and a few round and fusiform bipolar interneurons (fig. 5b). In DL1, layers II and III contained more fusiform bipolar and bitufted neurons than in CL2 (fig. 5d, 216


e). Layer IV had very few CR-ir interneurons and no pyramidal neurons. Layer V had a large number of pyramidal neurons that outnumbered stained interneurons (fig. 5a, c). CR-ir interneurons in layer V were more numerous than in layer III and consisted primarily of round bipolar neurons, with very few fusiform bipolar neurons and round multipolar neurons (fig. 5f). Layer VI was generally similar to layer V but had very few pyramidal neurons and fewer stained interneurons. As observed for CB, there were no specific staining patterns that could be associated with Rindenkerne in area CL2. Parvalbumin PV immunostaining of interneurons in both regions was sparse (fig. 3c, h). In contrast to CB- and CR-immunostained sections, there were very few lightly stained pyramidal neurons. In both regions, the neuropil was stained more darkly in layers II and IV, and the predominant type of interneuron was multipolar. Layer I had no PV-ir neurons, while layers II and III contained a few multipolar neurons (fig. 6c, e). Layers IV, V and VI had an increased number of multipolar neurons compared to the supragranular layers. Lower layer IV had the highest density of PV-ir interneurons and contained mainly small and medium multipolar neurons. Layers V and VI in particular had round and triangular multipolar neurons (fig. 6a, b, d). Layer V also had a darkly stained neuropil and was similar to layer IV and a few very lightly stained pyramidal neurons (fig. 6a). In CL2, layer VI showed no specific staining patterns related to Rindenkerne. In DL1, the neuropil at the boundary between layer VI and the white matter was more darkly stained than in CL2, similar to neuropil staining in layers II and IV. Neuropeptide Y In both regions, NPY staining was fairly uniform throughout the neuropil, and layer I was only slightly lighter than the other cortical layers. Both regions also showed very sparse NPY immunoreactivity for interneurons throughout the cortex (fig. 3d, i). Layer I did not have any NPY-expressing neurons. Layers III and V contained very lightly stained pyramidal neurons. Layers III– VI had an increase in darkly stained interneurons compared to layer II, particularly in layer III. Most of the NPY-ir interneurons in layers II–VI were round multipolar neurons (fig. 7a, b, d, e), and there were few neurons of other types. DL1, however, did have a few bitufted neurons in layer V (fig. 7c). There were no interneurons stained in layer VI that were associated with Rindenkerne in CL2. Reyes/Stimpson/Gupta/Raghanti/Hof/ Reep/Sherwood


Comparisons with Afrotherians and Xenarthrans The results obtained from the manatee S1 were compared with results from other afrotherians and xenarthrans, including previously published data for total cerebral cortical neuron density in African elephants [Haug, 1987] and S1 neuron density in a sample of other afrotherians and xenarthrans [Sherwood et al., 2009], and previously published data for NPNFP-ir neuron percentage in a sample of other afrotherians and xenarthrans, excluding the twotoed sloth [Sherwood et al., 2009]. Measurements of brain mass used in the analyses were taken from Boddy et al. [2012]. The data were log transformed, and linear least-squares regressions were performed between log brain mass and log neuron density measurements, and log brain mass and log NPNFP-ir neuron percentages. All statistical analyses were performed using R [R Core Team, 2013].

a

c

Fig. 5. CR-ir neurons in areas CL2 (a, b) and DL1 (c–f). Pyramidal staining was d

b

e

f

GAD67 Both regions had a darkly stained neuropil, with many immunoreactive synaptic terminals and GAD67-ir neurons (fig. 3e, j). Layer I contained mostly round multipolar and a few bipolar neurons. Layer II had an increase in the density of interneurons, including the same types as layer I. Layer III included round multipolar and bipolar interneurons. Layer IV appeared somewhat lighter stained than the surrounding layers, though it contained the same neuron morphologies as layers I–III. Layer V was similar to layer III and contained both bipolar and multipolar neurons (fig. 8b). Layer VI had the most interneurons of any layer, especially at the border with white matter. These interneurons were primarily round multipolar neurons, but there were also round bipolar interneurons and a particular increase in fusiform bipolar neurons in DL1 (fig. 8a, c–e). In CL2, layer VI did not contain any specific staining patterns that were associated with Rindenkerne.

Nonphosphorylated Neurofilament Protein In both regions, staining for NPNFP was robust, although DL1 was generally more darkly stained than CL2 (fig. 9a, b). Layers I and II did not contain any NPNFPstained neurons, though the neuropil of layer I was more lightly stained than layer II. Layer III had sparse, but darkly stained pyramidal neurons along with other smaller neurons. Layer IV appeared devoid of neuronal somata and contained mostly axons and dendrites from layers III and V. Layer V included many darkly stained pyramidal and atypical principal neurons (fig. 9a, b). Layer VI had fewer stained neurons than layer V except for the border with the white matter, where there were many darkly stained atypical principal neurons. In addition to pyramidal neurons (fig. 9c), there were four types of NPNFP-ir atypical principal neurons identified in layer V of both regions: inverted (fig. 9d), multipolar (fig. 9e), extraverted (fig. 9f) and fusiform (fig. 9g).



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darker than for other stains, as seen in layer V in CL2 (a) and DL1 (c). CR-ir interneuron types had much less variety than in other regions. Shown here from CL2 are round and fusiform bipolar neurons in layer III (b). Shown from DL1 are a bitufted (d) and fusiform bipolar (e) neuron from layer III, and a round multipolar neuron (f) from layer V. Scale bars = 25 μm.

b

a

d

e

Fig. 6. PV-ir neurons in areas CL2 (a, b) and DL1 (c–e). PV-ir interneurons primarily consisted of multipolar neurons. Smaller neurons were observed in supragranular and granular layers, while larger neurons were seen in infragranular layers. Shown here from

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CL2 are multipolar neurons from layer V (a, b). Shown from area DL1 are multipolar neurons from layer III (c, e) and layer V (d). Scale bars = 25 μm.



c

a

c

b

d

Fig. 7. NPY-ir neurons in areas CL2 (a, b) and DL1 (c–e). NPY-ir interneurons pri-

e

The pyramidal neurons identified by NPNFP immunoreactivity had a single large apical dendrite that extended upward into supragranular layers, with a short skirt of basilar dendrites. Inverted neurons had a similar morphology except they were reversed so that the basilar den-

drites were oriented toward the pial surface. Multipolar neurons had a number of dendrites extending from the soma and were highly variable in shape. Extraverted neurons were also identified and had wide somata with two dendrites extending toward the pial surface from either



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marily consisted of multipolar neurons. Shown here from CL2 are multipolar neurons from layer III (a) and layer V (b). Shown from area DL1 are a bitufted neuron from layer V (c) and multipolar (d, e) neurons from layer III. Scale bars = 25 μm.

end. Fusiform neurons had a long and narrow soma that was vertically situated, with long dendrites extending from either end. The proportion of each of these neuron types relative to total layer V neurons is discussed below.

a

d

c

e

Fig. 8. GAD67-ir neurons in areas CL2 (a, b) and DL1 (c–e). Neu-

ron staining in CL2 was generally very light. Shown here are round bipolar and multipolar neurons from layer VI (a) and round bipolar and multipolar neurons from layer V (b) in CL2. Staining was much darker in area DL1. Shown here are fusiform bipolar (c, d) and multipolar neurons from layer VI (c) and a round bipolar neuron from layer V (e) in DL1. Lightly stained pyramidal cells can also be seen in layer VI of DL1 (c). Scale bars = 25 μm.

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Inhibitory Interneuron Density Densities of CB-, CR-, PV- and NPY-ir interneurons were low in both S1 regions (fig. 12; table 1). In both regions combined, CR-ir interneurons had the highest density (66 neurons/mm3, SD = 56), although CL2 exhibited a relatively high PV-ir interneuron density in layer IV (221 neurons/mm3) that was not observed in layer IV of DL1 (8 neurons/mm3) or in other cortical layers. There was also a relatively homogeneous density of NPY-ir interneurons across layers in S1 (16 neurons/mm3, SD = 9). Because the density of these interneuron subtypes was extremely low, additional immunohistochemical staining against GAD67 protein was performed to detect the total population of GABAergic interneurons. A substantially Reyes/Stimpson/Gupta/Raghanti/Hof/ Reep/Sherwood


b

Neuron Density Neuron densities in both regions were quantified in Nissl-stained sections (fig. 10). These regions were identified based on previous cytoarchitectural descriptions [Reep et al., 1989; Marshall and Reep, 1995]. Neurons were identified as distinct from glial cells based on their morphology; neurons consisted of a translucent cell body with a visible nucleus, while glia were more opaque and did not have a visible nucleus. Both regions had similar neuron densities across layers. Neuron density was lowest in layer I, highest in layer II, and declined progressively through layers III, IV, V and VI. DL1 had higher neuron density in layers II and III than CL2, but both followed a similar pattern of neuron density relative to the other layers. Overall density was 35,617 neurons/mm3. Densities for each region across each layer along with measurements of layer thickness are shown in table 1. A direct comparison between manatee neuron density and that of other afrotherians and xenarthrans was performed to determine if manatee neuron density followed the previously observed scaling relationship with brain mass for this group. In this sample, allometric scaling indicates that as brain mass increases, neuron density tends to decrease with a scaling exponent of –0.38, similar to the neuron density scaling exponent reported for afrotherians based on the isotropic fractionator method [Neves et al., 2014]. The mean neuron density of S1 in the manatee falls within the 95% prediction intervals for afrotherians and xenarthrans, although it falls towards the upper end of the prediction intervals (fig. 11a).

showed a similar pattern of low density in layer I (1,251 neurons/mm3, SD = 434) and high density in layer VI (5,395 neurons/mm3, SD = 2,137). GAD67-ir interneuron density in layer VI of CL2 (6,906 neurons/mm3) was also higher than in DL1 (3,884 neurons/mm3) and may be linked with Rindenkerne. Percentage of Layer V NPNFP-ir Neurons In layer V, DL1 had a higher percentage of NPNFP-ir neurons (86.7%) than did CL2 (62.9%). Generally in S1, the majority of NPNFP-ir neurons were pyramidal neurons (75.4%), with smaller percentages of inverted (6.2%), fusiform (4.9%), extraverted (3.3%) and other multipolar (10.1%) neuron types (fig. 13). DL1 had less diversity in NPNFP-ir neurons, with more pyramidal neurons, and fewer fusiform, extraverted and multipolar neurons than CL2. To compare NPNFP-ir neuron percentage in layer V of the manatee S1 with its close living relatives, a linear least-squares regression was performed on previously published data for NPNFP-ir neuron percentage in a sample of other afrotherians and xenarthrans, excluding the two-toed sloth [Sherwood et al., 2009]. The manatee S1 had the highest percentage of NPNFP-ir neurons in layer V of all afrotherian and xenarthran species measured. When plotted relative to brain mass (fig. 11b), the proportion of NPNFP-ir neurons out of total neurons (measured from Nissl staining) in the manatee was lower than what would be expected for brain mass but fell just within the lower 95% prediction interval.

a

b

Discussion d

e

f

g

Fig. 9. NPNFP-ir neurons in layer V of CL2 (a) and DL1 (b). NPNFP staining was robust and stained both somata and dendritic processes in both areas, but was darker in DL2 (b). Examples of pyramidal (c), inverted (d), multipolar (e), extraverted (f) and fusiform (g) neurons are shown. Pyramidal neurons formed the majority of cells in layer V for both regions, and other types were less common. a, b Scale bars = 50 μm; c–g scale bars = 25 μm.

The present study investigated the neuronal organization of two regions (CL2, DL1) of S1 in Florida manatees based on the measurement of neuron density, interneuron densities and NPNFP-ir neuron proportion and morphology in layer V. To understand this pattern of features in the manatee S1 within an evolutionary context, the data were compared with previous findings in other afrotherians and closely related xenarthrans.

greater density of interneurons was immunoreactive for GAD67 for the combined S1 (2,857 neurons/mm3, SD = 1,590) than the sum total for the other interneuron subtype markers. GAD67-ir interneuron density was slightly higher in CL2 (3,622 neurons/mm3, SD = 1,786) compared with DL1 (2,092 neurons/mm3, SD = 982), but both

Comparison with Afrotherians and Xenarthrans Neuron Density Although the manatee brain mass is absolutely larger than many of the afrotherians and xenarthrans, its neuron density was similar to that observed in brains approximately 10-fold smaller (fig. 11a) and is substantially higher than what is observed in the African elephant iso-



221


c

I

I

II

II

III

III

IV

IV V V

Fig. 10. Nissl-stained sections from areas CL2 (a) and DL1 (b). Both areas have all six

cortical layers, although the layers are more clearly visible in area DL1. Area DL1 is also characterized by radial striations that extend through layers V and VI. In area CL2, layer V is larger than in DL1, and layer VI contains Rindenkerne, shown by black arrowheads. Roman numerals indicate cortical layers. Scale bar = 100 μm for both a and b.

VI

VI

a

b

Region Layer

Neuron density, neurons/mm3

CB-ir neuron density, neurons/mm3

CR-ir neuron density, neurons/mm3

PV-ir neuron density, neurons/mm3

NPY-ir neuron density, neurons/mm3

GAD67-ir neuron density, neurons/mm3

Mean layer thickness ± SD, mm

CL2

I II III IV V VI Mean ± SD

6,731 53,548 43,067 41,862 34,985 23,796 33,998 ± 16,582

2 36 81 62 21 18 37 ± 32

33 123 84 41 33 24 56 ± 39

0 0 40 221 36 0 50 ± 86

0 23 32 20 18 21 19 ± 11

1,558 3,716 3,000 2,905 3,646 6,906 3,622 ± 1,786

0.58 ± 0.03 0.13 ± 0.02 0.68 ± 0.03 0.51 ± 0.01 0.62 ± 0.01 2.09 ± 0.02 0.77 ± 0.06

DL1


5,076 69,614 49,553 40,546 34,039 23,194 37,004 ± 22,146

7 82 81 15 12 11 35 ± 36

21 120 201 57 43 10 75 ± 73

0 0 13 8 6 0 5±5

0 18 16 18 15 13 13 ± 7

944 2,012 2,155 1,569 1,990 3,884 2,092 ± 982

0.44 ± 0.02 0.11 ± 0.01 0.64 ± 0.02 0.44 ± 0.02 0.74 ± 0.01 1.69 ± 0.02 0.68 ± 0.06

S1


5,903 ± 1,169 61,581 ± 11,359 46,309 ± 4,586 41,204 ± 930 34,511 ± 668 23,495 ± 425 35,617 ± 18,719

5±4 59 ± 33 81 ± 0 39 ± 33 17 ± 6 15 ± 5 36 ± 32

27 ± 8 122 ± 2 143 ± 83 49 ± 11 38 ± 7 17 ± 10 66 ± 56

0±0 0±0 27 ± 19 115 ± 151 21 ± 21 0±0 27 ± 60

0±0 21 ± 4 24 ± 11 19 ± 1 17 ± 2 17 ± 6 16 ± 9

1,251 ± 434 2,864 ± 1,205 2,578 ± 598 2,237 ± 945 2,818 ± 1,171 5,395 ± 2,137 2,857 ± 1,590

0.51 ± 0.09 0.12 ± 0.03 0.66 ± 0.05 0.48 ± 0.02 0.68 ± 0.03 1.89 ± 0.25 0.72 ± 0.4

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Table 1. Neuron density, interneuron density and layer thickness in the manatee S1

Giant elephant shrew

Lesser anteater Two-toed sloth

4.5

Manatee

Giant anteater

4.0 African elephant

Regression 95% prediction interval

3.5 0

a

Rock hyrax

1

2 3 log brain mass (g)

0

r 2 = 0.93 p < 0.001

–0.5

Manatee

Giant anteater

–1.0 Lesser anteater Rock hyrax

–1.5 Giant elephant shrew

–2.0

Two-toed sloth

Regression 95% prediction interval

–2.5 4

5

Fig. 11. Comparisons of S1 neuron density NPNFP-ir neuron per-

centage of all layer V neurons between manatees and other afrotherians and xenarthrans [Sherwood et al., 2009]. a Logarithmic total cortical neuron density for the African elephant [Haug, 1987] and S1 neuron density for afrotherians, xenarthrans [Sherwood et al., 2009] and the manatee were plotted against log brain mass. The least-squares regression line was calculated excluding the manatee; slope = –0.38 and intercept = 5.33. The manatee falls within the

cortex. Nonetheless, manatee S1 neuron density falls within the prediction intervals based on scaling to brain mass for afrotherians and xenarthrans. This reflects previous findings that manatee neuron density is within the range of other mammals [Charvet et al., 2015].

0.5

b

1.0

1.5 2.0 log brain mass (g)

2.5

3.0

95% prediction intervals. b Logarithmic NPNFP-ir neuron percentage in S1 layer V plotted against log brain mass for the twotoed sloth, lesser anteater, giant anteater, rock hyrax, elephant shrew and manatee (average S1 NPNFP-ir neuron percentage). The least-squares regression line was calculated excluding the manatee and sloth; slope = 0.36 and intercept = 0.97. The dotted lines indicate 95% prediction intervals. The manatee falls within the 95% prediction intervals.

Interneuron Density Based on a comparison of inhibitory interneurons between manatees and other afrotherians and xenarthrans, we found that manatees have a different proportion of interneuron types and pattern of interneuron distribution in S1. The percentage of inhibitory interneuron subtypes labeled by immunohistochemistry against calciumbinding proteins and NPY in layers II–VI were substantially lower in manatees compared to xenarthrans and other afrotherians (table 2) [Sherwood et al., 2009]. The greater percentage of GAD67-ir interneurons than for the other interneuron subtypes suggests that the majority of GABAergic interneurons in the manatee S1 do not express high levels of CB, CR, PV or NPY. Notably, the percentage of GAD67-ir interneurons in manatees was comparable to the proportion of inhibitory interneurons cal-

culated from totals for CB-, CR-, PV- and NPY-ir neurons in the other afrotherian and xenarthran species, although this overall proportion of inhibitory interneurons was still lower than that previously reported for CB-, CR-, PVand NPY-ir interneurons in cetaceans, rodents and primates [Glezer et al., 1993; Gabbott and Bacon, 1996; Gabbott et al., 1997; Gonchar and Burkhalter, 1997; Hof et al., 2000; DeFelipe et al., 2002]. GAD67-ir staining was strongest in CL2 of manatees and exhibited the highest density of interneurons in layer VI. High GAD67 immunoreactivity in layer VI of CL2 was associated with the presence of Rindenkerne. CL2 also showed an overall higher density of GAD67-ir interneurons than did CL2, which may potentially indicate increased sensory processing in this portion of the S1. It is unlikely that low density for CB-, CR-, PV- and NPY-ir interneurons in the manatee S1 is due to tissue preservation artifact, as GAD67 and NPNFP proteins were detected by immunohistochemistry in the same tissue. The density of calcium-binding protein- and NPY-ir interneurons differs substantially between manatees and other afrotherians and xenarthrans, and may signify a de-



223


5.0

r 2 = 0.93 p < 0.001

log average NPNFP-ir neuron percentage (NPNFP-ir neurons/total layer V neurons)

log average neuron density (neurons/mm3)

5.5

GAD67

6,000 5,000

4,500

Average S1 interneuron density

4,000

CR

PV

7,000 5,000

2,500

4,000

2,000

2,000

3,000

1,500

2,000

1,000

1,000

500

1,000

250

250

250

200

200

200

150

150

150

100

100

100

50

50

50

0

I

II

III

IV

V

VI

0

CL2 interneuron density

6,000

3,000

3,000

NPY

8,000

DL1 interneuron density

3,500

4,000

Density (neurons/mm3)

CB

I

II

III

IV

V

VI

0

I

II

III

IV

V

VI

Layer

100 Relative frequency (%)

90 80 70 60 50 40 30 20 10 0

Average S1

Pyramidal Inverted Fusiform

DL1

CL2

Extraverted Other multipolar

Fig. 13. Relative frequency of the different morphological classes

of NPNFP-ir neurons in layer V of manatee S1. Pyramidal neurons were the predominant type in both S1 areas, though they were more common in DL1. Area CL2 had more variety of NPNFP-ir neuron types in layer V than did area DL1, particularly other multipolar types.

224


substantially higher than that of the other stains, and the pattern of neuron density was consistent between areas. In both areas, layer VI had the highest density of GAD67-ir interneurons, a pattern not observed for the other stains.

rived state in manatees relative to these taxa. Relatively low densities of these specific interneuron subtypes suggest that manatees may have different interneuron subtypes that do not express the calcium-binding proteins typically seen in the isocortex of other mammals. Compared with monotremes, marsupials and other eutherians, manatees have many of the same interneuron morphological types (e.g. round, bipolar, bitufted) that are observed across all mammals [Hof et al., 1999]. Manatees, however, display a characteristic pattern of interneuron distribution as shown by GAD67-ir neurons, with a predominance of interneurons in infragranular layers, particularly in layer VI. A further investigation of interneuron density in additional manatee species and closely related dugongs is thus necessary to determine whether this pattern of interneuron density and distribution is common to all sirenians. NPNFP-ir Neurons A comparison of NPNFP-ir neuron percentage in layer V of the manatee S1 with other afrotherians and xenarthrans indicated that manatee NPNFP-ir neuron percentage was lower than expected based on scaling for Reyes/Stimpson/Gupta/Raghanti/Hof/ Reep/Sherwood


Fig. 12. Density of interneuron subtypes in the manatee primary somatosensory cortex. Overall, density was low in S1 for CB-, CR-, PV- and NPY-ir interneurons. CR-ir interneurons had the highest density across all layers in both areas. CL2 also had a relatively high PV-ir interneuron density in layer IV. GAD67 neuron density was

Table 2. The percentage of inhibitory interneuron subtypes in layers II–VI of Xenarthra and Afrotheria

Superorder

Species

CB-ir interneurons

CR-ir interneurons

PV-ir interneurons

NPY-ir interneurons

GAD67-ir interneurons

Xenarthra

Two-toed sloth Lesser anteater Giant anteater

2.40 4.33 3.84

1.99 2.99 3.07

4.65 7.19 2.71

2.99 3.12 3.20

− − −

Afrotheria

Rock hyrax Elephant shrew Florida manatee

2.61 1.58 0.07

2.36 2.71 0.13

5.16 5.89 0.06

5.71 3.38 0.04

− − 7.37

Possible Factors Influencing Manatee Neuron Types and Distribution Metabolism Manatees exhibit a pattern of traits in their S1 that has not been observed in other afrotherians. These could be linked to a low basal metabolic rate (BMR). Afrotherian taxa have relatively low BMRs compared to other placental mammals [McNairn and Fairall, 1984; O’Shea and Reep, 1990; Milner and Harris, 1999; Goto et al., 2008], a trend also seen in xenarthran taxa [Fowler and Cubas, 2001; Vizcaíno and Loughry, 2008]. These taxa also have lower BMR scaling than most other placental mammals; therefore, as their body mass increases, their BMR increases at a relatively low rate [White et al., 2009]. Even compared to dugongs and other aquatic mammals, manatees have a low BMR [Gallivan and Best, 1980; Irvine, 1983; Goto et al., 2008; Varela-Lasheras et al., 2011], and there is evidence that a low BMR can affect a variety of systems in the body.


A low BMR has been linked with changes in the body of manatees and sloths, a xenarthran also with an extremely low BMR [Galis and Metz, 2007; Varela-Lasheras et al., 2011]. Manatees and sloths also share similarities such as slow movement, and a relatively low-quality diet [O’Shea and Reep, 1990; Vizcaíno and Loughry, 2008; Siegal-Willott et al., 2010]. Changes in the number of cervical vertebrae and ribs, and a high incidence of body asymmetry have been associated with low BMR in both of these species, possibly due to pleiotropy [Varela-Lasheras et al., 2011]. The BMR has also been associated with energetic trade-offs. The brain is energetically expensive and requires more glucose than other body tissues while at rest [Mink et al., 1981]. A metabolic trade-off between other energetically expensive tissues in the gut or testes and the brain has been proposed in lineages such as primates and bats, in which brain size increases without a corresponding increase in BMR [Aiello and Wheeler, 1995; Pitnick et al., 2006]. The reduced expression of calcium-binding proteins and NPY among interneurons in manatees could be linked to the energetic requirements of the different classes of interneurons in the context of overall brain metabolism. Calcium-binding proteins such as CB, CR and PV transport calcium ions and have the capacity to regulate calcium levels throughout the neuronal cytoplasm as an important second messenger for neurotransmission and synaptic plasticity [Schwaller et al., 2002; Yáñez et al., 2012]. Calcium regulation and transport are critical processes in neurons and require a significant amount of energy [Gleichmann and Mattson, 2011]. In vitro studies in rat tissue have indicated that PV-positive GABAergic inhibitory interneurons in the hippocampus, particularly basket cells, are an integral part of neuronal circuits and are associated with a high energetic demand [Kann et al., 2014; Hu et al., 2014]. In this regard, a reduction in PVcontaining interneurons may be one strategy for an overBrain Behav Evol 2015;86:210–231 DOI: 10.1159/000441964

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brain mass but was within the 95% prediction intervals (fig. 11). The manatee also showed a similar diversity of NPNFP-ir neuron types that is observed in other afrotherians, with ∼25% atypical principal neurons including inverted, fusiform, extraverted and other multipolar shapes. The manatee, however, exhibited 3% extraverted neurons, which is a higher percentage than observed in other afrotherians. The variety of NPNFP-ir neuron subtypes exhibited by the manatee appears to be related to phylogeny; most mammalian taxa exhibit greater diversity in NPNFP-ir neuron morphology in the cortex than is observed in the boreoeutherian lineage (e.g. primates, rodents, carnivores and cetartiodactyls), where NPNFPir neurons tend to be more limited to pyramidal morphologies [Sherwood et al., 2009; Jacobs et al., 2011; Butti et al., 2014, 2015; Jacobs et al., 2015].

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Aquatic Adaptations Sirenians occupy a unique aquatic niche compared to other afrotherians and show adaptive changes to their overall body plan [Hartman, 1979; Reidenberg, 2007]. Such changes include a streamlined body with modified limbs for swimming, a loss of fur and development of large amounts of fat, decreased reliance on vision and increased use of auditory or tactile stimulation, specialized feeding and digestive adaptations, and an increase in bone density to regulate depth under water [Buchholtz et al., 2007; Reidenberg, 2007; Reep et al., 2001; Bauer et al., 2012]. Many of these changes are convergent with those seen in other aquatic mammals from different mammalian lineages [Buchholtz et al., 2007; Reidenberg, 2007]. Despite a wealth of evidence for physical changes, however, not much is known about specific brain adaptations that are necessary for life under water. Some have suggested that the relatively large brains of cetaceans might be an adaptation for an aquatic lifestyle and particularly for echolocation, while others have suggested that large brain size appears more closely linked with increased sociality. An increase in relative brain size is not associated with other aquatic mammals, including the manatee [Marino, 2007]. A highly gyrified brain is also observed in cetaceans, but this feature is also unlikely to be solely attributed to an aquatic adaptation. The cetacean brain is more gyrified than that of the manatee, which is considered lissencephalic [Edinger, 1933, 1939; Reep et al., 1989; Reep and O’Shea, 1990; Marino, 2007]. The degree of gyrification observed in the brains of these two taxa is more likely related to cortical thickness and development rather than an aquatic habitat; cetaceans have a thin cerebral cortex that folds more than the thick cerebral cortex of the manatee [Mota and HerculanoHouzel, 2012, 2015; Striedter et al., 2015]. The cause of these differences in cortical thickness across taxa is currently unknown, but manatee lissencephaly may be influenced by a number of factors including diet and maternal metabolism during development [Reep and O’Shea, 1990; Siegal-Willott et al., 2010]. There are a few specific regions of the brain that do appear to be specially adapted in aquatic mammals, although they tend to vary by species. Many of these adaptations include changes of interneuron type and density in auditory and somatosensory areas that are increasingly relied upon under water [Glezer et al., 1992a, b, 1998] and a reduction in olfactory areas [Mackay-Sim et al., 1985; Oelschläger, 1992; Marino, 2007]. Manatee brains contain Rindenkerne, or clusters of neurons in layer VI of specific cluster cortex regions in the S1 [Reep et al., Reyes/Stimpson/Gupta/Raghanti/Hof/ Reep/Sherwood


all reduction in energy expenditure [Karbowski, 2009]. NPY has also been linked with metabolic processes, particularly energy regulation [White, 1993], and fewer NPY-ir interneurons in the manatee brain may also be linked with reduced metabolism. The low-quality diet and low metabolic rate of the manatee may therefore limit the amount of energy available to the brain and could restrict the spiking dynamics of interneurons that are correlated with the expression of these calcium-binding proteins and NPY. Neurofilament proteins are associated with stabilizing the axon cytoskeleton and are found in neurons with large axons and heavy myelination [Morris and Lasek, 1982; Lawson and Waddell, 1991]. Therefore, greater numbers of NPNFP-ir neurons are associated with connectivity across long distances, and exhibit higher density in regions with intrahemispheric and callosal connections in macaque monkeys [Campbell and Morrison, 1989; Campbell et al., 1991; Hof et al., 1995, 1996; Nimchinsky et al., 1996] and more intense staining in neurons with more intracortical connectivity in rats [Kirkcaldie et al., 2002]. Because neurofilament proteins support connectivity both within and between cortical regions, an increase in brain size may call for more NPNFP expression in neurons due to a corresponding increase in connectivity [Sherwood et al., 2009]. In the sloth, the NPNFP-ir neuron percentage in layer V of S1 is much lower than expected for brain mass scaling and falls well outside the 95% prediction intervals (fig. 11b). Manatees also have relatively reduced NPNFP-ir proportions compared to what would be expected at their brain mass. This suggests that metabolism may also be a limiting factor for the formation of NPNFP-ir neurons in the manatee S1. It should also be noted that the sloth does not share a reduction in calcium-binding protein-ir and NPY-ir interneurons [Sherwood et al., 2009], and it is possible that this particular feature is unique to the manatee and its close relatives. This reduction in calcium-binding protein and NPY-ir interneuron density in the manatee brain might be a pleiotropic effect, especially since such effects are prevalent in these species with low metabolism. It is also possible that both of these traits represent different energetic trade-offs that these species have made to cope with low metabolism and a low-quality diet. A more in-depth comparative investigation of interneurons and NPNFP-ir neurons in taxa with a low metabolism is thus warranted to assess any effect the metabolic rate might have on their appearance.

1989; Loerzel and Reep, 1991]. These Rindenkerne are hypothesized to be associated with facial vibrissae that are involved in oripulation [Marshall et al., 1998; Reep et al., 1998, 2001; Sarko et al., 2007] and might also be involved in processing information from tactile hairs on the body of the manatee that may act as hydrodynamic receptors, a mammalian version of the lateral line system [Reep et al., 2002; Sarko et al., 2007; Gaspard et al., 2013]. There are a few similarities between manatee interneuron density and that seen in the brains of other aquatic mammals, particularly cetaceans. Like cetaceans, the predominant interneuron subtype in the manatee S1 were CR-ir, and to a lesser extent, CB-ir interneurons [Glezer et al., 1998]. However, this feature of cetaceans was also shared with closely related artiodactyls as well as insectivorous bats and hedgehogs, and differed from the pattern seen in rodents and primates [Glezer et al., 1992a, b, 1998; Hof et al., 1999]. This indicates that a larger amount of CB- and CR-ir interneurons relative to PV-ir interneurons may be a feature shared across Laurasiatheria and may be an ancestral trait [Glezer et al., 1998; Hof et al., 1999]. The low densities of the calcium-binding protein-ir and NPY-ir interneurons found in the manatee S1 have not been observed in any of the aquatic mammals that have been investigated thus far. As discussed previously, an increase in GAD67-ir interneurons in CL2 relative to DL1 also indicates that the manatee may have increased sensory processing in CL2 associated with Rindenkerne in layer VI that could be linked with facial vibrissae and oripulation [Marshall et al., 1998; Reep et al., 1998, 2001; Sarko et al., 2007]. This provides further evidence that these patterns of interneuron type and density may be derived features in manatees and possibly all sirenians, although a wider sample of both manatee and dugong neural specimens is necessary to test this hypothesis. Similarities between manatees and cetaceans are present in NPNFP-ir neuron distribution. In both taxa, NPNFP-ir neuron staining in sensory regions was restricted to specific layers. The manatee mainly exhibited light staining in layers IIIc and parts of VI, and heavy staining in layer V, while cetaceans have heavy staining in the combined layer IIIc/V [Hof et al., 1992]. This pattern differs from what is seen in primates, rodents and carnivores, where staining is more diffusely distributed throughout the cortex wherever there are pyramidal neurons [Campbell and Morrison, 1989; Hof et al., 1992; Hof and Morrison, 1995; Chaudhuri et al., 1996; Hof et al., 1996; Nimchinsky et al., 1997; Preuss et al., 1997; Budinger et al., 2000; Tsang et al., 2000; Van der Gucht et al., 2001; Sherwood et al., 2004; Baldauf, 2005; Boire et al.,

2005; Bourne et al., 2005; Hof and Sherwood, 2005; Bourne et al., 2007; Van der Gucht et al., 2007] but is similar to what is also observed in afrotherians, xenarthrans, monotremes and marsupials [Ashwell et al., 2005; Hassiotis et al., 2005; Sherwood et al., 2009]. Primates, rodents and carnivores also have different patterns of NPNFP-ir neuron distribution in different cortical areas [Campbell and Morrison, 1989; Campbell et al., 1991; Hof and Nimchinsky, 1992; Hof and Morrison, 1995; Budinger et al., 2000; Van der Gucht et al., 2001, 2007], which is not as clearly variable across the cortex in afrotherians, xenarthrans, monotremes and marsupials [Hof et al., 1992; Ashwell et al., 2005; Hassiotis and Paxinos, 2004; Hassiotis et al., 2005; Sherwood et al., 2009]. Although only the manatee S1 has been investigated in this study, the restriction of NPNFP-ir neurons to specific layers in two different cortical regions is similar to that seen in the majority of mammalian taxa and differs from the more derived pattern of certain groups such as primates, rodents and carnivores. The pattern observed in manatees and cetaceans is therefore not likely the result of adapting to an aquatic environment but appears to be a primitive trait among many mammals.



Conclusion

227


The manatee has a distinctive collection of architectural features in its S1. Overall neuron density is relatively high for its brain mass but is within 95% prediction intervals scaling relationships calculated from other afrotherians and xenarthrans. Densities for isocortex calcium-binding protein-ir and NPY-ir interneurons are substantially lower than in other species. However, the manatee’s overall interneuron density as shown by GAD67 immunostaining is comparable to afrotherians and xenarthrans, suggesting that they have different subtypes of interneurons than is seen in these other species. Because calcium-binding proteins and NPY are expressed in metabolically expensive physiological functions, these specific subtypes of interneurons may have been reduced as a result of low metabolism in the manatee. The percentage of NPNFP-ir neurons in layer V also falls below the prediction line based on values from other afrotherians and xenarthrans, indicating that a low metabolism might also have an effect on this aspect of cortical structure. Although this particular pattern of features sets the manatee apart from other afrotherians and xenarthrans, the manatee cerebral cortex does not appear to be specifically adapted for an aquatic habitat. Many of the features that

are shared between manatees and cetaceans, such as the predominance of CB- and CR-ir interneurons and the laminar distribution of NPNFP-ir neurons, are also shared with a diverse array of terrestrial mammals and likely represent highly conserved neural features. Further investigation of cerebral cortical architecture in different manatee species as well as dugongs is necessary to determine whether these traits are specific to one or more of the manatee species or can be generalized to all sirenians.

Acknowledgments This work was supported by the James S. McDonnell Foundation (220020293). We would like to thank the faculty, students and staff of the Laboratory for Evolutionary Neuroscience and the Center for the Advanced Study of Human Paleobiology at the George Washington University for their support in completing this paper. We would also like to thank Dr. Bob Jacobs and Tessa Harland for comments and suggestions during the preparation of the manuscript, as well as the reviewers and the editor, Dr. Georg Striedter, for their comments and edits that improved this article.

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Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida.

Conservative management of pneumothorax and pneumoperitoneum in two Florida manatees (Trichechus manatus latirostris).

ESTABLISHMENT OF ECHOCARDIOGRAPHIC PARAMETERS OF CLINICALLY HEALTHY FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

The presence of ovarian cysts in a captive Antillean manatee (Trichechus manatus manatus L. 1758).

Digestive efficiencies of ex situ and in situ West Indian manatees (Trichechus manatus latirostris).

Intussusception in a Florida manatee.

Somatosensory cortex of the neonatal pig: I. Topographic organization of the primary somatosensory cortex (SI).

Properties of the primary somatosensory cortex projection to the primary motor cortex in the mouse.

Evolution of cytoarchitectural landscapes in the mammalian isocortex: Sirenians (Trichechus manatus) in comparison with other mammals.

The mammary glands of the Amazonian manatee, Trichechus inunguis (Mammalia: Sirenia): morphological characteristics and microscopic anatomy.

Disinhibition of the primary somatosensory cortex in patients with fibromyalgia.

Early integration of bilateral touch in the primary somatosensory cortex.

Sharing social touch in the primary somatosensory cortex.

Florida Red Tides, Manatee Brevetoxicosis, and Lung Models.

Corticocortical connections of cat primary somatosensory cortex.

Rapid-rate paired associative stimulation over the primary somatosensory cortex.

Prediction of primary somatosensory neuron activity during active tactile exploration.

Somatosensory cortex of the neonatal pig: II. Topographic organization of the secondary somatosensory cortex (SII).

First attempt to monitor luteinizing hormone and reproductive steroids in urine samples of the Amazonian manatee (Trichechus inunguis).

Projections from the primary somatosensory cortex to basal ganglia and thalamus in the monkey.

Primary somatosensory cortex hand representation dynamically modulated by motor output.

Image-guided transcranial focused ultrasound stimulates human primary somatosensory cortex.

Functional regions within the map of a single digit in raccoon primary somatosensory cortex.

Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans.