Neuroanatomy and Volumes of Brain Structures of a Live California

THE ANATOMICAL RECORD 292:1523–1547 (2009)

Neuroanatomy and Volumes of BrainStructures of a Live California Sea Lion(Zalophus californianus) From Magnetic

Resonance ImagesERIC W. MONTIE,1* NICOLA PUSSINI,2 GERALD E. SCHNEIDER,3

THOMAS W.K. BATTEY,4 SOPHIE DENNISON,2,5 JEROME BARAKOS,6

AND FRANCES GULLAND2

1College of Marine Science, University of South Florida, Florida2The Marine Mammal Center, Sausalito, California

3Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts4Eckerd College, Galbraith Marine Science Center, St. Petersburg, Florida

5School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin6California Pacific Medical Center, University of California, San Francisco, California

ABSTRACTThe California sea lion (Zalophus californianus) has been a focal point

for sensory, communication, cognition, and neurological disease studies inmarine mammals. However, as a scientific community, we lack a noninva-sive approach to investigate the anatomy and size of brain structures inthis species and other free-ranging, live marine mammals. In this article,we provide the first anatomically labeled, magnetic resonance imaging-based atlas derived from a live marine mammal, the California sea lion.The brain of the California seal lion contained more secondary gyri andsulci than the brains of terrestrial carnivores. The olfactory bulb was pres-ent but small. The hippocampus of the California sea lion was foundmostly in the ventral position with very little extension dorsally, quiteunlike the canids and the mustelids, in which the hippocampus is presentin the ventral position but extends dorsally above the thalamus. In con-trast to the canids and the mustelids, the pineal gland of the Californiasea lion was strikingly large. In addition, we report three-dimensionalreconstructions and volumes of cerebrospinal fluid, cerebral ventricles,total white matter (WM), total gray matter (GM), cerebral hemispheres(WM and GM), cerebellum and brainstem combined (WM and GM), andhippocampal structures all derived from magnetic resonance images.These measurements are the first to be determined for any pinniped spe-cies. In California sea lions, this approach can be used not only to relatecognitive and sensory capabilities to brain size but also to investigate theneurological effects of exposure to neurotoxins such as domoic acid. AnatRec, 292:1523–1547, 2009. VVC 2009 Wiley-Liss, Inc.

Grant sponsor: Subaward through the University ofCalifornia Davis Wildlife Health Center and the UniversityCorporation of Atmospheric Research (UCAR) from theNational Oceanic and Atmospheric Administration (NOAA),U.S. Department of Commerce; Grant number:NA06OAR4310119; Grant sponsors: The Marine MammalCenter, the US National Marine Fisheries Service, and Dr.David Mann (College of Marine Science, University of SouthFlorida).

*Correspondence to: Eric W. Montie, College of Marine Sci-ence, University of South Florida, 140 Seventh Avenue South,KRC 2107, St. Petersburg, FL 33701.E-mail: [email protected]

Received 26 March 2009; Accepted 28 April 2009

DOI 10.1002/ar.20937Published online in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

Keywords: California sea lion; Zalophus californianus; pin-niped; magnetic resonance imaging; brain;hippocampus; MRI

INTRODUCTION

Since the 1960s, the California sea lion (Zalophus cali-fornianus) has been a model organism for sensory, com-munication, and cognition studies in marine mammals.Behavioral research on this animal has focused on visualacuity and discrimination between objects (Kastak andSchusterman, 2002a; Schusterman et al., 1965; Schus-terman and Balliet, 1970), hearing sensitivity (Schuster-man, 1974; Reichmuth et al., 2007), vocalizations andcommunication (Schusterman et al., 1966; Schustermanand Balliet, 1969; Hanggi and Schusterman, 1990;Gisiner and Schusterman, 1991), and memory (Schuster-man et al., 1993; Kastak and Schusterman, 2002a).However, to the best of our knowledge, no formal studieshave focused on the anatomy and size of brain struc-tures in this species.

In the wild, California sea lions can be exposed todomoic acid (DA), a natural marine neurotoxin producedby diatoms belonging to the Pseudo-nitzschia genus. DAacts as an excitotoxin by binding to the a-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid and kainatesubclasses of neuronal glutamate ionotropic receptors(Jeffrey et al., 2004). This binding causes massive celldepolarization, resulting in dysfunction and death ofcells expressing these receptors (Olney et al., 1979). In1998, more than 400 sea lions were exposed to DAthrough contaminated prey (Scholin et al., 2000). Sealions that died acutely and contained detectable levels ofDA in blood and urine exhibited lesions in the limbicsystem (Scholin et al., 2000). These lesions were charac-terized by neuronal necrosis in the hippocampal forma-tion, specifically granule cells in the dentate gyrus andpyramidal cells in sectors CA4, CA3, and CA1 of thecornu ammonis (Silvagni et al., 2005).

A second novel neurological syndrome has been identi-fied in California sea lions associated with chronic expo-sure to sublethal levels of DA (Goldstein et al., 2008).Sea lions examined alive by magnetic resonance imaging(MRI) exhibited varying degrees of hippocampal atrophy,thinning of the parahippocampal gyrus, and increases inthe size of the temporal horn of the lateral ventricle(Goldstein et al., 2008). This examination was based onsubjective analysis and not on volumetric measure-ments. Now, there is concern that low levels of DA expo-sure in the developing fetus and neonate, viaconcentration in the amniotic fluid and mammaryglands, respectively, may cause subtle changes in thebrain that may result in long-term cognitive impairment(as reviewed by Ramsdell and Zabka, 2008). As humanscan also be exposed to DA in seafood, there is a need tounderstand the range of effects that DA may have onnaturally exposed mammals, so effects on humans canbe predicted and prevented.

With the widespread exposure of wild sea lions to DA,there is a need to learn more about the normal brain ofthe California sea lion to accurately identify changes inthe brain due to DA toxicity. MRI has been used recently

to study the anatomy of cetacean brains that wereremoved from the skull and formalin fixed (Marinoet al., 2001a,b,c, 2003a,b, 2004a,b) and that were freshlyintact within the skull and attached to the body (Montieet al., 2007, 2008). For the Atlantic white-sided dolphin(Lagenorhynchus acutus), MRI has been used to acquireimages of the brain to calculate the volumes of whitematter (WM), gray matter (GM), cerebellum WM andGM, and the hippocampus at different developmentalstages (Montie et al., 2008). Using these methods, MRIprovides a means to evaluate normal brain structureand determine the size of brain regions in live Californiasea lions. This approach would be a valuable tool inassessing the effects of DA neurotoxicity in exposedanimals.

Our goal in this study was to create a neuroanatomi-cal atlas and establish a quantitative approach to deter-mine the size of brain structures from MR images of alive California sea lion. Specifically, the objectives wereto (a) present an anatomically labeled MRI-based atlasof a neurologically normal brain; (b) provide detailedlabeling of hippocampal structures; (c) determine theWM and GM volumes of the total brain, cerebellum andbrainstem combined, and cerebral hemispheres; and (d)determine the volumes of the left and right hippocampusand associated structures.

MATERIALS AND METHODSAnimal Information

The female California sea lion used in this study(Accession number ¼ CSL7775; Name ¼ ‘‘Kirina’’) wasrescued on 25 July, 2008 by the Marine Mammal Center,Sausalito, CA from Pico Point, San Simeon, CA(35�36046.799400N, �121�907.200W) due to a fracturedright hind flipper. Hematological and serum biochemicalparameters were within the normal range for this spe-cies, other than an elevated white blood cell count. Aneurological assessment (posture, mentation, body move-ments other than use of the right hind flipper, cranialnerve reflexes, and responsiveness to audible stimuliand visual approach) appeared normal. At the time ofMRI, the body length was 117 cm, and the weight was34.5 kg. These measurements along with the stage oftooth development indicated that the animal wasapproximately 1 year old (Greig et al., 2005). Radiogra-phy and MRI were used to evaluate the flipper andbrain of the sea lion to determine its prognosis. MR ex-amination did not reveal any brain pathologies; thebrain appeared grossly normal.

Magnetic Resonance Data Acquisition

Radiographs of the whole body nullified any suspicionof metallic foreign bodies present in the animal. The ani-mal was imaged at IAMs Pet Imaging Center, San Fran-cisco, CA, following anesthesia with isoflurane. MRI

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TABLE 1. List of anatomical nomenclature

Nomenclature used insea lion atlas Alternate names

Accumbens nucleus Nucleus accumbens1

Alveus Alveus hippocampi1

Amygdala Corpus amygdaloideum1

Anterior olfactory nucleus Nucleus olfactorius anterior1

Basilar artery Arteriae basilaris1

Calcarine sulcus Sulcus calcarinus1

Caudal cerebellar peduncle Pedunculus cerebellaris caudalis1

Caudal cerebral artery Arteriae cerebri caudalis1, posterior cerebral artery2

Caudal colliculus Colliculus caudalis1, inferior colliculus2

Caudal commissure Commisura caudalis1, commisura posterior3

Caudate nucleus Nucleus caudatus1

Central canal Canalis centralis1

Cerebellum (hemisphere) Hemispherium cerebelli1

Cerebellum (nodulus)Cerebellum (vermis)Cerebral falx Falx cerebri1

Choroid plexus Plexus choroideus1

Cingulate gyrus Gyrus cinguli1

Cingulate sulcusCochleaCollateral sulcusCornu ammonis Hippocampus proper2; Ammon’s horn2; CA1, CA2,

CA3, CA4 2

Coronal gyrus Gyrus coronalis1

Coronal sulcus Sulcus coronalis1

Corpus callosum (body) Truncus corporis callosi1

Corpus callosum (genu) Genu corporis callosi1

Corpus callosum (splenium) Splenium corporis callosi1

Dentate gyrus Gyrus dentatus1

Dentate nucleus Nucleus dentatus1

Dorsal venous sinusExternal capsule Capsula externa1

Fimbria Fimbria fornicus3

FornixInterpeduncular Fossa Fossa interpeduncularis1

Fourth ventricle Ventriculus quartus1

Fourth ventricle (lateral recess) Recessus lateralis ventriculi quarti1

Frontal gyrus Gyrus frontalis1

Frontal lobeGlobus pallidus Pallidum1

Habenular nucleus Nucleus habenulares1

Hippocampal sulcus Sulcus hippocampi1

HippocampusHorizontal sulcus Sulci cerebelli1

HypothalamusInfundibulumInternal capsule Capsula interna1

Interventricular foramen Foramen interventriculare1

Lateral geniculate body Corpus geniculatum laterale1

Lateral lemniscus Lemniscus lateralis1

Lateral ventricle (caudal horn) Ventriculus lateralis1, lateral ventricle (posterioror occipital horn)2

Lateral ventricle (central) Ventriculus lateralis1

Lateral ventricle (rostral horn) Ventriculus lateralis1, lateral ventricle (anterioror frontal horn)2

Lateral ventricle (ventral horn) Ventriculus lateralis1, lateral ventricle (inferioror temporal horn)2

Longitudinal cerebral fissure Fissura longitudinalis cerebri1

Mammillary body Corpus mamillare1

Medial geniculate body Corpus geniculatum mediale1

Medulla oblongata Pyramis1

Mesencephalic aqueduct Aqueductus mesencephali1

Middle cerebellar peduncle Pedunculus cerebellaris medius1

ObexOccipital lobeOlfactory bulb Bulbus olfactorius1

BRAIN MRI OF A LIVE CALIFORNIA SEA LION 1525

scanning of the brain was completed with a 1.5-T Sie-mens Magnetom Symphony scanner (Siemens, Munich,Germany) equipped with a CP Extremity Coil. Followingthe localizer scan, T1-weighted images in the sagittalplane were acquired using a spoiled gradient echo(FLASH) sequence with the following parameters: TR ¼22 ms; TE ¼ 10 ms; FOV ¼ 200 � 200 mm; slice thick-ness ¼ 1 mm; and voxel size ¼ 0.3 � 0.3 � 1 mm. Two-dimensional proton density (PD) and T2-weightedimages in the transverse plane were acquired using aturbo spin-echo (TSE) sequence with the following pa-rameters: TR ¼ 3650 ms and TE ¼ 14/98 ms for PD andT2, respectively; slice thickness ¼ 2.5 mm; FOV ¼ 150 �150 mm; and voxel size ¼ 0.3 � 0.3 � 2.5 mm. Addition-ally, two-dimensional PD- and T2-weighted images in

the oblique plane (i.e., perpendicular to the long axis ofthe sylvian fissure and temporal lobe) were acquiredusing a TSE sequence with the following parameters:TR ¼ 5470 ms and TE ¼ 14/98 ms for PD and T2,respectively; slice thickness ¼ 2.5 mm; FOV ¼ 160 �160 mm; and voxel size ¼ 0.3 � 0.3 � 2.5 mm. Theoblique orientation was selected to optimize viewing ofthe hippocampus, as previously described (Goldsteinet al., 2008).

After radiography and MR examination, the animalwas euthanized because of poor prognosis for rehabilita-tion and release due to the severity of osteomyelitis inthe hind flipper. A necropsy was completed. The brainwas removed, weighed, and archived whole in 10% neu-tral buffered formalin.

TABLE 1. List of anatomical nomenclature (continued)

Nomenclature used insea lion atlas Alternate names

Olfactory peduncle Pedunculus olfactorius1, olfactory tract2

Olfactory tubercle Tuberculum olfactorium1

Optic chiasm Chiasma opticum1

Optic nerve Nervi opticus1, cranial nerve II2

Parahippocampal gyrus Gyrus parahippocampalis1, lobus piriformis3

Periaqueductal gray matter Substantia grisea centralis1

Pineal gland Glandula pinealis1

Pituitary gland Hypophysis or Glandula pituitaria1

PonsPrecommisural septal fibers Commisura fornicis dorsalis1, fibrae

precommissurales septi3

Presylvian sulcus Sulcus presylvius1

Proreal gyrus Gyrus proreus1

PulvinarPutamenPyramidal tract Tractus pyramidalis1

Reticular formation Formatio reticularis1

Rostral cerebellar artery Arteriae cerebelli rostralis1, superiorcerebellar artery2

Rostral cerebellar peduncle Pedunculus cerebellaris rostralis1

Rostral cerebral artery Arteriae cerebri rostrallis1, anterior cerebral artery2

Rostral colliculus Colliculus rostralis1, superior colliculus2

Rostral commissure Commisura rostralis1, commisura anterior3

Rostral rhinal fissure Fissura rhinalis rostralis1

Septal nucleus Nucleus septalis1

Septum Septum telencephali1

Spinal cordStraight gyrus Gyrus rectus3

Stria medullarisSubiculum Subiculum1

Sulcus cruciatus Sulcus cruciatus1

Suprasylvian gyrus Gyrus suprasylvius1

Sylvian fissure Fissura sylvia or lateralis cerebri1

Temporal lobeTentorium cerebelliThalamusThird ventricle Ventriculus tertius1

Transverse pontine fibers Fibrae pontis transversae1

Trigeminal ganglion Ganglion trigeminale1

Trigeminal nerve Nervi trigeminus1, cranial nerve V2

Trigeminal nucleusTurbinate bones Ethmoturbinalia1

Vestibulocochlear nerve Nervi vestibulocochlearis1, cranial nerve VIII2

1Nomenclature reported in Nomina Anatomica Veterinaria (fifth edition).2Nomenclature by Nolte and Angevine (2000).3Nomenclature by Dua-Sharma et al. (1970).

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Anatomic Labeling and Nomenclature

Anatomical structures were identified using the brainatlas of the domestic dog (Beagle, Canis familiaris) (Dua-Sharma et al., 1970) and human (Nolte and Angevine,2000) and labeled using nomenclature adopted from theEnglish translation of Nomina Anatomica Veterinaria(ICVGAN, 2005) (Table 1). Comparisons of the Californiasea lion brain to other species in the Order Carnivorawere made using the Comparative Mammalian Brain Col-lection website (http://www.brainmuseum.org/index.html)prepared by the University of Wisconsin, Michigan StateUniversity and the National Museum of Health and Medi-cine (Welker et al., 2009). Segmentation (i.e., assigningpixels to particular structures) and three-dimensional(3D) reconstructions of the brain surface were performedusing the software program AMIRA 4.1.1 (Mercury Com-puter Systems, San Diego, CA). To create a 3D reconstruc-tion of the brain surface, the pixels in the native (i.e., noimage processing) T1-weighted images from the FLASHsequence were selected manually and defined as cere-brum, cerebellum, spinal cord, olfactory bulb and tract,and optic nerves.

The atlases of the brain and hippocampus of theCalifornia sea lion were constructed from native (i.e., noimage processing) transverse and oblique T2-weightedimages, respectively. These images were created using

eFilm Lite 2.1.2 (Merge Healthcare, Milwaukee, WI) fromthe Digital Imaging and Communication in Medicine(DICOM) files saved during the TSE sequences. T2-weighted images were used in the label schematics becausethese images are very sensitive to minute changes in waterconcentration and are therefore useful in illustrating pa-thology within the brain. The oblique T2-weighted imageswere selected for the hippocampal atlas because theseimages were exceptional in defining hippocampal anatomy,as previously described (Goldstein et al., 2008).

Volume Analysis of Brain Structures

Visualization of MR images was completed first on theMRI unit. Post-processing, segmentation, 3D reconstruc-tions, and volume analysis were also performed usingthe software program AMIRA 4.1.1 (Mercury ComputerSystems, San Diego, CA). 3D reconstructions and vol-umes of brain structures that were determined includedwhole brain; cerebrospinal fluid (CSF) of the total, left,and right brain; CSF of the total, left, and right cerebralventricles; GM and WM of the entire brain; GM andWM of the total, left, and right cerebral hemispheres;and GM and WM of the cerebellum including the brain-stem. Volumes of the left and right hippocampus andassociated structures that were determined included

Fig. 1. 3D reconstruction of the brain surface of a California sealion, in which MRI was performed live. (A) Side view of brain recon-struction with parasagittal section through surrounding head struc-tures. (B) Reconstruction with head structures removed. Orange,cerebral hemisphere gray matter (GM); white, cerebellum; red, spinal

cord; aqua blue, olfactory bulb and tract; purple, optic nerves. Scalebars ¼ 10 cm. (C) Side view of brain removed and fixed in formalin.Scale bar ¼ 1 cm. Yellow arrows demarcate the position of the Sylvianfissure.


lateral ventricle (ventral horn); hippocampal sulcus; hip-pocampus and parahippocampal gyrus combined; hippo-campus alone; parahippocampal gyrus alone; andparahippocampal gyrus WM and GM. The WM of theparahippocampal gyrus most likely contained fibers ofthe subiculum because of the inability to identify boun-daries between WM of the parahippocampal gyrus andWM of the subiculum. Segmenting brain structures, cre-ating 3D reconstructions, and volume analysis used

methods similar to those described by Montie et al.(2008) with modifications explained below.

Brain CSF, GM, and WM. To determine the vol-umes of brain structures, a brain surface mask createdfrom the native T2-weighted transverse images was pro-duced to determine edges for digital removal of nearbyblubber, muscle, skull, and other head structures. The

Fig. 2. (Legend on page 1537)


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mask was constructed by manually tracing the surfaceof the brain and deleting all pixels outside this trace foreach MR image. The whole brain volume was calculatedby integrating the area of the selected tissue for eachslice of the brain surface mask. The caudal boundary ofthe brain was defined by the caudal aspect of the fora-men magnum. Virtual brain weight was calculated bymultiplying the total brain segmented volume by theassumed specific gravity of brain tissue, 1.036 g/cm3

(Stephan et al., 1981).

Total brain CSF volumes were determined by thresh-old segmentation of the brain surface mask (obtainedfrom the native T2-weighted transverse images) followedby manual editing of each slice. Specifically, this proce-dure involved thresholding for signal intensity rangesthat captured the boundaries of brain CSF followed byvisual inspection and manual editing to ensure that CSFwas properly defined. From this segmentation, pixelsrepresenting CSF of the left and right hemispheres weremanually selected and defined as a new label field (i.e.,




a set of images where pixels are selected to representdifferent anatomical structures). Pixels of the left andright ventricular system that captured the lateral, third,and fourth ventricles were manually selected anddefined as another label field. The volumes of thesestructures (i.e., total CSF, left and right CSF, total ven-tricles, left and right ventricles) were determined threeseparate times. The mean volume (cm3) and standarddeviation of these structures were reported.

Total GM and WM volumes were determined by acombination of manual and threshold segmentation ofprocessed, transverse PD-weighted images. Processinginvolved deleting the pixels outside the brain surfacemask and deleting the pixels of each CSF label fieldfrom the native PD-weighted transverse images. Fromthe processed PD-weighted images, the segmentationprocedure involved thresholding for signal intensityranges that captured the boundaries of GM and WM



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followed by visual inspection and manual editing toensure that GM and WM were properly defined. The vol-ume of total GM and WM was then determined. Fromthis segmentation, pixels representing cerebral GM andWM and pixels representing cerebellum and brainstemGM and WM were manually selected and defined as anew label field. The volumes of total cerebral GM andWM and cerebellum plus brainstem GM and WM weredetermined. The pixels defining GM and WM of the cere-bellum were combined with the pixels representing GM

and WM of the brainstem because the boundaries of thecerebellum and brainstem were not easily recognized.Pixels defining the left and right cerebral GM and WMwere manually selected and the volumes of these struc-tures determined. For each structure, volumes weredetermined three times. The mean volume (cm3) andstandard deviation of these structures were reported.

Hippocampus. Volumes of the left and right hippo-campus and associated structures were determined by




manual segmentation of native, oblique T2-weightedimages. The T2-weighted images were used because theywere better at highlighting fluid structures surroundingthe hippocampus compared with the PD images. Thesefluid structures served as boundaries of the hippocam-pus and were defined by higher signal intensities. Theatlas by Dua-Sharma et al. (1970) and the hippocampusatlas we constructed served as guides for segmentingthe left and right hippocampus and associated struc-

tures. For these volume calculations, the various struc-tures of the hippocampal formation (i.e., subiculum,cornu ammonis, and the dentate gyrus) could not beadequately distinguished. These brain regions and thefimbria and alveus were collectively grouped andreferred to as the hippocampus. Structure volumes weredetermined three times, separately. The mean volume(mm3) and standard deviation of these structures werereported.



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The percentage of brain occupied by the left or righthippocampal structure was calculated by dividing thatstructure’s volume (i.e., from the native oblique T2-weighted images) by the sum of the WM and GM vol-umes of the whole brain (i.e., from the processed PD-weighted images) multiplied by 100. The percentage ofcerebral hemisphere occupied by the left or right hippo-campal structure was calculated by dividing the struc-ture volume (i.e., from native T2 weighted images) bythe sum of the left or right cerebral WM and GM vol-

umes (i.e., from processed PD weighted images) multi-plied by 100.

RESULTS AND DISCUSSION3D Reconstruction of the Brain Surface fromMagnetic Resonance Images

Comparing the 3D reconstruction of the Californiasea lion brain to photographs of formalin-fixed brainsof other mammals (i.e., from the Comparative




Mammalian Brain Collection website, http://www.brainmuseum.org/index.html) showed the resem-blance of the sea lion brain to the brains of other spe-cies belonging to the Order Carnivora, specificallycarnivores within the Suborder Caniformia (Fig. 1).The California sea lion brain was similar in shape torepresentative species in the Family Canidae [e.g., thedomestic beagle (Canis familiaris), the wolf (Canislupus), and the coyote (Canis latrans)]; the FamilyUrsidae [e.g., the polar bear (Ursus maritimus)]; and

the Family Mustelidae [e.g., the American mink (Neo-vison vison) and the American badger (Taxidea taxus)].Compared with the canids, ursids, and mustelids, theCalifornia sea lion brain was expanded laterally, withlarge frontal, temporal, and occipital lobes. Qualitativecomparisons of the degree of neocortical folding (or gyr-ification) among these carnivores indicated that thebrain of the California sea lion contained more second-ary folds and sulci than the American mink, the Amer-ican badger, the domestic beagle, the wolf, the coyote,



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and the polar bear. In addition, the pattern of foldsand fissures in the sea lion brain was very differentfrom these carnivores. For example, the Sylvian fissurein the sea lion brain was perpendicular to the ventralsurface of the brain, whereas in the domestic dog, thesulcus was obtuse (�135�) to the ventral surface. TheSylvian fissure in the sea lion also extended deeperinto the brain towards the longitudinal cerebral fissurecompared with the shallower fissures in the domesticdog.

The increase in gyri and sulci in the California sealion compared with canids and mustelids may be bestexplained by the larger brain size in the sea lion. Neo-cortical folding has been shown to correlate with brainsize in large-brained mammals in many different line-ages, as reviewed by Striedter (2005). It has been specu-lated that the neocortex tends to fold in large brainsbecause the most efficient way to increase the area ofthe neocortex without dramatically increasing neocortexthickness is for the neocortex to fold inward [as reviewed




by Striedter (2005)]. If the neocortex were to have bal-looned outward, the expansion without this foldingwould have yielded very large heads and long intracorti-cal connections, both unfavorable factors during naturalselection [as reviewed by Striedter (2005)].

Neuroanatomical Atlas from MagneticResonance Images

Figures 2–21 display a rostral-to-caudal sequence of T2-weighted, 2.5 mm-thick transverse MR brain sections at 5

mm intervals. Panels A display a sagittal section showingthe orientation and level at which the T2 section wastaken; Panels B illustrate the position of the brain in thetransverse plane relative to surrounding head structuresof the T2-weighted image; panels C show labeled versionsof each brain section. The right side of the images corre-sponds to the left side of the brain, which is the traditionalmethod in showing radiological images. These figuresdemonstrate undisturbed spatial relationships amongbrain structures and surrounding head anatomy obtain-able by MR imaging of live animals.



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Figures 22–35 display a ventral-to-dorsal sequence ofT2-weighted, 2.5-mm thick oblique MR brain sections at2.5 mm intervals of the hippocampal region. Panels A dis-play a sagittal section showing the orientation and levelat which the T2 section was taken; panels B illustrate theposition of the brain in the oblique plane relative to sur-

rounding head structures of the T2-weighted image; pan-els C show labeled versions of each brain section. Theright side of the images corresponds to the left side of thebrain. The images obtained in the oblique plane provideda better view of the hippocampus compared with theimages obtained in the transverse plane.


Fig. 21. Fig 2-21. Rostral-to-caudal sequence of transverse T2-weighted images of the brain of a California sea lion imaged live. (A)Sagittal MR images illustrating the orientation of the section. Dottedlines represent the plane of section. Scale bar ¼ 10 cm. (B) Native T2-

weighted 2.5 mm-thick transverse MR brain sections at 5 mm inter-vals with surrounding head structure. Scale bar ¼ 5 cm. (C) Labeledbrain section with head structures removed.


Telencephalon. The MR images illustrate distin-guishing features of the California sea lion telencepha-lon. The size of the olfactory bulb relative to the brain issmall (Figs. 2C–3C); comparisons indicated that the bulbin canids, ursids, and mustelids are larger. The decreasein size of this brain structure in California sea lions maybe related to the possibility that sea lions rely less on ol-factory signals to detect prey and predators than terres-trial carnivores. It is very possible that pinnipeds do nothave active olfaction underwater (Denhardt, 2002).

The neocortex was highly convoluted and containedmany secondary gyri and sulci that were not identifiableusing the domestic dog atlas (Figs. 2C–21C). The supra-sylvian gyrus was expanded laterally (Figs. 5C, 6C),more so than the gyrus of the domestic dog. Despite thelarge hemispheres, the corpus callosum appears to besmall (Figs. 8C–13C).

Structures of the basal ganglia (i.e., the caudate nu-cleus, the putamen, and the globus pallidus) were identi-fied (Figs. 7C–12C). Interestingly, the caudate nucleus in



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the California sea lion seems to have a very small tail.The putamen was difficult to recognize and seemed to bemixed with fiber bundles.

The amygdala was evident in the rostral portion of thetemporal lobes (Figs. 10C, 22C), whereas the hippocam-pus was located near the center of the sections in themedial wall of the temporal lobes (Figs. 11C–13C, 23C–33C). The boundaries of the hippocampus were bestobserved in native T2-weighted images rather than PD-

weighted images. This finding can be best explained bythe CSF surrounding the hippocampus, as observed bythe hyperintensity of the ventral horns of the lateralventricles (lateral and dorsal borders) (Figs. 11C–13C,23C–33C) and the hyperintensity of the hippocampalsulcus (medial border) (Figs. 11C–12C, 22C–29C). Thestructures of the hippocampal formation were bestvisualized in the oblique T2-weighted images (Figs.23C–33C). These structures included the cornu ammonis




or hippocampus proper (Figs. 23C–33C), the subiculum(Figs. 24C–29C), and the dentate gyrus (Figs. 26C–30C).The small size of the dentate gyrus made its identifica-tion very difficult in some of the sections. WM tracts ofthe hippocampus, the alveus and fimbria, were alsoidentified (Figs. 24C–32C). The parahippocampal gyruswas easily recognized in the MR images (Figs. 11C–13C,23C–33C).

One very interesting finding was that the Californiasea lion hippocampus was found mostly in the ventral

position with very little extension dorsally (Figs. 11C–13C, 23C–33C). However, in canids and mustelids, thehippocampus is present in the ventral position but alsoextends dorsally above the thalamus (Dua-Sharmaet al., 1970; Welker et al., 2009).

Diencephalon. The MR images revealed a large dien-cephalon in the California sea lion. The hypothalamus(Figs. 10C–11C) and thalamus (Figs. 10C–14C) were



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easily recognized in the MR images. The optic nerves(Figs. 5C–8C), optic chiasm (Fig. 9C), and pituitarygland (Figs. 10C–12C) were also observed. The mammil-lary body was visualized, protruding downward towardsthe pituitary gland (Fig. 11C). The pineal gland wasvery prominent, located in the midline above the caudalcommissure (Fig. 13C). This finding was expected, as thepineal gland is known to be exceptionally large in pinni-peds (Cuello and Tramezzin, 1969; Turner, 1888). The

habenular nucleus was also located just underneath thepineal gland (one on each side) (Fig. 12C).

Mesencephalon. The rostral colliculus (i.e., superiorcolliculus in bipeds) was easily identified, as was thecaudal colliculus (i.e., inferior colliculus in bipeds) (Fig.14B). Neither of these structures appeared to be particu-larly enlarged.




Rhombencephalon. The MR images showed typicalcharacteristics of the carnivore metencephalon and mye-lencephalon. Auditory pathways were observed, includ-ing the cochlea (Fig. 15C), the vestibulocochlear nerve(Fig. 15C), and the lateral lemniscus (Fig. 14C). The tri-geminal ganglion (Figs. 10C–12C) and trigeminal nerve(Figs. 13C–15C) were identified, as well as the trigemi-nal nucleus (Fig. 17C). The cerebellum was large, andGM and WM were easily distinguishable (Figs. 15C–

21C). Hindbrain structures including the pons (Figs.13C–15C), reticular formation (Figs. 13C–20C), pyrami-dal tract (Figs. 16C–17C, 20C–21C), and medulla oblon-gata were identified (Figs. 20C, 21C).

CSF and Cerebral Ventricles. The three majorprocesses of the lateral ventricles were easily recognizedincluding the rostral or frontal horn (Figs. 7C–14C), thecaudal or occipital horn (Fig. 15C), and the ventral or



1542 MONTIE ET AL.

temporal horn (Figs. 11C–14C). The third ventricleappeared as a thin slit between the two thalami or hypo-thalami (Figs. 10C–12C). The interventricular foramen(i.e., a fluid connection where CSF flows from the lateralventricles to the third ventricle) was observed (Fig. 11C).The mesencephalic aqueduct (i.e., a thin, fluid connec-tion where CSF flows from the third to the fourth ventri-cle) was identified but characterized by a signal void(Figs. 13C–14C). The lack of CSF signal in the mesence-phalic aqueduct represents a flow artifact secondary to

pulsatile CSF flow (Feinberg and Mark, 1987; Malkoet al., 1988; Lisanti et al., 2007). The fourth ventriclewas visualized as a tent-like structure just dorsal of thereticular formation and ended at the obex (Figs. 16C–19C). The lateral recess of the fourth ventricle (i.e., asite where CSF leaves the ventricular system and entersthe subarachnoid space) was also observed (Fig. 18C).The choroid plexus (i.e., vascular tufts responsible forCSF production) in the lateral ventricles (Figs. 10C–12C) and the fourth ventricle (Fig. 18C) were recognized.




Choroid plexus appears to be present in the third ventri-cle as well (Fig. 12C).

Volumes of Brain Structures

Brain CSF, GM, and WM. Segmentation of the trans-verse T2-weighted images was used to delineate the brain

surface and calculate whole brain volume (Table 2). Thevolume of the entire brain was 301.71 cm3, which includedCSF of the subarachnoid space and cerebral ventricles.The virtual brain weight (calculated by multiplying themeasured volume by the specific gravity of brain tissue)was 312.6 g. This estimate was very similar to the actualbrain weight measured after fixation (i.e., 306 g).


Fig. 35. Fig. 22-35. Ventral-to-dorsal sequence of oblique T2-weighted images of the hippocampal region of a California sea lionimaged live. A: Sagittal MR images illustrating the orientation of thesection, which was perpendicular to the temporal lobe and sylvian fis-sure. Dotted blue lines represent the plane of section. Scale bar ¼ 7

cm. B: Native T2-weighted 2.5-mm thick oblique MR brain sections at2.5-mm intervals with surrounding head structure. Scale bar ¼ 1 cm.The dotted rectangle represents the magnified image in panel C. C:Labeled brain section with head structures removed. Scale bar ¼ 1cm.

1544 MONTIE ET AL.

The volumes of CSF and cerebral ventricles weredetermined from segmentations of the transverse T2-weighted images, after digital removal of nearby blub-

ber, muscle, skull, and other head anatomy (Table 2).The CSF volumes of the left and right hemispheres wereapproximately equal (Table 2). In addition, the CSF vol-umes in the cerebral ventricles of the left and righthemispheres were similar (Table 2).

The volumes of GM and WM of the brain were esti-mated from segmentations of the transverse PD-weighted images, after digital removal of nearby blub-ber, muscle, skull, and other head anatomy and removalof total CSF (Table 2). The volumes of either the GM orthe WM of the left and right cerebral hemispheres wereapproximately equal (Table 2). 3D reconstructions of GMand CSF of the subarachnoid space of the left and righthemispheres were constructed (Fig. 36A). The GM andsubarachnoid CSF in the 3D reconstruction were thenremoved to reveal the underlying WM of the left andright hemispheres (Fig. 36B).

Hippocampus. The volumes of the ventral horn ofthe lateral ventricles, the hippocampal sulcus, the hippo-campus (including alveus and fimbria, dentate gyrus,cornu ammonis, and subiculum), and the parahippocam-pal gyrus were determined from segmentations ofoblique T2-weighted images (Table 3). The volumes of

Fig. 36. 3D reconstructions of brain structures of the California sealion, in which MRI was performed live. A: Rostral view of the 3D recon-struction of the brain. Surface of left hemisphere, dark pink; surface ofright hemisphere, light pink; left cerebrospinal fluid (CSF), dark purple;right CSF, light purple. B: Rostral view of the 3D reconstruction of theWM, with the GM and exterior CSF removed. The CSF of the third andlateral ventricles are visible (aqua blue), as well as the left (green) andright (red) hippocampi. WM of left hemisphere, orange; WM of right

hemisphere, tan; WM of cerebellum and brainstem, yellow. C: Rostralview of the lateral ventricles and hippocampi, with the WM strippedaway. Lateral ventricles, aqua blue; green, left hippocampus; red, righthippocampus; white, fimbria; brown, alveus; yellow, fornix; bright pink,mammillary bodies. D: Rostral view of the hippocampi, with the lateralventricles removed. Green, left hippocampus; red, right hippocampus;white, fimbria; brown, alveus; yellow, fornix; aqua blue, septal nucleus;bright pink, mammillary bodies. Scale bars ¼ 8 cm.

TABLE 2. Brain volumes of the Californiasea lion

Structure Volume (cm3)

Whole brain 301.71Total cerebrospinal fluid 38.96 � 2.08Left cerebrospinal fluid 19.94 � 1.06Right cerebrospinal fluid 19.02 � 1.03Total ventricles 7.59 � 0.12Left ventricles 3.76 � 0.05Right ventricles 3.83 � 0.07Total gray matter 164.34 � 6.57Total white matter 80.07 � 2.62Total cerebral gray matter 129.39 � 5.22Left cerebral gray matter 64.82 � 2.84Right cerebral gray matter 64.06 � 2.12Total cerebral white matter 54.42 � 1.87Left cerebral white matter 27.00 � 1.12Right cerebral white matter 27.43 � 0.89Cerebellum and brainstem gray matter 25.65 � 1.13Cerebellum and brainstem white matter 34.94 � 1.45


the left and right hippocampus were approximatelyequal (Table 3). In addition, the left and right parahippo-campal gyri were approximately equal in volume (Table3). Both the volumes of the left and right hippocampuswere 0.84% of the volumes of the left and right cerebralhemispheres (Table 3). Both the volumes of the left andright parahippocampal gyrus were approximately 1.6%of the volumes of the left and right cerebral hemispheres(Table 3). 3D reconstructions of the cerebral ventriclesand their association with the hippocampi were created(Fig. 36C). The ventricles were then removed to revealthe underlying hippocampi, fornix, septal nucleus, andmammillary bodies (Fig. 36D). The 3D reconstruction ofthe California sea lion hippocampus revealed its ventralposition with very little extension dorsally (Fig. 36D), aspreviously mentioned. In the sea lion, only the fornixwas found above the thalamus. This finding in the sealion is different from the domestic dog and the Americanmink (see the Comparative Mammalian Brain Collectionby Welker et al. (2009), http://www.brainmuseum.org/);in these terrestrial relatives, the hippocampus is presentin the ventral position but also extends dorsally abovethe thalamus.

CONCLUSIONS

This article presents the first anatomically labeledMRI-based atlas of a pinniped brain. It is different fromprevious MRI-based atlases of marine mammals in thatit was created from imaging a live animal. This studyalso presents a quantitative approach to determine thesize of brain structures, such as the hippocampus, fromMR images of live California sea lions.

Live MRI scanning coupled with volumetric analysiscan be used not only as a tool to study brain evolution inpinnipeds but also to investigate the impacts of biologi-cal, chemical, and physical agents on marine mammalhealth (Montie, 2006). Of particular concern are theacute, chronic, and possible developmental effects of DA

on the brain of California sea lions (Silvagni et al., 2005;Goldstein et al., 2008; as reviewed by Ramsdell andZabka, 2008). The MRI atlas presented here of a neuro-logically normal California sea lion will allow us to bet-ter evaluate the hippocampus in the oblique plane anddetermine the volumes of brain structures. Thisapproach will help in deciphering the acute, chronic, anddevelopmental effects of DA exposure, or exposure toother pollutants, in wild California sea lions, other ma-rine mammals, and wildlife in general.

ACKNOWLEDGMENTS

The authors thank all the staff and volunteers at theMarine Mammal Center, especially Elizabeth Wheelerfor performing the necropsy of this animal. They alsothank Dr. Cheryl Cross for providing gross images ofthis California sea lion brain, and Dr. Heather Harrisand Dr. Felicia Nutter for their veterinary assistance.They thank the University of Wisconsin and MichiganState Comparative Mammalian Brain Collection, andthe National Museum of Health and Medicine for pro-viding gross and histological images of carnivore brains.The preparations of those images were funded by theNational Science Foundation, as well as the NationalInstitutes of Health.

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TABLE 3. Volumes of the hippocampus and associated structures in the California sea lion

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Left lateral ventricle (ventral horn) 514.218 � 8.753Right lateral ventricle (ventral horn) 492.816 � 7.486Left hippocampal sulcus 313.788 � 5.494Right hippocampal sulcus 348.373 � 15.542Left hippocampus & parahippocampal gyrus 2232.157 � 27.933 0.913 � 0.018 2.432 � 0.054Right hippocampus & parahippocampal gyrus 2265.195 � 45.655 0.927 � 0.019 2.476 � 0.043Left hippocampus 771.286 � 16.381 0.316 � 0.011 0.840 � 0.033Right hippocampus 772.914 � 17.786 0.316 � 0.002 0.845 � 0.006Left parahippocampal gyrus 1463.475 � 27.099 0.599 � 0.010 1.594 � 0.031

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Neuroanatomy and Volumes of Brain Structures of a Live California