Abstract
To demonstrate the kinds of data that can be obtained non-destructively and non-invasively from preserved museum specimens using modern imaging technology the head region of a whole body fetal specimen of the common dolphin, Delphinus delphis, aged 8-9 months post-conception, was scanned using Magnetic Resonance Imaging (MRI). Series of scans were obtained in coronal, sagittal and horizontal planes. A digital three-dimensional reconstruction of the whole brain was prepared from the coronal series of scans. Sectional areas and three-dimensional volumes were obtained of the cerebral hemispheres and of the brainstemplus-cerebellum. Neuroanatomical features identified in the scans include the major sulci of the cerebral hemispheres, well-differentiated regions of gray and white matter, the mesencephalic, pontine, and cervical flexures, the "foreshortened'' appearance of the forebrain, and the large auditory inferior colliculi. These findings show that numerous features of the fetal common dolphin brain can be visualized and analyzed from MRI scans.
Introduction
Cetaceans (whales, dolphins, and porpoises) have undergone a number of evolutionary modifications from their terrestrial ancestral state. One of the more dramatic transformations occurred within the suborder Odontoceti (toothed whales, dolphins, and porpoises) in brain size, neuroanatomical structure and organization. Compared with the brains of other mammalian orders, odontocete brain morphology is unusual in many respects. Researchers have stated that "..the lobular formations in the dolphin brain are organized in a pattern fundamentally different from that seen in the brains of primates or carnivores'' (Morgane et al. 1980, pp 105) . Because of the fiftyfive million year divergence between cetaceans and other mammals, odontocete brains are a blend of early mammalian features along with unique derived characteristics (Ridgway 1986 (Ridgway , 1990 Glezer et al. 1988; Manger et al. 1998) . There have been numerous published studies of adult odontocete brains. These studies have documented the differences between odontocete and other mammalian brains at the level of cortical cytoarchitecture and immunohistochemistry (Garey et al. 1985; Garey and Leuba 1986; Glezer et al. , 1992a Hof et al. 1992; Hof et al. 1995; Glezer et al. 1998) , cortical surface morphology (Jacobs et al. 1979; Morgane et al. 1980; Haug 1987) , and noncortical structures and features (Tarpley and Ridgway 1994; Glezer et al. 1995) . In contrast, there have been relatively few published studies of cetacean prenatal neuroanatomy. These studies have been restricted to a few odontocete species such as the sperm whale, Physeter macrocephalus (Oelschlager and Kemp 1998) and harbour porpoise, Phocoena phocoena (Oelschlager and Buhl 1985; Buhl and Oelschlager 1988) . To date, no published studies exist on the prenatal neuroanatomy of the common dolphin, Delphinus delphis.
The relative dearth of neuroanatomical studies on prenatal cetaceans is due to both the relative rarity of such specimens and the difficulties involved in accessing and preparing the brain for conventional histological analysis. Thus, it would be extremely beneficial to take advantage of those specimens that already exist in museum and stranding facility collections. Neuroimaging, and Magnetic Resonance Imaging (MRI) in particular, provides a nondestructive and versatile way to visualize, measure, and reconstruct postmortem fetal cetacean brains in situ.
In this paper we demonstrate the use of MRI for visualizing, measuring, and reconstructing the postmortem brain of a fetal common dolphin that had been fluid-preserved in a museum collection for 19 years.
Materials and methods

Specimen
The specimen is a male fetal common dolphin (Delphinus delphis) from the Marine Mammal Collection at the Smithsonian Institution. The specimen was found freshly dead in utero on Curritock Beach in North Carolina, where its mother stranded. Upon removal from the mother, the specimen was pressure perfused with 10% buffered formalin and placed in a container of 70% alcohol with brain intact. The body length is 53 cm, which is consistent with an estimated prenatal age of 8-9 months (Ferrero and Walker 1995; Perrin et al. 1977) . Full gestation requires 11 months when the neonate is approximately 82 cm in length (Ferrero and Walker 1995) . 
MRI protocol
After 19 years in fluid preservative, magnetic resonance images of the entire brain in situ were acquired in the coronal, sagittal, and horizontal planes with a General Electric 1.5 Tesla scanner at the Dept. of Radiology at Walter Reed Army Medical Center. T1-weighted coronal images were obtained with the following protocol: pulse sequence=fast spin echo, slice thickness=1.5 mm, slice interval=1.5 mm, TR=3,800 ms, TE=85 s, field of view=140 mm, matrix=256×256 pixels. T1-weighted sagittal images were acquired with two different spin echo pulse sequences. The first series was obtained with the following parameters: slice thickness=3 mm, slice interval=3 mm, TR=400 ms, TE=9 ms, field of view=120 mm, matrix=256×256 pixels. The second series was obtained with the same parameters as the first except TR=500 ms and TE=14 ms. T1-weighted horizontal scans were obtained with the following spin echo protocol: slice thickness=3 mm, slice Three-dimensional reconstruction and reformatting Three-dimensional digital reconstruction images were created by Timothy L. Murphy, using VoxelView/E and VoxelMath programs (Vital Images) at the Laser Scanning Microscopy Laboratory at Michigan State University, Joanne Whallon, Director.
Anatomical labeling and nomenclature
Identifiable neuroanatomical structures were labeled using the nomenclature from Morgane et al. (1980) . 
Results

Qualitative anatomical descriptions
Figures 1a, b and 2a, b display three-dimensional reconstructions of the specimen from a ventral and lateral viewpoint, respectively. Each MR image is accompanied by an anatomically labeled schematic drawing. These images show a number of major features of the specimen, including the almost vertically-oriented Sylvian cleft (Fig. 2a, b) and the large cerebellum (Figs. 1a, b,  2a, b) . Figure 2a , b most clearly displays the "pronated boxing glove'' shape typical of odontocete brains and noted by Morgane et al. (1980) in reference to adult odontocete brains. Notable is the presence of structures that may be construed as residual olfactory bulbs (Fig. 1a, b) . Despite the marked regression detectability of these olfactory structures at this relatively late fetal stage is surprising given that olfactory nerves and bulbs are lacking in adult odontocetes altogether (Breathnach 1960 ) with very few exceptions such as the sperm whale (Ries and Langworthy 1937; Kojima 1951) . Therefore, these structures in the present specimen must be interpreted with caution. Secondary olfactory structures, such as the olfactory lobe, are present as expected (Morgane et. al., 1980) . Figure 3a , b display a three-dimensional reconstruction of the brain, viewed from the right front of the brain, with the "orbital'' lobes "dissected away'' by a coronal section, to show the sulcal pattern on the surface and the depths of the sulci within the section. Major sulcal features are identified in Fig. 3b . Figure 4a , b are similar views of the posterior surface of the reconstructed brain, viewed from the right posterior aspect. The coronal section revealing Fig. 4a is a few mm rostral to that of Fig. 4b . These figures show that the cetacean three-tiered pattern of major clefts (Morgane et. al., 1980 ) is already well-established by the mid-late fetal stage. Figure 5a -l is a posterior-to-anterior selection of originally-acquired coronal scans, 1.5 mm thick, taken from the entire sequence of images, along with corresponding labeled illustrations. The MR images demonstrate that detectable differentiation of gray matter and white matter can be achieved with MRI in postmortem fetal cetacean brain specimens preserved for almost two decades. Figure 6a , b displays one of the originally-acquired 3-mm-thick horizontal scans and a labeled schematic version. Prominent in this image are the large inferior colliculi and the extensive lateral development of the (Breathnach 1960; Ridgway 1990 ). The inferior colliculus appears to already have taken on adult proportions as it is clearly larger than the superior colliculus in size. In contrast, the inferior colliculus does not surpass the superior colliculus in size in very late fetal stages in the harbour porpoise (Phocoena phocoena) (Buhl and Oelschlager 1988) . Figure 7a -f displays examples of originally-acquired 3-mm-thick sagittal sections and corresponding labeled illustrations. These images clearly show some of the more distinctive features of the dolphin brain pertaining to the position of the brain in the cranial cavity with respect to the longitudinal body axis (beakfluke axis). The pronounced pontine and cervical flexures typical of odontocete brains and the mesencephalic flexure, which largely gives the brain its "boxing glove'' appearance, are evident in the images of Fig. 6 . These flexures are found in the brains of other mammals, including humans, but only during the embryonic state. In the dolphin, these flexures are maintained in the adult brain. The dolphin forebrain is also foreshortened, giving the appearance that the cerebral hemispheres are rotated on the transverse axis 90 degrees from the beak-fluke axis. This appearance is also characteristic of the adult dolphin brain. Because of the distinctive orientation of the brain, the most anterior cortical lobe is called "orbital'' instead of "frontal'' (Morgane et al. 1980 ).
Volumetric measurements
Volumetric measurements indicate that whole brain volume for the present specimen is 194.91 cc. There is no substantial difference in volume between the two cerebral hemispheres. The left hemisphere volume is 84.02 cc and the right hemisphere volume 83.37 cc. Hindbrain volume (which includes the cerebellum) is 27.51 cc. The hindbrain made up 14.1 percent of the whole brain volume. Ridgway and Brownson (1984) reported a brain weight of 476 g for a neonatal common dolphin. Because the specific gravity of brain tissue is close to one, weight units can be reasonably substituted for volume units. When this is done, the brain weight reported in Ridgway and Brownson (1984) is almost 2.5 times the brain "weight'' reported in the present study for a specimen of 8-9 months prenatal age. However, because actual ages of the specimens are unknown, this comparison might not be especially meaningful. Rapid prenatal brain growth and some shrinkage of tissue may account for any real discrepancy. The large relative size of the hindbrain is consistent with reports of a relatively large cerebellum in odontocetes that apparently develops its large size after the embryonic stage in dolphins (Ridgway 1990) . Because much of the hindbrain volume includes the cerebellum, the fact that the entire hindbrain constitutes 14.1% of the whole brain volume suggests that the cerebellum in the present fetal specimen forms a slightly lower proportion of total brain volume than in adults, in which the cerebellum constitutes 15% of the entire brain volume (Marino et al. 2000) . 
Discussion
The study of prenatal cetacean brain development, and cetacean neuroanatomy in general, has been constrained by the lack of enough data to formulate a database on the normative range of morphometric values for whole brains and substructures in cetaceans. This situation can be resolved by using MRI to analyze the numerous postmortem specimens available in museums and similar facilities. The present study demonstrates that useful neuroanatomical and morphometric data can be obtained non-invasively and efficiently from postmortem prenatal cetacean specimens that have been in fixative for long periods of time. MRI images can be produced that allow anatomical labeling, volumetric measuring, and three-dimensional reconstruction of the brain. MRI also allows for the preservation of spatial aspects of internal struc- tures and their relationship to one another to a degree that cannot be readily achieved through conventional histological techniques. This enables accurate three-dimensional reconstruction that then offers the flexibility to view any part of the brain from any angle and in any plane of sectioning while maintaining the intactness of the specimen.
Comparative analyses of prenatal developmental patterns in and among cetaceans and other mammals are extremely promising for elucidating the history of phylogenetic divergence among cetacean species and between cetaceans and other mammals. The importance of including prenatal specimens in comparative evolutionary studies is achieving wide recognition: there has been a recent upsurge of contributions to the literature that point to the value of examining developmental patterns in studies of evolution. One of the questions that may benefit substantially from studies of cetacean prenatal brain development is that of the phylogenetic relationship among the various families and species of cetaceans. There are still unresolved issues in the literature about various aspects of the cetacean phylogenetic tree. Perhaps on an even more profound level, studies of cetacean brain development may provide valuable data that will assist efforts to reconstruct the evolutionary history of cetaceans since their divergence from terrestrial ancestors. This enterprise, recently dubbed "evo-devo'', requires the kinds of data presented in this paper and in the few other existing studies on cetacean prenatal brain development. MRI studies provide a way to more efficiently collect and analyze these data in the future.

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