The brain of the common vampire bat, Desmodus rotundus murinus (Wagner, 1840): a cytoarchitectural atlas/O cerebro do morcego vampiro comum, Desmodus rotundus murinus (Wagner, 1840): um atlas citoarquitetural.
Of the three species of vampires (Desmodus, Diaemus, and Diphylla), Desmodus is the most common and best known as a pest and a scourge. Desmodus is unique among bats in having the largest neocortical volume (size index 1024) amongst the nearly 276 species of bats investigated (Baron et al., 1996a; b, c). For this characteristic alone Desmodus is considered one of the most evolved bats studied thus far. Monographic works on chiropteran brain anatomy are those of Schneider (1957; 1966), Mann (1963), Henson (1970), and McDaniel (1976). An unparalleled, three-volume, encyclopedic work on the neurobiology of Chiroptera authored by Georg Baron, Heinz Stephan and Heiko Frahm appeared in 1996. While this work is priceless for data on brains of bats, and includes brain atlases of a megabat (Rousettus) and a microbat (Myotis), there have been no systematic studies on the vampire brain. Several other chiropteran brain atlases have also been available in the literature (Table 1).
For the Latin American countries, Desmodus carries a special threat. It is called 'thief of the night', and causes substantial loss of cattle from transmitted rabies (Greenhall et al., 1983). Its importance is further enhanced in the newly discovered use of the clot-buster, plasminogen activator (desmoteplase or DSPA) in its saliva (Hawkey, 1966) in prevention of strokes in the human (Fauber, 2003). It is not an overstated conclusion that knowledge of the brain cytoarchitectonics on this species is essential to have.
The association of the vampire behavioral complexity with its voluminous neocortex has been established. (Baron et al., 1996a; b; c). These features make Desmodus an ideal bat species for neuroanatomical and neurophysiological research. This study was, therefore, undertaken to analyze the serial brain anatomy of the common vampire bat.
2. Materials and Methods
Adult Desmodus rotundus were captured live from a wild colony in Veracruz, Mexico through the courtesy of the late Professor William Wimsatt, and William Lopez-Forment. Five females and two males were transported to the author's laboratory in Louisville and remained in good health in captivity for several years.
2.2. Brain preparation
Bats were deeply anesthetized with ether and intracardiacally perfused first with saline in nitrite solution with the right atrium punctured. Perfusion was then continued using one of the three solutions: Bouin's, 4% glutaraldehyde, or 10% buffered formalin. The skin and muscles were dissected away from the cranium and the neck. The head was severed at the neck and kept immersed in the fixative for several hours, after which the calvarium and the dorsal half of the upper cervical vertebrae were chipped away. The exposed brain and the spinal cord were further left in the fixative for several more hours. The olfactory nerves were transected and the frontal lobes were gently lifted, gradually severing other cranial nerves. The hypophysis was gently pushed out of the sella turcica. When the entire brain (Figure 1) was thus freed, it was kept in the fixative for 24-48 hours, washed, and processed in graded ethyl alcohol. Paraffin embedding as well as cellosolve (ethylene-glycolmonoethyl ether) procedures were followed for the individual brains.
[FIGURE 1 OMITTED]
For the cellosolve infiltration, the brain was processed through four changes of cellosolve, leaving it overnight in the last change. Three changes of one hour duration each in benzene, followed by three one-hour changes in paraffin were made under vacuum at 58[degrees]C. The infiltrated brain was then embedded in paraffin. Serial frontal (coronal) sections were cut at 20 [micro]m thickness, arranged on albumen-coated slides which were kept at 58[degrees]C in an oven for drying. Staining was achieved with lugol fast blue overnight, differentiated in 70% alcohol, rinsed, and counterstained with cresyl echt violet for 5-7 minutes, cleared in xylene and mounted in permount.
Another 10 [micro]m thick serial section series of the decalcified heads of several specimens, perfused with Bouin's solution, was prepared. These were stained with the one-step Gomori trichrome procedure (Bhatnagar and Kallen, 1974; Bhatnagar, 1980). A partly incomplete, 20 [micro]m thick, stained serial series made from a female Desmodus rotundus murinus brain was received from the late Professor William A. Wimsatt, Cornell University, Ithaca, New York.
2.3. Brain analysis
Using a macrophotography apparatus, selected sections, primarily from the lugol fast blue-cresyl echt violet series, beginning with the olfactory bulb and ending with the medulla oblongata, were photographed with gaps of about 300-560 [micro]m between sections. A total of 710 sections, each 20 [micro]m thick, was prepared. Even though over 100 sections were photographed, studied and labeled, 26 sections were finally selected for this atlas, representing the entire brain, without unduly repeating. In this process of selecting representative sections, a few structures may be found missing. These were purposely left out to conserve space.
Desmodus brain cytoarchitecture was studied in comparison with the brain anatomy of the human (Villiger-Ludwig-Rasmussen, 1951), rat (Paxinos and Watson, 1997), muskrat (Panneton and Watson, 1991), cat, hamster, guinea pig, and other bat species (Table 1). Structures were localized and identified on a camera lucida drawing, an exact match control of the right half of the photographed section (Atlas Figures 2-27).
3. Results and Discussion
The primary objective of this investigation was to prepare an atlas of the brain of the vampire, Desmodus rotundus and identify the structures, such as the nuclear groups, ascending and descending fiber tracts, cranial nerve nuclei, cortical regions, and brainstem components. This species, being so commonly available and so unique in its behavioral characteristics, has long deserved detailed cytoarchitectural studies of its brain. Desmodus has the largest neocortical volume compared to 276 species of bats (Table 2; see Baron et al., 1996a; b; c). Most early studies on the vampire brain anatomy are on Desmodus rotundus (see Mann, 1960, 1963). Plenty of data are provided in the three-volume work "Comparative Neurobiology in Chiroptera" by Baron et al. (1996a; b; c). The superior olivary complex was investigated by Kuwabara and Bhatnagar (1999). They also reported that the lateral lemniscus is columnar and similar as in other echolocating bats (Kuwabara and Bhatnagar, 2000). Data on the three species of vampire brains were reviewed in detail by Bhatnagar (1988a) Ultrastructural observations on the pineal gland of Desmodus were also reported (Bhatnagar, 1988b). The accessory olfactory bulb in Desmodus, though short in height, extends nearly to the full diameter of the main olfactory bulb (Cooper and Bhatnagar, 1976), an observation that has not been made in other mammalian olfactory bulbs, including those of bats. There is an olfactory ventricle. In one of the brains, prepared from a wild caught Desmodus (see Figure 2), isolated telencephalic regions were devoid of brain tissue, as though it had been punched out. It would be interesting to investigate if this strange morphological finding bears any relationship to the rabies virus carrying characteristics of the vampire.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
The Desmodus brain is short, lissencephalic (smooth; sulci lacking on the outer surface), with high hemispheres (Figure 2), and a rostral sulcus. Parafloculus is unexposed. The pons is large, and the pyramidal tracts are huge until they cross. Accurately detailed and painstakingly measured volumes on the vampire brains (Desmodus rotundus, and including another vampire species, Diphylla ecaudata) are provided in Baron et al. (1996a; b; c). These are summarized in Table 3. For the sake of space, those specifically not included in Table 3 are the volumes of subdivisions and parts of medulla oblongata, mesencephalic components, cerebellar nuclei, and lateral geniculate body. For the data on these structures and others, the reader is directed to Baron et al. (1996a; b; c).
[FIGURE 7 OMITTED]
Even though major senses (vision, hearing, echolocation and olfaction) are well developed in the vampire, it is not practical for the bat to use phyllostomid type of sonar for locating its prey. Statements have been made in the literature that vampires use olfaction to a greater extent in feeding (Mann, 1963). If greater sizes or volumes are related to greater sense of acuity, then the size of the olfactory and the vomeronasal organs are indicative of an olfactory sense acuter than in most bat species. Even by cursory examination, of the three vampires, Diaemus appears to have larger volumes dedicated to the olfactory and the vomeronasal (accessory) systems than Desmodus. Diphylla appears to occupy the last position amongst vampires in this regard.
Mann (1963) has described ten rhinencephalic circuits using Desmodus as an example. They are summarized as follows: for detection and discrimination of odors, reflex motor responses to olfactory stimuli with or without the intervention of lower pallidal sectors, high level reflex motor responses through nigropallidial pathways (the development of these circuits depends upon the development of the anterior olfactory nucleus). Extrapyramidal functions, olfactory tubercle, pale opallium and the synaptic cascades of its sectors, cortical involvement of instinctive action patterns and responses based on neocortical associations are the remaining circuitary. The pursuit of the main and the accessory olfactory systems in Desmodus is likely to provide the neuroanatomical explanation of its behavioral complexities.
[FIGURE 8 OMITTED]
[FIGURE 9 OMITTED]
[FIGURE 10 OMITTED]
[FIGURE 11 OMITTED]
[FIGURE 12 OMITTED]
[FIGURE 13 OMITTED]
[FIGURE 14 OMITTED]
[FIGURE 15 OMITTED]
[FIGURE 16 OMITTED]
[FIGURE 17 OMITTED]
[FIGURE 18 OMITTED]
[FIGURE 19 OMITTED]
[FIGURE 20 OMITTED]
[FIGURE 21 OMITTED]
[FIGURE 22 OMITTED]
[FIGURE 23 OMITTED]
[FIGURE 24 OMITTED]
[FIGURE 25 OMITTED]
[FIGURE 26 OMITTED]
[FIGURE 27 OMITTED]
For neurological information on Desmodus, the reader is directed to Greenhall et al. (1983, species account), Mann (1960, neurobiology), Escobar et al. (1968, inferior olivary nucleus), Yamamoto et al. (1955, abducens nucleus, pyramidal tract and the vestibular nucleus), Palacios Pru and Briceno (1972, glial cell and neuronal dendrite ultrastructural relationship in the olfactory bulb), and Palacios Pru (1970, ultrastructure of mossy fibers).
The cytoarchitectural organization of the Desmodus brain deserves to be compared in parallel with that of other vampire species, bats of other families (especially megachiroptera), and some other representative mammals. When nuclear groups, fiber tracts, and their interconnections are thus compared and contrasted, the neuroanatomical basis of behavior in the vampire is more likely to become apparent. This atlas has been presented with exactly that future goal in mind.
Acknowledgements--A serial section series of the vampire brain prepared by Lois Copeland was kindly gifted to the author by the late Professor William Abel Wimsatt of Cornell University, Ithaca, New York. Without his help and encouragement, not only this atlas, but the author's other studies in the field of bat biology and neuroanatomy would not have become a reality. As a tribute to a great mentor and comparative morpho- physiologist, this work is dedicated to Professor William Wimsatt. Cathy Caple at Louisville spent long hours in the dark room to create the best photographic images from the serial sections. Lucinda Schultz helped with histological preparations. Grateful thanks are due to the Editor-in-Chief, Dr Takako Matsumara-Tundisi, and Dr. Paula Matvienko-Sikar, of the Brazilian Journal of Biology[R], for encouragement and many courtesies. Dr. Wilson Uieda and Dr. Jader MarinhoFilho kindly provided the Scholarly support.
Received July 19, 2006--Accepted February 2, 2007--Distributed August 31, 2008 (With 27 figures)
BARON, G., STEPHAN, H. and FRAHM, H., 1996a. Comparative Neurobiology in Chiroptera: macromorphology, brain structures, tables and atlases. Birkhauser Verlag: Basel. vol. 1, p. 1-529.
-, 1996b. Comparative Neurobiology in Chiroptera: brain characteristics in taxonomic units. Birkhauser Verlag: Basel. vol. 2, p. 533-1074.
-, 1996c. Comparative Neurobiology in Chiroptera: brain haracteristics in functional systems, echoethological adaptation, adaptive radiation and evolution. Birkhauser Verlag: Basel. vol. 3, p. 1075-1596.
BHATNAGAR, KP., 1980. The chiropteran vomeronasal organ: its relevance to the phylogeny of bats. In Wilson, DE. and Gardner, AL. (Eds.). Proceedings of the Fifth International Bat Research Conference. Lubbock: Texas Tech Press. p. 289-315.
-, 1988a. Anatomy. In Greenhall, AM. and Schmidt, U. (Eds.). Natural History of Vampire Bats. Boca Raton, Florida: CRC Press. p. 41-70.
-, 1988b. Ultrastructure of the pineal body of the common vampire bat, Desmodus rotundus. Am. J. Anat., vol. 181, no. 2, p. 163-178.
BHATNAGAR, KP., and KALLEN, FC., 1974. Morphology of the nasal cavities and associated structures in Artibeus jamaicensis and Myotis lucifugus. Am. J. Anat., vol. 139, no. 2, p. 167-190.
BHATNAGAR, KP., FRAHM, HD. and STEPHAN, H., 1990. The megachiropteran pineal organ: a comparative morphological and volumetric investigation with special emphasis on the remarkably large pineal of Dobsonia praedatrix. J. Anat. (Lond.), vol. 168, p. 143-166.
BRAUER, K., and SCHOBER, W., 1970. Catalogue of Mammalian Brains. Part I. Jena: Fischer.
-, 1976. Catalogue of Mammalian Brains. Part II. Jena: Fischer.
COOPER, JG. and BHATNAGAR, KP., 1976. Comparative anatomy of the vomeronasal complex in bats. J. Anat. (Lond.), vol. 122, no. 3, p 571-601.
ESCOBAR, A., SAMPEDRO, ED. and DOW, RS., 1968. Qualitative data on the inferior olivary nuclei in man, cat and vampire bat. J. Comp. Neur., vol. 132, p. 397-404.
FAUBER, J., 2003. Vampire bats' saliva may help in treating blood clots, stroke. Milwaukee Journal Sentinel, January 10.
GREENHALL, AM., JOERMANN, G., and SCHMIDT, U., 1983. Desmodus rotundus. Mammalian Species, vol. 202, p. 1-6.
HAWKEY, CM., 1966. Plasminogen activator in the saliva of the vampire bat, Desmodus rotundus. Nature, vol. 211, p. 434-435.
HENSON, OW., 1970. The central nervous system. In Wimsatt, WA. (Ed.). Biology of Bats. vol. II. New York: Academic. p. 57-152.
HUMPHREY, T., 1936. The telecephalon of the bat. I. The noncortical nuclear masses and certain pertinent fiber connections. J. Comp. Neur., vol. 65, no. 1, p. 603-711.
IGRASHI, S. and KAMIYA, T., 1972. Atlas of the vertebrate brain: morphological evolution from cyclostomes to mammals. Baltimore: University Park Press. 126 p.
KUWABARA, N. and BHATNAGAR, KP., 1999. The superior olivary complex of the vampire bat, Desmodus rotundus (Chiroptera: Phyllostomidae). Acta Chiropterologica, vol. 1, no. 1, p. 81-92.
-, 2000. The nuclei of the lateral leminiscus of the vampire bat, Desmodus rotundus. Association for Research in Otolaryngology, vol. 23, p. 180. (abstract).
McDANIEL, VR., 1976. Brain anatomy. In Baker, RJ., Jones, JK. and Carter, DC. (Eds.). Biology of Bats of the New World Family Phyllostomatidae. Part I. Lubbock: Mus. Texas Tech Univ. Spec. Publ. vol. 10, p. 147-200.
MANN, G., 1960. Neurobiologia de Desmodus rotundus. Invest. Zool. Chil., vol. 6, p. 79-99.
-, 1963. The rhinencephalon of Chiroptera. Invest. Zool. Chil., vol. 9, p. 1-93.
PALACIOS-Pru, EL., 1970. Aspectos estructurales sobre fibras musgosas: studio comparative Cavia cobaya y Desmodus rotundus. Metodo de Golgi y microscopia electronica. Acta Cient. Venezolana, vol. 21, p. 226-233.
PALACIOS-Pru, EL. and BRICENO, RVM., 1972. An unusual relationship between glial cells and neuronal dendrites in olfactory bulbs of Desmodus rotundus. Brain Res., vol. 36, no. 2, p. 404-408.
PANNETON, WM. and WATSON, BJ., 1991. Stereotaxic atlas of the brainstem of the muskrat, Ondatra zibethicus. Brain Res. Bull., vol. 26, no. 4, p. 479-509.
PAXINOS, G. and WATSON, C., 1997. The rat brain in stereotaxic coordinates. New York: Academic.
SCHNEIDER, R., 1957. Morphologische Untersuchungen am Gehirn der Chiropteren (Mammalia). Abh. Senckenb. Naturforsch. Ges., vol. 495, p. 1-92.
-, 1966. Das Gehirn von Rousettus aegyptiacus (E. Geoffroy 1810) (Megachiroptera, Chiroptera, Mammalia). Ein mit Hilfe mehrerer Schnittserien ersteller Atlas. Abh. Senckenb. Naturforsch. Ges. vol. 513, p. 1-160.
TAMURA, I., 1950. Comparative anatomical studies on the brain stem, with special references to the reticular formation and its relating nuclei of Chiroptera. Acta Anat. Niigata, Ensia Niigata, vol. 50, p. 65-98.
WAGNER, JA., 1840. Die Saugethiere in Abbildungen nach der Natur. Suppl. 1. Abt. Die Affen und Flederthiere, Munchen. 558 p.
VILLIGER, E., LUDWIG, E. and RASMUSSEN, AT., 1951. Atlas of the cross section anatomy of the brain. New York: Blakiston.
YAMAMOTO, S., SHIMODA, B., MOMMA, R. and SAI, H., 1955. Studies on brain stem. IX. Comparative anatomical study of brain stems of some species of bats. Tohoku J. Exp. Med., vol. 61, no. 4, p. 339-344.
Bhatnagar, KP. *
Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville Louisville, Kentucky 40292, USA
* e-mail: firstname.lastname@example.org
Table 1. A partial list of atlases and monographs on the brains of bats. Species (Family) Reference 1. Rousettus aegyptiacus Schneider, 1966 (PTEROPODIDAE) 2. Rousettus amplexicaudatus Baron et al., 1996 (PTEROPODIDAE) 3. Pteropus giganteus Igrashi and Kamiya, 1972 (PTEROPODIDAE) 4. Dobsonia praedatrix Bhatnagar et al., 1990 (PTEROPODIDAE) 5. Desmodus rotundus Bhatnagar, this study (PHYLLOSTOMIDAE) 6. Pteronotus parnellii Henson, 1970 (MORMOOPIDAE) 7. Tonatia bidens McDaniel, 1976 (PHYLLOSTOMIDAE) 8. Tadarida mexicana Humphrey, 1936 (MOLOSSIDAE) 9. Myotis montivagus Baron et al., 1996 (VESPERTILIONIDAE) 10. Miniopterus schreibersii Igrashi and Kamiya, 1972 (VESPERTILIONIDAE) 11. Rhinolophus ferrumequinum, Tamura, 1950 Pipistrellus abramus, Plecotus auritus (RHINOLOPHIDAE, VESPERTILIONIDAE) Species (Family) Notes 1. Rousettus aegyptiacus Frontal, sagittal, and horizontal (PTEROPODIDAE) series; Weigert's hematoxylin- eosin; Heidenhain's hematoxylin 2. Rousettus amplexicaudatus Frontal series; Gallocyanin (PTEROPODIDAE) 3. Pteropus giganteus Six frontal sections (PTEROPODIDAE) 4. Dobsonia praedatrix One sagittal section (PTEROPODIDAE) 5. Desmodus rotundus Lugol-fast blue-cresyl echt violet (PHYLLOSTOMIDAE) 6. Pteronotus parnellii Line drawings of 14 frontal (MORMOOPIDAE) sections 7. Tonatia bidens Photos of 18 coronal sections (PHYLLOSTOMIDAE) 8. Tadarida mexicana Line drawings of the telencephalic (MOLOSSIDAE) region in 16 figures 9. Myotis montivagus Frontal series, Gallocyanin (VESPERTILIONIDAE) 10. Miniopterus schreibersii Six frontal sections (VESPERTILIONIDAE) 11. Rhinolophus ferrumequinum, Reticular formation of the entire Pipistrellus abramus, brainstem in cross sections Plecotus auritus (RHINOLOPHIDAE, VESPERTILIONIDAE) Other monographs on bat brains: Schneider, 1957; two species of megachiroptera and 22 species of microchiroptera, Brauer and Schober, 1970, 1976; sixty-five species of phyllostomids, McDaniel, 1976. Table 2. Average percentages and range for the five main regions of the brain of 47 megachiropteran and 225 microchiropteran species including the Desmodontidae. Data on Desmodus and Diphylla have been calculated and shown separately. The values for human brain are provided for comparison. (After Baron et al., 1996a, p. 89). Megachiroptera Microchiroptera % (Range) % (Range) Medulla oblongata 7.1 (5.3-8.9) 13.1 (7.8-19.7) Cerebellum 13.5 (11.5-16) 19.5 (15.3-28.9) Mesencephalon 5.4 (4.2-6.9) 9.8 (5.5-14.6) Diencephalon 8.6 (7.9-9.4) 8.0 (6.7-10.6) Telencephalon 65.3 (60.1-70.2) 49.6 (37.7-63.4) Desmodus Diphylla Human (%) (%) (%) Medulla oblongata 8.5 8.3 0.8 Cerebellum 17.6 17.4 11.0 Mesencephalon 6.2 7.0 0.6 Diencephalon 7.9 8.4 2.7 Telencephalon 59.8 58.9 85.0 Table 3. Neurobiological data on Desmodus rotundus (from Baron et al., 1996a; b; c). Brain length 16.80 mm Standard brain volume 964 [mm.sup.3] Net brain volume 944 [mm.sup.3] Brain weight 999 mg Body weight 36.3 g Volumes of brain parts, [mm.sup.3] Telecephalon 564.8 Neocortex 312.5 main olf bulb 33.0 acc. olf bulb 0.355 Diencephalon 74.9 Mesencephalon 58.3 Cerebellum 166.4 Medulla oblongata 80.0