A Stereotaxic Atlas of the Brain of the Jungle Crow (Corvus macrorhynchos)


Ei-Ichi Izawa E Shigeru Watanabe


This material is a part of a book chapter published by Ei-Ichi Izawa and Shigeru Watanabe in eIntegration of Comparative Neuroanatomy and Cognition (Eds. S. Watanabe & M.A. Hofman, Keio University Press, in press).f


Materials and Methods
Six wild-caught Japanese jungle crows (3 males and 3 females; Corvus macrorhynchos) were used for the present study. These subjects were provided from Tokyo Ueno Zoo. Sex was determined by DNA analysis of sex chromosome according to a method introduced by Fridolfsson and Ellegren (1999). Average bodyweight was 680 g in males and 570 g in females. Estimated age was over-1-year old (i.e., subadults) based on their mouth color (Kitagawa 1990).
Under deep anesthesia with an overdose injection of veterinary pentobarbital (Nembutal, 0.45?0.6 ml / bird), birds were perfused with 0.9% saline containing hepaline (0.2 ml / 50 ml saline) followed by 4% paraformaldehyde in 0.1M PBS. The head was then placed in the stereotaxic holder which was customized for crows (KS-0041, Narishige Co.). This stereotaxic holder allowed to hold the head in the standard orientation with placing the anterior fixation point (i.e., beak bar position) 45 below the horizontal axis of the instrument, which has been conventionally used in the previous brain atlases since Karten and Hodos (1967; also Stokes et al. 1974, Youngren and Phillips 1978, Kuenzel and Masson 1988). In this proper position, the average distance between the tip of the ear bar and the beak bar was 34.0 mm (see Fig. 1).
The brain was blocked arbitrarily in sagital or coronal planes and removed from the skull. After post-fixation over one night in the 4% paraformaldehyde, the brain was transferred to a 30% sucrose solution for 4-5 days before sectioning. The brain then sectioned at 50 m by using a cryostat. In either sagital or coronal sectioning, every tenth section (i.e., 0.5 mm distance) was collected and mounted on gelatinized slides. Mounted sections were counterstained with cresyl violet for Nissle substance and luxol fast blue for myeline sheath. We used 1 male and 2 females for coronal sections, and also 2 males and 1 female for sagital sections. Left-hemisphere was basically used for all birds.
Drawings were made on every second stained section (i.e., 1 mm distance) throughout the brain from L 1.0 to L 21.0 for the sagital plane and from P 2.0 to A 27.0 for the coronal plane. We used digital images scanned by a photo scanner (GT-X900, EPSON) to outline the major cell nuclei and fiber pathways. Correction of brain size was performed based on the degree of shrinkage which was calculated by comparing overall brain sizes measured on perfused tissue and estimated from section series. Mean size of perfused brain were 30.8 mm along anterio-posterior axis and 19.1 mm along medio-lateral axis. In comparison with these measured values from perfused tissue, less than 8 % shrinkage in size was found in stained sections.
Although the number of samples was small in this study, no significant sex differences was not found in size (AP length, ns, p = 0.72, df = 4, t = -0.38; mean, males, 31.4mm, female, 31.0mm; ML length, ns, p = 0.11, df = 4, t = -2.01; mean, males, 19.4mm, female, 18.8mm), nor in weight of whole brain (ns, p = 0.31, df = 4, t = -1.16; mean, males, 12.8g, female, 11.6g) comprising forebrain, midbrain, cerebellum, and hindbrain. Note that brain size was based on the measurement of left hemisphere. In respect to brain nuclei, there seemed to be no sex difference at the level of cytoarchitecture. It might be noteworthy that both male and female crows have so-called vocal nuclei, referred to as MAN, HVC, X, RA in typical song birds, in the telencephalon. This was also found in other corvid species, Australian magpies (Gymnorhina tibicen; Deng et al., 2001). Comparing size of nucleus and their connections between sexes should be investigated to understand their functional similarity or difference between sexes, which might provide the insight for evolution of vocal nuclei.

Acknowledgements
We would like to acknowledge Tokyo Ueno Zoo for their kind provision of captured birds. Note that the zoo provided the captured birds to us with permission from the environmental office of Tokyo (Permission No.-698). This research was supported by grants from the Japan Society for the Promotion of Science (JSPS) to E-I. Izawa (grant-in-aid for young scientists, #167493) and to S. Watanabe (the 21st Century COE Program, D-1), and also supported by a grant by Keio Gijuku Academic Development Funds to S. Watanabe.



References

Deng, C., Kaplan, G., Rogers, J.L., 2001. Similarity of the song nuclei of male and female Australian magpies (Gymnorhina tibicen). Behav Brain Res 123, 89-102.
Fridolfsson, A.-K., Ellegren, H., 1999. A simple and universal method for molecular sexing of non-ratite birds. J Avian Biol 30, 116-121.
Karten, H., Hodos, W., 1967. A Stereotaxic Atlas of the Brain of the Pigeon (Columbia livia). Johns Hopkins Press, Baltimore.
Kitagawa T., 1980. Four seasons of the Japanese jungle crow (Corvus macrorhynchos). Wild birds 45, 416-421 (in Japanese).
Kuenzel W.J., Masson, M., 1988. A Stereotaxic Atlas of the Brain of the Chick (Gallus domesticus). Johns Hopkins Press, Baltimore.
Stokes, T.M., Leonard, C.M., Nottebohm, F., 1974. The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates. J Comp Neurol 156, 337-374.
Youngren O.M., Phillips, R.E., 1978. A stereotaxic atlas of the brain of the three-day-old domestic chick. J Comp Neurol 181, 567-600.
Crow brain image album