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ABSTRACT. Substance P-immunoreactive neurons projecting from the midbrain to the spinal cord of the chicken were examined by the use of the retrograde tract-tracing method combined with immunohistochemical techniques. Many small neurons were densely clustered in the rostral midline area of the midbrain (RMA), and showed substance P-immunoreactivity. These substance P-immunoreactive neurons sent axons to the intermediomedial cell column (avian autonomic preganglion) and its vicinity in the lumbar spinal segments. On the basis of the strong neuroanatomical analogy in the cytoarchitectural features, immunoreactivity, and fiber connections, the RMA was assumed to be the avian homologue of the anteromedian nucleus in the mammalian midbrain.KEY WORDS: autonomic nervous system, bird, substance P.J. Vet. Med. Sci. 62(8): 909-911, 2000 In the course of our study on substance P (SP)-immunoreactive (SP-IR) neurons in the central nervous system of the chicken, we found that many SP-IR neurons were clustered in the midline area of the rostral midbrain [9]. Cytoarchitecturally, this neuron cluster was identified as a clearly isolated cell group comprised of small neurons deeply stained with cresylviolet (outlined area on the right side of the section in Fig. 1A). We call this neuron cluster the rostral midline area of the midbrain (RMA) tentatively [9]. On the other hand, it has been known that a small neuron group called the anteromedian nucleus (AM) exists in the midline area of the mammalian midbrain. This neuron group in mammals also contains many SP-IR neurons [10,11], and these neurons send their axons to the spinal cord [14]. In this paper, we attempted to reason about the homology between the avian RMA and the mammalian AM on the basis of the neuroanatomical analogy in these small areas. Kuenzel and Masson's atlas [3] was used for the cytoarchitectural definition of the avian midbrain.In the first experiment, we examined the cytoarchitectural extent of the RMA and its immunoreactivity. Three chickens were injected with 2.4% colchicine (10 µl/kg). At 24 hr after injection, they were sacrificed by perfusion of 3% paraformaldehyde-15% saturated picric acid. Transverse cryosections (30 µm in thickness) of the midbrain were treated by the peroxidase-antiperoxidase method as follows: 1) 0.3% hydrogen peroxide in 0.01 M phosphate buffer containing 0.75% saline and 0.2% Triton X-100 (PBS-T, pH 7.2; 30 min), 2) 2.5% normal goat serum in PBS-T (30 min), 3) rabbit SP-antiserum in PBS-T (1:2000, Genosys Biotechnologies, Cambridge, UK; 48 hr, 4°C), 4) goat anti-rabbit IgG in PBS-T (1:100; 45 min, 4°C), 5) rabbit peroxidase-antiperoxidase complex in PBS-T (1:200, Polyscience, Los Angeles, U.S.A.; 60 min). Finally, the sections were treated with 0.05% 3,3'-diaminobenzidine (DAB)-0.003% H 2 O 2 in 0.05 M Tris-HCl buffer (pH 7.4; 5-10 min).In the second experiment, 12 chickens (male White Leghorn, weighing 1.8-2.8 kg) were used under the sodium pentobarbital anesthesia (20 mg/kg) to examine whether or not immunoreactive RMA neurons...

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We attempted some methods to solve the technical problems of anesthetization, perfusion of fixative, and stereotaxic adjustment in chickens.1) An intermittent application of a diluted Nembutal solution through the fixed vein catheter permits a long time operation (5-6hrs). 2) We recommend the researchers to make an invariable weight of chickens and to prepare a brain map appropriate to those chickens and using stereotaxic apparatus.3) Merits and demerits in three methods of the perfusion were evaluated.

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