Brain Research Bulletin, Vol. 9, pp. 383-389, 1982.Printed in the U.S.A. Dopaminergic Periglomerular Cells in the Turtle Olfactory Bulb N. HAL&Z,’ M. NOWYCKY, T. HOKFELT, G. M. SHEPHERD, K. MARKEY AND M. GOLDSTEIN Section of Neuroanatomy, Yale University, School of Medicine, New Haven, CT 06510 Department of Histology, Karolinska Institute, 104 01 Stockholm, Sweden and Department of Psychiatry, New York University Medical Center, New York, NY 10016 HAL&Z, N., M. NOWYCKY, T. HGKFELT, G. M. SHEPHERD, K. MARKEY AND M. GOLDSTEIN. Dopa- minergic periglomerular cells in the turtle olfactory zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA bulb. BRAIN RES. BULL. 9(1-6) 383-389, 1982.-With the indirect immunofluorescence technique using antisera to three catecholamine synthesizing enzymes, labeled periglomerular cells as well as their intraglomerular processes were observed in the turtle olfactory bulb. These cells could also be recognized in the EPL and the glomerular layer. Unlabeled periglomerular cells were also seen. Thick labeled processes (presumably dendrites) entered the glomerular neuropil, and there formed a dense network, with numerous terminal varicosities. These results support the existence of a unique, homologous dopaminergic subdivision of the periglomerular intemeurons throughout classes of vertebrates. In addition, a second type of weakly tyrosine hydroxylase immunoreactive neurons was observed in the outer part of the granule layer. Dopamine P-hydroxylase positive fibers were seen in the granule, mitral and external plexiform layers. Olfactory bulb Turtle Dopaminergic cells Immunohistochemistry Autoradiography STUDIES in the isolated turtle olfactory bulb have shown that olfactory nerve and lateral olfactory tract stimulation elicit a characteristic series of field potential responses [2,3]. Recently we have shown that these responses are re- producably modified by the addition of exogenous dopamine (DA) to the bath 1171. In earlier studies, periglomerular cate- cholamine (CA) cells were demonstrated in the mammalian [5] and amphibian 131 olfactory bulb, and it was subsequently shown by immunohistochemical techniques that in the rat, these cells are dopaminergic [lo]. We therefore initiated a series of histochemical experiments to determine if a similar neurochemical system is reponsible for the observed physi- ological effects of dopamine in the turtle olfactory bulb. Turtle olfactory bulbs were processed for fluorescence immunohistochemical visualization [4] of three CA synthe- sizing enzymes [l l] as well as for autoradiography of tritiated dopamine (3H-DA). In this preliminary report, labeled periglomerular cells as well as a second previously unidentified cell type will be described briefly and discussed. A more detailed account, including the physiological effects of DA on field potentials, is in preparation and will be pub- lished elsewhere. METHOD Immunohistochemistry Eight turtles (Pseudemys scripta, 6-8 inches long) were processed for the indirect immunofluorescence technique. Some animals were pretreated with colchicine (Sigma) to prevent intracellular transport mechanisms and in this way increase enzyme levels in the cell somata. Animals were anesthetized with Diabutal (0.3-1.5 ml of a 60 mg/ml solu- tion) and a burr hole was drilled above each olfactory bulb. Five to 10 ~1 of a 1% colchicine solution were delivered into each olfactory bulb ventricle via stereotaxically lowered Hamilton syringe (delivery rate of 0.1 pl/min). The success of the placement of the needle was determined after perfu- sion. Perfusion of colchicine-injected and non-injected turtles was performed under Diabutal anesthesia. Plastrons of anesthetized animals were removed, the heart opened and a perfusion cannula introduced into the common carotid- brachial artery through the heart. Turtles were perfused with 50 ml of Ca-free Tyrode solution (pH 7.4) followed by 30 min perfusion with 250-400 ml of 4% parafonnaldehyde dis- solved in Sorensen buffer. The perfusion was carried out with the turtle lying on ice and both solutions were chilled. The brains were then dissected out, and the dura and arachnoid membranes were removed. Following isolation of the bulbs, an additional postfixation was carried out by let- ting the brains sit in the same futative for a total time of two hours from the initiation of the perfusion. The dissected pieces were then rinsed and stored in Sorensen-sucrose (5%) solution at 4°C and then sent to Stockholm for immunohis- tochemical analysis. Present address: Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary. *Send reprint requests to Dr. G. M. Shepherd, Section of Neuroanatomy, Yale University, School of Medicine, New Haven, CT 06510. Copyright 0 1982 ANKHO International Inc.-0361-9230/82/070383-07$03.00/O