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We have used bromodeoxyuridine (BrdU) single and BrdU-arginine vasopressinoxytocin (BrdU-AVP-OT) double and triple label immunohistochemistry to characterize postnatal neurogenesis of the supraoptic and paraventricular nuclei in the Brazilian opossum. Developing pups received a single injection of BrdU between days 1 and 11 postnatally. All brains were collected on day 60 of postnatal life (60 PN). Single label BrdU immunohistochemistry revealed that an injection at 1 PN resulted in heavy labelling in the hypothalamus including the area of the paraventricular nucleus, whereas only approximately one third of the cells in the supraoptic nucleus were labelled. Analysis of data indicated that neurogenesis of the supraoptic and paraventricular nuclei is completed by days 5 and 7 PN, respectively. Double and triple label immunohistochemistry demonstrated that following BrdU injection on day 1 or 2 PN, few of the AVP and OT secreting cells in the supraoptic nucleus were double labelled with either peptide and BrdU, and no double labelled cells were seen following BrdU injection on day 5 PN. Similarly, in the paraventricular nucleus most of the AVP and OT secreting magnocellular cells were not double labelled with either peptide and BrdU. Whereas several double labelled cells were observed in the parvicellular 13 part following BrdU injection on day 1 or 2 PN. No double labelled cells were present in any component of the paraventricular nucleus following injection on day 7 PN or later. These results indicate that the majority of the AVP and OT secreting magnocellular neurons are bom prenatally and the OT and AVP parvicellular group of neurons are bom during postnatal life. Our results also demonstrate that in contrast to that of eutherian rodents such as the rat and mouse, neurogenesis in the opossum hypothalamus continues into the postnatal period and provides a unique opportunity to study the neuroanatomical development of diverse regions such as the paraventricular nucleus. Introduction The paraventricular and supraoptic nuclei (PVN, SON respectively) are positioned in the anterior hypothalamus and constitute the hypothalamo-neurohypophyseal system. The PVN consists of magnocellular and parvicellular groups of neurons, whereas the SON is composed of magnocellular neurons^'. The magnocellular neurons of the PVN and SON project to the neurohypophysis and median eminence, while the parvicellular neurons of the PVN project to the brainstem and spinal cord and are involved in the control of autonomic homeostasis 6.48.49,50.51 ^rginine vasopressin (AVP) and oxytocin (OT) are two closely related peptides synthesized and secreted by the PVN and SON. Immunohistochemical studies have demonstrated that many other neuropeptides including galanin, corticotrophin releasing factor, thyrotropin releasing factor, enkephalin, cholecystokinin, and neurotensin are also localized in the parvicellular neurons of the PVN and magnocellular neurons of the PVN and SON in the Due to the presence of a wide variety of neuropeptides in the 14 neurons forming the PVN and SON, and the different functions of these neuronal populations, the time periods of neurogenesis and proliferation of these neurons in relation to their neurochemical phenotype, neurosecretions, and involvement in autonomic functions may differ as compared to that of other hypothalamic neurons. Since the introduction of tritiated thymidine autoradiography several investigators have studied neurogenesis of the central nervous system in multiple mammalian species including the rat and mouse 2.3.4.5.21.23.25.27.32,41,46,55.56 Neurogenesis of the diencephalon of rodents is generally considered a prenatal event. Tritiated thymidine autoradiography has demonstrated that neurogenesis in the hypothalamic SON and PVN, both in the rat and mouse, is completed between day 13 and 15 postfertilization, (eight to ten days prior to birth) 2 3.27,46 Using tritiated thymidine autoradiography the migratory patterns of posonitotic cells have also been documented^. Such studies have shown that neurons forming the PVN and SON originate from the germinal zone of the neuroepithelium of the third ventricle. The cells destined for the SON complete their lateral migration by day 15 postfertilization, while those forming the PVN remain in the periventricular area Although previous autoradiographic studies have given a detailed account of the time of completion of neurogenesis of the diencephalon in the rat, the development of the PVN and SON, and specifically the possible existence of time differences in the formation of magnocellular and parvicellular neurons has not been fully investigated 2.3.23.27.46 purther, neurons that express a specific neurochemical phenotype and have a specific function may have different time periods of formation. In this study, we have used the Brazilian gray short-tailed opossum as an 15 experimental model to study postnatal neurogenesis of the PVN and SON. The Brazilian gray short-tailed opossum, Monodelphis domestica, is a small pouchless marsupial. Brazilian opossum young are bom after 14 days of gestation in an extremely immature and sexually undifferentiated state The absence of the pouch in Monodelphis makes the fetus-like pups very accessible for developmental studies 9.13.14,15.18,19.32.37,41,43.45,54,57 To study postnatal neurogenesis in the paraventricular and supraoptic nuclei, we have utilized bromodeoxyuridine (BrdU) immunohistochemistry. Bromodeoxyuridine is a thymidine analog which has recently become an alternative to tritiated thymidine for the study of developmental events in the central nervous system Following exogenous adminisb'ation, BrdU is incorporated into the DNA during the synthesizing phase (S phase) of the cell cycle. Like tritiated thymidine it remains in the cell permanently and the BrdU containing cells can then be detected by using an antibody directed against BrdU and standard immunohistochemical protocols^". In order to determine: (1) if neurogenesis of the PVN and SON continues during the postnatal period, (2) if neurons of the PVN and SON complete neurogenesis at a different time point as compared to other neurons in the surrounding regions of the brain, (3) if AVP and OT secreting cells have the same birth dates as that of the surrounding neurons in the SON and PVN, and (4) if neurogenesis of magnocellular and parvicellular neurons is distinct, we have utilized BrdU single label, BrdU-AVP double label, and BrdU-OT-AVP triple label immunohistochemistry. 16 Materials and Methods Animals Developing Brazilian opossum pups were obtained from a colony maintained at Iowa State University. The animals were housed individually in plastic cages, maintained at a constant temperature (26'C) and a 14:10 h light-dark cycle. The animals were provided with water and food ad libitum (Reproduction Fox Chow; Milk Specialties Products, Madison,WI). For breeding purposes, male and female opossums were housed together and separated after a two week period. Subsequently the females were checked daily for the presence of pups. The day of birth of the pups was considered as day 1 of postnatal (PN) life. The animals and procedures used in this study were in accordance with those approved by the Iowa State University Conmiittee on animal care. BrdU injection and tissue collection On each of the days between 1 and 11 PN, a different litter of pups was injected with BrdU. Individual pups were injected without separation from their mother. Each pup was given a subcutaneous injection along the dorsal midline of 20 ^1 of BrdU solution (Sigma; 1 mg/ml), dissolved in 0.9% saline. Preliminary results demonstrated that this method is quite effective in delivering BrdU throughout the brain^". All animals in each litter were weighed, examined for presence of any abnormalities, and killed at 60 PN by etherization. At that time, the animals were perfused transcardially with 0.9% saline followed by Zamboni's fixative solution for 10 minutes as has been reported previously in the opossum . Brains were removed from the calvaria and postfixed for 48 hours at room temperature 17 in the same Hxative solution. Subsequently brains were sunk in 30% sucrose solution, and 20 fim thick coronal sections were cut on a cryostat (Reichert Instruments) at -20*C. Tissue sections were thaw mounted onto poly-L-lysine coated slides, kept on a slide warmer at 37'C overnight, and stored at 4'C until immunohistochemistiy was conducted. Immunohistochemistry The protocol utilized for single label BrdU immunohistochemistry was a modification of that which we have been reported previously for BrdU' and immunohistochemical detection of neuropeptides'" '®. Pretreatment and DNA denaturation The slide mounted tissue sections were rinsed with 50 mM potassium phosphate buffered saline (KPBS), and pretreated with trypsin (0.06% trypsin bovine type III; Sigma, and 0.06% calcium chloride dissolved in 300 ml of KPBS) for 30 minutes at 37*C. After washing for 10 minutes with KPBS, tissue sections were treated with .IN HCl (ice cold) for 10 minutes followed by incubation in 2N HCl at 37'C for 30 minutes. The tissue sections were then neutralized in basic KPBS (pH 8.5) for 10 minutes and followed by routine immunohistochemistry. Single label immunohistochemical staining Tissue sections were rinsed in KPBS, incubated with a 0.3 % hydrogen peroxide solution to remove endogenous peroxidase activity, exposed to normal horse serum as blocking agent 18 (Vector; 1:67), and followed by an overnight incubation in BrdU antisera (1:200, Mouse monoclonal, Dako) at room temperature. After adequate washing, the tissue sections were incubated in horse anti-mouse IgG (Vector; 1:200) for 2 hours at room temperature, rinsed, and reacted with avidin-biotin complex (ABC; Vector Elite Kit, 1:50) at room temperature for an additional hour. After rinsing, the tissue sections were exposed to a substrate composed of 0.04% 3, 3' diaminobenzidi

Characterization of arginine vasopressin and oxytocin containing systems in the brain of the adult and developing Brazilian opossum, Monodelphis domestica