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The development of the facial and hypoglossal motor nuclei were examined in the neonatal Brazilian opossum (Monodelphis domestica), a marsupial in which postnatal central nervous system development has been well characterized. In this study, we utilized postnatal injection of the retrograde tracer cholera toxin subunit B (CtB) to characterize the formation of the facial and hypoglossal motor nuclei in the developing neonatal opossum brainstem. Injections of CtB were made into the cheek/lip region or tongue of opossum pups to retrogradely label the facial or hypoglossal motor nuclei, respectively. Following a 2 h survival time, facial motoneurons in newborn opossum pups (1 PN) exhibited CtB labeling, with their cell bodies localized near the developing cranial abducens nucleus. At 3 and 5 PN, following a 48 h survival time, CtB-labeled facial motoneurons were observed in and migrating to the region of the adult facial motor nucleus in the rostral medulla. Between 7 and 10 PN, almost all facial motoneurons had migrated to their destination within the facial motor nucleus. Hypoglossal motoneurons also exhibited CtB labeling from 1 PN; however, their cell bodies were localized within the hypoglossal motor nucleus at the earliest age examined. Double label studies, to examine guidance of facial motoneurons during migration, demonstrated that CtB-labeled facial motoneurons are in close proximity to vimentin-like immunostained radial glial fibers during migration. These results suggest: (1) migration of facial motoneurons to the facial motor nucleus is a postnatal event, (2) efferent projections from facial and hypoglossal motoneurons project into the peripheral region of their target muscles from the day of birth, and (3) facial motoneurons migrate to their destination in the brainstem thereafter, in close association with radial glial fibers.

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We are utilizing the Brazilian short-tailed opossum, Monodelphis domestica, to study the development of the vasopressinergic system. Earlier studies demonstrated that arginine vasopressin-like immunoreactivity was present very early in the Brazilian opossum brain, suggesting a role for vasopressin in the developing central nervous system of mammals. In this study, we have utilized [3H]arginine vasopressin autoradiography to describe the distribution of arginine vasopressin binding sites in adult and developing Brazilian opossum brains. In general, arginine vasopressin binding patterns in adult opossum brains resembled those of other species. However, we found very few labelled areas in neonatal Brazilian opossum brains. At birth, only the ventral tegmental area and the nucleus of the solitary tract were labelled. Binding was not evident in the forebrain until 25 days of postnatal age. The anterior pituitary was heavily labelled from birth onward, but binding in the brain itself remained at low levels until 35 days postnatal. Heavy binding was observed in only a few areas of the brain in adults, including the dorsal part of the lateral septal nucleus, the suprachiasmatic nucleus, the dorsal and median raphe, the nucleus of the solitary tract, and the caudal part of the spinal trigeminal nucleus. Surprisingly, arginine vasopressin binding sites in the Brazilian opossum appeared much later than arginine vasopressin immunoreactivity and, in many cases, after neurogenesis was complete. These findings suggest that the arginine vasopressin binding sites are not playing a developmental role in opossums, although the peptide is present at an early age.

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In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial, undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins synaptotagmin, Rab3A, synaptophysin and synaptosomal-associated protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively undifferentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and synaptotagmin-, Rab3A-, synaptophysin- and SNAP-25-like immunoreactivities exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibits developmentally regulated changes in its cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.

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The characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei were examined in the Brazilian opossum (Monodelphis domestica). Immunohistochemical markers utilized in this study were the synaptic vesicle-associated proteins synaptophysin and synaptotagmin; a synaptic membrane protein, plasma membrane-associated protein of 25 kDa (SNAP-25); a growth cone protein, growth-associated phosphoprotein-43 (GAP-43); and the microtubule-associated proteins axonal marker tau and dendritic marker microtubule-associated protein-2 (MAP-2). In this study, we have found that, during the first 10 postnatal days (1-10 PN), the facial motor nucleus lacked immunoreactivity for synaptophysin, synaptotagmin, GAP-43, tau, and SNAP-25. After 10 PN, immunoreactivity increased in the facial motor nucleus for synaptophysin, synaptotagmin, GAP-43, and tau, whereas immunoreactivity for SNAP-25 was not evident until between 15 and 25 PN. Conversely, immunoreactivity for MAP-2, was present in the facial motor nucleus from the day of birth. In contrast, the hypoglossal motor nucleus displayed immunoreactivity from 1 PN for synaptophysin, synaptotagmin, SNAP-25, GAP-43, tau, and MAP-2. These results suggest that the facial motor nucleus of the opossum may not receive afferent innervation as defined by classical synaptic markers until 15 PN and, further, that characteristic mature synapses are not present until between 15 and 25 PN. These results indicate that there may be a delay in synaptogenesis in the facial motor nucleus compared to synaptogenetic events in the hypoglossal motor nucleus. Because the facial motor nucleus is active prior to completion of synaptogenesis, we suggest that the facial motoneurons are regulated in a novel or distinct manner during this time period.

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The neuropeptide oxytocin (OT) has been shown to function as a neurotransmitter and/or neuromodulator in addition to its hormonal function in the periphery in the adult central nervous system (CNS). Previously, we have studied the postnatal neurogenesis of the paraventricular and supraoptic nuclei and ontogeny of arginine vasopressin-like immunoreactivity in the Brazilian opossum brain, Monodelphis domestica. In this study, we have described the ontogeny of oxytocin-like immunoreactivity (OT-IR) in the opossum brain. As a marsupial, opossum pups are in an extremely immature state, with neurogenesis and morphogenesis continuing into the second week of postnatal life. Thus, opossum pups are a good model for developmental studies. In the adult opossum brain, OT-IR was localized in regions as reported for the adult rat and other species, except for a few differences. These findings suggest similar functional roles for OT in the adult opossum brain as in other mammals. Unlike the prenatal expression of arginine vasopressin, OT-IR was first detected in the forming median eminence on day 1 of postnatal life (1 PN). Between 3 and 5 PN, OT-IR was present in the hypothalamic supraoptic and paraventricular nuclei and posterior pituitary. At this time, neurogenesis of these nuclei is not completed. By 10 to 15 PN, OT-IR was seen in several brain areas, and begins to resemble that of the adult between 45 and 60 PN. These results indicate that the time course of appearance of the OTnergic system does not directly parallel the early expression of the vasopressinergic system. However, the expression of OT-IR in the opossum brain before neurogenesis and morphogenesis is completed suggests a potential role for OT in developmental events. Similar to arginine vasopressin, oxytocin may also be involved in the regulation of autonomic functions that are essential for the opossum's adaptation to an ex utero environment. Future studies utilizing experimental manipulations of the OTnergic system will help determine the significance of this peptide in the neonatal opossum.

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Androgens are involved in a variety of centrally mediated functions after binding to their intracellular receptors. In the present report, we have employed the androgen receptor antibody, PG-21, and indirect immunohistochemistry to examine the distribution of cells containing androgen receptor-like immunoreactivity (AR-IR) in the intact adult male Brazilian opossum brain and pituitary. Additional adult males were castrated to examine the effects of withdrawal of circulating androgens and testosterone replacement on AR-IR. Immunoblots and immunohistochemical controls demonstrated that the androgen receptor in the opossum brain and peripheral tissues are of a similar molecular mass as to has been reported for the rat. Cells containing AR-IR were widely distributed throughout the brain of intact adult males. The highest number of immunoreactive cells were present in the dorsal and ventral nuclei of the lateral septum, medial division of the bed nucleus of the stria terminalis, medial preoptic area, median preoptic nucleus, nucleus of the lateral olfactory tubercle, central amygdaloid nucleus, anterior cortical amygdaloid nucleus, posterior amygdaloid nucleus, subiculum, ventromedial hypothalamic nucleus, arcuate-median eminence region, and ventral premammillary nucleus. The anterior pituitary gland also contained a high number of cells containing AR-IR. The general distribution of AR-IR both in the brain and anterior pituitary gland resembled that reported for other mammalian species. Castration of the adult males four days prior to perfusion eliminated androgen receptor immunostaining throughout the brain except for a few lightly immunostained cells in the ventral nucleus of the lateral septum and stria terminalis. Androgen receptor immunostaining was decreased in the anterior pituitary gland following castration and became cytoplasmic. Testosterone administration 2 h before perfusion restored AR-IR both in the brain and anterior pituitary gland. These data suggested that immunohistochemical detection of bound (nuclear) androgen receptors as seen with PG-21 antibody in the brain and anterior pituitary gland of the opossum is dependent upon circulating androgens. Further, the wide distribution and similarity in localization of androgen receptors in the opossum brain and anterior pituitary gland to that of other species suggests that androgen receptors might be involved in similar functions in the opossum as has been reported for other species.

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The neuropeptide arginine vasopressin is involved in many centrally mediated functions and brain development. In this study, we have examined the ontogeny of arginine vasopressin-like immunoreactivity (AVP-IR) in the Brazilian opossum (Monodelphis domestica) brain to further understand the involvement of AVP in the forming central nervous system. Monodelphis is a small pouchless marsupial and its pups are born in an extremely immature state before neurogenesis is completed. In the adult brain, cell bodies containing AVP-IR were found in several nuclear groups and areas, and immunoreactive fibers were found to be widely distributed throughout the brain. The distribution of AVP-IR in the adult opossum brain generally resembled that reported for other species including the rat, however, some differences in localization of immunoreactive cells were observed. In the developing opossum brain, AVP-IR was first seen in the mesencephalon and diencephalon between embryonic days 12 and 13. Subsequently, a distinct group of AVP immunoreactive cells was present in the forming supraoptic nucleus on day 1 of postnatal life (1 PN) and at 3 PN in the paraventricular nucleus. Between 1 and 3 PN, a few cells transiently expressed AVP-IR in the forming thalamus and tegmental area. At these ages a few immunoreactive fibers were also detected in the forming cerebellum. These fibers were not seen at later ages in these areas. By 5 PN, an increased expression of AVP-IR was seen in the forming supraoptic and paraventricular hypothalamic nuclei, median eminence, and posterior pituitary. At 7 PN, immunoreactive cells and fibers were seen in several forebrain areas. The distribution pattern of AVP-IR became adult-like by 60 PN. A sex difference in the amount of AVP-IR in the lateral septum was also observed in the opossum brain at 60 PN. This difference persisted in the adult brain. Due to the early presence of AVP-IR in the Monodelphis brain before neurogenesis and morphogenesis is completed, we suggest that AVP may be involved in morphogenesis of the central nervous system. In addition, AVP may have a significant physiological function in regard to homeostasis before the forebrain contributes to these control mechanisms. Further studies, including physiological and developmental manipulations, will define the significance of the early presence of AVP during the differentiation and maturation of the central nervous system in Monodelphis.

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We have used bromodeoxyuridine (BrdU) single and BrdU-arginine vasopressin-oxytocin (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 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 born prenatally and the OT and AVP parvicellular group of neurons are born 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.

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Cells of glial origin are involved in the morphogenesis of the mammalian central nervous system (CNS). Characterization of glial-associated proteins during neurogenesis and differentiation may aid in understanding the complexity of CNS development. We have utilized immunoblotting and immunohistochemistry to characterize the developmental profiles of glial fibrillary acidic protein (GFAP) and vimentin (VIM) in the brain of the Brazilian opossum, Monodelphis domestica. Typical of marsupials, CNS morphogenesis and neurogenesis in the opossum extend well into the postnatal period. Opossum GFAP and VIM were found as single bands at molecular weights consistent with those reported for other species, thus indicating conservation of the VIM and GFAP proteins through mammalian evolution. Differential developmental trends were observed for both proteins with relative VIM levels decreasing and GFAP levels increasing with age. Vimentin-like immunoreactivity (VIM-IR) was present at day 1 of postnatal life throughout the brain. The density of VIM-IR was maximal at 10 and 15 days postnatal (especially in radial glial elements) and decreased slightly by 25 days postnatal. In the adult brain, VIM-IR was markedly reduced compared to that of younger ages. In contrast, GFAP-like immunoreactivity (GFAP-IR) in the brain of Monodelphis increased dramatically with age. No GFAP-IR was observed in the 1 and 5 day postnatal brains. By 25 days postnatal, the pattern of GFAP-IR in the brainstem resembled that of the adult. In the forebrain, more GFAP-IR was present than at younger ages. The adult distribution of GFAP-IR was very similar to that reported for other mammalian species. These results indicate that GFAP and VIM are reciprocally related during periods of morphogenesis and differentiation of the opossum brain.

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