Opossum Brain Research Articles

A three-dimensional stereotaxic atlas of the gray short-tailed opossum (Monodelphis domestica) brain

The gray short-tailed opossum (Monodelphis domestica) is a small marsupial gaining recognition as a laboratory animal in biomedical research. Despite numerous studies on opossum neuroanatomy, a consistent and comprehensive neuroanatomical reference for this species is still missing. Here we present the first three-dimensional, multimodal atlas of the Monodelphis opossum brain. It is based on four complementary imaging modalities: high resolution ex vivo magnetic resonance images, micro-computed tomography scans of the cranium, images of the face of the cutting block, and series of sections stained with the Nissl method and for myelinated fibers. Individual imaging modalities were reconstructed into a three-dimensional form and then registered to the MR image by means of affine and deformable registration routines. Based on a superimposition of the 3D images, 113 anatomical structures were demarcated and the volumes of individual regions were measured. The stereotaxic coordinate system was defined using a set of cranial landmarks: interaural line, bregma, and lambda, which allows for easy expression of any location within the brain with respect to the skull. The atlas is released under the Creative Commons license and available through various digital atlasing web services.

Paternal X inactivation does not correlate with X chromosome evolutionary strata in marsupials.

BackgroundX chromosome inactivation is the transcriptional silencing of one X chromosome in the somatic cells of female mammals. In eutherian mammals (e.g. humans) one of the two X chromosomes is randomly chosen for silencing, with about 15% (usually in younger evolutionary strata of the X chromosome) of genes escaping this silencing. In contrast, in the distantly related marsupial mammals the paternally derived X is silenced, although not as completely as the eutherian X. A chromosome wide examination of X inactivation, using RNA-seq, was recently undertaken in grey short-tailed opossum (Monodelphis domestica) brain and extraembryonic tissues. However, no such study has been conduced in Australian marsupials, which diverged from their American cousins ~80 million years ago, leaving a large gap in our understanding of marsupial X inactivation.ResultsWe used RNA-seq data from blood or liver of a family (mother, father and daughter) of tammar wallabies (Macropus eugenii), which in conjunction with available genome sequence from the mother and father, permitted genotyping of 42 expressed heterozygous SNPs on the daughter’s X. These 42 SNPs represented 34 X loci, of which 68% (23 of the 34) were confirmed as inactivated on the paternally derived X in the daughter’s liver; the remaining 11 X loci escaped inactivation. Seven of the wallaby loci sampled were part of the old X evolutionary stratum, of which three escaped inactivation. Three loci were classified as part of the newer X stratum, of which two escaped inactivation. A meta-analysis of previously published opossum X inactivation data revealed that 5 of 52 genes in the old X stratum escaped inactivation.ConclusionsWe demonstrate that chromosome wide inactivation of the paternal X is common to an Australian marsupial representative, but that there is more escape from inactivation than reported for opossum (32% v 14%). We also provide evidence that, unlike the human X chromosome, the location of loci within the oldest evolutionary stratum on the marsupial X does not correlate with their probability of escape from inactivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0267-z) contains supplementary material, which is available to authorized users.

Distribution and function of TrkB receptors in the developing brain of the opossum Monodelphis domestica

The expression, development pattern, spatiotemporal distribution, and function of TrkB receptors were investigated during the postnatal brain development of the opossum. Full-length TrkB receptor expression was detectable in the newborn opossum, whereas three different short forms that are expressed in the adult brain were almost undetectable in the newborn opossum brain. The highest level of full-length TrkB receptor expression was observed at P35, which corresponds to the time of eye opening. We found that in different brain structures, TrkB receptors were localized in various compartments of cells. The hypothalamus was distinguished by the presence of TrkB receptors not only in cell bodies but also in the neuropil. Double immunofluroscent staining for TrkB and a marker for the identification of the cell phenotype in several brain regions such as the olfactory bulb, hippocampus, thalamus, and cerebellum showed that unlike in eutherians, in the opossum, TrkB receptors were predominantly expressed in neurons. A lack of TrkB receptors in glial cells, particularly astrocytes and oligodendrocytes, provides evidence that TrkB receptors can play a functionally different role in marsupials than in eutherians. The effects of TrkB signaling on the development of cortical progenitor cells were examined in vitro using shRNAs. Blockade of the endogenous TrkB receptor expression induced a decrease in the number of progenitor cells proliferation, whereas the number of apoptotic progenitor cells increased. These changes were statistically significant but relatively small. In contrast, TrkB signaling was strongly involved in regulation of the cortical progenitor cell differentiation process.

Expression of TrkC receptors in the developing brain of the Monodelphis opossum and its effect on the development of cortical cells.

In this study, we investigated the distribution, localization and several various functions of TrkC receptors during development of the Monodelphis opossum brain. Western blotting analysis showed that two different forms of the TrkC receptor, the full-length receptor and one of its truncated forms, are abundantly expressed in the opossum brain. The expression of TrkC receptors was barely detected in the brain of newborn opossums. At postnatal day (P) 3, the expression of full-length TrkC remained at low levels, while moderate expression of the TrkC truncated form was detected. The expression levels of both forms of this protein gradually increased throughout development, peaking at P35. We found that in different neocortical areas located both at the rostral and caudal regions of the cortex, up to 98% of BrdU-labeled cells forming cortical layers (II-VI) had prominently expressed TrkC. To assess which developmental processes of cortical cells are regulated by TrkC receptors, three different shRNAs were constructed. The shRNAs were individually tested in transfected cortical progenitor cells grown on culture plates for 2 days. The effects of the shRNA-TrkC constructs were similar: blockade of TrkC receptors decreased the number of Ki67-positive and apoptotic cells, and it did not change the number of TUJ-positive neurons in vitro. Thus, the lack of TrkC receptors in cultured progenitor cells provided insight on the potential role of these receptors in the regulation of proliferation and cell survival but not in the differentiation of cortical cells.

Differential changes in the cellular composition of the developing marsupial brain.

Throughout development both the body and the brain change at remarkable rates. Specifically, the number of cells in the brain undergoes dramatic nonlinear changes, first exponentially increasing in cell number and then decreasing in cell number. Different cell types, such as neurons and glia, undergo these changes at different stages of development. The current investigation used the isotropic fractionator method to examine the changes in cellular composition at multiple developmental milestones in the short-tailed opossum, Monodelphis domestica. Here we report several novel findings concerning marsupial brain development and organization. First, during the later stages of neurogenesis (P18), neurons make up most of the cells in the neocortex, although the total number of neurons remains the same throughout the life span. In contrast, in the subcortical regions, the number of neurons decreases dramatically after P18, and a converse relationship is observed for nonneuronal cells. In the cerebellum, the total number of cells gradually increases until P180 and then remains constant, and then the number of neurons is consistent across the developmental ages examined. For the three major structures examined, neuronal density and the percentage of neurons within a structure are highest during neurogenesis and then decrease after this point. Finally, the total number of neurons in the opossum brain is relatively low compared with other small-brained mammals such as mice. The relatively low number of neurons and correspondingly high number of nonneurons suggests that in the marsupial brain nonneurons may play a significant role in signal processing.

Multimodal stereotactic template of the gray short-tailed opossum's brain

Multimodal stereotactic template of the gray short-tailed opossum's brain

Deformable coregistration of multimodal imaging data of gray short-tailed opossum brain

Frontiers Events is a rapidly growing calendar management system dedicated to the scheduling of academic events. This includes announcements and invitations, participant listings and search functionality, abstract handling and publication, related events and post-event exchanges. Whether an organizer or participant, make your event a Frontiers Event!

Architectonic subdivisions of the amygdalar complex of a primitive marsupial ( Didelphis aurita)

The architecture of the amygdaloid complex of a marsupial, the opossum Didelphis aurita, was analyzed using classical stains like Nissl staining and myelin (Gallyas) staining, and enzyme histochemistry for acetylcholinesterase and NADPH-diaphorase. Most of the subdivisions of the amygdaloid complex described in eutherian mammals were identified in the opossum brain. NADPH-diaphorase revealed reactivity in the neuropil of nearly all amygdaloid subdivisions with different intensities, allowing the identification of the medial and lateral subdivisions of the cortical posterior nucleus and the lateral subdivision of the lateral nucleus. The lateral, central, basolateral and basomedial nuclei exhibited acetylcholinesterase positivity, which provided a useful chemoarchitectural criterion for the identification of the anterior basolateral nucleus. Myelin stain allowed the identification of the medial subdivision of the lateral nucleus, and resulted in intense staining of the medial subdivisions of the central nucleus. The medial, posterior, and cortical nuclei, as well as the amygdalopiriform area did not exhibit positivity for myelin staining. On the basis of cyto- and chemoarchitectural criteria, the present study highlights that the opossum amygdaloid complex shares similarities with that of other species, thus supporting the idea that the organization of the amygdala is part of a basic plan conserved through mammalian evolution.

Development of the facial and hypoglossal motor nuclei in the neonatal Brazilian opossum brain

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.

Autoradiographic localization of arginine vasopressin binding sites in the brain of adult and developing Brazilian opossums.

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.

The regional distribution of neuronal glycogen in the opossum brain, with special reference to hypothalamic systems.

Neurons that accumulate glycogen have been identified in the opossum brain stem and diencephalon by a modified histochemical method using alcoholic solutions and fuchsin proper (pararosanilin) rather than the Schiff reagent (leucosulphite derivative). Several of the glycogen-positive cell groups such as the mesencephalic trigeminal nucleus and the brainstem somatic and special visceral efferent nuclei have been previously detected in the developing brain of small, common laboratory mammals. Scattered glycogen-containing neurons also appear in the dorsal thalamus and basal forebrain. A conspicuous, often Golgi-like accumulation of glycogen has been found in neurons of the magnocellular and parvocellular hypothalamic systems. Together with available data on the metabolic rate of marsupials, our results suggest that the patterns of glycogen deposition may be common to several vertebrates and may be a constant although not exclusive property of cells with axonal endings outside the blood-brain barrier.

Ontogeny of oxytocin-like immunoreactivity in the Brazilian opossum brain

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 born 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.

Androgen receptor-like immunoreactivity in the Brazilian opossum brain and pituitary: distribution and effects of castration and testosterone replacement in the adult male

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 immunuhistochemistry 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 immunohistochemscal 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.

Ontogeny of arginine vasopressin-like immunoreactivity in the Brazilian opossum brain

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 bom 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.

CNPase expression in the developing opossum brain stem and cerebellum.

Immunoreactivity to 2',3' cyclic nucleotide 3' phosphohydrolase (CNPase; EC 3.1.4.37) was studied in the developing opossum brain stem and cerebellum. Regional differences were found in oligodendrocytes concerning the time of appearance (early: medical longitudinal fascicle [mlf]; intermediate: inferior colliculus [IC], deep layers of the superior colliculus [SC] and white matter of cerebellar folia; late: optic layer of SC) and duration of immunoreactivity (short: optic layer of SC; intermediate: mlf; long: cerebellar folia, etc). The results suggest that regional heterogeneities in CNPase expression are linked to intrinsic properties of local and afferent axons.

Developmental distribution of GFAP and vimentin in the Brazilian opossum brain.

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.

Localization of cholecystokinin binding sites in the adult and developing Brazilian opossum brain.

Cholecystokinin (CCK) is now recognized as one of the most abundant peptides in the mammalian central nervous system. We have previously used immunohistochemistry to localize CCK in the adult and developing Brazilian opossum brain. However, little is known about the distribution of CCK binding sites in the developing mammalian brain. Therefore, to further our knowledge of the sites of action for CCK during development, we initiated a series of studies to localize CCK binding sites in the adult and developing Brazilian opossum. This species was chosen because pups are born in a fetus-like state. Receptor autoradiography was performed on coronally sectioned brains of 1 to 60 day postnatal (PN) animals and adults with 125I-Bolton Hunter-CCK-8 as the radioligand. Binding is evident in the 1PN opossum brainstem and is observed in the developing forebrain by 5PN. Region-specific binding increases during development, and binding in the 35PN brain resembles the adult pattern. Binding is evident prior to the detection of CCK-like immunoreactivity in many areas. The facial motor nucleus is identifiable and exhibits high levels of binding in Brazilian opossum pups of 10 to 35 days of age. However, binding is undetectable in the facial motor nucleus of 45 and 60PN pups. In general, the binding patterns for CCK in the adult opossum resemble those of other mammals and likely mediate similar physiological functions. However, some cholecystokininergic pathways appear to be unique to neonatal mammals.

Neural aromatase activity in a marsupial, the gray short-tailed opossum ( Monodelphis domestica): ontogeny during postnatal development and androgen regulation in adulthood

Neural aromatase activity (AA) was measured in gray short-tailed opossums ( Monodelphis domestica) on the day of birth and at selected ages through adulthood. In adulthood, regulation by testicular androgens was examined. In mixed-sex samples of whole brain, AA was present at birth and increased until postnatal day (PD) 16. In hypothalamus-preoptic area (HPOA), significantly higher levels of AA were seen in animals on PD 16 than on PD 30 and PD 30 animals had higher levels of AA than all older ages including adults. Significant sex differences in HPOA AA with male levels higher, were seen only on PD 16 and in adulthood. While lower overall than in HPOA, AA was present also in cerebral cortex (CX). In CX, AA was higher on PDs 16 and 30 than at older ages. Significant sex differences in CX AA were observed only in adulthood. One week following castration in adulthood, AA dropped significantly in CX but not in HPOA. These findings are compared with those obtained from other marsupial and eutherian mammals with reference to the possible significance of AA in sexual differentiation of the gray opossum brain.

Differential down-regulation of estrogen receptor-like immunoreactivity by estradiol in the female Brazilian opossum brain

The effects of estrus and prolonged estradiol benzoate (EB) treatment on estrogen receptor-like immunoreactivity (ER-LI) were studied in the female Brazilian opossum ( Monodelphis domestica) brain. In general, both estrus animals and EB treated animals had reduced ER-LI in specific brain regions when compared to ovariectomized controls. These results support a down-regulatory role for estrogen in relation to its receptor in the brain, and indicate that ER-LI is differentially regulated in the Monodelphis forebrain.

Purification and characterization of fibroblast growth factors from the opossum, Monodelphis domestica, brain.

An extract from the brain of the opossum Monodelphis domestica was fractionated by heparin affinity chromatography. A major peak of mitogenic activity (heparin binding growth factor 2, HBGF-2) eluted from heparin-Sepharose between 1.7 and 2.0 M NaCl. Antisera specific for bovine bFGF detected four polypeptides of 17.5-23 kDa in opossum brain HBGF-2 preparations. Opossum brain heparin binding growth factor 1 (HBGF-1), a minor peak of activity, eluted from heparin-Sepharose at 1.1 NaCl and contained a 16.2 kDa protein that cross-reacted with antiserum against bovine aFGF.