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Marsupials belonging to the Didelphis genus are widely distributed in the American Continent, and Didelphis albiventris and Didelphis aurita, are common in all of their areas of distribution in Brazil. Here we describe the bacterial and viral diversity of samples from opossums captured in three forest fragments in the State of São Paulo, Brazil. Microbiomes from the same body site were more similar across species and sampling sites while oral swabs presented higher bacterial diversity than rectal swabs. We also identified sequences related to bacterial species involved in zoonotic diseases. The detection of pathogens in such abundant mammal species warns for the possibility of emergence in other species. [ABSTRACT FROM AUTHOR]

The opossum (Monodelphis domestica), with its sequenced genome, ease of laboratory care and experimental manipulation, and unique biology, is the most used laboratory marsupial. Using the mammalian methylation array, we generated DNA methylation data from n = 100 opossum samples from the ear, liver, and tail. We contrasted postnatal development and later aging effects in the opossum methylome with those in mouse (Mus musculus, C57BL/6 J strain) and other marsupial species such as Tasmanian devil, kangaroos, and wallabies. While the opossum methylome is similar to that of mouse during postnatal development, it is distinct from that shared by other mammals when it comes to the age-related gain of methylation at target sites of polycomb repressive complex 2. Our immunohistochemical staining results provide additional support for the hypothesis that PRC2 activity increases with later aging in mouse tissues but remains constant in opossum tissues. We present several epigenetic clocks for opossums that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (opossum and human). Two dual-species human-opossum pan-tissue clocks accurately measure chronological age and relative age, respectively. The human-opossum epigenetic clocks are expected to provide a significant boost to the attractiveness of opossum as a biological model. Additional epigenetic clocks for Tasmanian devil, red kangaroos and other species of the genus Macropus may aid species conservation efforts. [ABSTRACT FROM AUTHOR]

The order Piroplasmida encompasses tick-borne pathogens of veterinary and medical importance positioned in two main families: Babesiidae and Theileriidae. Even though previous studies carried out in Brazil recorded the occurrence of piroplasmid species circulating in small mammals, 18S RNA gene sequences were only partially sequenced, preventing the assessment of their phylogenetic positioning. The current study aimed to detect and characterize, using morphological, molecular, and bioinformatic approaches, piroplasmids from wild mammals and associated ticks sampled in Central-Western Brazil. Out of 67 Didelphis albiventris sampled, 22 (16.4%) were positive for piroplasmids by PCR. In contrast, none of the 48 small rodents and 14 capybaras (Hydrochoerus hydrochaeris) was PCR-positive. Four Amblyomma dubitatum ticks—one from Rattus rattus, one from H. hydrochaeris, and two from D. albiventris—out of 114 Amblyomma spp. DNA samples were positive for piroplasmids by PCR. The phylogenetic inference performed using the near-complete 18S rRNA gene positioned the putative novel piroplasmid species detected in D. albiventris and associated A. dubitatum ticks near to Babesia sensu lato clade (Western group—cluster III) and distant from the Australian marsupial-associated piroplasms. Phylogenetic inferences based on two additional molecular markers, namely hsp-70 and cox-1, supported the near-complete 18S rRNA gene phylogenetic inference. Finally, the partial 18S rRNA gene sequences detected in ticks from rodents (R. rattus and H. hydrochaeris) showed 97.2–99.4% identity with the Piroplasmida previously detected in a capybara from Brazil, raising evidence that a still uncharacterized piroplasmid species has been identified in the capybara, the largest rodent species from South America. [ABSTRACT FROM AUTHOR]

In mammals, the superior olivary complex (SOC) of the brainstem is composed of nuclei that integrate afferent auditory originating from both ears. Here, the expression of different calcium-binding proteins in subnuclei of the SOC was studied in distantly related mammals, the Mongolian gerbil ( Meriones unguiculatus) and the gray short-tailed opossum ( Monodelphis domestica) to get a better understanding of the basal nuclear organization of the SOC. Combined immunofluorescence labeling of the calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k, and calretinin as well as pan- neuronal markers displayed characteristic distribution patterns highlighting details of neuronal architecture of SOC nuclei. Parvalbumin was found in almost all neurons of SOC nuclei in both species, while calbindin and calretinin were restricted to specific cell types and axonal terminal fields. In both species, calbindin displayed a ubiquitous and mostly selective distribution in neurons of the medial nucleus of trapezoid body (MNTB) including their terminal axonal fields in different SOC targets. In Meriones, calretinin and calbindin showed non-overlapping expression patterns in neuron somata and terminal fields throughout the SOC. In Monodelphis, co-expression of calbindin and calretinin was observed in the MNTB, and hence both CaBPs were also co-localized in terminal fields within the adjacent SOC nuclei. The distribution patterns of CaBPs in both species are discussed with respect to the intrinsic neuronal SOC circuits as part of the auditory brainstem system that underlie the binaural integrative processing of acoustic signals as the basis for localization and discrimination of auditory objects. [ABSTRACT FROM AUTHOR]

A fundamental question in biology is 'how is growth differentially regulated during development to produce organs of particular sizes?' We used a new model system for the study of differential organ growth, the limbs of the opossum ( Monodelphis domestica), to investigate the cellular and molecular basis of differential organ growth in mammals. Opossum forelimbs grow much faster than hindlimbs, making opossum limbs an exceptional system with which to study differential growth. We first used the great differences in opossum forelimb and hindlimb growth to identify cellular processes and molecular signals that underlie differential limb growth. We then used organ culture and pharmacological addition of FGF ligands and inhibitors to test the role of the Fgf/Mitogen-activated protein kinases ( MAPK) signaling pathway in driving these cellular processes. We found that molecular signals from within the limb drive differences in cell proliferation that contribute to the differential growth of the forelimb and hindlimbs of opossums. We also found that alterations in the Fgf/MAPK pathway can generate differences in cell proliferation that mirror those observed between wild-type forelimb and hindlimbs of opossums and that manipulation of Fgf/MAPK signaling affects downstream focal adhesion-extracellular matrix (FA-ECM) and Wnt signaling in opossum limbs. Taken together, these findings suggest that evolutionary changes in the Fgf/MAPK pathway could help drive the observed differences in cell behaviors and growth in opossum forelimb and hindlimbs. [ABSTRACT FROM AUTHOR]

The aim of this study was to provide information of the occurrence of Rickettsia felis in wild mammals from three municipalities in Yucatan, Mexico. The reactivity of rodent serum to Rickettsia antigens was detected in 80.9% (17 of 21) samples using immunofluorescence assay. Polymerase chain reaction identified rickettsial DNA in spleens of 43.5% (10 of 23) rodents and 57.1% (4 of 7) opossums. The identification of the rickettsial DNA was confirmed as R. felis by restriction fragment length polymorphism and DNA sequencing. This study comprises the first report of R. felis detection in wild mammals in Yucatan. [ABSTRACT FROM AUTHOR]

The histophysiology of both brown and yellow paracloacal glands of control animals from wild populations was analyzed in the breeding and non-breeding seasons and in oophorectomized animals. The effect of estrogens on the scent glands of female Metachirus nudicaudatus was investigated. Radioimmunoassay indicated that estradiol levels in the breeding season were high, while those in the non-breeding season and oophorectomized had minute amounts. No apparent change in gland volume was observed in the various animal groups. Light microscopy showed a wide variation in the number of layers of the holocrine secretory epithelium of the major (brown) gland between the breeding (1-7) and non-breeding season (8-19) and in oophorectomized (8-18) animals. The minor (yellow) gland showed the most evident variation: the holocrine epithelium was restricted to the basal layer in the breeding season, but was restored and exhibited up to eight layers in the non-breeding season and in oophorectomized females. The mean cellular area of the secretory holocrine epithelium in the breeding season was smaller than in the non-breeding period and in castrated animals. On the other hand, the mean cellular areas of tubular glands inserted in both yellow and brown glands in the breeding season were larger than those in the non-breeding season and after castration. The results consistently indicate that the glandular volumes of yellow and brown glands do not depend on estrogens, whereas the holocrine secretory epithelium and tubular glands of both are estrogenic modulation. [ABSTRACT FROM AUTHOR]

NKG2D is a major activating receptor of natural killer cells. Its ligands are major histocompatibility complex (MHC) class I-like molecules whose expression is induced by cellular stresses such as infections and tumorigenesis. Humans have two families of NKG2D ligands (NKG2DL): MHC class I-related chains (MIC) encoded in the MHC and UL16-binding proteins (ULBP) encoded outside the MHC. By contrast, mice have only the latter family of ligands; instead, they have non-MHC-encoded MILL molecules that are closely related to MIC, but do not function as NKG2DL. To gain insights into the origin and evolution of MIC, ULBP, and MILL gene families, we conducted comparative genomic analysis of NKG2DL family genes in five mammalian species. In the opossum MHC, we identified a ULBP-like gene adjacent to a previously described MIC-like gene, suggesting that ULBP genes were originally encoded in the MHC. The opossum genome also contained a transcribed MILL-like gene in a region syntenic to the rodent regions encoding MILL molecules. These observations indicate that MIC-, ULBP-, and MILL-like genes emerged before the divergence of placental and marsupial mammals. Comparison of the human, cattle, rat, mouse, and opossum genomes indicates that after emigration from the MHC, ULBP genes underwent extensive duplications in each species. In mice, some of the ULBP genes appear to have been translocated telomerically on the same chromosome, forming a major cluster of existent NKG2DL genes. [ABSTRACT FROM AUTHOR]

Mammalian ALDH3 isozymes participate in peroxidic and fatty aldehyde metabolism, and in anterior eye tissue UV-filtration. BLAT analyses were undertaken of the opossum genome using rat ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2 amino acid sequences. Two predicted opossum ALDH3A1-like genes and an ALDH3A2-like gene were observed on chromosome 2, as well as an ALDH3B-like gene, which showed similar intron–exon boundaries with other mammalian ALDH3-like genes. Opossum ALDH3 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for rat ALDH3A1. Eleven glycine residues were conserved for all of the opossum ALDH3-like sequences examined, including two glycine residues previously located within the stem of the rat ALDH3A1 active site funnel. Phylogeny studies of human, rat, opossum, and chicken ALDH3-like sequences indicated that the common ancestor for ALDH3A- and ALDH3B-like genes predates the appearance of birds during vertebrate evolution. [ABSTRACT FROM AUTHOR]

Evidence is presented for six opossum ALDH1A genes, including four ALDH1A1-like genes on chromosome 6 and ALDH1A2- and ALDH1A3-like genes on chromosome 1. Predicted structures for the opossum aldehyde dehydrogenase (ALDH) subunits and the intron–exon boundaries for opossum ALDH genes showed a high degree of similarity with other mammalian ALDHs. Phylogenetic analyses supported the proposed designation of these opossum class 1 ALDHs as ALDH1A-like, ALDH1A2-like, and ALDH1A3-like and are therefore likely to play important roles in retinal and peroxidic aldehyde metabolism. Alignments of predicted opossum ALDH1A amino acid sequences with sheep ALDH1A1 and rat ALDH1A2 sequences demonstrated conservation of key residues previously shown to participate in catalysis and coenzyme binding. Amino acid substitution rates observed for family 1A ALDHs during vertebrate evolution indicated that ALDH1A2-like genes are evolving slower than ALDH1A1- and ALDH1A3-like genes. It is proposed that the common ancestor for ALDH1A genes predates the appearance of birds during vertebrate evolution. [ABSTRACT FROM AUTHOR]

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Swallowing is an important defense mechanism against reflux esophagitis as it helps clear refluxed gastric contents from the esophagus, while bicarbonate in the saliva acts to neutralize acid. The purpose of the present study was to examine the effect of esophagitis on the deglutition reflex in anesthetized opossums. Animals perfused with either an acidified pepsin solution for 45 min or with 100 mM hydrochloric acid for 45 min on each of three consecutive days exhibited a significantly impaired deglutition reflex in comparison to baseline. Control animals perfused with 0.9% saline showed no impairment. Bilateral cervical vagotomy in animals perfused with acidified pepsin attenuated the impaired deglutition reflex. Taken together, these results suggest that esophagitis causes an impairment in the deglutition reflex that is mediated by vagal afferent pathways. [ABSTRACT FROM AUTHOR]

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the α-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified. [ABSTRACT FROM AUTHOR]

Complete ureteral obstruction, in fetal opossum kidneys has been used as an experimental method to induce tubulointerstitial damage and interstitial fibrosis. However, the molecular events underlying extracellular matrix deposition are currently unknown. Cytokines such as plateletderived growth factor (PDGF) are considered possible participants in these processes. In this study we used a marsupial model of ureteral obstruction to examine the expression of PDGF-A and type I procollagen mRNAs by in situ hybridization. Complete unilateral ureteral obstruction was performed in six animals at midtrimester human equivalent. Obstructed kidneys, as well as the contralateral and age-matched sham kidneys, were harvested at 1, 3, 5, 10, and 20 days post obstruction. Morphological assessment of the obstructed kidneys harvested between 1 and 5 days post obstruction showed mild tubulocystic changes, interstitial fibrosis, and inflammation compared with controls. Kidneys harvested at days 10 and 20 showed moderate tubulointerstitial damage compared with kidneys harvested after 1 and 5 days. PDGF-A mRNA signal of low abundance was detected within renal interstitial cells urothelial cells of the pelvis, and focally within epithelial cells of immature distal convoluted tubules in non-obstructed kidneys. Type I procollagen mRNA expression was spatially co-distributed with PDGF-A-expressing interstitial cells. PDGF-A and type I procollagen signal intensities in obstructed kidneys harvested 10 and 20 days post obstruction were increased several fold compared with controls and kidneys harvested 1-5 days post obstruction. Both PDGF-A and type I procollagen mRNA increases correlated with morphological features of tubulointerstitial damage. Our results suggest that PGDF-A may participate in this form of fetal kidney damage. [ABSTRACT FROM AUTHOR]

Melanosomal metabolism, including both formation and degradation of melanosomes, was studied in the retinal pigmented epithelium (RPE) of the adult opossum. The majority of the observations were made on a transitional zone between the tapetal and non-tapetal RPE, the region where melanosome metabolism was at its highest level. Formation of melanosomes, demonstrated ultrastructurally by the presence of stage-II and -III premelanosomes, was also examined autoradiographically following the incorporation of the melanin precursor, dihydroxyphenylalanine. The autoradiographic evidence indicated that many newly formed melanosomes were rapidly incorporated into complexes. Ultrastructural observations suggested that melanosome complexes were formed by at least two methods, via the fusion of melanosomes with phagosomes derived from outer segments of photoreceptors, or by the sequestration of melanosomes by cisternae. A central finding of this study, supported by both ultrastructural and histochemical data, is that there are specialized cellular regions that vary in melanosomal formation and lysosomal activity. Stage-II premelanosomes were observed only in the basal parts of the RPE cells, whereas stage-III and -IV melanosomes were found primarily in the apical RPE. Both ultrastructural and cytochemical observations indicated that degradation of melanosomes occurs only in the basal RPE. These findings are interpreted in terms of the expression of both tapetal and nontapetal characteristics in transitional cells. Finally, this study illustrates the role of lysosomal enzymes in shaping the pattern of pigmentation, and shows that the association of lysosomal activity with melanosomes depends on the functional state of the melanosome. [ABSTRACT FROM AUTHOR]

Cardiac muscle cells in the right ventricle of the postnatal opposum were studied ultrastructurally, with particular attention to the developmental stage of T-tubule formation. Animals ranging from 5.5cm (day 29 of postnatal life) to 41 cm (young adult) in body length were used. Typical T-tubules were first recognized in a few myocardial cells of the 7.5cm (day 43) opossum. T-tubules increased in number as cardiac muscle cells continued to differentiate until 22cm in body length (about 105 days after birth). At this stage of development most of the T-tubules were completely established. The general mode of differentiation and development of cardiac muscle cells appeared essentially the same as in other mammals. [ABSTRACT FROM AUTHOR]

In the pineal region of the opossum, Didelphis virginiana, two types of cells predominate: 1) pinealocytes, and 2) fibrous astrocytes. Pinealocytes are characterized by the presence of prominent Golgi bodies, numerous clear and dense-cored vesicles, sensory cilia (9+0), vesicle-crowned rods, and condensation of a material that was always associated with the rough endoplasmic reticulum. In addition, two other cell types are occasionally seen. These include 1) neuron-like cells, and 2) darker staining cells of unknown identity. The endoplasmic reticulum of the darker staining cells is typically expanded and filled with an amorphous substance. Although the pineal region is small in size, the present findings suggest that pinealocytes in this species are metabolically active cells displaying a secretory function. Moreover, the presence of sensory cilia (9+0) and vesicle-crowned rods indicates that pinealocytes of the opossum are phylogenetically related to the photoreceptor cells found in the pineal organ of lower vertebrates. [ABSTRACT FROM AUTHOR]

The phylogenetic relationships among Primates (human), Artiodactyla (cow), Cetacea (whale), Carnivora (seal), and Rodentia (mouse and rat) were estimated from the inferred amino acid sequences of the mitochondrial genomes using Marsupialia (opossum), Aves (chicken), and Amphibia ( Xenopus) as an outgroup. The overall evidence of the maximum likelihood analysis suggests that Rodentia is an outgroup to the other four eutherian orders and that Cetacea and Artiodactyla form a clade with Carnivora as a sister taxon irrespective of the assumed model for amino acid substitutions. Although there remains an uncertainty concerning the relation among Artiodactyla, Cetacea, and Carnivora, the existence of a clade formed by these three orders and the outgroup status of Rodentia to the other eutherian orders seems to be firmly established. However, analyses of individual genes do not necessarily conform to this conclusion, and some of the genes reject the putatively correct tree with nearly 5% significance. Although this discrepancy can be due to convergent or parallel evolution in the specific genes, it was pointed out that, even without a particular reason, such a discrepancy can occur in 5% of the cases if the branching among the orders in question occurred within a short period. Due to uncertainty about the assumed model underlying the phylogenetic inference, this can occur even more frequently. This demonstrates the importance of analyzing enough sequences to avoid the danger of concluding an erroneous tree. [ABSTRACT FROM AUTHOR]

The Harderian gland of the North American opossum ( Didelphis virginiana) is large and well developed, despite the absence of a nictitating membrane in the adult of this species. The elongate glands are surrounded by a delicate connective tissue capsule from which thin septae extend, subdividing the gland into numerous lobules. The secretory units of the opossum Harderian gland are drained by a well defined but not extensive intralobular and interlobular duct system. Most of the secretory end pieces consist of tubuloalveolar units with widely dilated lumina filled with secretory product. Numerous intact lipid vesicles suspended within an amorphous material constitute the luminal contents. Cells lining the tubuloalveolar secretory endpieces are usually columnar in shape, and characterized by numerous lipid-containing secretory vesicles and aggregations of polytubular complexes 40-60 nm in diameter. In addition, these cells contain numerous large irregularly shaped mitochondria, whose matrix is of considerable electron density. Intralobular and interlobular ducts are lined by electron-lucent epithelial cells that lack both the lipid-containing vesicles and the large mitochondria, although typical smaller mitochondria are found scattered within the cytoplasm. Both secretory endpieces and ductal elements are invested by an abundance of myoepithelial cells. A second, smaller serous type of secretory unit may occur near the centre of some Harderian gland lobules. In these units secretory tubules and acini are compactly arranged surrounding a narrow lumen. Serous cells are pyramidal in shape and the cytoplasm is characterized by numerous electron-dense secretory granules and scattered profiles of rough endoplasmic reticulum. Basolateral cell membranes show extensive infoldings and intercellular canaliculi are present. The overall size of cells forming the serous secretory units is much less than that comprising the tubuloalveolar secretory endpieces. [ABSTRACT FROM AUTHOR]

The nasal cavities of opossums prior to and shortly after birth were examined by scanning electron microscopy. Numerous morphologically mature olfactory receptor neurons are observed in the dorso-rostralmost extent of the olfactory epithelium positioned adjacent to the opening of the nares in all prenatal stages and newborn animals examined. The remainder of the olfactory epithelium, occupying a more dorso-caudal position within the nasal cavity, is undifferentiated, and lacks morphologically mature receptor neurons. A short transition zone of stratified squamous epithelium lies between the epithelium lining the nares and olfactory epithelium. It forms an abrupt junction with the latter. The remainder of the nasal cavity in this group of animals is lined by a non-ciliated pseudostratified type (undifferentiated respiratory) of epithelium. By the end of the second postnatal week the morphologically mature olfactory epithelium is no longer observed in the vestibular area of the nasal cavity, which is lined by stratified squamous epithelium at this time. Mature receptor neurons are now observed within the olfactory epithelium lining the roof of the nasal cavity and covering the turbinates. The greater part of the nasal cavity is lined by a ciliated respiratory epithelium. It is proposed that the precocious differentiation of mature olfactory receptor neurons within the rostral-most extent of the olfactory epithelium just prior to birth is important in guiding the newborn young to the pouch. [ABSTRACT FROM AUTHOR]

The development of the neocortex of the marsupial Monodelphis domestica has been studied from birth until adulthood. Monodelphis is born after a gestational period of 14 days, a time when the neocortex is still at a two-layered 'embryonic' stage of development, that is equivalent to a 13-14 day rat embryo or 6 week human embryo. The cortical plate does not begin to appear until 3 to 5 days postnatal. Thus the whole of neocortical development is a postnatal phenomenon in this species, as has been previously described in other marsupials. The general pattern of development of the characteristic layers of the immature neocortex and the subsequent development of a six-layered adult neocortex is similar to that found in eutherian species. However there are some differences. The depth of the immature cortical plate when compared to the thickness of the neocortical wall is less than in eutherians and the subplate zone is much deeper in Monodelphis; this transient subplate zone consists of widely spaced rows of cells that are aligned parallel to the cortical surface. Unlike eutherians there appears to be no secondary proliferative zone in the subventricular zone of the dorso-lateral neocortical wall. Maturation of the neocortex is apparent by 45 days postnatal and by 60 days (around the time of weaning) the characteristic six-layered adult neocortex is clearly present. The neuronal marker PGP 9.5 was used to define neuronal populations in the adult brain. The density of neurons in Monodelphis appears to be considerably less than in eutherians such as the rat. The suitability of postnatal Monodelphis for studies of neocortical development is discussed. [ABSTRACT FROM AUTHOR]

We have studied the early development of subcortical projections to presumptive somatic sensory-motor areas of neocortex in the North American opossum Didelphis virginiana. The opossum is born in a very immature state, 12-13 days after conception, and climbs into an external pouch where it is available for experimental manipulation. Using the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, we have obtained evidence that axons from the dorsal raphe and superior central nuclei, the substantia nigra, the locus coeruleus and the parabrachial nuclei reach presumptive somatic sensory-motor areas of neocortex by at least postnatal day (PND) 10. Axons showing serotonin-like immunoreactivity, presumably from the dorsal raphe and/or superior central nuclei, and axons containing tyrosine hydroxylase immunoreactivity, presumably from the substantia nigra and/or locus coeruleus, are present in the same areas at birth or shortly thereafter. Thalamic axons do not grow into comparable areas of neocortex until after PND 10. Such axons reach the subplate region of ventrolateral neocortex first and then proceed dorsomedially; by estimated PD (EPND) 21, they are present in presumptive layers I, V and VI, but they do not innervate an identified layer IV until EPND 48. The developmental sequences suggested by our study are compared with those reported for other species and are discussed in light of their importance in the formation of major sensory and motor circuits. [ABSTRACT FROM AUTHOR]

In the present study, a temporal analysis of the pattern of distribution of serotoninergic fibers and varicosities within the cerebellum of pouch young opossums was carried out. Particular attention was focused on animals ranging in age from postnatal day (PD) 21-PD 72, because there is a transient expression of serotonin immunoreactivity in the cerebellar cortex during that interval. Between PD 1-33, there is a progressive increase in serotoninergic immunoreactivity throughout the cerebellar cortex. After PD 33, there is a decrease in the relative number of immunostained fibers followed by a reorganization into the adult pattern of distribution. A double labeling paradigm, in which horseradish peroxidase, used as a retrograde marker, combined with serotonin immunohistochemistry was employed to localize serotoninergic neurons that project to the developing cerebellum. Initially (PD 9), serotoninergic cells in the medullary reticular formation and dorsolateral pontine tegmentum are double labeled. After PD 77, only neurons in the medullary reticular formation were double labeled. The course taken by serotoninergic axons from the brainstem to the cerebellum also was analyzed. Between PD 1 and PD 42, serotoninergic axons enter the cerebellum via four different routes: 1) the inferior cerebellar peduncle; 2) a pathway located lateral and rostral to the inferior cerebellar peduncle; this bundle of serotonin axons contains immunoreactive fibers that also enter the tectum (this tract is referred to as the tecto-cerebellar bundle in this report); 3) the medial aspect of the superior cerebellar peduncle; and 4) the tela choroidea. After PD 40, the latter two pathways are the primary routes by which serotoninergic fibers enter the cerebellum. The loss of serotoninergic fibers in the first two pathways coincides with the decrease in serotoninergic immunoreactivity seen in the cerebellar cortex described above. In summary, the results suggest that the serotoninergic projection to the opossum's cerebellum is remodelled during development. It is proposed that the serotonin fibers present at early stages of development may play a role in regulating specific events in cerebellar maturation. In contrast, the serotoninergic axons which have a more restricted pattern of distribution later in development, and in the adult, likely modulate neuronal activity within the cerebellum. [ABSTRACT FROM AUTHOR]

The North American opossum does not have a corpus callosum; neocortical commissural axons are contained entirely within the anterior commissure. We have used axonal transport techniques to study the origin and distribution of commissural axons from somatic motor-sensory cortex in developing and adult opossums. Neocortical axons grow into the anterior commissure by postnatal day (PND) 12, the contralateral external capsule by approximately PND 19, the area deep to the contralateral homotypic cortex by approximately PND 26 and the cortex proper by approximately PND 35. Commissural neurons were first demonstrated at about PND 26, when they form a fairly continuous band in the cortical subplate (presumptive layers V-VI). By at least PND 37, commissural neurons are also present in layers II and III, where they form a continuous band, and in layer IV, where they are sparse. In older pouch young and adult opossums the bands of commissural neurons, especially in layers V-VI, are interrupted, and commissural neurons are rare in layer IV. In general, commissural axons in both pouch-young and adult opossums innervate areas containing commissural neurons as well as layer I. In the acallosal opossum as well as in the callosal rat, the development of commissural connections from somatic motor-sensory cortex is characterized by pauses during the growth of axons into the opposite cortex, by a general inside-out-gradient, and by a transition from continuous bands to patchy, radial columns of commissural neurons and axons. This suggests that similar mechanisms govern the formation of commissural connections in the two species. [ABSTRACT FROM AUTHOR]

The development of the cerebellar cortex in the opossum was analyzed in Nissl-stained sections using qualitative and quantitative methods. The young of the opossum are born 12-13 days after conception and mature for approximately 85 days in an external pouch providing an excellent model for embryological studies. Qualitative observations of cerebellar growth were made from birth to postnatal day (PN) 19. At birth the opossum cerebellar anlage can be divided into two layers, a ventricular layer and an intermediate layer; histologically this is comparable to the rat cerebellar anlage at embryonic day 13 (Altman and Bayer 1978) and the human cerebellar anlage prior to the seventh embryonic week (Rakic and Sidman 1970). By PN 3 the cerebellar anlage consists of five layers: the ventricular layer, the ventral intermediate layer, the acellular layer, the dorsal intermediate layer and the marginal layer. The external granular layer begins migrating over the dorsal surface of the cerebellum at PN 5. The immature Purkinje cell layer is first seen at PN 12 and is subsequently arranged as four clusters between PN 12 and PN 22. At PN 19 the opossum cerebellum is comparable to the rat cerebellum at birth (Korneliussen 1968c). A quantitative analysis of cerebellar growth was performed between PN 17 and PN 77 using vermal sections. The area and thickness of each of the cortical layers was determined from five vermal sagittal sections using two methods: a Zeiss Videoplan and a point counting system. The external granular layer increases in area from PN 17 to PN 75, however its maximal width is achieved between PN 19 and PN 33. The persistence of the EGL until after PN 105 suggests that synaptic contacts between granule cell axons and Purkinje cells may continue to form after PN 77 when the Purkinje cell is mature based on Golgi and fine structural features (Laxson and King 1983). Between PN 17 and PN 77 the area of the molecular layer and the area of the internal granular layer increase at a more rapid rate than the other cerebellar layers. The maturation of the cerebellum in the opossum is a lengthy process lasting approximately 77 days in comparison to rodent cerebellar growth which requires about 25 days (Korneliussen 1968c). Also, the entire process of cortical lamination occurs after birth while the opossum is maturing in an external pouch. [ABSTRACT FROM AUTHOR]

Pre- and postsynaptic elements within the developing inferior olive (IO) of both control and experimental opossums were examined via electron microscopy. Electron dense boutons identified di-/mesencephalic, cerebellar and spinal afferents within the IO of 8-71 day old animals, which survived 4-48 hours following either midbrain hemisections or spinal transections. During its initial stage of development ( 3-22 days) the neuropil of the IO is segregated into fields of small diameter neurites or flocculent profiles. Within the fields of flocculent profiles, synaptic interactions are established, which are both infrequent and immature. Although some flocculent profiles are presynaptic, most are postsynaptic and emanate from olivary somata and dendrites. Synaptic contacts also occur with olivary somata, dendritic shafts, spines and dendritic varicosities. Clear round vesicles (crv's; 40 μm) predominate within all boutons, normal ones as well as those which degenerate after di-/mesencephalic, cerebellar and spinal lesions; however, larger (70 μm) dense cored vesicles (dcv's) are occasionally observed within some boutons. Degenerating terminals from all three sources primarily contact flocculent profiles and dendritic shafts. As the opossum matures ( 42 days) dramatic increases occur in the number and complexity of both pre- and postsynaptic elements. Marked variations are observed in the matrix density of dendritic shafts. Although all terminal boutons predominantly contain crv's, the number of dcv's within the population of presynaptic elements increases markedly. Concurently, olivary neurons are profusely studded with spines. Simple dendritic spines and spiny appendages as well as dendritic shafts are the most frequent postsynaptic structures within the principal nucleus (PO). Olivary somata and their spines, however, are postsynaptic to degenerating de-/mesencephalic afferents within the PO. Flocculent profiles, which persist within the accessory nuclei, and dendritic shafts are postsynaptic to degenerating spinal boutons. By 70 days of age synaptic contacts appear more mature and more nearly approximate those seen in the adult (King 1980). Few somatic contacts, opaque dendrites, dendritic varicosities, and flocculent profiles are evident within the PO. Dendritic shafts and spines are the principal postsynaptic structures. Many di-/mesencephalic and cerebellar afferents synapse within maturing synaptic clusters on spines between which a rare gap junction is observed. Other di-/mesencephalic and cerebellar endings in the PO as well as spinal endings in the accessory nuclei are presynaptic to dendritic shafts and spines external to synaptic clusters. This predilection for contacting more specific loci on olivary neurons provides good evidence for synaptic remodeling. As the olivary nuclei develop further, the incidence of gap junctions increases and pleomorphic vesicles appear within boutons. The glial investment of neuronal elements, including synaptic clusters, also becomes more extensive. In conclusion, early di-/mesencephalic, cerebellar and spinal synaptic contacts appear qualitatively uniform in their synaptic features and postsynaptic interactions. As olivary development proceeds, however, the distinguishing synaptic features of the nuclear complex become more apparent. Synaptic remodeling occurs as some midbrain and cerebellar terminals are localized within synaptic clusters. The ultrastructural features characteristic of the adult IO are finally achieved by 80 days of age. [ABSTRACT FROM AUTHOR]

We have employed degeneration techniques to study the ontogeny of major projections to the inferior olivary nucleus in the North American opossum, a species which is born 12 days after conception and which enjoys a protracted development in an external pouch. Subsequent to spinal lesions a small amount of axonal degeneration can be produced within the edge of the olive before subnuclei can be distinguished (7 days after birth, 24 mm, snout-rump length). Degenerating axons are present more deeply within the olive in animals operated 12 days after birth (30 mm, snout-rump length) and by at least day 16 (36 mm, snout-rump length), they are found in all of the regions they occupy in the adult animal. Subsequent to lesions which undercut all descending mesencephalic and diencephalic systems, a small amount of axonal degeneration is found at the dorsolateral edge of the olive by day 7 (23 mm, snout-rump length). Degenerating axons fill more of the olive, particularly caudally, after comparable lesions in older animals and by day 17 (38 mm, snout-rump length), degeneration is present in all of the olivary regions innervated by midbrain and thalamic axons in the adult opossum. There is some evidence that spinal, mesencephalic and diencephalic axons follow a caudal to rostral gradient in their intraolivary growth. Lesions which undercut neurites growing out of the cerebellum produce evidence for cerebello-olivary connections by day 17. Axons from the cerebral cortex reach their olivary targets considerably later than those from either the spinal cord, mesencephalon, diencephalon or cerebellum. It is not until approximately postnatal day 30 (55 mm, snout-rump length) that degenerating axons can be traced into the olive after lesions of the cortical mantle. These data indicate that the inferior olive receives major connections early in development and that there is an orderly sequence to their growth. [ABSTRACT FROM AUTHOR]

Evidence is presented for an early appearance of monoaminergic neurites within the spinal cord of the developing opossum. They are present within the marginal zone before hindlimb movements begin (stage I) and they start to grow into the intermediate zone by the time hindlimb movements are first observed (stage II). Monoaminergic neurites grow first into the dorsolateral intermediate zone and the intermediolateral cell column where they can be found by the beginning of stage II. Shortly thereafter, fluorescent varicosities can be traced into the area dorsal to the central canal presumed to become lamina X. Fluorescent processes extend in to the ventral intermediate zone (ventral horn) somewhat later in development. Monoaminergic axons have grown into all of the areas they occupy in the adult animal, except for laminae I and II, by the time immature hindlimb movements can be altered by cutting all brainstem projections to the lumbosacral cord (stage III). Monoaminergic innervation of laminae I and II is the last to develope, but it is present by the time thoracic transection produces complete spinal shock. [ABSTRACT FROM AUTHOR]

As part of a study on X chromosomes, metaphase cell synchrony and chromosome isolation methods were developed for the opossum ( Didelphis virginiana) kidney epithelial cell line (OK). The cell synchrony yielded large amounts of metaphase cells using a relatively simple method in which a key feature was a calcium- and magnesium-free balanced salt wash. A neutral pH chromosome isolation method was developed for the kidney epithelial cells, because they were somewhat difficult to disrupt fully by other methods. FACS IV flow microfluorometric analysis of OK chromosomes confirms a clear difference between the sizes of opossum X chromosomes and autosomes. [ABSTRACT FROM AUTHOR]

In the present work, we characterize electrophysiologically a commissural subcortical pathway which is related to binocular interactions in the nucleus of the optic tract (NOT) of the opossum. The main role played by the circuit comprising this pathway seems to be in relaying information coming from the ipsilateral eye to the NOT. The strongest evidence comes from experiments in which lidocaine was injected into the NOT and the ensuing effects in the opposite nucleus were monitored under ipsilateral monocular stimulation. It was consistently observed that during action of lidocaine the directional response normally elicited by stimulation of the ipsilateral eye did not take place in the NOT opposite to the silenced nucleus. This effect was reverted in a few minutes after recovery of the injected NOT. The response to stimulation of the contralateral eye, though, was not affected by this procedure. [ABSTRACT FROM AUTHOR]

Binocular visual responses can be recorded in two regions of the superficial layers of the superior colliculus of the opossum. The direct binocular region (DBR) represents the binocular portion of the contralateral hemifield whereas the rostral pole (RP) represents the binocular portion of the ipsilateral hemifield. In the present study single units from both of these regions were tested with binocular and monocular stimulation. Most cells in both regions showed response facilitation when both eyes were simultaneously stimulated and, when tested with different binocular disparities, most cells showed broadly-tuned disparity selectivity. DBR units usually preferred disparities near zero whereas RP units had a wider range of preferred disparities, with a tendency toward positive (crossed) values. This data indicates that the superior colliculus of the opossum could provide a neural substrate for a coarse analysis of depth and also might help control vergence eye movements. The different ranges of disparity selectivity of DBR and RP are consistent with the previously reported monocular receptive-field data and suggest that DBR and RP analyze different depths of the 3-dimensional visual scene. [ABSTRACT FROM AUTHOR]

Single-units were recorded from the nucleus of the optic tract. Most of the units showed excitation in response to random check patterns presented on a tangent screen to the contralateral eye, moving in a temporal to nasal direction and/or inhibition in the opposite direction. The excitatory response to the temporal to nasal movement, observed in most units, was unchanged throughout the range of speeds tested, except for a decrease at the slowest (0.6 deg/s) and fastest (150 deg/s) speeds. On the other hand, the inhibitory responses evoked by a nasal to temporal movement, had a peak between 3 and 16 deg/s which decreased towards both extremes. An average of 45% of the units were influenced by the stimulation of the ipsilateral eye. In one third of them the response was very weak. In the remainder, the mean frequency of spikes in one direction of the horizontal movement was more than twice that in the opposite stimulus direction. In the great majority of these units, stimulation of each eye yielded the same overall pattern of directionality, that is, movement of the stimulus towards the recording side led to excitation and/or movement in the reverse direction led to inhibition. Inhibition was stronger than excitation in most ipsilaterally responding units. Excitatory responses elicited by the ipsilateral eye were always weaker than those by the contralateral but in a few cases the ipsilateral inhibitory component was more prominent than the contralateral one. [ABSTRACT FROM AUTHOR]

Reference axes for the visuotopic study of the opossum's striate cortex were estimated from corresponding binocular response fields using multi-unit recording. These central binocular axes (CBA) were derived from experimental data based on the concept that corresponding receptive fields for each eye should be mostly in register under natural conditions. Vertical reference meridians, orthogonal to these axes, define a contralateral and an ipsilateral field for each eye with respect to the recording site. An ipsilateral field representation was observed for both eyes in the striate cortex at the transition zone with peristriate. Maximal values for the center and border of ipsilateral receptive fields were, respectively, 8 and 20 degrees for the contralateral eye and 6 and 14 degrees for the ipsilateral eye. An equivalent ipsilateral field representation was found in animals that had the anterior commissure cut prior to the recording session. This suggests that the ipsilateral field of both eyes may be represented in the striate cortex via the ipsilateral optic tract. Additionally, it was observed that the region of higher ganglion cell density in the retina shows a flattened distribution and that the CBA intersects the retina at the temporal aspect of this region. [ABSTRACT FROM AUTHOR]

The uniocular visual field representations on the superior colliculus (SC), as estimated from multiunit response field centres about the horizontal meridian, were compared in midpontine pretrigeminal opossums ( Didelphis marsupialis aurita Wied 1826). Recordings from the rostral pole (RP) and its caudal neighbour, the direct binocular region (DBR), as defined by Rocha-Miranda et al. (1978), were distinguished by the histological control. The results showed that while the hemifield contralateral to the recording site was well represented on the DBR by both eyes, the ipsilateral hemifield was generously represented at the RP only by the contralateral eye. At the RP the ipsilateral eye usually conveyed information about the vertical meridian, bringing about an expanded representation of the central visual space. Distinct patterns of representation were also recognized on graphs which relate recording sites along the AP axis of the SC with the azimuths of response field centres. The representation of the vertical reference meridian upon this axis on an oculocentric system was estimated from the DBR data and localized in the RP, at about 500 μm from the rostral end, for the ipsilateral eye (Vo') and in the DBR, at about 800 μm for the other eye (Vo). Similarly, plots of the magnification factor against the AP collicular axis indicated different strategies of representation for each eye. At the segment between 500 and 800 μm on this axis the magnification factors of the ipsilateral eye were usually much higher than those of the other eye. Furthermore, horizontal disparities between field centres were shown to have distinct distributions along the AP axis within the RP and DBR regions. At the latter a constant crossed disparity value (median=5.3°) was present along the AP axis while at the former greater variability and higher central disparity values were detected. An argument is developed based on this data to suggest that under our conditions the central binocular axis of the opossum are convergent with respect to the visual axis and their representation centred about the RP/DBR boundary. The different strategy of representation adopted by each eye at these two regions argue against a redundancy in the processing of visual information at the RP and the DBR on the opposite side, both of which bear a representation of the same visual space when considering only the information conveyed by the eye contralateral to the RP. The possible roles of this organization and the relevancy of these findings for studies of plastic rearrangement are discussed. [ABSTRACT FROM AUTHOR]

Visual receptive fields of single units in the superior colliculus of the opossum were analysed in regions representing the ipsilateral (rostral pole - RP) and the contralateral hemifields (direct binocular region - DBR) close to and including the representation of the horizontal meridian. Of 242 units half had receptive field centres of the contralateral eye at the ipsilateral hemifield and the other half at the contralateral hemifield. The proportion of units driven by the ipsilateral eye was considerably smaller at the RP than at the DBR and the frequency of unit response to this eye diminished within the ipsilateral hemifield with progressively more rostral penetrations in the RP. The midline was unequally represented by the two eyes in RP units: while 24 out of 26 receptive fields of the ipsilateral eye straddled the vertical meridian, only 50 out of 120 of the contralateral eye did so. Furthermore, at the RP receptive fields of units responsive to the contralateral eye extended up to 46.8 ° into the ipsilateral visual field while those mapped for the ipsilateral eye extended only up to 26 °. As a result of this tendency for the receptive fields of the ipsilateral, but not the contralateral eye, to overlap the vertical meridian, progressively more rostral recordings yielded an increase in the binocular convergent disparity as a function of the eccentricity for the receptive field of the contralateral eye. Since the disparity didn't vary with the eccentricity in the DBR units, we suggest that stimuli at the midline and close to the animal might be the natural ones to drive large disparity units present in the RP. This investigation has not detected significant differences in the responses of RP and DBR neurones to stationary stimuli, different velocities and directions of movement and variations in stimulus dimensions. The mean spontaneous activity was relatively low for neurons at both collicular regions. [ABSTRACT FROM AUTHOR]

The size, shape and laminar position of efferent neurons in the parietal cortex of the Virginia opossum were identified using the method of retrograde transport of horseradish peroxidase (HRP). Injection of HRP into the spinal cord, dorsal column nuclei or pontine nuclei leads to labeling of cells in layer V and occasionally in layer VI, while a large injection of HRP in the dorsal thalamus labels many cells in layer VI, with fewer cells in layer V. HRP injections in the SSM cortex label cells in layers II-VI of ipsilateral and contralateral cortical areas. However, the majority of these cortico-cortical cells are found in the supragranular layers. Examination of the size, shape and laminar position of retrogradely labeled layer V neurons after injections in each of these areas suggests that none of these features can be used to predict accurately the projection target of individual neurons. We conclude that the laminar organization of efferent cells of the opossum parietal cortex is very similar to that seen in the neocortex of other mammals, despite the complete coalescence of somatic sensory (SI) and motor (MI) areas in the opossum. [ABSTRACT FROM AUTHOR]

Although degeneration techniques suggest that cerebello-olivary fibers are limited in their origin and distribution, horseradish peroxidase and autoradiographic experiments make it clear that they arise within all cerebellar nuclei and project to most, if not all, areas of the contralateral inferior olive. Autoradiographic preparations show that cerebello-olivary fibers are highly ordered and suggest that the dentate nucleus projects primarily to the principal olive, the interpositus anterior relays particularly heavy to the dorsal accessory nucleus and the interpositus posterior distributes extensively to the medial accessory complex. Evidence for a small projection from the fastigial nucleus to the caudal medial accessory nucleus is also available. However, it appears clear that neither the dentate nor the interpositus nuclei project to just one subdivision of the olive. For example, although dentate fibers end extensively within the principal nucleus some of them also distribute to portions of the medial accessory nucleus and perhaps the dorsal accessory nucleus as well. The medial accessory olive is particularly complex and at rostral levels receives input from both interposed and dentate nuclei, whereas more caudally it receives a projection from the fastigial nucleus. Olivary fibers from both the interposed and dentate nuclei traverse the brachium conjunctivum descendons and distribute primarily to the rostral 2/3 to 3/4 of the olive, whereas those from fastigial neurons take a different route and end more caudally. Experiments utilizing horseradish peroxidase as a retrograde tracer suggest that cerebello-olivary fibers from both the interpositus anterior and dentate nuclei take origin from a population of generally small neurons. [ABSTRACT FROM AUTHOR]

The distributions of transthyretin and albumin in the choroid plexus during brain development have been compared. The South American opossum was chosen because the young are born around the time of choroid plexus formation. Previous work showed that in the adult opossum, transthyretin is expressed in the choroid plexus cells. However, systematic studies of transthyretin in the choroid plexus during development have not been carried out before. Transthyretin was present in 90–95% of the choroidal cells from birth to adulthood. In most cells, transthyretin immunoreactivity was concentrated in the apical region of the cytoplasm. Double labelling of choroid plexus sections with antibodies to albumin and transthyretin showed that 1–2% of cells were positive for both proteins. These findings suggest that from the very earliest stage of choroid plexus formation most epithelial cells both synthesize and contain transthyretin, and a few of these transthyretin-synthesizing cells also contain albumin that is probably being transferred from blood to the cerebrospinal fluid. [ABSTRACT FROM AUTHOR]

We identified a site suitable for venipuncture on the tail of the Virginia opossum ( Didelphis virginiana) that is reliably and easily located. The prominent hemal arch associated with the ventral surface of caudal vertebra 5 serves as an easily palpated anatomical landmark for locating the ventral caudal vein for blood collection. Because this venipuncture site is only thinly covered by fur and visualization of the vein is not necessary for its location, site preparation and total animal handling time for routine venipuncture are minimal. Blood may be collected from immature and adult male and female animals, and the technique is easily taught to new technicians with minimal danger of injury to the animal. [ABSTRACT FROM AUTHOR]

The key factors that influence the population growth rate can be identified with sensitivity analysis. Caswell presented an algorithm based on vector calculus that speeds up, and improves the practicality of the application of the sensitivity analysis, by calculating the derivatives of the explicit elements from the population Leslie matrix. Despite the usefulness of the sensitivity analysis, it is still barely applied to empirical data, partially due to requirements of high quality demographic data and failure of compatibility between time intervals of field data collection and organism's life stage timing. This can result in the absence of a certain vital rate as a separated element in the population projection matrix, and thus intricacy in obtaining its sensitivity. In this note we call the attention for the applied value of sensitivity analysis, and also we point to a simple formula that is easily implemented in Caswell Matlab algorithm, to calculate the sensitivity of a vital rate that is not explicit as a matrix element. [ABSTRACT FROM AUTHOR]