Rolling of the jaw is essential for mammalian chewing and tribosphenic molar function.

Over the past two centuries, mammalian chewing and related anatomical features have been among the most discussed of all vertebrate evolutionary innovations1-3. Chief among these features are two characters: the dentary-only mandible, and the tribosphenic molar with its triangulated upper cusps and lower talonid basin3-5. The flexible mandibular joint and the unfused symphysis of ancestral mammals-in combination with transformations of the adductor musculature and palate-are thought to have permitted greater mobility of each lower jaw, or hemimandible6,7. Following the appearance of precise dental occlusion near the origin of the mammalian crown8,9, therians evolved a tribosphenic molar with a craggy topography that is presumed to have been used to catch, cut and crush food. Here we describe the ancestral tribosphenic therian chewing stroke, as conserved in the short-tailed opossum Monodelphis domestica: it is a simple symmetrical sequence of lower tooth-row eversion and inversion during jaw opening and closing, respectively, enacted by hemimandibular long-axis rotation. This sequence is coupled with an eversion-inversion rotational grinding stroke. We infer that the ancestral therian chewing stroke relied heavily on long-axis rotation, including symmetrical eversion and inversion (inherited from the first mammaliaforms) as well as a mortar-and-pestle rotational grinding stroke that was inherited from stem therians along with the tribosphenic molar. The yaw-dominated masticatory cycle of primates, ungulates and other bunodont therians is derived; it is necessitated by a secondarily fused jaw symphysis, and permitted by the reduction of high, interlocking cusps10-12. The development of an efficient masticatory system-culminating in the tribosphenic apparatus-allowed early mammals to begin the process of digestion by shearing and crushing food into small boli instead of swallowing larger pieces in the reptilian manner, which necessitates a long, slow and wholly chemical breakdown. The vast diversity of mammalian teeth has emerged from the basic tribosphenic groundplan13.

Translating the Timing of Developmental Benchmarks in Short-Tailed Opossums (Monodelphisdomestica) to Facilitate Comparisons with Commonly Used Rodent Models.

INTRODUCTION: The gray short-tailed opossum, Monodelhis domestica (M. domestica), is a widely used marsupial model species that presents unique advantages for neurodevelopmental studies. Notably their extremely altricial birth allows manipulation of postnatal pups at timepoints equivalent to embryonic stages of placental mammals. A robust literature exists on the development of short-tailed opossums, but many researchers working in the more conventional model species of mice and rats may find it daunting to identify the appropriate age at which to conduct experiments. METHODS: Here, we present detailed staging diagrams taken from photographic observations of 40 individual pups, in 6 litters, over 25 timepoints across postnatal development. We also present a comparative neurodevelopmental timeline of short-tailed opossums (M. domestica), the house mouse (Mus musculus), and the laboratory rat (Rattus norvegicus) during embryonic as well as postnatal development, using timepoints taken from this study and a review of existing literature, and use this dataset to present statistical models comparing the opossum to the rat and mouse. RESULTS: One aim of this research was to aid in testing the generalizability of results found in rodents to other mammalian brains, such as the more distantly related metatherians. However, this broad dataset also allows the identification of potential heterochronies in opossum development compared to rats and mice. In contrast to previous work, we found broad similarity between the pace of opossum neural development with that of rats and mice. We also found that development of some systems was accelerated in the opossum, such as the forelimb motor plant, oral motor control, and some aspects of the olfactory system, while the development of the cortex, some aspects of the retina, and other aspects of the olfactory system are delayed compared to the rat and mouse. DISCUSSION: The pace of opossum development is broadly similar to that of mice and rats, which underscores the usefulness of this species as a compliment to the more commonly used rodents. Many features that differ the most between opossums and rats and mice were either clustered around the day of birth and were features that have functional importance for the pup immediately after or during birth, or were features that have reduced functional importance for the pup until later in postnatal development, given that it is initially attached to the mother.

On the prenatal initiation of T cell development in the opossum Monodelphis domestica.

Thymus-dependent lymphocytes (T cells) are a critical cell lineage in the adaptive immune system of all jawed vertebrates. In eutherian mammals the initiation of T cell development takes place prenatally and the offspring of many species are born relatively immuno-competent. Marsupials, in contrast, are born in a comparatively altricial state and with a less well developed immune system. As such, marsupials are valuable models for studying the peri- and postnatal initiation of immune system development in mammals. Previous results supported a lack of prenatal T cell development in a variety of marsupial species. In the gray short-tailed opossum, Monodelphis domestica, however, there was evidence that αßT cells were present on postnatal day 1 and likely initiated development prenatally. Demonstrated here is the presence of CD3ε+ lymphocytes in late-stage embryos at a site in the upper thoracic cavity, the site of an early developing thymus. CD3ε+ cells were evident as early as 48 h prior to parturition. In day 14 embryos, where there is clear organogenesis, CD3ε+ cells were only found at the site of the early thymus, consistent with no extra-thymic sites of T cell development in the opossum. These observations are the first evidence of prenatal T cell lineage commitment in any marsupial.

Rate of evolutionary change in cranial morphology of the marsupial genus Monodelphis is constrained by the availability of additive genetic variation.

We tested the hypothesis that the rate of marsupial cranial evolution is dependent on the distribution of genetic variation in multivariate space. To do so, we carried out a genetic analysis of cranial morphological variation in laboratory strains of Monodelphis domestica and used estimates of genetic covariation to analyse the morphological diversification of the Monodelphis brevicaudata species group. We found that within-species genetic variation is concentrated in only a few axes of the morphospace and that this strong genetic covariation influenced the rate of morphological diversification of the brevicaudata group, with between-species divergence occurring fastest when occurring along the genetic line of least resistance. Accounting for the geometric distribution of genetic variation also increased our ability to detect the selective regimen underlying species diversification, with several instances of selection only being detected when genetic covariances were taken into account. Therefore, this work directly links patterns of genetic covariation among traits to macroevolutionary patterns of morphological divergence. Our findings also suggest that the limited distribution of Monodelphis species in morphospace is the result of a complex interplay between the limited dimensionality of available genetic variation and strong stabilizing selection along two major axes of genetic variation.

Molecular phylogeny of short-tailed opossums (Didelphidae: Monodelphis): taxonomic implications and tests of evolutionary hypotheses.

Short-tailed opossums (genus Monodelphis) represent one of the most speciose clades of New World marsupials, with 26 currently recognized species that collectively range from eastern Panama to northern Argentina. Here we present the first phylogenetic analyses of the genus based on dense taxonomic sampling and multiple genes. From most sampled species we obtained >4800bp of DNA sequence from one mitochondrial gene (CYTB), two autosomal exons (IRBP exon 1, BRCA1 exon 11), one autosomal intron (SLC38 intron 7), and one X-linked intron (OGT intron 14). Maximum-parsimony, maximum-likelihood and Bayesian analyses of these data strongly support the monophyly of Monodelphis and recover six major clades within the genus. Additionally, our analyses support previous suggestions that several nominal taxa are synonyms of other species (M. "sorex" of M. dimidiata, M. "theresa" of M. scalops, M. "rubida" and M. "umbristriata" of M. americana, and M. "maraxina" of M. glirina). By contrast, four unnamed lineages recovered by our analyses may represent new species. Reconstructions of ancestral states of two discrete characters-dorsal pelage color pattern and habitat-suggest that the most recent common ancestor of Monodelphis was uniformly colored (with unpatterned dorsal pelage) and inhabited moist forest. Whereas some dorsal pelage patterns appear to have evolved homoplastically in Monodelphis, dorsal stripes may have had a unique historical origin in this genus.

The mammary hair of Monodelphis domestica and homology of the mammary pilosebacous unit.

The unitary mammary gland is a synapomorphy of therian mammals and is thought to have evolved from the pilosebaceous organ in the mammalian stem lineage from which the lactogenic patch of monotremes is also derived. One of the key lines of evidence for the homology of the nipple and the lactogenic patch is that marsupials have retained a transient hair associated with developing mammary glands. However, these structures have not been documented since the early 20th-century drawings of Ernst Bresslau. In this study, we examine the developing mammary organs of Monodelphis domestica and document the presence of mammary hairs in 12-week-old females, as well as their absence after 18 weeks of age. Histochemical staining for cystine confirms the structures as keratinized hairs. Milk ducts of both juvenile and adult nipples show a division between KRT18+ luminal epithelium and KRT14+ ACTA2+ myoepithelium. These patterns match those in eutherians and suggest a conserved ductal morphology and mechanism of milk expulsion. Finally, PTHLH, a peptide hormone which promotes homeotic transformation of hairy skin into hairless nipples in the mouse, was detected in the Monodelphis milk duct during the mammary hair stage, suggesting that the mutual exclusivity of "hairless nipple" and "hair" organ identity is derived in eutherian mammals. These results reveal shared characteristics of the M. domestica nipple with both the eutherian nipple and the pilosebaceous organ, consistent with the evolutionary derivation of the mammary gland from an ancestral hair organ via developmental individualization of pilosebaceous and mammary identities.

The second known specimen of Monodelphis unistriata (Wagner) (Mammalia: Didelphimorphia), with redescription of the species and phylogenetic analysis.

Very little information exists relevant to the species grouping and phylogenetic relationships of the opossum genus Monodelphis Burnett. Of the clearly distinct named species, the least information is available for M. unistriata (Wagner), one of the world's most poorly known species of mammals. Extant specimens consist of the Brazilian holotype of a skin now without a skull and dating from almost 200 years ago, and a second specimen with skin and incomplete skull dating from over a hundred years ago and from Argentina. The most recent published notes on the holotype date from well over half a century ago and, all told, such notes, the earliest dating from 1842, add up to a highly fragmentary and contradictory picture. No observations whatsoever have ever been published for the second and more complete specimen. Also, no hypotheses have ever been made concerning the intrageneric affinities of M. unistriata and such affinities have also been obscure throughout the genus. Herein, we provide a detailed redescription of M. unistriata, the first published images of specimens, and the first account, beyond the previous few most vague and incomplete remarks, of the morphology of the skull. In an effort to ascertain the phylogenetic affinities of M. unistriata, we performed a combined molecular (cytochrome b) and nonmolecular (postcranial, cranial, integument, and karyotypic characters) parsimony analysis incorporating 27 species of didelphids, including 11 of Monodelphis. Our results strongly support the monophyly of Monodelphis, and place M. unistriata as sister group to M. iheringi, among the included species.

The subventricular zone is the developmental milestone of a 6-layered neocortex: comparisons in metatherian and eutherian mammals.

The major lineages of mammals (Eutheria, Metatheria, and Monotremata) diverged more than 100 million years ago and have undergone independent changes in the neocortex. We found that adult South American gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii) possess a significantly lower number of cerebral cortical neurons compared with the mouse (Mus musculus). To determine whether the difference is reflected in the development of the cortical germinal zones, the location of progenitor cell divisions was examined in opossum, tammar wallaby, and rat. The basic pattern of the cell divisions was conserved, but the emergence of a distinctive band of dividing cells in the subventricular zone (SVZ) occurred relatively later in the opossum (postnatal day [P14]) and the tammar wallaby (P40) than in rodents. The planes of cell divisions in the ventricular zone (VZ) were similar in all species, with comparable mRNA expression patterns of Brn2, Cux2, NeuroD6, Tbr2, and Pax6 in opossum (P12 and P20) and mouse (embryonic day 15 and P0). In conclusion, the marsupial neurodevelopmental program utilizes an organized SVZ, as indicated by the presence of intermediate (or basal) progenitor cell divisions and gene expression patterns, suggesting that the SVZ emerged prior to the Eutherian-Metatherian split.

Distribution of the neuronal gap junction protein Connexin36 in the spinal cord enlargements of developing and adult opossums, Monodelphis domestica.

We use opossums Monodelphis domestica to study the development of mammalian motor systems. The immature forelimbs of the newborn perform rhythmic and alternating movements that are likely under spinal control. The hindlimbs start moving in the second week. Chemical synapses are scant in the spinal enlargements of neonatal opossums and the presence of electrochemical synapses has not been evaluated in this species or in other marsupials. As a first step aiming at evaluating the existence of such synapses in the neonatal spinal cord, we have investigated the presence of the exclusively neuronal gap junction protein connexin36 (Cx36) by immunohistochemistry in light microscopy. At birth, Cx36 immunoreactivity is moderate in the presumptive gray matter in both enlargements. Thereafter, it decreases gradually, except in the superficial dorsal horn where it increases to a plateau between P10 and P20. Cx36 labeling is detected in the presumptive white matter at birth, but then decreases except in the dorsal part of the lateral funiculus, where it is dense between P10 and P20. Cx36 has become virtually undetectable by P52. The presence of Cx36 in the spinal enlargements of postnatal opossums suggests that neurons might be linked by gap junctions at a time when chemical synapses are only beginning to form. The greater abundance of Cx36 observed transiently in the superficial dorsal horn suggests a stronger involvement of this protein in spinal sensory systems than in direct motor control of the limbs.

Cellular transfer of macromolecules across the developing choroid plexus of Monodelphis domestica.

Choroid plexus epithelial cells secrete cerebrospinal fluid (CSF) and transfer molecules from blood into CSF. Tight junctions between choroidal epithelial cells are functionally effective from early in development: the route of transfer is suggested to be transcellular. Routes of transfer for endogenous and exogenous plasma proteins and dextrans were studied in Monodelphis domestica (opossum). Pups at postnatal (P) days 1-65 and young adults were injected with biotinylated dextrans (3-70 kDa) and/or foetal protein fetuin. CSF, plasma and brain samples were collected from terminally anaesthetized animals. Choroid plexus cells containing plasma proteins were detected immunocytochemically. Numbers of plasma protein-positive epithelial cells increased to adult levels by P28, but their percentage of plexus cells declined. Numbers of cells positive for biotinylated probes increased with age, while their percentage remained constant. Colocalization studies showed specificity for individual proteins in some epithelial cells. Biotinylated probes and endogenous proteins colocalized in about 10% of cells in younger animals, increasing towards 100% by adulthood. Injections of markers into the ventricles demonstrated that protein is transferred only from blood into CSF, whereas dextrans pass in both directions. These results indicate that protein and lipid-insoluble markers are transferred by separate mechanisms present in choroid plexuses from the earliest stage of brain development, and transfer of proteins from plasma across choroid plexus epithelial cells contributes to the high protein concentration in CSF in the immature brain.

An architectonic study of the neocortex of the short-tailed opossum (Monodelphis domestica).

Short-tailed opossums (Monodelphis domestica) belong to the branch of marsupial mammals that diverged from eutherian mammals approximately 180 million years ago. They are small in size, lack a marsupial pouch, and may have retained more morphological characteristics of early marsupial neocortex than most other marsupials. In the present study, we used several different histochemical and immunochemical procedures to reveal the architectonic characteristics of cortical areas in short-tailed opossums. Subdivisions of cortex were identified in brain sections cut in the coronal, sagittal, horizontal or tangential planes and processed for a calcium-binding protein, parvalbumin (PV), neurofilament protein epitopes recognized by SMI-32, the vesicle glutamate transporter 2 (VGluT2), myelin, cytochrome oxidase (CO), and Nissl substance. These different procedures revealed similar boundaries among areas, suggesting that functionally relevant borders were detected. The results allowed a fuller description and more precise demarcation of previously identified sensory areas, and the delineation of additional subdivisions of cortex. Area 17 (V1) was especially prominent, with a densely populated layer 4, high myelination levels, and dark staining of PV and VGluT2 immunopositive terminations. These architectonic features were present, albeit less pronounced, in somatosensory and auditory cortex. The major findings support the conclusion that short-tailed opossums have fewer cortical areas and their neocortex is less distinctly laminated than most other mammals.

Suprasternal gland secretion of male short-tailed opossum induces IP3 generation in the vomeronasal organ.

Chemical communication is an important component of mammalian social behaviors. Gray short-tailed opossums (Monodelphis domestica) communicate by scent marking. The male opossum possesses a prominent suprasternal scent gland, extracts of which strongly attract female opossums. This attractivity remains unaltered following repeated lyophilization. The suprasternal gland secretion functions in a sexually dimorphic manner, i.e., it elicits elevated levels of IP(3) in the vomeronasal (VN) sensory epithelium of female opossums, but suppressed the levels of IP(3) in the VN sensory epithelium of male opossums. The elevated levels of IP(3) induced by suprasternal gland secretion in female vomeronasal sensory epithelium is inhibited by the G(i/o) specific inhibitor, NF023, but not its inactive analogue, NF007. It is also suppressed by specific antibodies to the alpha subunits of G(i) and G(o) proteins, by the phospholipase C inhibitor, U73122, as well as by GDPbetaS. Surprisingly, GDPbetaS itself enhances basal levels of IP(3) in female VN sensory epithelium. This GDPbetaS-induced increase in levels of IP(3) is reduced by the PLC inhibitor, U73122, but not by the G(i/o) inhibitor, NF023. In addition, GDP also enhances basal levels of IP(3). GDPbetaS, a known inhibitor of G-protein activation, thus appears to have dual functions: as both stimulator and inhibitor of IP(3) production in the VN sensory epithelium of opossums. In contrast, this nucleotide analogue functions as an inhibitor in the VN sensory epithelium of mice. The mechanism of signal transduction underlying the suprasternal gland secretion-elicited signals in the VN sensory epithelium of opossums appears to involve signals that are generated through activation of G-protein-coupled receptors and transduced via activation of G(i/o)-proteins and the effector, phospholipase C, resulting in an increased production of the second messenger, IP(3). The extracellular signals are thus amplified.

Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling.

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.

Thalamic nuclei in the opossum Monodelphis domestica.

We investigated nuclear divisions of the thalamus in the gray short-tailed opossum (Monodelphis domestica) to gain detailed information for further developmental and comparative studies. Nissl and myelin staining, histochemistry for acetylcholinesterase and immunohistochemistry for calretinin and parvalbumin were performed on parallel series of sections. Many features of the Monodelphis opossum thalamus resemble those in Didelphis and small eutherians showing no particular sensory specializations, particularly in small murid rodents. However, several features of thalamic organization in Monodelphis were distinct from those in rodents. In the opossum the anterior and midline nuclear groups are more clearly separated from adjacent structures than in eutherians. The dorsal lateral geniculate nucleus (LGNd) starts more rostrally and occupies a large part of the lateral wall of the thalamus. As in other marsupials, two cytoarchitectonically different parts, alpha and beta are discernible in the LGNd of the opossum. Each of them may be subdivided into two additional bands in acetylcholinesterase staining, while in murid rodents the LGNd consists of a homogeneous mass of cells. Therefore, differentiation of the LGNd of the Monodelphis opossum is more advanced than in murid rodents. The medial geniculate body consists of three nuclei (medial, dorsal and ventral) that are cytoarchitectonically distinct and stain differentially for parvalbumin. The relatively large size of the MG and LGNd points to specialization of the visual and auditory systems in the Monodelphis opossum. In contrast to rodents, the lateral dorsal and lateral posterior nuclei in the opossum are poorly differentiated cytoarchitectonically.

Cell birth and death in the developing retina of the Brazilian opossum, Monodelphis domestica.

The purpose of this study was to characterize cytogenesis and apoptosis in the developing retina of the Brazilian opossum, Monodelphis domestica. Monodelphis is a small pouchless marsupial whose young undergo a protracted period of postnatal development. Moreover, the Monodelphis retina represents a unique in vivo compartment for investigating cellular interactions that occur during early neural development and is an important system to study plasticity of neural stem cells following transplantation. Using bromodeoxyuridine (BrdU) labeling of newly generated cells, double-labeling immunohistochemistry and TUNEL labeling of apoptotic cells we have performed a detailed analysis of cell birth and death in the Monodelphis retina from fetal development through early postnatal life. Pregnant opossums or pups received a single injection of BrdU between gestational day 12 and postnatal day 35 (35PN), eyes were collected two hours after injection or on day 15, 30, or 60 of postnatal life. BrdU-labeled cells were visualized immunohistochemically. Cells were classified according to their morphology, location and immunoreactivity for cell-type specific antibodies. Cell genesis in the opossum retina begins at E13 and was near completion by 25PN. Apoptotic retinal cells were identified using the TUNEL technique for labeling of fragmented DNA. Apoptosis covered a relatively broad period of postnatal development, beginning around 10PN, peaking at 30PN, and concluding before 60PN. These results demonstrate that the retina of Monodelphis, a polyprotodont marsupial, is generated in a similar pattern to the wallaby, a diprotodont marsupial, and to eutherian species.

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.

Placental function in two distantly related marsupials.

The biochemical composition of uterine and fetal fluids during pregnancy of the grey short-tailed opossum was compared with new and published data on the tammar wallaby. In the grey short-tailed opossum, there are three main phases of embryonic nourishment. During the first phase, the embryo obtains nutrients from uterine secretion transferred into the yolk sac. The amount of uterine secretion declines during the second phase up to the time of shell coat rupture. As a result, the protein concentration in yolk sac fluid also declines. During phase three, which begins with shell coat rupture, nutrients are predominantly available from the maternal blood. In the grey short-tailed opossum that lacks a vesicular, fluid-filled allantois, waste products such as urea are apparently stored in the yolk sac and from there pass into the maternal circulation across the invasive yolk sac placenta. In contrast, in the tammar wallaby, the main source of nutrients available to the late term fetus is glandular secretion that is complemented by substances from the maternal circulation via the chorio-vitelline placenta, and waste products are stored in the large, fluid-filled allantois.

Distribution of the parvalbumin, calbindin-D28K and calretinin immunoreactivity in globus pallidus of the Brazilian short-tailed opossum (Monodelphis domestica).

This study describes the topography, borders and divisions of the globus pallidus in the Brazilian short-tailed opossum (Monodelphis domestica) and distribution of the three calcium binding proteins, parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR) in that nucleus. The globus pallidus of the opossum consists of medial and lateral parts that are visible with Nissl or Timm's staining and also in PV and CR immunostained sections. Neurons of the globus pallidus expressing these proteins were classified into three types on the basis of size and shape of their soma and dendritic tree. Type 1 neurons had medium-sized fusiform soma with dendrites sprouting from the opposite poles. Neurons of the type 2 had medium-to-large, multipolar soma with scarce, thin dendrites. Cell bodies of type 3 neurons were small and either ovoid or round. Immunostaining showed that the most numerous were neurons expressing PV that belonged to all three types. Density of the PV-immunopositive fibers and puncta correlated with the density of the PV-labeled neurons. Labeling for CB resulted mainly in the light staining of neuropil in both parts of the nucleus, while the CB-expressing cells (mainly of the type 2) were scarce and placed only along the border of the globus pallidus and putamen. Staining for calretinin resulted in labeling almost exclusively the immunoreactive puncta and fibers that were distributed with medium-to-high density throughout the nucleus. Close to the border of globus pallidus with the putamen these fibers (probably dendrites) were long, thin and varicous, while more medially bundles of thick, short and smooth fibers predominated. Single CR-ir neurons (all of the type 3) were scattered through the globus pallidus. Colocalization of two calcium binding proteins in one neuron was. never observed. The CB-ir puncta (probably terminals of axons projecting to the nucleus) frequently formed basket-like structures around the PV-ir neurons. Therefore, the globus pallidus in the opossum, much as that in the rat, consists of a heterogeneous population of neurons, probably playing diversified functions.

The Unique Paired Retinal Vessels of the Gray Short-Tailed Opossum (Monodelphis domestica) and Their Relationship to Astrocytes and Microglial Cells.

In marsupials that possess a retinal vasculature, the arterial and venous segments, down to the smallest calibre capillaries, have been shown to occur in pairs. This pattern is seen in the marsupial central nervous system (CNS) but not in other tissues in this group or in any tissues in eutherian mammals. The present study aimed to determine if the gray short-tailed opossum (Monodelphis domestica), a south American marsupial, possesses double retinal vessels. Secondly, we investigated the relationship between vessels and astrocytes and microglia, which are known to play pivotal roles in the blood retinal barrier and immune surveillance respectively. Eyes from M. domestica between 2 months and 33 months of age were examined by bright field and fluorescein angiography, resin histology, and wholemount immunostaining. Retinal vessels in this marsupial always occur in closely related pairs with the arteriolar limb usually on the vitread aspect. Branches penetrate the retina to form layers of paired capillaries as far as the outer nuclear layer. Dense networks of GFAP+ astrocytes enveloped the vitread aspect of vessels. No particularly strong association with blood vessels and ramified Iba1+ and Ib4+ microglia was noted. M. domestica possessed the unusual paired vasculature and capillary loops arrangement previously described in the marsupial CNS. These observations in a small laboratory-friendly marsupial open up new frontiers to investigate the factors that regulate paired blood vessel development and the functional significance of this arrangement when compared to the anastomotic pattern observed in the retina of eutherian mammals. Anat Rec, 300:1391-1400, 2017. © 2017 Wiley Periodicals, Inc.

Phenotypic diversity is the cornerstone of evolution: variation in cortical field size within short-tailed opossums.

Natural selection operates on phenotypic variation that exists within a population. Variable aspects of cortical organization, such as the size and connections of a cortical field, can generate differences in behavior, which is a target of natural selection. Yet studies pertaining to within-species variability in cortical organization are limited. In the present investigation, we examined variation in brain size, cortical sheet size, and primary sensory cortical field sizes in the adult short-tailed opossum (Monodelphis domestica). Within individuals, we found no significant difference between the right and left hemispheres in the overall size of the dorsolateral cortex or in primary cortical field sizes. Between individuals, we found relatively little intraspecies variation in brain weight, brain volume, and cortical sheet area for the dorsolateral neocortex and pyriform cortex; however, we observed a large degree of variability in body weight and primary sensory cortical field size, as defined by myeloarchitecture. Further, we found that the size of each cortical field correlated with the size of the other cortical fields as well as with the total size of the dorsolateral cortex. Here we discuss the possible sources of variation and examine the relationship between cortical field size and sensory processing abilities and behaviors across species. Since behavior is the target of natural selection, variation in cortical field size across individuals may supply the raw material necessary for cortical field evolution.