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.

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.

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.

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.

Angioarchitecture and morphology of temporomandibular joint of Monodelphis domestica.

The opossum Monodelphis domestica presents movement of the temporomandibular joint (TMJ) reflecting adaptation to eating habits similar to movement in humans, but the structure of the TMJ is not yet known. Thus, nine young M. domestica, of both sexes were weighed, anesthetized with xylazine (10 mg kg(-1) ), and ketamine (70 mg kg(-1) ) and processed for: 1. The analyses of the macroscopic angioarchitecture after latex injection, as well as the topography of the TMJ; 2. The analysis of microvascularization after injection of Mercox resin and corrosion of soft tissue with NaOH using scanning electron microscopy and; 3. The histological evaluation of the TMJ with an optical microscope. Macroscopic analysis of the latex injected vessels revealed the distribution of the arteries from the common carotid artery, receiving branches of the superficial temporal and maxillary arteries. The mandibular condyle has the long axis in the lateral-lateral direction, and is convex in the anterior-posterior direction. Its topography was determined in relation to the eye and external acoustic meatus. With scanning electron microscopy, microvascularization consists of arterioles of varying diameter (85-15 µm) of the meandering capillary network in the retrodiscal region, and a network of straight capillaries in the TMJ anterior region. Via light microscopy the TMJ has similar histological features to those of humans. These macroscopic, microscopic and ultrastructural data from TMJ of the M. domestica could be a suitable model for TMJ physiology and pathophysiology studies for then speculate on possible human studies. Microsc. Res. Tech. 79:806-813, 2016. © 2016 Wiley Periodicals, Inc.

The evolution of whisker-mediated somatosensation in mammals: Sensory processing in barrelless S1 cortex of a marsupial, Monodelphis domestica.

Movable tactile sensors in the form of whiskers are present in most mammals, but sensory coding in the cortical whisker representation has been studied almost exclusively in mice and rats. Many species that possess whiskers lack the modular "barrel" organization found in the primary somatosensory cortex (S1) of mice and rats, but it is unclear how whisker-related input is represented in these species. We used single-unit extracellular recording techniques to characterize receptive fields and response properties in S1 of Monodelphis domestica (short-tailed opossum), a nocturnal, terrestrial marsupial that shared its last common ancestor with placental mammals over 160 million years ago. Short-tailed opossums lack barrels and septa in S1 but show active whisking behavior similar to that of mice and rats. Most neurons in short-tailed opossum S1 exhibited multiwhisker receptive fields, including a single best whisker (BW) and lower magnitude responses to the deflection of surrounding whiskers. Mean tuning width was similar to that reported for mice and rats. Both symmetrical and asymmetrical receptive fields were present. Neurons tuned to ventral whiskers tended to show broad tuning along the rostrocaudal axis. Thus, despite the absence of barrels, most receptive field properties were similar to those reported for mice and rats. However, unlike those species, S1 neuronal responses to BW and surround whisker deflection showed comparable latencies in short-tailed opossums. This dissimilarity suggests that some aspects of barrel cortex function may not generalize to tactile processing across mammalian species and may be related to differences in the architecture of the whisker-to-cortex pathway. J. Comp. Neurol. 524:3587-3613, 2016. © 2016 Wiley Periodicals, Inc.

Differential morphology of the superior olivary complex of Meriones unguiculatus and Monodelphis domestica revealed by calcium-binding proteins.

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.

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 cellular composition of the marsupial neocortex.

In the current investigation we examined the number and proportion of neuronal and non-neuronal cells in the primary sensory areas of the neocortex of a South American marsupial, the short-tailed opossum (Monodelphis domestica). The primary somatosensory (S1), auditory (A1), and visual (V1) areas were dissected from the cortical sheet and compared with each other and the remaining neocortex using the isotropic fractionator technique. We found that although the overall sizes of V1, S1, A1, and the remaining cortical regions differed from each other, these divisions of the neocortex contained the same number of neurons, but the remaining cortex contained significantly more non-neurons than the primary sensory regions. In addition, the percent of neurons was higher in A1 than in the remaining cortex and the cortex as a whole. These results are similar to those seen in non-human primates. Furthermore, these results indicate that in some respects, such as number of neurons, the neocortex is homogenous across its extent, whereas in other aspects of organization, such as non-neuronal number and percentage of neurons, there is non-uniformity. Whereas the overall pattern of neuronal distribution is similar between short-tailed opossums and eutherian mammals, short-tailed opossum have a much lower cellular and neuronal density than other eutherian mammals. This suggests that the high neuronal density cortices of mammals such as rodents and primates may be a more recently evolved characteristic that is restricted to eutherians, and likely contributes to the complex behaviors we see in modern mammals.

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.

Microscopic anatomy of the lower respiratory tract of the grey short-tailed opossum (Monodelphis domestica).

The respiratory tracts of seven grey short-tailed opossums were histologically examined. Six opossums were prepared by perfusion with buffered formalin. Opossum seven was perfused with gluteraldehyde. Samples taken from the respiratory passages and lungs of specimens 1-6 were stained with haematoxylin and eosin. A mixture of methylene and azure blue was used for specimen 7. The trachea and right and left principal bronchi are lined with a pseudostratified ciliated columnar epithelium with occasional goblet cells. The secondary and tertiary bronchi and the primary and secondary bronchioles are lined by a simple ciliated columnar epithelium. The terminal bronchioles and a portion of the respiratory bronchioles are lined by a simple ciliated cuboidal epithelium. The terminal portion of the respiratory bronchioles and the alveolar ducts are lined with simple squamous epithelium. Alveoli are lined by type I and II pneumocytes. Tracheal glands are present in the tela submucosa. The fibromusculocartilaginous tunic of the trachea consists of c-shaped cartilage rings and the trachealis muscle. A lamina muscularis mucosa begins in the intrapulmonary portion of the principal bronchus and continues into the respiratory bronchioles. Bronchial glands are present in the propria submucosa and tela submucosa of the principal bronchi. The musculocartilaginous tunic is localized to the extrapulmonary portion of the principal bronchus. The bronchial cartilages are irregular shaped plates and limited to the extrapulmonary portion of the principal bronchus. The visceral pleura is a simple squamous mesothelium covering the outer surface of the lung.

Normal organ weights, serum chemistry, hematology, and cecal and nasopharyngeal bacterial cultures in the gray short-tailed opossum (Monodelphis domestica).

Gray short-tailed opossums (Monodelphis domestica) currently are used in genetic, developmental, oncology, and neurologic research. Little is known about their natural flora or potential for pathogenic infectious disease. The present study aims to improve existing comparative normal blood and organ weight values available to researchers and to describe flora of clinically normal M. domestica to obtain an understanding of potential pathogenic flora in clinically abnormal animals. For evaluation of serum hematology and serum chemistry, clinically normal animals were assigned to 1 of 6 groups stratified by age (younger than 1 y, 1 to 2 y, and 2 to 3 y) and sex. Hemoglobin and phosphorus levels were higher in male than female opossums, whereas monocyte and eosinophil counts were greater in females than males. Hemoglobin concentration decreased with increasing age. The youngest group had significantly higher levels of serum alkaline phosphatase and lower serum protein levels compared with older age groups. Liver and kidney weights of adult animals (1 to 3 y) were greater in female than male opossums. The predominant nasopharyngeal flora in 20 clinically normal animals from the 2- to 3-y-old group were Streptococcus viridans, Escherichia coli, and coagulase-negative Staphylococcus spp.; predominant cecal organisms were Escherichia coli and Citrobacter spp. The availability of reference hematologic values and flora for Monodelphis domestica will aid researchers in comparisons and analysis of experimental data and in diagnosis and evaluation of potential pathogens in clinically ill animals.

Ontogenetic variation in the bony labyrinth of Monodelphis domestica (Mammalia: Marsupialia) following ossification of the inner ear cavities.

Ontogeny, or the development of an individual from conception to death, is a major source of variation in vertebrate morphology. All anatomical systems are affected by ontogeny, and knowledge of the ontogenetic history of these systems is important to understand when formulating biological interpretations of evolutionary history and physiology. The present study is focused on how variation affects the bony labyrinth across a growth series of an extant mammal after ossification of the inner ear chambers. Digital endocasts of the bony labyrinth were constructed using CT data across an ontogenetic sequence of Monodelphis domestica, an important experimental animal. Various aspects of the labyrinth were measured, including angles between the semicircular canals, number of turns of the cochlea, volumes of inner ear constituents, as well as linear dimensions of semicircular canals. There is a strong correlation between skull length and age, but from 27 days after birth onward, there is no correlation with age among most of the inner ear measurements. Exceptions are the height of the arc of the lateral semicircular canal, the angular deviation of the lateral canal from planarity, the length of the slender portion of the posterior semicircular canal, and the length of the canaliculus cochleae. Adult dimensions of several of the inner ear structures, such as the arcs of the semicircular canals, are achieved before the inner ear is functional, and the non-ontogenetic variation in the bony labyrinth serves as an important source for behavioral, physiological, and possibly phylogenetic information.

The divergent development of the apical ectodermal ridge in the marsupial Monodelphis domestica.

Marsupials give birth after short gestation times to neonates that have an intriguing combination of precocial and altricial features, based on their functional necessity after birth. Perhaps most noticeably, marsupial newborns have highly developed forelimbs, which provide the propulsion necessary for the newborn's crawl to the teat. To achieve their advanced state at birth, the development of marsupial forelimbs is accelerated. The development of the newborn's hind limb, which plays no part in the crawl, is not accelerated, and is likely even delayed. Given the large differences in the rate of limb outgrowth among marsupials and placentals, we hypothesize that the pathways underlying the early development and outgrowth of marsupial limbs, especially that of their forelimbs, will also be divergent. As a first step toward testing this, we examine the development of one of the two major signaling centers of the developing limb, the apical ectodermal ridge (AER), in a marsupial, Monodelphis domestica. We found that, while both opossum limbs have reduced physical AER's, in the opossum forelimb this reduction has been taken to the extreme. Where the M. domestica forelimb should have an AER, it instead has only a few patches of disorganized cells. These results make the marsupial, M. domestica, the only known amniote (without reduced limbs) to exhibit no morphological AER. However, both M. domestica limbs normally express Fgf8, a molecular marker of the AER.

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.

The developmental reduction of the marsupial coracoid: a case study in Monodelphis domestica.

During their embryogenesis, marsupials develop a unique structure, the shoulder arch, which provides the structural and muscle-attachment support necessary for the newborn's crawl to the teat. One of the most pronounced and important aspects of the shoulder arch is an enlarged coracoid. After marsupial newborns reach the teat, the shoulder arch is remodeled and the coracoid is reduced to a small process on the scapula. Although an understanding of marsupial coracoid reduction has the potential to provide insights into both, marsupial evolution and the origin of mammals, little is known about the morphological and cellular processes controlling this process. To remedy this situation, this study examined the morphological and cellular mechanisms behind coracoid reduction in the gray short-tailed opossum, Monodelphis domestica. A quantitative, morphometric study of shoulder girdle development revealed that the coracoid is reduced in size relative to other aspects of the shoulder girdle by growing at a slower rate. Using a series of molecular assays for cell death, no evidence was found for programmed cell death playing a role in the reduction of coracoid size in marsupials (in contrast to hypotheses of previous researchers). Although it is likely the case that coracoid growth is reduced through a relatively lower rate of cellular proliferation, differences in proliferative rates in the coracoid and scapula were not great enough to be quantified using standard molecular assays.

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.

Developmental expression of spontaneous activity in the spinal cord of postnatal opossums, Monodelphis domestica: an anatomical study.

Using Sulforhodamine-101 (SR101) labeling and calcium imaging on in vitro preparations, we investigated the development of spontaneous activity in the spinal enlargements of a marsupial born more immature than eutherian mammals, the opossum Monodelphis domestica. Following the retrograde transport of Calcium Green dye from the limb nerves, we observed the occurrence of spontaneous calcium waves activating the motor columns of the cervical enlargement of opossums aged from P3 to P15 (day of birth: P0) and of the lumbar enlargement from at least P6 to P12. In other preparations, SR101 was added to the bath to identify the active cells. In P1 opossums, only a few SR101-labeled cells were observed in the cervical enlargement and none in the lumbar enlargement. At P5, their number increased cervically and they appeared in the lumbar enlargement. Motoneurons were the major cell type labeled by SR101 but dye leakage made their quantification inaccurate. SR101-labeled cells also occurred elsewhere in the ventral and dorsal grey. Their number increased until P12-14 in both enlargements and then decreased to disappear by P21, the last age examined. Thus in contrast to eutherian mammals, in which spontaneous activity is mostly prenatal, spontaneous activity occurs predominantly postnatally in opossums. It increases at the time when connections from the brain begin to impinge on spinal neurons and when the limbs, especially the hindlimbs, start moving and then decreases as the systems mature.

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.