Astrocytes of the Anterior Commissure Regulate the Axon Guidance Pathways of Newly Generated Neocortical Neurons in the Opossum Monodelphis domestica.

In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the corpus callosum, that interconnects both hemispheres. In this study, we aimed to examine neurogenesis during the formation of cortical upper layers, including their morphological maturation in a marsupial species, namely the opossum (Monodelphis domestica). Furthermore, we studied how the axons of upper layers neurons pass through the anterior commissure of the opossum, which connects neocortical areas. We showed that upper-layer II/III neurons were generated within at least seven days in the opossum neocortex. Surprisingly, these neurons expressed special AT-rich sequence binding protein 2 (Satb2) and neuropilin 1 interacting protein (Nrp1), which are proteins known to be essential for the formation of the corpus callosum in eutherians. This indicates that extrinsic, but not intrinsic, cues could be key players in guiding the axons of newly generated cortical neurons in the opossum. Although oligodendrocyte precursor cells were present in the neocortex and anterior commissure, newly generated upper-layer neurons sent unmyelinated axons to the anterior commissure. We also found numerous GFAP-expressing progenitor cells in both brain structures, the neocortex and the anterior commissure. However, at P12-P17 in the opossums, a small population of astrocytes was observed only in the midline area of the anterior commissure. We postulate that in the opossum, midline astrocytes allow neocortical axons to be guided to cross the midline, as this structure resembles the glial wedge required by fibers to cross the midline area of the corpus callosum in the rodent.

Effects of Brain Size on Adult Neurogenesis in Shrews.

Shrews are small animals found in many different habitats. Like other mammals, adult neurogenesis occurs in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus (DG) of the hippocampal formation. We asked whether the number of new generated cells in shrews depends on their brain size. We examined Crocidura russula and Neomys fodiens, weighing 10-22 g, and Crocidura olivieri and Suncus murinus that weigh three times more. We found that the density of proliferated cells in the SVZ was approximately at the same level in all species. These cells migrated from the SVZ through the rostral migratory stream to the olfactory bulb (OB). In this pathway, a low level of neurogenesis occurred in C. olivieri compared to three other species of shrews. In the DG, the rate of adult neurogenesis was regulated differently. Specifically, the lowest density of newly generated neurons was observed in C. russula, which had a substantial number of new neurons in the OB compared with C. olivieri. We suggest that the number of newly generated neurons in an adult shrew's brain is independent of the brain size, and molecular mechanisms of neurogenesis appeared to be different in two neurogenic structures.

Adult Neurogenesis in the Mammalian Hypothalamus: Impact of Newly Generated Neurons on Hypothalamic Function.

In mammals, adult neurogenesis was first demonstrated in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus of the hippocampal formation. Further research showed that adult neurogenesis persists in other brain structures, such as the cerebral cortex, piriform cortex, striatum, amygdala, and hypothalamus. However, the origin of newly generated cells in these structures is not clear. Accumulating evidence indicates that newly generated neurons in the striatum or amygdala are derived from the SVZ, while in the adult hypothalamus, the proliferation of progenitor cells occurs in the ependymal cells lining the third ventricle, which give rise to new neurons. The heterogeneous cellular organization of the ependymal layer of the hypothalamus leads to different conclusions regarding the type of hypothalamic progenitor cells. In addition, adult hypothalamic neurogenesis occurs at low levels. Based on comparative and functional approaches, we synthesize the knowledge of newly generated cells in the adult hypothalamus. The aim of this review is to provide new insights on adult neurogenesis in the mammalian hypothalamus, with particular attention given to marsupial species. We highlight the number of adult-born neurons in various hypothalamic nuclei, debating whether their low number has an impact on hypothalamic function.

Postnatal and Adult Neurogenesis in Mammals, Including Marsupials.

In mammals, neurogenesis occurs during both embryonic and postnatal development. In eutherians, most brain structures develop embryonically; conversely, in marsupials, a number of brain structures develop after birth. The exception is the generation of granule cells in the dentate gyrus, olfactory bulb, and cerebellum of eutherian species. The formation of these structures starts during embryogenesis and continues postnatally. In both eutherians and marsupials, neurogenesis continues in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus of the hippocampal formation throughout life. The majority of proliferated cells from the SVZ migrate to the olfactory bulb, whereas, in the dentate gyrus, cells reside within this structure after division and differentiation into neurons. A key aim of this review is to evaluate advances in understanding developmental neurogenesis that occurs postnatally in both marsupials and eutherians, with a particular emphasis on the generation of granule cells during the formation of the olfactory bulb, dentate gyrus, and cerebellum. We debate the significance of immature neurons in the piriform cortex of young mammals. We also synthesize the knowledge of adult neurogenesis in the olfactory bulb and the dentate gyrus of marsupials by considering whether adult-born neurons are essential for the functioning of a given area.

Impaired olfactory neurogenesis affects the performance of olfactory-guided behavior in aged female opossums.

Increasing evidence has indicated that adult neurogenesis contributes to brain plasticity, although function of new neurons is still under debate. In opossums, we performed an olfactory-guided behavior task and examined the association between olfactory discrimination-guided behavior and adult neurogenesis in the olfactory bulb (OB). We found that young and aged opossums of either sex learned to find food buried in litter using olfactory cues. However, aged females required more time to find food compared to aged males and young opossums of both sexes. The levels of doublecortin, that is used as a marker for immature neurons, were the lowest in the OB of aged female opossums. Another protein, HuD that is associated with learning and memory, was detected in all layers of the OB, except the granule cell layer, where a high density of DCX cells was detected. The level of HuD was higher in aged opossums compared to young opossums. This indicates that HuD is involved in plasticity and negatively regulates olfactory perception. The majority of 2-year-old female opossums are in the post-reproductive age but males of this age are still sexually active. We suggest that in aged female opossums neural plasticity induced by adult neurogenesis decreases due to their hormonal decline.

Downregulation of TrkC Receptors Increases Dendritic Arborization of Purkinje Cells in the Developing Cerebellum of the Opossum, Monodelphis domestica.

In therian mammals, the cerebellum is one of the late developing structures in the brain. Specifically, the proliferation of cerebellar granule cells occurs after birth, and even in humans, the generation of these cells continues during the first year of life. The main difference between marsupials and eutherians is that the majority of the brain structures in marsupials develop after birth. Herein, we report that in the newborn laboratory opossum (Monodelphis domestica), the cerebellar primordium is distinguishable in Nissl-stained sections. Additionally, bromodeoxyuridine birthdating experiments revealed that the first neurons form the deep cerebellar nuclei (DCN) and Purkinje cells, and are generated within postnatal days (P) 1 and 5. Three weeks after birth, progenitors of granule cells in the external germinal layer (EGL) proliferate, producing granule cells. These progenitor cells persist for a long time, approximately 5 months. Furthermore, to study the effects of neurotrophic tropomyosin receptor kinase C (TrkC) during cerebellar development, cells were obtained from P3 opossums and cultured for 8 days. We found that TrkC downregulation stimulates dendritic branching of Purkinje neurons, which was surprising. The number of dendritic branches was higher in Purkinje cells transfected with the shRNA TrkC plasmid. However, there was no morphological change in the number of dendritic branches of granule cells transfected with either control or shRNA TrkC plasmids. We suggest that inhibition of TrkC activity enables NT3 binding to the neurotrophic receptor p75NTR that promotes dendritic arborization of Purkinje cells. This effect of TrkC receptors on dendritic branching is cell type specific, which could be explained by the strong expression of TrkC in Purkinje cells but not in granule cells. The data indicate a new role for TrkC receptors in Monodelphis opossum.

Aged Opossums Show Alterations in Spatial Learning Behavior and Reduced Neurogenesis in the Dentate Gyrus.

In many mammalian species including opossums, adult neurogenesis, the function of which is not completely understood, declines with aging. Aging also causes impairment of cognition. To understand whether new neurons contribute to learning and memory, we performed experiments on young and aged laboratory opossums, Monodelphis domestica, and examined the association between spatial memory using the Morris water maze test and the rate of adult neurogenesis in the dentate gyrus (DG). Modification of this test allowed us to assess how both young and aged opossums learn and remember the location of the platform in the water maze. We found that both young and aged opossums were motivated to perform this task. However, aged opossums needed more time to achieve the test than young opossums. Classical parameters measuring spatial learning in a water maze during a probe test showed that young opossums spent more time in the platform zone crossing it more often than aged opossums. Additionally, hippocampal neurogenesis was lower in the aged opossums than in the young animals but new neurons were still generated in the DG of aged opossums. Our data revealed individual differences in the levels of doublecortin in relation to memory performance across aged opossums. These differences were correlated with distinct behaviors, particularly, aged opossums with high levels of DCX achieved high performance levels in the water maze task. We, therefore suggest that new neurons in the DG of Monodelphis opossums contribute to learning and memory.

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.

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.

Development and plasticity of the retina in the opossum Monodelphis domestica.

We investigated the rate of cell proliferation and death in the retina of the Monodelphis opossum during its postnatal development and the influence of early monocular enucleation on these processes. Our results show that in the opossum, as in other marsupials, the peak of the retinal cells divisions occurs postnatally and that generation of retinal cells continues till the time of eye opening (P34), except of the marginal rim, where it continued till P60. Ganglion and amacrine cells are generated between postnatal days (P) P4 and P9, while bipolar cells and photoreceptors are generated simultaneously between P14 and P25. The peak of ganglion cell death as detected by the TUNEL method occurs around P14-19 in the center of retina. The second peak of apoptosis appears in the inner nuclear layer (INL) at P19-25. Gliogenesis takes place between P25 and P34. We also found that monocular enucleation performed during the early period of retinal development (P0-P7) did not influence proliferation, developmental apoptosis or other developmental processes in the retina of the remaining eye.

Interleukin 6 deficiency affects spontaneous activity of mice in age- and sex-dependent manner.

We analyzed the role of interleukin 6 (IL-6) in modulation of the pattern of mice spontaneous activity. Wild type (WT) and IL-6 deficient mice of both sexes, young and aging, were housed individually and various types of their activity were recorded and analyzed with the Phenorack system in their home cages during 72 hours-long sessions. All investigated groups of mice were active mainly during the dark phase of the 24-hours cycle. Generally, the IL-6 deficient animals were more active than their WT controls and females of both genotypes more active than males. Aging mice were less active than the sex and genotype-matched young animals. The independent variables (age, sex and genotype) strongly interacted, which suggests that the modulatory influence of IL-6 on mice behavior may be different in males and females and that it changes during aging. We conclude that under normal physiological conditions signaling of IL-6 via its receptor participates in modulation of the basic pattern of activity. This modulation differs in males and females and changes with aging.

Correlation between dopaminergic phenotype and expression of calretinin in the midbrain nuclei of the opossum (Monodelphis domestica): an immunohistological study.

We investigated distribution and morphology of neurons of the midbrain nuclei: the ventral tegmental area (VTA), substantia nigra (SN) and periaqueductal gray (PAG) of the adult grey short-tailed opossums that were double immunolabeled for the presence of calretinin (CR) and/or tyrosine hydroxylase (TH). The majority of TH-immunopositive neurons and fibers were located in the VTA, SN, and only scarce population of small neurons expressing TH was present in the PAG. In the SN 80 percent of TH-expressing neurons had large cell bodies, and only a small fraction had small perikarya. In the PAG populations of large and medium sized neurons were equal and 20 percent of neurons had small perikarya. Much scarcer population of TH-immunoreactive neurons in the PAG consisted of large or small neurons in its dorsal part (PAGd) and almost exclusively small neurons in the ventral part (PAGv). Distribution of neurons expressing TH and their types in the opossum are similar to those in rodents. The majority of CR-immunolabeled neurons were found in the VTA. In its subdivision, the parabrachal pigmented nucleus (PBP) cells expressing CR were approximately 28 percent more numerous than cells expressing TH. In spite of that, only 42 percent of TH-expressing neurons coexpressed CR. The high degree of colocalization TH and CR was observed in the SN. We propose that a higher percentage of TH/CR colocalization, which is observed in the opossums SN, may give them the ability to adapt to changes in their motor functions.

Circadian rhythm of outside-nest activity in wild (WWCPS), albino and pigmented laboratory rats.

The domestication process of the laboratory rat has been going on for several hundred generations in stable environmental conditions, which may have affected their physiological and behavioural functions, including their circadian system. Rats tested in our ethological experiments were laboratory-bred wild Norway rats (WWCPS), two strains of pigmented laboratory rats (Brown Norway and Long Evans), and two strains of albino rats (Sprague-Dawley and Wistar). Rats were placed in purpose-built enclosures and their cycle of activity (time spent actively outside the nest) has been studied for one week in standard light conditions and for the next one in round-the-clock darkness. The analysis of circadian pattern of outside-nest activity revealed differences between wild, pigmented laboratory, and albino laboratory strains. During daytime, albino rats showed lower activity than pigmented rats, greater decrease in activity when the light was turned on and greater increase in activity when the light was switched off, than pigmented rats. Moreover albino rats presented higher activity during the night than wild rats. The magnitude of the change in activity between daytime and nighttime was also more pronounced in albino rats. Additionaly, they slept outside the nest more often during the night than during the day. These results can be interpreted in accordance with the proposition that intense light is an aversive stimulus for albino rats, due to lack of pigment in their iris and choroid, which reduces their ability to adapt to light. Pigmented laboratory rats were more active during lights on, not only in comparison to the albino, but also to the wild rats. Since the difference seems to be independent of light intensity, it is likely to be a result of the domestication process. Cosinor analysis revealed a high rhythmicity of circadian cycles in all groups.

Different strategies of exploration and phenotypic variability of the locomotor behavior in new environment: Comparative study of the laboratory opossum (Monodelphis domestica) and Wistar rat (Rattus norvegicus).

Spontaneous locomotor activity of opossums and Wistar rats during a two-hour session in the open field has been recorded, assessed and behavior of individuals of the two species compared. Afterwards, groups of highly active (HA) and low active (LA) opossums and rats were selected on the basis of the distance traveled in the test. Differences between the selected groups were evaluated. Opossums were generally more active, moving faster and covering longer distance. They spent more time in the central part of the open field and traveled across the center more times than rats, therefore they showed also a lower level of anxiety. These data confirm our previous results indicating that opossums preferentially use the risky exploration strategy while rats mainly rely on the defensive behavior. Opossums showed a higher variability of the volume of locomotor activity than rats. Comparison of the HA and LA groups of opossums and rats showed that in each species they differed on another principle: the level of anxiety in Wistar rats and level of locomotor activity in opossums. Therefore results of the open field test might measure different parameters in different species.

Response to novelty in the laboratory Wistar rat, wild-captive WWCPS rat, and the gray short-tailed opossum (Monodelphis domestica).

Behavior of the laboratory gray short-tailed opossums (Monodelphis domestica), Warsaw Wild Captive Pisula Stryjek rats (WWCPS) and laboratory rats (Wistar) has been registered in the period of familiarization with a new environment and consecutive confrontation with a novel, innocuous object placed in that familiarized environment. In the new environment the sequence of anxiety, investigation, and habituation was shortest in the opossum, longer in the laboratory rat and longest in the WWCPS rat. When placed in it, gray short-tailed opossums investigated the new environment with the shortest delay and most intensity. In reaction to novel objects, opossums and laboratory rats prolonged the time spent in the proximity of the new object, while the WWCPS rat did not show that reaction. Both opossums and laboratory rats increased the number of contacts with the new object, whereas WWCPS rats reduced those contacts. Behavior of all three species and lines grouped in different clusters. Some other quantitative and qualitative differences in behavior of the investigated animals are also described, showing a higher level of anxiety in both lines of rats than in the opossum. Behavioral differences between species and lines of animals used in this study may be attributed to different ecological adaptations of rats and opossums and to the effect of domestication in the laboratory rats. These behavioral differences make comparisons of opossums vs rat, and wild rat vs laboratory rat interesting models for studying the brain mechanisms of anxiety and neotic motivations.