MDPV (3,4-methylenedioxypyrovalerone) administered to mice during development of the central nervous system produces persistent learning and memory impairments.

BACKGROUND: Synthetic cathinones (SC) constitute the second most frequently abused class of new psychoactive substances. They serve as an alternative to classic psychostimulatory drugs of abuse, such as methamphetamine, cocaine, or 3,4-methylenedioxymethamphetamine (MDMA). Despite the worldwide prevalence of SC, little is known about their long-term impact on the central nervous system. Here, we examined the effects of repeated exposure of mice during infancy, to 3,4-methylenedioxypyrovalerone (MDPV), a SC potently enhancing dopaminergic neurotransmission, on learning and memory in young adult mice. METHODS: All experiments were performed on C57BL/6J male and female mice. Animals were injected with MDPV (10 or 20 mg/kg) and BrdU (bromodeoxyuridine, 25 mg/kg) during postnatal days 11-20, which is a crucial period for the development of their hippocampus. At the age of 12 weeks, mice underwent an assessment of various types of memory using a battery of behavioral tests. Afterward, their brains were removed for detection of BrdU-positive cells in the dentate gyrus of the hippocampal formation with immunohistochemistry, and for measurement of the expression of synaptic proteins, such as synaptophysin and PSD95, in the hippocampus using Western blot. RESULTS: Exposure to MDPV resulted in impairment of spatial working memory assessed with Y-maze spontaneous alternation test, and of object recognition memory. However, no deficits in hippocampus-dependent spatial learning and memory were found using the Morris water maze paradigm. Consistently, hippocampal neurogenesis and synaptogenesis were not interrupted. All observed MDPV effects were sex-independent. CONCLUSIONS: MDPV administered repeatedly to mice during infancy causes learning and memory deficits that persist into adulthood but are not related to aberrant hippocampal development.

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.

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.

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.

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.

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

[This corrects the article DOI: 10.3389/fnana.2020.00056.].

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.

Roles of the exon junction complex components in the central nervous system: a mini review.

The exon junction complex (EJC) consists of four core proteins: Magoh, RNA-binding motif 8A (Rbm8a, also known as Y14), eukaryotic initiation factor 4A3 (eIF4A3, also known as DDX48), and metastatic lymph node 51 (MLN51, also known as Casc3 or Barentsz), which are involved in the regulation of many processes occurring between gene transcription and protein translation. Its main role is to assemble into spliceosomes at the exon-exon junction of mRNA during splicing. It is, therefore, a range of functions concerning post-splicing events such as mRNA translocation, translation, and nonsense-mediated mRNA decay (NMD). Apart from this, proteins of the EJC control the splicing of specific pre-mRNAs, for example, splicing of the mapk transcript. Recent studies support essential functions of EJC proteins in oocytes and, after fertilization, in all stages of zygote development, as well as the growth of the embryo, including the development of the nervous system. During the development of the central nervous system (CNS), the EJC controls mitosis, regulating both symmetric and asymmetric cell divisions. Reduced levels of EJC components cause microcephaly. In the adult brain, Y14 and eIF4A3 appear to be involved in synaptic plasticity and in learning and memory. In this review, we focus on the involvement of EJC components in brain development and its functioning under normal conditions.

CacyBP/SIP, a Hsp90 binding chaperone, in cellular stress response.

CacyBP/SIP interacts with Hsp90 and is able to protect proteins from denaturation and/or aggregation induced by elevated temperature. In this work we studied the influence of different stress factors on CacyBP/SIP level in HEp-2 cells. We have found that H2O2 and radicicol treatment resulted in a significant increase (up to 40%) in the CacyBP/SIP level. We have also found that HEp-2 cells overexpressing CacyBP/SIP were more resistant to stress-induced death. Further studies have revealed that the Hsf1 transcription factor binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression under stress conditions. To check whether the CacyBP/SIP protein might play a role in stress responses in vivo, we analyzed its level in selected brain structures of control and stressed mice. We have found that the level of the CacyBP/SIP protein was higher in the thalamus/hypothalamus, hippocampus and brainstem of stressed mice. Thus, the presented results clearly indicate that CacyBP/SIP is involved in cellular stress response.

Stress-Dependent Changes in the CacyBP/SIP Interacting Protein S100A6 in the Mouse Brain.

The CacyBP/SIP target S100A6 is widely present in the nervous system, and its up-regulation is associated with certain neurodegenerative diseases. Here, we examined the involvement of S100A6 protein in stress responses in mice. Using Western blotting, we observed a marked change in brainstem structures, whereby stressed mice showed approximately one-third the protein level produced in the control group. A decreased level of S100A6 protein in stressed animals was also detected in the olfactory bulb and the cerebellum and stress-related structures such as the hippocampus and the hypothalamus. Additionally, using immunohistochemistry, high levels of S100A6 expression were observed in astrocytes localized in the border zones of all brain ventricles, tanycytes of the ventro-lateral walls of the hypothalamus, including the arcuate nucleus (ARH) and low levels of this protein were in neurons of the olfactory bulb, the hippocampus, the thalamus, the cerebral cortex, the brainstem and the cerebellum. Although S100A6-expressing cells in all these brain structures did not change their phenotype in response to stress, the intensity of immunofluorescent labeling in all studied structures was lower in stressed mice than in control animals. For example, in the ARH, where extremely strong immunostaining was observed, the number of immunolabeled fibers was decreased by approximately half in the stressed group compared with the controls. Although these results are descriptive and do not give clue about functional role of S100A6 in stress, they indicate that the level of S100A6 decreases in several brain structures in response to chronic mild stress, suggesting that this protein may modify stress responses.

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

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

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

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

Lipopolysaccharide injected to pregnant mice affects behavior of their offspring in adulthood.

We studied consequences of maternal immune response on the course of pregnancy and the behavior of adult offspring. Mice in late gestation (day 16-17) were injected with lipopolysaccharide (LPS). Treatment of pregnant mice with high doses of LPS resulted in fetal resorption or stillbirths. Pregnant mice treated with low doses (100 or 300 micrograms/kg) of LPS gave birth to normal numbers of pups. However, behavior of the offspring was altered. Adult offspring of dams injected at a dose of 300 micrograms/kg of LPS traveled longer distances in the open field and spent more time in the central part of the arena, than mice in the control group. Female mice of this group spent more time in open arms of the elevated plus maze, in comparison to female control mice. Results of the Morris water maze test showed impairment of spatial learning and memory in male offspring born to LPS-injected dams. Furthermore, in the nest building test adult mice born from LPS challenged pregnancies constructed worse quality nests, which points to the presence of hippocampal dysfunction. These findings indicate that maternal bacterial infections during pregnancy may alter offspring behavior in adult life.

Neurotrophins and their receptors in early development of the mammalian nervous system.

Neurotrophins belonging to the class of growth factors and including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are widely recognized as essential factors in the developing central nervous system (CNS). Neurotrophins are synthesized as precursor forms (proneurotrophins). Mature forms of neurotrophins exert their effect by binding to specific tyrosine kinases receptors (TrkA, TrkB and TrkC) as well as via the p75 receptor, a member of the tumor necrosis factor receptor superfamily while proneurotrophins interact with the receptor p75 or co-receptor complex of p75 and sortilin, that is a Vps10p domain-containing transmembrane protein. Expression of neurotrophins corresponds with the onset of neurogenesis in developing mammalian species. BDNF is low in early embryonic stages of development, while NT-3 highly expresses in the developing CNS. Expression of neurotrophins receptors mainly overlaps at early development. Data concerning early distribution of neurotrophins and their receptors in the nervous system and results in mice with targeted disruptions of neurotrophin or receptor genes show that neurotrophins and their receptors play distinct roles in control and regulation of the most crucial developmental processes such as proliferation, migration, differentiation, survival, apoptosis and synaptic plasticity.

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.

Generation recruitment and death of brain cells throughout the life cycle of Sorex shrews (Lipotyphla).

Young shrews of the genus Sorex that are born in early summer reduce their body size before wintering, including a reduction of brain weight of 10-30%. In the spring they mature sexually, double their body weight and regain about half of the loss in brain weight. To investigate the mechanisms of brain weight oscillations we studied the rate of cell death and generation in the brain during the whole life cycle of the common shrew (Sorex araneus) and pygmy shrew (S. minutus). After weaning, shrews generate new brain cells in only two mammalian neurogenic zones and approximately 80% of these develop into neurones. The increase of the shrew brain weight in the spring did not depend on recruitment of new cells. Moreover, adult Sorex shrews did not generate new cells in the dentate gyri. Injections of 5-HT1A receptor agonists in the adult shrews induced neurogenesis in their dentate gyri, showing the presence of dormant progenitor cells. Generation of new neurones in the subventricular zone of the lateral ventricles and their recruitment to olfactory bulbs continued throughout life. TUNEL labelling showed that the rate of cell death in all brain structures, including the proliferation zones and olfactory bulb, was very low throughout life. We conclude that neither cell death nor recruitment significantly contributes to seasonal oscillations and the net loss of brain weight in the Sorex shrews. With the exception of dentate gyrus and olfactory bulb, cellular populations of brain structures are stable throughout the life cycle of these shrews.

Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development.

Increasing evidence indicates that development of embryonic central nervous system precursors is tightly regulated by extrinsic cues located in the local environment. Here, we asked whether neurotrophin-mediated signaling through Trk tyrosine kinase receptors is important for embryonic cortical precursor cell development. These studies demonstrate that inhibition of TrkB (Ntrk2) and/or TrkC (Ntrk3) signaling using dominant-negative Trk receptors, or genetic knockdown of TrkB using shRNA, caused a decrease in embryonic precursor cell proliferation both in culture and in vivo. Inhibition of TrkB/C also caused a delay in the generation of neurons, but not astrocytes, and ultimately perturbed the postnatal localization of cortical neurons in vivo. Conversely, overexpression of BDNF in cortical precursors in vivo promoted proliferation and enhanced neurogenesis. Together, these results indicate that neurotrophin-mediated Trk signaling plays an essential, cell-autonomous role in regulating the proliferation and differentiation of embryonic cortical precursors and thus controls cortical development at earlier stages than previously thought.

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.