Processes of Proliferation and Apoptosis in the Brain of the Amur Sturgeon

Using techniques of immunoperoxidase staining of proliferating cell nuclear antigen (PCNA) and TUNEL labeling of fragmented DNA, we studied sites of proliferation and apoptosis in the myelencephalon, cerebellum, tectum opticum, thalamus, and hypothalamus of the Amur sturgeon (Acipenser schrenckii). We found that the processes of proliferation and apoptosis are maintained in the brain of 3-year-old sturgeon individuals; the ratio of these processes in different cerebral regions varied significantly. The maximum intensity of proliferative activity was found in the periventricular zone of the myelencephalon (proliferation index, on average, 21.0 ± 1.3%). This fact allows us to consider this cerebral region a most important zone were adult neurogenesis occurs in the sturgeon. In the medial reticular formation, dorsal thalamic nuclei, inner fibrous layer of the tectum, and lateral hypothalamus, the maximum numbers of apoptotic elements were found. Therefore, these zones in the brain of the sturgeon correspond, apparently, to the regions where postmitotic neuroblasts are localized. In sensory centers (tectum and nuclei of the V, VII, and X nerves), significantly varying ratios of intensities of proliferation and apoptosis were found; this is indicative of dissimilar rates of growth and differentiation in visual and chemosensory centers of the sturgeon brain. The high proliferative activity in sensory and motor cerebral centers of the sturgeon allows us to hypothesize that a neotenic pattern is preserved in these CNS regions of adult sturgeons over a long period after the embryogenesis has been completed.     

Mapping neurogenesis onset in the optic tectum of Xenopus laevis

Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior‐posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increasing restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1328–1341, 2016     

Neurogenesis and neuronal regeneration in the adult fish brain

Fish are distinctive in their enormous potential to continuously produce new neurons in the adult brain, whereas in mammals adult neurogenesis is restricted to the olfactory bulb and the hippocampus. In fish new neurons are not only generated in structures homologous to those two regions, but also in dozens of other brain areas. In some regions of the fish brain, such as the optic tectum, the new cells remain near the proliferation zones in the course of their further development. In others, as in most subdivisions of the cerebellum, they migrate, often guided by radial glial fibers, to specific target areas. Approximately 50% of the young cells undergo apoptotic cell death, whereas the others survive for the rest of the fish’s life. A large number of the surviving cells differentiate into neurons. Two key factors enabling highly efficient brain repair in fish after injuries involve the elimination of damaged cells by apoptosis (instead of necrosis, the dominant type of cell death in mammals) and the replacement of cells lost to injury by newly generated ones. Proteome analysis has suggested well over 100 proteins, including two dozen identified ones, to be involved in the individual steps of this phenomenon of neuronal regeneration.     

Optic nerve input‐dependent regulation of neural stem cell proliferation in the optic tectum of adult zebrafish

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial‐type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU‐positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU‐positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli‐dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Neurogenesis during optic tectum regeneration in Xenopus laevis

The regenerative neurogenesis of the optic tectum of larval Xenopus laevis has been studied analyzing the proliferative and morphogenetic phases of the regeneration process after removal of one optic lobe. To this end, short‐term and long‐term pulses were carried out using the thymidine analog BrdU, selectively incorporated into cells during the S phase of the cell cycle. Results indicate that while in early larvae (stage 49/50, according to Nieuwkoop & Faber 1967 ) regeneration occurs mainly at the expense of the stem cells present in extensive proliferation zones (“matrix areas”) of the midbrain, in late larvae (stage 55/56) regeneration occurs at the expense of stem cells present in very limited matrix areas of the brain and of quiescent cells, which re‐enter the cell cycle following trauma. Moreover, in early larvae, morphogenesis of the optic tectum is carried out according to a precise spatio‐temporal order from rostro‐caudal to latero‐medial. By contrast, in late larvae, the topographical order of the regenerative morphogenesis of the optic lobe is completely altered. As a consequence, the regenerated optic tectum in early larvae has an apparently normal structure, while the regenerated optic tectum in late larvae lacks stratification.     

Quantitative Ultrastructural Investigations on Synaptogenesis in the Cerebellum and the Optic Tectum of Light-Reared and Dark-Reared Rainbow Trout (Salmo gairdneri Rich.)

The morphogenetic differentiation of synapses in the cerebellum and the optic tectum of darkand light- reared rainbow trout was investigated at critical stages of development. During normal differentiation the cerebellum is characterized by the appearance of 'indented', spinelike synapses. This type of synapses increases with age and prevails from day 60 on. At the same time the number of 'flat' synapses decreases. In the cerebellum the highest synaptic density (123 ± 12 synapses/1,000 μm²) is reached 30 days after hatching when the larvae begin to swim. The optic tectum is characterized by a preponderance of flat synapses in early postnatal and adult life; maximal synaptic density (66 ± 5 synapses/1,000 μm²) is reached 60 days after hatching when the larvae have reached optimal visual acuity.
Light deprivation causes a considerable and significant reduction in the number of synapses per unit area in the cerebellum and the optic tectum. The length of synaptic contacts do not change. If light-deprived, the density of synaptic vesicles decreases significantly in the optic tectum of a 25-day-old trout (74 ± 3 instead of 132 ± 7 vesicles/μm²). In the cerebellum this effect is absent.     

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Proliferative response of the mesencephalic matrix areas in the reparation of the optic tectum of Triturus cristatus carnifex

The localization and proliferative response of optic tectum matrix cells has been studied in adult newt following an experimental lesion on an optic lobe. The results show that 15 days after the lesion the cells in division, autoradiographically labelled, are located in the periventricular layer. Thirty days after the lesion the labelled cells are also found in the innermost grey layers; at 90 days the injured optic tectum regains the cytoarchitecture characteristic of this centre, with labelled cells, whether in the external or in the internal pyriform layers. In all the stages the labelled cells are also found in the periventricular layers of the controlateral optic tectum, in the dorsal pallium and in the striatum. The quantitative data exhibit the existence of a direct relationship between the number of proliferating cells in the injured optic lobe and the extent of the lesion. These data show the possibility of active cellular proliferation for the reconstruction of the lesioned nervous area and for restoration of the characteristic histological structure.     

Histochemical distribution of acid mucopolysaccharides during circadian rhythm in the cerebellum of Serranus scriba.

Histochemical analysis of AMPS (AB-staining materials) in Serranus scriba brain showed its irregular distribution in different brain regions. The highest AMPS content was observed in the optocoele and ependymal cells lining it, above the marginal telencephalon and optic tectum as well as the molecular cerebellum layer. Particularly significant AMPS concentrations were noted in Purkinje cell regions. The telencephalon and optic tectum showed AB-negative-staining materials. Following histochemical changes during a 24 h period, AMPS concentrations were shown to follow circadian variations, especially in the Purkinje cell region. The highest AMPS content was noted during the morning and the lowest during the evening hours. The above changes were discussed in the context of the role of AMPS in neural functions.     

The effect of hyperthermia on the induction of cell death in brain, testis, and thymus of the adult and developing rat

Stressful stimuli can elicit 2 distinct reactive cellular responses, the heat shock (stress) response and the activation of cell death pathways. Most studies on the effects of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of Hsps, which play roles in repair and protective mechanisms. This study examines the effect of hyperthermia on the induction of cell death via apoptosis, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and active caspase 3 cytochemistry, in the adult rat brain, testis, and thymus. Results show that a fever-like increase in temperature triggered apoptosis in dividing cell populations of testis and thymus, but not in mature, postmitotic cells of the adult cerebellum. These differential apoptotic responses did not correlate with whole-tissue levels of Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and the neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were highly susceptible to hyperthermia-induced apoptosis, suggesting that actively dividing cell populations are more prone to cell death induced by hyperthermia than fully differentiated postmitotic neural cells.     

FGF2 Delays Tectal Neurogenesis,Increases Tectal Cell Numbers,and Alters Tectal Lamination in Embryonic Chicks

Intraventricular injections of the fibroblast growth factor 2 (FGF2) are known to increase the size of the optic tectum in embryonic chicks. Here we show that this increase in tectum size is due to a delay in tectal neurogenesis, which by definition extends the proliferation of tectal progenitors. Specifically, we use cumulative labeling with the thymidine analog EdU to demonstrate that FGF2 treatment on embryonic day 4 (ED4) reduces the proportion and absolute number of unlabeled cells in the rostroventral tectum when EdU infusions are begun on ED5, as one would expect if FGF2 retards tectal neurogenesis. We also examined FGF2′s effect on neurogenesis in the caudodorsal tectum, which is born 2-3 days after the rostroventral tectum, by combining FGF2 treatment on ED4 with EDU infusions beginning on ED8. Again, FGF2 treatment reduced the proportion and number of EdU-negative (i.e., unlabeled) cells, consistent with a delay in neurogenesis. Collectively, these data indicate FGF2 in embryonic chicks delays neurogenesis throughout much of the tectum and continues to do so for several days after the FGF2 injection. One effect of this delay in neurogenesis is that tectal cell numbers more than double. In addition, tectal laminae that are born early in development become abnormally thin and cell-sparse after FGF2 treatment, whereas late-born layers remain unaffected. Combined with the results of prior work, these data indicate that FGF2 delays tectal neurogenesis and, thereby, triggers a cascade of changes in tectum size and morphology.     

The <Emphasis Type="Italic">disarrayed</Emphasis> mutation results in cell cycle and neurogenesis defects during retinal development in zebrafish

Background  

The vertebrate retina is derived from proliferative neuroepithelial cells of the optic cup. During retinal development, cell proliferation and the processes of cell cycle exit and neurogenesis are coordinated in neuroepithelial progenitor cells. Previous studies have demonstrated reciprocal influences between the cell cycle and neurogenesis. However the specific mechanisms and exact relationships of cell cycle regulation and neurogenesis in the vertebrate retina remain largely unknown.     

Effect of Nutrient Availability on Progenitor Cells in Zebrafish (Danio Rerio)

In zebrafish brains, populations of continuously proliferating cells are present during an entire life span. Under normal conditions, stem cells give rise to rapidly proliferating progenitors that quickly exit the cell cycle and differentiate. Hence fish are favorable models to study what regulates postembryonic neurogenesis. The aim of this study was to determine if optic tectum (OT) cell proliferation is halted during nutritional deprivation (ND) and whether or not it can be restored with refeeding. We examined the effect of ND on the proliferation of Neuroepithelial/Ependymal Progenitor cell (NeEPC) and transitory‐amplifying progenitors (TAPs). Following ND, no PCNA immunostaining was found in OT of starved fish, while positive cell populations of PCNA positive progenitors are found at its periphery in control fish. This indicated that active proliferation stopped. To label retaining progenitor cells, BrdU was applied and a chase‐period was accompanied by ND. Positive NeEPCs were detected in the external tectum marginal zone of starved fish suggesting that these progenitors are relatively immune to ND. Moreover in the internal tectum marginal zone labeled retaining cells were observed leaving the possibility that some arrested TAPs were present to readily restart proliferation when nutrition was returned. Our results suggest that neurogenesis was maintained during ND and that a normal proliferative situation was recovered after refeeding. We point to the mTOR pathway as a necessary pathway in progenitors to regulate their mitosis activity. Thus, this study highlights mechanisms involved in neural stem and progenitor cell homeostatic maintenance in an adverse situation. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 26–38, 2017     

Histochemical localization of acetylcholinesterase in the cerebellum and optic tectum of four freshwater teleosts.

A histochemical study has been carried out on the localization of acetylcholinesterase (AChE) in the cerebellum and optic tectum of four species of freshwater teleosts. AChE distribution in the cerebellar cortex of teleosts shows differences among the species examined and, in the trout, also differences between different cerebellar areas. This uneven kind of enzyme distribution corresponds to a similar variety of AChE patterns noticed in other vertebrates, especially mammals. AChE distribution in the optic tectum shows a prevalent pattern characterized by precise laminar distribution of enzymatic activity which is alternatively strong, weak or absent in the different tectal layers. The results suggest that most of sensitive imput and many systems of stimuli propagation may be mediated by cholinergic mechanisms in the optic tectum of telecosts.     

HDAC1 Regulates the Proliferation of Radial Glial Cells in the Developing Xenopus Tectum

In the developing central nervous system (CNS), progenitor cells differentiate into progeny to form functional neural circuits. Radial glial cells (RGs) are a transient progenitor cell type that is present during neurogenesis. It is thought that a combination of neural trophic factors, neurotransmitters and electrical activity regulates the proliferation and differentiation of RGs. However, it is less clear how epigenetic modulation changes RG proliferation. We sought to explore the effect of histone deacetylase (HDAC) activity on the proliferation of RGs in the visual optic tectum of Xenopus laevis. We found that the number of BrdU-labeled precursor cells along the ventricular layer of the tectum decrease developmentally from stage 46 to stage 49. The co-labeling of BrdU-positive cells with brain lipid-binding protein (BLBP), a radial glia marker, showed that the majority of BrdU-labeled cells along the tectal midline are RGs. BLBP-positive cells are also developmentally decreased with the maturation of the brain. Furthermore, HDAC1 expression is developmentally down-regulated in tectal cells, especially in the ventricular layer of the tectum. Pharmacological blockade of HDACs using Trichostatin A (TSA) or Valproic acid (VPA) decreased the number of BrdU-positive, BLBP-positive and co-labeling cells. Specific knockdown of HDAC1 by a morpholino (HDAC1-MO) decreased the number of BrdU- and BLBP-labeled cells and increased the acetylation level of histone H4 at lysine 12 (H4K12). The visual deprivation-induced increase in BrdU- and BLBP-positive cells was blocked by HDAC1 knockdown at stage 49 tadpoles. These data demonstrate that HDAC1 regulates radial glia cell proliferation in the developing optical tectum of Xenopus laevis.     

Development of the cerebellum and cerebellar neural circuits

The cerebellum, a structure derived from the dorsal part of the most anterior hindbrain, is important for integrating sensory perception and motor control. While the structure and development of the cerebellum have been analyzed most extensively in mammals,recent studies have shown that the anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost) species, including zebrafish. In the mammalian and teleost cerebellum,Purkinje and granule cells serve, respectively, as the major GABAergic and glutamatergic neurons. Purkinje cells originate in the ventricular zone (VZ), and receive inputs from climbing fibers. Granule cells originate in the upper rhombic lip (URL) and receive inputs from mossy fibers. Thus, the teleost cerebellum shares many features with the cerebellum of other vertebrates, and isa good model system for studying cerebellar function and development. The teleost cerebellum also has features that are specific to teleosts or have not been elucidated in mammals, including eurydendroid cells and adult neurogenesis. Furthermore, the neural circuitry in part of the optic tectum and the dorsal hindbrain closely resembles the circuitry of the teleost cerebellum; hence,these are called cerebellum-like structures. Here we describe the anatomy and development of cerebellar neurons and their circuitry, and discuss the possible roles of the cerebellum and cerebellum-like structures in behavior and higher cognitive functions. We also consider the potential use of genetics and novel techniques for studying the cerebellum in zebrafish.     

Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate

Lifelong neurogenesis in vertebrates relies on stem cells producing proliferation zones that contain neuronal precursors with distinct fates. Proliferation zones in the adult zebrafish brain are located in distinct regions along its entire anterior-posterior axis. We show a previously unappreciated degree of conservation of brain proliferation patterns among teleosts, suggestive of a teleost ground plan. Pulse chase labeling of proliferating populations reveals a centrifugal movement of cells away from their places of birth into the surrounding mantle zone. We observe tangential migration of cells born in the ventral telencephalon, but only a minor rostral migratory stream to the olfactory bulb. In contrast, the lateral telencephalic area, a domain considered homologous to the mammalian dentate gyrus, shows production of interneurons and migration as in mammals. After a 46-day chase, newborn highly mobile cells have moved into nuclear areas surrounding the proliferation zones. They often show HuC/D immunoreactivity but importantly also more specific neuronal identities as indicated by immunoreactivity for tyrosine hydroxylase, serotonin and parvalbumin. Application of a second proliferation marker allows us to recognize label-retaining, actively cycling cells that remain in the proliferation zones. The latter population meets two key criteria of neural stem cells: label retention and self renewal.