Fish are a convenient model for the study of reparative and post-traumatic processes of central nervous system (CNS) recovery, because the formation of new cells in their CNS continues throughout life. After a traumatic injury to the cerebellum of juvenile masu salmon, Oncorhynchus masou, the cell composition of the neurogenic zones containing neural stem cells (NSCs)/neural progenitor cells (NPCs) in the acute period (two days post-injury) changes. The presence of neuroepithelial (NE) and radial glial (RG) neuronal precursors located in the dorsal, lateral, and basal zones of the cerebellar body was shown by the immunohistochemical (IHC) labeling of glutamine synthetase (GS). Progenitors of both types are sources of neurons in the cerebellum of juvenile O. masou during constitutive growth, thus, playing an important role in CNS homeostasis and neuronal plasticity during ontogenesis. Precursors with the RG phenotype were found in the same regions of the molecular layer as part of heterogeneous constitutive neurogenic niches. The presence of neuroepithelial and radial glia GS+ cells indicates a certain proportion of embryonic and adult progenitors and, obviously, different contributions of these cells to constitutive and reparative neurogenesis in the acute post-traumatic period. Expression of nestin and vimentin was revealed in neuroepithelial cerebellar progenitors of juvenile O. masou. Patterns of granular expression of these markers were found in neurogenic niches and adjacent areas, which probably indicates the neurotrophic and proneurogenic effects of vimentin and nestin in constitutive and post-traumatic neurogenesis and a high level of constructive metabolism. No expression of vimentin and nestin was detected in the cerebellar RG of juvenile O. masou. Thus, the molecular markers of NSCs/NPCs in the cerebellum of juvenile O. masou are as follows: vimentin, nestin, and glutamine synthetase label NE cells in intact animals and in the post-traumatic period, while GS expression is present in the RG of intact animals and decreases in the acute post-traumatic period. A study of distribution of cystathionine β-synthase (CBS) in the cerebellum of intact young O. masou showed the expression of the marker mainly in type 1 cells, corresponding to NSCs/NCPs for other molecular markers. In the post-traumatic period, the number of CBS+ cells sharply increased, which indicates the involvement of H2S in the post-traumatic response. Induction of CBS in type 3 cells indicates the involvement of H2S in the metabolism of extracellular glutamate in the cerebellum, a decrease in the production of reactive oxygen species, and also arrest of the oxidative stress development, a weakening of the toxic effects of glutamate, and a reduction in excitotoxicity. The obtained results allow us to consider H2S as a biologically active substance, the numerous known effects of which can be supplemented by participation in the processes of constitutive neurogenesis and neuronal regeneration.

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10In the cerebellum of juvenile Oncorhynchus masou, proliferating BrdU+ and HuCD+ cells and 11 constitutive neurogenic niches were detected in different zones; the largest number of labeled 12 cells were found in the dorsal part of the molecular layer and the dorsal matrix zone (DMZ). 13Cells labeled with glutamine synthetase (GS) and radial glia were also present in the intact O. 14 masou cerebellum. The most intensive proliferation was detected in the rostral part of 15 cerebellum. This part is assumed to contain active zones of constitutive neurogenesis. After an 16 injury inflicted to the cerebellum, the number of BrdU+ and HuCD+ cells increased 17 significantly. The number of BrdU+ cells after this type of injury was much greater than after a 18 telencephalon trauma. A quantitative analysis revealed that after the cerebellum injury the 19 proliferative activity in the caudal part of CCb is increased compared to that in the control. A 20 reactivation of neurogenic and neuroepithelial niches and their transformation into reactive 21 neurogenic domains, with an increased distribution density of intensely labeled HuCD+ cells of 22 different types, were observed. The increase in the number of HuCD+ differentiated cells in the 23 basal area suggests that the processes of neuronal differentiation are intensified in the cerebellum 24 of juvenile O. masou after injury. The number of GS positive cells (GS+) and fibers increased in 25 all the zones of cerebellum. The most intensive astrocytic response was noted in the dorsal part 2 26 of cerebellum. The data of the enzyme immunoassay confirm the multiple variations in the level 27 of GS after a traumatic injury to cerebellum in O. masou. 28

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The fish brain has a unique feature of vertebrates-it grows with the growth of body over a lifetime. In this regard, fishes are a convenient model for the study of embryonic and postembryonic development of the central nervous system and of the influence of different factors on these processes. Currently, the mechanisms of adult brain morphogenesis of fish, which retain larval stage for a long time, are poorly understood. This is particularly true for participation of radial glia during morphogenesis of the brain, as well as the presence and distribution of the proliferative zone in the adult fish brain. Another interesting and little known aspect is the posttraumatic ability of fish to form active neurogenic niches. Investigation of the structural organizations of neurogenic niches and special conditions of the extracellular environment, as well as the interactions between neighboring cells in a neurogenic niche, is interesting and relevant direction in the study of the neuronal stem cells biology. Injury of fish brain creates special conditions for the implementation of genetic programs aimed at strengthening the proliferation of progenitor cells, as well as the activation and proliferation activity in the neuronal stem cells.

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Corpus cerebelli in juvenile chum salmon is a multiprojective region of the brain connected via afferent and efferent projections with the higher regions of the brainstem and synencephalon, as well as with multiprojection regions of the medulla oblongata and spinal cord. During the postembryonic development of the cerebellum in chum salmon, Oncorhynchus keta, the lateral part of the juvenile cerebellum gives rise to the caudomedial part of the definitive cerebellum, which is consistent with the data reported for zebrafish and mouse cerebellum. Thus, the topographic organization of the cerebellum and its efferents are similar between fish (chum salmon and zebrafish) and mammals, including mice and humans. The distributions of recombinant adeno-associated viral vectors (rAAVs) after an injection of the base vector into the cerebellum have shown highly specific patterns of transgene expression in bipolar neurons in the latero-caudal lobe of the juvenile chum tectum opticum. The distribution of rAAVs in the dorsal thalamus, epithalamus, nucleus rotundus, and pretectal complex indicates the targeted distribution of the transgene via the thalamo-cerebellar projections. The detection of GFP expression in the cells of the epiphysis and posterior tubercle of juvenile chum salmon is associated with the transgene’s distribution and with the cerebrospinal fluid flow, the brain ventricles and its outer surface. The direct delivery of the rAAV into the central nervous system by intracerebroventricular administration allows it to spread widely in the brain. Thus, the presence of special projection areas in the juvenile chum salmon cerebellum, as well as outside it, and the identification of the transgene’s expression in them confirm the potential ability of rAAVs to distribute in both intracerebellar and afferent and efferent extracerebellar projections of the cerebellum.

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