The proliferation of neural stem cells (NSCs)/neuronal precursor cells (NPCs) and the occurrence of postmitotic neuroblasts in the mesencephalic tegmentum of intact juvenile chum salmon, Oncorhynchus keta, and at 3 days after a tegmental injury, were studied by immunohistochemical labeling. BrdU+ constitutive progenitor cells located both in the periventricular matrix zone and in deeper subventricular and parenchymal layers of the brain are revealed in the tegmentum of juvenile chum salmon. As a result of traumatic damage to the tegmentum, the proliferation of resident progenitor cells of the neuroepithelial type increases. Nestin-positive and vimentin-positive NPCs and granules located in the periventricular and subventricular matrix zones, as well as in the parenchymal regions of the tegmentum, are revealed in the mesencephalic tegmentum of juvenile chum salmon, which indicates a high level of constructive metabolism and constitutive neurogenesis. The expression of vimentin and nestin in the extracellular space, as well as additionally in the NSCs and NPCs of the neuroepithelial phenotype, which do not express nestin in the control animals, is enhanced during the traumatic process. As a result of the proliferation of such cells in the post-traumatic period, local Nes+ and Vim+ NPCs clusters are formed and become involved in the reparative response. Along with the primary traumatic lesion, which coincides with the injury zone, additional Nes+ and Vim+ secondary lesions are observed to form in the adjacent subventricular and parenchymal zones of the tegmentum. In the lateral tegmentum, the number of doublecortin-positive cells is higher compared to that in the medial tegmentum, which determines the different intensities and rates of neuronal differentiation in the sensory and motor regions of the tegmentum, respectively. In periventricular regions remote from the injury, the expression of doublecortin in single cells and their groups significantly increases compared to that in the damage zone.

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To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, Oncorhynchus keta. AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile O. keta was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.

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