Zebrafish embryonically exposed to valproic acid present impaired retinal development and sleep behavior.

Prenatal exposure to valproic acid (VPA), a drug widely used to treat epilepsy and bipolar disorder, is an environmental risk factor for autism spectrum disorder (ASD). VPA has been used to reproduce the core symptoms of ASD in animal model organisms, including zebrafish. Visual system functioning is essential in the interpretation of social conditions and plays an important role of several behavioral responses. We hypothesized that behavioral deficits displayed by ASD patients may involve impaired visual processing. We used zebrafish as model organism to investigate the visual system after embryonic exposure to VPA using histological, behavioral and gene expression analysis. We analyzed the pineal gland of zebrafish and sleep-like behavior to study how VPA exposure alters photo-sensibility of zebrafish. VPA-exposed zebrafish showed a delay in the development of the retina and optic nerve, which normalized at five days post fertilization. At larval stage, VPA-exposed zebrafish showed sleep disturbances associated with a reduced number of serotonin-producing cells of the pineal gland. In addition, the number of hypocretin/orexin (hcrt) expressing neurons in the rostral hypothalamus at 6 and 14 days post fertilization was reduced. In conclusion, we demonstrated that although VPA exposure leads to a delay in visual system development, it does not affect larval visual function. The novel finding that VPA alters significantly cells involved in sleep regulation and the sleep-like state itself may be relevant for understanding sleep disturbances in ASD patients.

Oligodendrocyte origin and development in the zebrafish visual system.

Oligodendrocytes are the myelinating cells in the central nervous system. In birds and mammals, the oligodendrocyte progenitor cells (OPCs) originate in the preoptic area (POA) of the hypothalamus. However, it remains unclear in other vertebrates such as fish. Thus, we have studied the early progression of OPCs during zebrafish visual morphogenesis from 2 days post fertilization (dpf) until 11 dpf using the olig2:EGFP transgenic line; and we have analyzed the differential expression of transcription factors involved in oligodendrocyte differentiation: Sox2 (using immunohistochemistry) and Sox10 (using the transgenic line sox10:tagRFP). The first OPCs (olig2:EGFP/Sox2) were found at 2 dpf in the POA. From 3 dpf onwards, these olig2:EGFP/Sox2 cells migrate to the optic chiasm, where they invade the optic nerve (ON), extending toward the retina. At 5 dpf, olig2:EGFP/Sox2 cells in the ON also colocalize with sox10:tagRFP. When olig2:EGFP cells differentiate and present more projections, they become positive only for sox10:tagRFP. olig2:EGFP/sox10: tagRFP cells ensheath the ON by 5 dpf when they also become positive for a myelin marker, based on the mbpa:tagRFPt transgenic line. We also found olig2:EGFP cells in other regions of the visual system. In the central retina at 2 dpf, they are positive for Sox2 but later become restricted to the proliferative germinal zone without this marker. In the ventricular areas of the optic tectum, olig2:EGFP cells present Sox2 but arborized ones sox10:tagRFP instead. Our data matches with other models, where OPCs are specified in the POA and migrate to the ON through the optic chiasm.

Doublecortin in the Fish Visual System, a Specific Protein of Maturing Neurons.

Doublecortin (DCX) is a microtubule associated protein, essential for correct central nervous system development and lamination in the mammalian cortex. It has been demonstrated to be expressed in developing-but not in mature-neurons. The teleost visual system is an ideal model to study mechanisms of adult neurogenesis due to its continuous life-long growth. Here, we report immunohistochemical, in silico, and western blot analysis to detect the DCX protein in the visual system of teleost fish. We clearly determined the expression of DCX in newly generated cells in the retina of the cichlid fish Astatotilapia burtoni, but not in the cyprinid fish Danio rerio. Here, we show that DCX is not associated with migrating cells but could be related to axonal growth. This work brings to light the high conservation of DCX sequences between different evolutionary groups, which make it an ideal marker for maturing neurons in various species. The results from different techniques corroborate the absence of DCX expression in zebrafish. In A. burtoni, DCX is very useful for identifying new neurons in the transition zone of the retina. In addition, this marker can be applied to follow axons from maturing neurons through the neural fiber layer, optic nerve head, and optic nerve.

Organization of radial glia reveals growth pattern in the telencephalon of a percomorph fish Astatotilapia burtoni.

In the brain of teleost fish, radial glial cells are the main astroglial cell type. To understand how radial glia structures are adapting to continuous growth of the brain, we studied the astroglial cells in the telencephalon of the cichlid fish Astatotilapia burtoni in small fry to large specimens. These animals grow to a standard length of 10-12 cm in this fish species, corresponding to a more than 100-fold increase in brain volume. Focusing on the telencephalon where glial cells are arranged radially in the everted (dorsal) pallium, immunocytochemistry for glial markers revealed an aberrant pattern of radial glial fibers in the central division of the dorsal pallium (DC, i.e., DC4 and DC5). The main glial processes curved around these nuclei, especially in the posterior part of the telencephalon. This was verified in tissue-cleared brains stained for glial markers. We further analyzed the growth of radial glia by immunocytochemically applied stem cell (proliferating cell nuclear antigen [PCNA], Sox2) and differentiation marker (doublecortin) and found that these markers were expressed at the ventricular surface consistent with a stacking growth pattern. In addition, we detected doublecortin and Sox2 positive cells in deeper nuclei of DC areas. Our data suggest that radial glial cells give rise to migrating cells providing new neurons and glia to deeper pallial regions. This results in expansion of the central pallial areas and displacement of existing radial glial. In summary, we show that radial glial cells can adapt to morphological growth processes in the adult fish brain and contribute to this growth.

Cultures of glial cells from optic nerve of two adult teleost fish: Astatotilapia burtoni and Danio rerio.

BACKGROUND: In vitro studies are very useful to increase the knowledge of different cell types and could be the key to understand cell metabolism and function. Fish optic nerves (ON) can recover visual functions by reestablishing its structure and reconnecting the axons of ganglion cells. This is because fish show spontaneous regeneration of the central nervous system which does not occur in mammals. In addition, several studies have indicated that glial cells of ON have different properties in comparison to the glial cells from brain or retina. Consequently, providing an in vitro tool will be highly beneficial to increase the knowledge of these cells. NEW METHOD: We developed a cell culture protocol to isolate glial cells from ON of two teleost fish species, Danio rerio and Astatotilapia burtoni. RESULTS: The optimized protocol allowed us to obtain ON cells and brain-derived cells from adult teleost fish. These cells were characterized as glial cells and their proprieties in vitro were analyzed.Comparison with Existing Method(s): Although it is striking that ON glial cells show peculiarities, their study in vitro has been limited by the only published protocol going back to the 1990s. Our protocol makes glial cells of different fish species available for experiments and studies to increase the understanding of these glial cell types. CONCLUSIONS: This validated and effective in vitro tool increases the possibilities on studies of glial cells from fish ON which implies a reduction in animal experimentation.

Sox2 expression in the visual system of two teleost species.

The visual system of teleost fish shows growth and regeneration capacities during the entire animal's life. Thus, the visual system of adult fish serves as a model for studying neurogenesis in the vertebrate central nervous system (CNS). Our study focused on the expression pattern of Sox2 in the fish visual system. Sox2 is a transcription factor known for its function in keeping stem cell properties, and as a regulator of cell fate during development, especially in the visual system. We used two different fish species: Astatotilapia burtoni and Danio rerio. In the visual system of fish, we identified Sox2 positive cells in the stem cell niche in the peripheral retina, in Müller cells and amacrine cells in the differentiated retina, and glial cells in the optic nerve (ON). We did not observe hardly any Sox2 expression in the optic nerve head (ONH). In the ON, Sox2 positive glial cells were lining the fascicles of new axons. Taking together, the broad spectrum of Sox2 expression indicates that this protein has different functions in the CNS of adult vertebrates. The results suggest that Sox2 has functions associated with the pathway of new axons from the retina. To understand the variety of cell types and subtypes and their plasticity potential in the visual system of fish will be essential to comprehend the growing and regenerating CNS in adult vertebrates.