Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock.

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.

Mitral cells in the olfactory bulb of adult zebrafish (Danio rerio): morphology and distribution.

The mitral cell is the primary output neuron and central relay in the olfactory bulb of vertebrates. The morphology of these cells has been studied extensively in mammalian systems and to a lesser degree in teleosts. This study uses retrograde tract tracing and other techniques to characterize the morphology and distribution of mitral cells in the olfactory bulb of adult zebrafish, Danio rerio. These output neurons, located primarily in the glomerular layer and superficial internal cell layer, had variable-shaped somata that ranged in size from 4-18 microm in diameter and 31-96 microm2 in cross-sectional area. The mitral cells exhibited two main types of morphologies with regard to their dendrites: the unidendritic morphology was a single primary dendrite with one or more tufts, but multidendritic cells with several dendritic projections also were seen. The axons of these cells projected to either the medial or the lateral olfactory tract and, in general, the location of the cell on the medial or lateral side of the bulb was indicative of the tract to which it would project. Further, this study shows that the majority of zebrafish mitral cells likely innervate a single glomerulus rather than multiple glomeruli. This information is contrary to the multiple innervation pattern suggested for all teleost mitral cells. Our findings suggest that mitral cells in zebrafish may be more similar to mammalian mitral cells than previously believed, despite variation in size and structure. This information provides a revised anatomical framework for olfactory processing studies in this key model system.

Changes in glutamate receptor subunit 4 expression in the deafferented olfactory bulb of zebrafish.

The distribution of ionotropic glutamate receptor subunit 4 (iGluR4) was examined in both normal and deafferented olfactory bulbs of adult zebrafish, Danio rerio. With the exception of the olfactory nerve layer, there was extensive labeling with antibodies to iGluR4 in the olfactory bulbs, specifically in juxtaglomerular cell bodies and their processes. These results are consistent with previous work, which has suggested differential distribution of glutamate receptors in the vertebrate olfactory system. Analysis of bulbs following olfactory organ removal revealed a significant loss of iGluR4 immunoreactivity by 24 h post-deafferentation. At 48 h after denervation, iGluR4 labeling had returned to normal levels and was retained through 3 weeks post-surgery. Thus, afferent input plays a role in reduced labeling of this protein immediately following injury, but return of immunoreactivity can occur even without sensory innervation.

Ruffed cells identified in the adult zebrafish olfactory bulb.

The morphology and distribution of ruffed cells was examined in the olfactory bulb of adult zebrafish, Danio rerio, using retrograde tract tracing and Golgi-Kopsch techniques. The neurons had variable-shaped soma that ranged in size from 7 to 15 microm in diameter. There was an obvious protrusion of the membrane, a ruff, near the initial portion of the axon, and the cells appeared to be distributed primarily in the glomerular layer and superficial internal cell layer. This cell type has been described for a number of teleosts, but not for other animal groups. While the presence of ruffed cells in all teleosts has been suggested, the existence of this cell type in zebrafish was uncertain until now. This new evidence may provide additional insight into olfactory coding and processing in this key model system.

Apoptosis following peripheral sensory deafferentation in the olfactory bulb of adult zebrafish.

Removal of the olfactory organ in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. The current study investigated the potential role of apoptosis in this phenomenon. It was hypothesized that cells in the adult olfactory bulb normally undergo minimal apoptosis and that apoptosis increases upon removal of sensory stimulation. By using both the terminal transferase-mediated deoxyuridine nick-end labeling method and bis-benzimide labeling, the current study showed that, in the normal adult olfactory bulb, cells exhibiting apoptotic profiles were scarce and were localized to the outer layers of the bulb. However, in deafferented animals, there was a significant increase in the number of apoptotic cells. The apoptotic response occurred in two phases and was confined to the rostral half of the bulb. The first phase of cell death peaked at 1 hour postsurgery. These apoptotic profiles appeared to be primarily nonneuronal in nature, in that they exhibited no immunohistochemical labeling to the neuron-specific protein Hu. The second phase of cell death peaked at 24 hours and declined to normal levels by 1 week. At the 24 hour time point, only a fraction of the apoptotic cells was neuronal in nature. Thus, apoptosis of nonneuronal and neuronal elements accounts for at least part of the deafferentation-induced volume decrease in the zebrafish olfactory bulb. This model of anterograde transneuronal degeneration will be useful in elucidating the afferent signals involved in survival and maintenance of mature brain neurons.