Deafferentation-induced alterations in mitral cell dendritic morphology in the adult zebrafish olfactory bulb.

The removal of afferent input to the olfactory bulb by both cautery and chemical olfactory organ ablation in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. To examine the effects of deafferentation at a cellular level, primary output neurons of the olfactory bulb, the mitral cells, were investigated using retrograde tract tracing with fluorescent dextran using ex vivo brain cultures. Morphological characteristics including the number of major dendritic branches, total length of dendritic branches, area of the dendritic arbor, overall dendritic complexity, and optical density of the arbor were used to determine the effects of deafferentation on mitral cell dendrites. Following 8 weeks of permanent deafferentation there were significant reductions in the total length of dendritic branches, the area of the dendritic arbor, and the density of fine processes in the dendritic tuft. With 8 weeks of chronic, partial deafferentation there were significant reductions in all parameters examined, including a modified Sholl analysis that showed significant decreases in overall dendritic complexity. These results show the plasticity of mitral cell dendritic structures in the adult brain and provide information about the response of these output neurons following the loss of sensory input in this key model system.

Compensatory network changes in the dentate gyrus restore long-term potentiation following ablation of neurogenesis in young-adult mice.

It is now well established that neurogenesis in the rodent subgranular zone of the hippocampal dentate gyrus continues throughout adulthood. Neuroblasts born in the dentate subgranular zone migrate into the granule cell layer, where they differentiate into neurons known as dentate granule cells. Suppression of neurogenesis by irradiation or genetic ablation has been shown to disrupt synaptic plasticity in the dentate gyrus and impair some forms of hippocampus-dependent learning and memory. Using a recently developed transgenic mouse model for suppressing neurogenesis, we sought to determine the long-term impact of ablating neurogenesis on synaptic plasticity in young-adult mice. Consistent with previous reports, we found that ablation of neurogenesis resulted in significant deficits in dentate gyrus long-term potentiation (LTP) when examined at a time proximal to the ablation. However, the observed deficits in LTP were not permanent. LTP in the dentate gyrus was restored within 6 wk and this recovery occurred in the complete absence of neurogenesis. The recovery in LTP was accompanied by prominent changes within the dentate gyrus, including an increase in the survival rate of newborn cells that were proliferating just before the ablation and a reduction in inhibitory input to the granule cells of the dentate gyrus. These findings suggest that prolonged suppression of neurogenesis in young-adult mice results in wide-ranging compensatory changes in the structure and dynamics of the dentate gyrus that function to restore plasticity.

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.

Conditional ablation and recovery of forebrain neurogenesis in the mouse.

Forebrain neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Several strategies have been employed to eliminate adult neurogenesis and thereby determine whether depleting adult-born neurons disrupts specific brain functions, but some approaches do not specifically target neural progenitors. We have developed a transgenic mouse line to reversibly ablate adult neural stem cells and suppress neurogenesis. The nestin-tk mouse expresses herpes simplex virus thymidine kinase (tk) under the control of the nestin 2nd intronic enhancer, which drives expression in neural progenitors. Administration of ganciclovir (GCV) kills actively dividing cells expressing this transgene. We found that peripheral GCV administration suppressed SVZ-olfactory bulb and DG neurogenesis within 2 weeks but caused systemic toxicity. Intracerebroventricular GCV infusion for 28 days nearly completely depleted proliferating cells and immature neurons in both the SVZ and DG without systemic toxicity. Reversibility of the effects after prolonged GCV infusion was slow and partial. Neurogenesis did not recover 2 weeks after cessation of GCV administration, but showed limited recovery 6 weeks after GCV that differed between the SVZ and DG. Suppression of neurogenesis did not inhibit antidepressant responsiveness of mice in the tail suspension test. These findings indicate that SVZ and DG neural stem cells differ in their capacity for repopulation, and that adult-born neurons are not required for antidepressant responses in a common behavioral test of antidepressant efficacy. The nestin-tk mouse should be useful for studying how reversible depletion of adult neurogenesis influences neurophysiology, other behaviors, and neural progenitor dynamics.