Large-scale identification of proteins involved in the development of a sexually dimorphic behavior.

Sexually dimorphic behaviors develop under the influence of sex steroids, which induce reversible changes in the underlying neural network of the brain. However, little is known about the proteins that mediate these activational effects of sex steroids. Here, we used a proteomics approach for large-scale identification of proteins involved in the development of a sexually dimorphic behavior, the electric organ discharge of brown ghost knifefish, Apteronotus leptorhynchus. In this weakly electric fish, the discharge frequency is controlled by the medullary pacemaker nucleus and is higher in males than in females. After lowering the discharge frequency by chronic administration of ß-estradiol, 2-dimensional difference gel electrophoresis revealed 62 proteins spots in tissue samples from the pacemaker nucleus that exhibited significant changes in abundance of >1.5-fold. The 20 identified protein spots indicated, among others, a potential involvement of astrocytes in the establishment of the behavioral dimorphism. Indeed, immunohistochemical analysis demonstrated higher expression of the astrocytic marker protein GFAP and increased gap-junction coupling between astrocytes in females compared with males. We hypothesize that changes in the size of the glial syncytium, glial coupling, and/or number of glia-specific potassium channels lead to alterations in the firing frequency of the pacemaker nucleus via a mechanism mediating the uptake of extracellular potassium ions from the extracellular space.

Upregulation of calbindin-D28k expression during regeneration in the adult fish cerebellum.

In contrast to mammals, fish are distinguished by their enormous potential for brain repair after injuries. This phenomenon has been well studied after application of stab-wound lesions to the corpus cerebelli, a cerebellar subdivision, in the teleost fish Apteronotus leptorhynchus. By combining this lesion paradigm with immunohistochemical staining, we examined the potential role of the calcium-binding protein calbindin-D(28k) in the process of regeneration. Calbindin-D(28k)-immunoreactive cell bodies and fibers were evident in the lesion path and the immediate vicinity of the lesion in the period between 16 h and 7 days after the lesion but absent from this region at shorter or longer postlesion survival times and in the intact brain. Both the number of immunolabeled cells and the intensity of the label were most pronounced 1-3 days postlesion. Analysis of the morphology of the immunostained cells by confocal microscopy suggested that most, and perhaps all of them, were granular neurons. Since the transient upregulation of calbindin-D(28k) is paralleled by a decline in the number of cells undergoing apoptotic cell death, we hypothesize that this protein exerts a neuroprotective function, probably by buffering free intracellular Ca(2+), whose concentration is elevated after brain insults.

Numerical chromosome variation and mitotic segregation defects in the adult brain of teleost fish.

Teleost fish are distinguished by their enormous potential for the generation of new cells in both the intact and the injured adult brain. Here, we present evidence that these cells are a genetic mosaic caused by somatic genomic alteration. Metaphase chromosome spreads from whole brains of the teleost Apteronotus leptorhynchus revealed an euploid complement of 22 chromosomes in only 22% of the cells examined. The rate of aneuploidy is substantially higher in brain cells than in liver cells, as shown by both metaphase chromosome spreads and flow cytometric analysis. Among the aneuploid cells in the brain, approximately 84% had fewer, and the remaining 16% more, than 22 chromosomes. Typically, multiple chromosomes were lost or gained. The aneuploidy is putatively caused by segregation defects during mitotic division. Labeling of condensed chromosomes of M-phase cells by phosphorylated histone-H3 revealed laggards, anaphase bridges, and micronuclei, all three of which indicate displaced mitotic chromosomes. Quantitative analysis has shown that in the entire brain on average 14% of all phosphorylated histone-H3-labeled cells exhibit such signs of segregation defects. Together with the recent discovery of aneuploidy in the adult mammalian brain, the results of the present investigation suggest that the loss or gain of chromosomes might provide a mechanism to regulate gene expression during development of new cells in the adult vertebrate brain.

New neurons for the injured brain: mechanisms of neuronal regeneration in adult teleost fish.

In contrast to mammals, teleost fish exhibit an enormous potential to continuously produce new neurons in many areas of the adult brain, and to regenerate neural tissue after brain injury. The regenerative capability of the teleost fish brain is based upon a series of well-orchestrated individual processes, including: elimination of damaged cells by apoptosis, removal of cellular debris by the action of microglia/macrophages, proliferation of endogenous neural precursor cells, radial glia-mediated migration of their progeny to the site of the lesion, neuronal differentiation, promotion of cellular survival, and integration of the new neurons into existing neural circuits. Combination of a well-defined cerebellar lesion paradigm with differential proteome analysis has demonstrated that identification of the multitude of proteins mediating the regenerative potential of the adult fish brain is feasible in the foreseeable future. A molecular understanding of brain regeneration in fish could help investigators to define novel strategies to stimulate endogenous neural precursor cells in the mammalian brain to undergo neurogenesis, thus forming the basis of a neuronal replacement therapy for brain injury or neurodegenerative diseases.

Proteome analysis identifies novel protein candidates involved in regeneration of the cerebellum of teleost fish.

In contrast to mammals, adult teleost fish exhibit an enormous potential to regenerate neuronal tissue after injuries to the CNS. By combining a well-defined cerebellar lesion paradigm with differential proteome analysis at a post-lesion survival time of 3 days, we screened for protein candidates involved in repair of the fish brain. Out of nearly 900 protein spots detected on 2-D gels, spot intensity was significantly increased at least twofold in 30 spots and decreased to at least half the intensity of control tissue in 23 spots. The proteins associated with 24 of the spots were identified by PMF and MS/MS fragmentation. The cellular localization and the spatiotemporal patterns of two of these proteins, beta-actin and beta-tubulin, were further characterized through immunohistochemistry. Comparison of the observed changes in protein abundance with the previously characterized events underlying regeneration of the cerebellum suggests that the proteins identified are especially involved in cellular proliferation and survival, as well as axonal sprouting.