Determination of relative age using growth increments of scales as a minimally invasive method in the tropical freshwater Apteronotus leptorhynchus.

This study describes a method for the determination of relative age in a tropical teleost, the brown ghost knifefish Apteronotus leptorhynchus. This method is based on identification of the maximum number of scale circuli, which is thought to be associated with the oldest scales, and thus to be the most indicative of the age of a given fish. Relative age can be inferred by relating differences in maximum circulus counts to the average rate of circulus addition, which was estimated at 34 circuli per year in adult fish through oxytetracycline marking. This method shows high inter-investigator reliability and has a limited effect on fish because of the low number of scales required in order to determine the maximum number of circuli with a sufficiently high confidence level. Analysis of the frequency distribution of the circulus counts revealed periodic patterns that are similar among fish, presumably reflecting the environmental life history of the individuals. Regression analysis and comparison of addition rates showed that scale circulus counts and otolith ring counts are equivalent approaches for age estimation, but scale analysis is superior because of its limited invasiveness and the lower demand in terms of technical skills and expensive instrumentation.

Proteome analysis reveals protein candidates involved in early stages of brain regeneration of teleost fish.

Exploration of the molecular dynamics underlying regeneration in the central nervous system of regeneration-competent organisms has received little attention thus far. By combining a cerebellar lesion paradigm with differential proteome analysis at a post-lesion survival time of 30 min, we screened for protein candidates involved in the early stages of regeneration in the cerebellum of such an organism, the teleost fish Apteronotus leptorhynchus. Out of 769 protein spots, the intensity of 26 spots was significantly increased by a factor of at least 1.5 in the lesioned hemisphere, relative to the intact hemisphere. The intensity of 9 protein spots was significantly reduced by a factor of at least 1.5. The proteins associated with 15 of the spots were identified by peptide mass fingerprinting and/or tandem mass spectrometry, resulting in the identification of a total of 11 proteins. Proteins whose abundance was significantly increased include: erythrocyte membrane protein 4.1N, fibrinogen gamma polypeptide, fructose-biphosphate aldolase C, alpha-internexin neuronal intermediate filament protein, major histocompatibility complex class I heavy chain, 26S proteasome non-ATPase regulatory subunit 8, tubulin alpha-1C chain, and ubiquitin-specific protease 5. Proteins with significantly decreased levels of abundance include: brain glycogen phosphorylase, neuron-specific calcium-binding protein hippocalcin, and spectrin alpha 2. We hypothesize that these proteins are involved in energy metabolism, blood clotting, electron transfer in oxidative reactions, cytoskeleton degradation, apoptotic cell death, synaptic plasticity, axonal regeneration, and promotion of mitotic activity.

Radial glia in the cerebellum of adult teleost fish: implications for the guidance of migrating new neurons.

In contrast to mammals, in teleost fish radial glia persist beyond early development. This persistence parallels the enormous potential of teleosts to continuously generate a large number of new neurons in dozens of specific proliferation zones in the adult brain. In the present study, we characterized in the teleost fish Apteronotus leptorhynchus the immunological properties of radial glia in the corpus cerebelli-a cerebellar subdivision with particularly high proliferative activity-and examined their possible function in the guidance of migrating young neurons. Radial glia stained immunopositive for glial fibrillary acidic protein (GFAP) and vimentin, and in most cases the two intermediate filament proteins co-localized. GFAP immunolabeling combined with immunohistochemistry against the mitotic marker 5-bromo-2'-deoxyuridine (BrdU) revealed an abundance of elongated BrdU-labeled nuclei closely apposed to, or localized within, GFAP-immunoreactive radial glia. The association of BrdU-labeled nuclei and GFAP-immunoreactive radial glial fibers was particularly pronounced 2 days after BrdU administration, when the migratory activity of the young cells is highest. When the new cells reach the granular layer, they start expressing the neuronal marker protein Hu C/D, but continue their close association with radial glial fibers. These results suggest the role of radial glia in the guidance of migrating adult-born neurons in the teleostean cerebellum. This function appears to be mediated both by somal translocation and by a glial-guided mode of locomotion.

Inhibition of caspase-3-mediated apoptosis improves spinal cord repair in a regeneration-competent vertebrate system.

Teleost fish exhibit an excellent potential for structural and functional recovery after CNS lesions. The function of apoptosis in the process of regeneration remains controversial. While some studies have identified this type of cell death as essential for successful regeneration, other investigations have suggested some degree of functional improvement after inhibition of apoptosis. In the present study, we examined whether inhibition of apoptosis immediately after injury can improve spinal cord regeneration. As a model system, we used Apteronotus leptorhynchus, a regeneration-competent weakly electric fish. To inhibit apoptosis, we employed 2,2'-methylenebis (1,3-cyclohexanedione) (M50054), a compound that prevents caspase-3 activation. Administration of this apoptosis inhibitor led to a significant reduction in the numbers of apoptotic cells at 24 h, 5 days, and 30 days after the lesion. Using triple immunolabeling, we identified a significant reduction in the level of apoptosis at 5 and 30 days after the lesion among the following cellular categories: cells generated shortly after the lesion, existing neurons, and newly differentiated neurons. This reduced rate of apoptosis led to an increase in the relative number of differentiating and surviving neurons at both 5 and 30 days post-injury, compared to the control groups. Functional regeneration, as indicated by the recovery rate of the amplitude of the electric organ discharge (EOD), was significantly improved within the first 20 days after the lesion in the fish treated with M50054. Our data provide the first evidence that modulation of caspase-3 activation can significantly improve neuroregeneration and functional recovery in a regeneration-competent organism.

Generation, long-term persistence, and neuronal differentiation of cells with nuclear aberrations in the adult zebrafish brain.

Zebrafish, like other teleosts, continuously produce new cells in numerous regions of the adult brain. Immunolabeling employing antisera against phosphorylated histone-H3 and 5-bromo-2'-deoxyuridine revealed that approximately 6%-7% of such cells exhibited nuclear aberrations. These aberrations, presumably the result of mitotic segregation defects, included single and multiple laggards (both during metaphase and anaphase) and anaphase bridges. Cells with such aberrations persisted long-term and comprised, when examined 7.5 months after their generation, approximately 2.5% of the total population of adult-born cells. The drop in relative frequency of aberrations in the course of further development appears to be caused by elimination of cells with nuclear aberrations, presumably by apoptotic cell death. The cells with nuclear aberrations that persisted long-term were capable of neuronal differentiation, as demonstrated by combining anti-5-bromo-2'-deoxyuridine immunohistochemistry with immunostaining against the neuronal marker protein Hu or the enzyme tyrosine hydroxylase, a marker of catecholaminergic neurons. We hypothesize that the alterations in chromosome number and/or chromosome structure caused by nuclear aberrations do not necessarily result in loss of vital functions or in tumorigenesis. Instead, cells with such aberrations are able to undergo what appears to be normal development.

Theodore H. Bullock: pioneer of integrative and comparative neurobiology.

Theodore H. Bullock (1905-2005) was a pioneer of integrative and comparative neurobiology and one of the founders of neuroethology. His work--distinguished by the tremendous number of different research themes and animal taxa studied--provided the basis for a comprehensive analysis of brain evolution. Among his major achievements are: one of the first physiological analyses of rhythmic central pattern generators; the first simultaneous recording from both the presynaptic and postsynaptic region of a chemical synapse; the demonstration of intercellular communication through graded potentials; and the discovery of two novel sensory organs formed by infrared receptors in pit vipers and electroreceptors in electric fish. He was also one of the first who applied computational tools to the analysis of complex neural signals and to perform a comparative analysis of cognitive events. His two-volume treatise "Structure and function in the nervous system of invertebrates" (with G. Adrian Horridge) remains the most comprehensive, authoritative review of this topic ever written. In addition to his research merits, his legacy is particularly based on his cosmopolitan way of thinking and acting, his large, worldwide school of students, and his committed advocacy for comparative and systems-oriented neurobiology.

Generation and long-term persistence of new neurons in the adult zebrafish brain: a quantitative analysis.

Zebrafish, like other teleosts, are distinguished by their enormous potential to produce new neurons in many parts of the adult brain. By labeling S-phase cells with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU), quantitative analysis demonstrated that, on average, 6000 new cells were generated in the entire adult brain within any 30 min period. This corresponds to roughly 0.06% of the total number of brain cells. Part of these cells underwent a second round of cell division a few days after their generation so that 10 days post-BrdU administration, when the cells have exited the mitotic cycle, approximately 10,000 BrdU-labeled cells were present in the entire brain. At post-BrdU survival times of 446-656 days, on average 4600 BrdU-labeled cells were found, suggesting that approximately 46% of the cells present at 10 days persisted in the adult zebrafish brain. Combination of BrdU-labeling of mitotic cells with immunostaining against Hu showed that roughly 47% of the BrdU-labeled cells that persisted in the brain expressed this neuronal marker protein. Taken together, the results of this investigation demonstrate that at least half of the cells generated in the adult zebrafish brain develop into neurons and are likely to persist for the rest of the fish's life.

Walter Heiligenberg: the jamming avoidance response and beyond.

Walter Heiligenberg (1938-1994) was an exceptionally gifted behavioral physiologist who made enormous contributions to the analysis of behavior and to our understanding of how the brain initiates and controls species-typical behavioral patterns. He was distinguished by his rigorous analytical approach used in both behavioral studies and neuroethological investigations. Among his most significant contributions to neuroethology are a detailed analysis of the computational rules governing the jamming avoidance response in weakly electric fish and the elucidation of the principal neural pathway involved in neural control of this behavior. Based on his work, the jamming avoidance response is perhaps the best-understood vertebrate behavior pattern in terms of the underlying neural substrate. In addition to this pioneering work, Heiligenberg stimulated research in a significant number of other areas of ethology and neuroethology, including: the quantitative assessment of aggressivity in cichlid fish; the ethological analysis of the stimulus-response relationship in the chirping behavior of crickets; the exploration of the neural and endocrine basis of communicatory behavior in weakly electric fish; the study of cellular mechanisms of neuronal plasticity in the adult fish brain; and the phylogenetic analysis of electric fishes using a combination of morphology, electrophysiology, and mitochondrial sequence data.

Neurogenesis and neuronal regeneration in the adult fish brain.

Fish are distinctive in their enormous potential to continuously produce new neurons in the adult brain, whereas in mammals adult neurogenesis is restricted to the olfactory bulb and the hippocampus. In fish new neurons are not only generated in structures homologous to those two regions, but also in dozens of other brain areas. In some regions of the fish brain, such as the optic tectum, the new cells remain near the proliferation zones in the course of their further development. In others, as in most subdivisions of the cerebellum, they migrate, often guided by radial glial fibers, to specific target areas. Approximately 50% of the young cells undergo apoptotic cell death, whereas the others survive for the rest of the fish's life. A large number of the surviving cells differentiate into neurons. Two key factors enabling highly efficient brain repair in fish after injuries involve the elimination of damaged cells by apoptosis (instead of necrosis, the dominant type of cell death in mammals) and the replacement of cells lost to injury by newly generated ones. Proteome analysis has suggested well over 100 proteins, including two dozen identified ones, to be involved in the individual steps of this phenomenon of neuronal regeneration.

Electric interactions through chirping behavior in the weakly electric fish, Apteronotus leptorhynchus.

The weakly electric fish Apteronotus leptorhynchus produces wave-like electric organ discharges distinguished by a high degree of regularity. Transient amplitude and frequency modulations ("chirps") can be evoked in males by stimulation with the electric field of a conspecific. During these interactions, the males examined in this study produced six types of chirps, including two novel ones. Stimulation of a test fish with a conspecific at various distances showed that two electrically interacting fish must be within 10 cm of each other to evoke chirping behavior in the neighboring fish. The chirp rate of all but one chirp type elicited by the neighboring fish was found to be negatively correlated with the absolute value of the frequency difference between the two interacting fish, but independent of the sign of this difference. Correlation analysis of the instantaneous rates of chirp occurrence revealed two modes of interactions characterized by reciprocal stimulation and reciprocal inhibition. Further analysis of the temporal relationship between the chirps generated by the two fish during electric interactions showed that the chirps generated by one individual follow the chirps of the other with a short latency of approximately 500-1,000 ms. We hypothesize that this "echo response" serves a communicatory function.

Fish somatostatin sst3 receptor: comparison of radioligand and GTPgammaS binding, adenylate cyclase and phospholipase C activities reveals different agonist-dependent pharmacological signatures.

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non-mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPgammaS binding, inhibition of forskolin-stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5). 2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G-protein-coupled fsst3 receptors. By contrast, [125I]LTT-SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non-coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT-SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg(-1)) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg(-1)). 3 SRIF14 (10 microm)-stimulated specific [35S]GTPgammaS binding by three-fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60-80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin-stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five- to six-fold, as determined by measuring total [3H] IP(x) accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml(-1), 21% inhibition), which suggests the G(q)-family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20-70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14-induced total [3H]-IP(x) production (pK(B) = 6.83), but failed to block competitively agonist-stimulated [35S]GTPgammaS binding or agonist-induced inhibition of FSAC activity. 4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPgammaS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410-0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPgammaS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75-0.83; 0.68-0.89). By contrast, the PLC stimulation and radioligand-binding profiles did not correlate. 5 Comparison of the functional data (GTPgammaS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17). 6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp-sensitivities determined with the four radioligands (agonists) are assumed to results from ligand-specific states of the fsst3-ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist-induced receptor trafficking.