Günther K.H. Zupanc

Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain

In contrast to mammals, fish exhibit an enormous potential to produce new cells in the adult brain. By labeling mitotically dividing cells with 5‐bromo‐2′‐deoxyuridine (BrdU), we have characterized the development of these cells in the zebrafish (Danio rerio). Proliferation zones were located in specific regions of the olfactory bulb, dorsal telencephalon (including a region presumably homologous to the mammalian hippocampus), preoptic area, dorsal zone of the periventricular hypothalamus, optic tectum, torus longitudinalis, vagal lobe, parenchyma near the rhombencephalic ventricle, and in a region of the medulla oblongata lateral to the vagal motor nucleus, as well as in all three subdivisions of the cerebellum, the valvula cerebelli, the corpus cerebelli, and the lobus caudalis cerebelli. In the valvula cerebelli and the corpus cerebelli, the young cells migrated from their site of origin in the molecular layers to the corresponding granule cell layers. By contrast, in the lobus caudalis cerebelli and optic tectum, no indication of a migration of the newly generated cells over wider distances could be obtained. BrdU‐labeled cells remained present in the brain over at least 292 days post‐BrdU administration, indicating a long‐term survival of a significant portion of the newly generated cells. The combination of BrdU immunohistochemistry with immunolabeling against the neural marker protein Hu, or with retrograde tracing, suggested a neuronal differentiation in a large portion of the young cells. J. Comp. Neurol. 488:290–319, 2005.

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.

Adult Neurogenesis and Neuronal Regeneration in the Central Nervous System of Teleost Fish

In contrast to mammals, teleost fish exhibit an enormous potential to produce new neurons in the adult central nervous system and to replace damaged neurons by newly generated ones. In the gymnotiform fish Apteronotus leptorhynchus, on average, 100,000 cells, corresponding to roughly 0.2% of the total population of cells in the adult brain, are in S-phase within any 2-h period. As in all other teleosts examined thus far, many of these cells are produced in specific proliferation zones located at or near the surface of ventricular, paraventricular, and cisternal systems, or in areas that are likely derived from proliferation zones located at ventricular surfaces during embryonic development. The majority of cells born in such proliferation zones migrate within the first few weeks following their generation to specific target areas. In the cerebellum, where approximately 75% of all brain cells are born during adulthood, cells originate from the molecular layers of the corpus cerebelli and the valvula cerebelli partes lateralis and medialis, as well as from the eminentia granularis pars medialis. From these proliferation zones, the young cells migrate to the associated granule cell layers or to the eminentia granularis pars posterior, respectively. In the course of their migration, the young cells appear to be guided by radial glial fibers. Upon arrival at their target region, approximately 50% of the young cerebellar cells undergo apoptosis. The remaining cells survive for the rest of the fish’s life, thus contributing to permanent brain growth. At least some cells differentiate into granule cell neurons. The potential to produce new neurons, together with the ability to guide the young cells to their target areas by radial glial fibers and to eliminate damaged cells through apoptosis, also forms the basis for the enormous regenerative capability of the central nervous system of Apteronotus, as demonstrated in the cerebellum and spinal cord. A factor involved in the cerebellar regeneration appears to be somatostatin, as the expression of this neuropeptide is up-regulated in a specific spatio-temporal fashion following mechanical lesions. Besides its involvement in neuronal regeneration adult neurogenesis in Apteronotus, and possibly teleost fish in general, appears to play a role in providing central neurons to match the growing number of sensory and motor elements in the periphery, and to establish the neural substrate to accommodate behavioral plasticity.

POSTEMBRYONIC DEVELOPMENT OF THE CEREBELLUM IN GYMNOTIFORM FISH

In contrast to adult mammals, adult teleost fish regularly generate new neurons and glial cells in many brain regions. A previous quantitative mapping of the proliferation zones in the brain of adult Apteronotus leptorhynchus (Teleostei, Gymnotiformes) has shown that 75% of all mitotically active cells are situated in the cerebellum (Zupanc and Horschke [1995] J. Comp. Neurol. 353:213–233). By employing the thymidine analogue 5‐bromo‐2′‐deoxyuridine, we have, in the present study, investigated the postembryonic development of this brain region in detail. In the corpus cerebelli and the valvula cerebelli, the vast majority of newborn cells originate in the respective molecular layers. Within the first few days of their life, these cells migrate toward specific target areas, namely, the respective granule cell layers. In the caudal part of the cerebellum, the granule cell layer of the eminentia granularis pars medialis displays the highest mitotic activity. From there, the cells migrate through the adjacent molecular layer to the granule cell layer of the eminentia granularis pars posterior. Combination of retrograde‐tracing techniques with immunohistochemistry for 5‐bromo‐2′‐deoxyuridine showed that at least a portion of the newly generated cells develop into granule neurons. Many of the newly generated cells survive for long periods of time. A large fraction of these cells is added to the population of already existing cells, thus resulting in a permanent growth of the target areas and their associated structures.

Potential role of radial glia in adult neurogenesis of teleost fish

Persistence of radial glia within the adult central nervous system is a widespread phenomenon among fish. Based on a series of studies in the teleost species Apteronotus leptorhynchus, we propose that one function of this persistence is the involvement of radial glia in adult neurogenesis, i.e., the generation and further development of new neurons in the adult central nervous system. In particular, evidence has been obtained for the involvement of radial glia in the guidance of migrating young neurons in both the intact and the regenerating brain; for a possible role as precursor cells from which new neurons arise; and for its role as a source of trophic substances promoting the generation, differentiation, and/or survival of new neurons. These functions contribute not only to the potential of the intact brain to generate new neurons continuously, and of the injured brain to replace damaged cells by newly generated ones, but they also provide an essential part of the cellular substrate of behavioral plasticity. GLIA 43:77–86, 2003.

Adult neurogenesis and neuronal regeneration in the central nervous system of teleost fish

Teleost fish are distinguished by their ability to constitutively generate new neurons in the adult central nervous system (‘adult neurogenesis’), and to regenerate whole neurons after injury (‘neuronal regeneration’). In the brain, new neurons are produced in large numbers in several dozens of proliferation zones. In the spinal cord, proliferating cells are present in the ependymal layer and throughout the parenchyma. In the retina, new cells arise from the ciliary marginal zone and from Müller glia. Experimental evidence has suggested that both radial glia and non‐glial cells can function as adult stem cells. The proliferative activity of these cells can be regulated by molecular factors, such as fibroblast growth factor and Notch, as well as by social and behavioral experience. The young cells may either reside near the respective proliferation zone, or migrate to specific target areas. Approximately half of the newly generated cells persist for the rest of the fish’s life, and many of them differentiate into neurons. After injury, a massive surge of apoptotic cell death occurs at the lesion site within a few hours. Apoptosis is followed by a marked increase in cell proliferation and neurogenesis, leading to repair of the tissue. The structural regeneration is paralleled by partial or complete recovery of function. Recent investigations have led to the identification of several dozens of molecular factors that are potentially involved in the process of regeneration.

NEURONAL CONTROL OF BEHAVIORAL PLASTICITY : THE PREPACEMAKER NUCLEUS OF WEAKLY ELECTRIC GYMNOTIFORM FISH

Abstract Gymnotiform fish of the genera Apteronotus and Eigenmannia provide an excellent vertebrate model system to study neural mechanisms controlling behavioral plasticity. These teleosts generate, by means of an electric organ, quasi-sinusoidal discharges of extremely stable frequency and waveform. Modulations consisting of transient rises in discharge frequency are produced during social encounters, and play an important role in communication. These so-called “chirps” exhibit a remarkable sexual dimorphism, as well as an enormous seasonal and individual variability. Chirping behavior is controlled by a subset of neurons in the complex of the central posterior/prepacemaker nucleus in the diencephalon. It is hypothesized that the plasticity in the performance of chirping behavior is, at least in part, governed by two mechanisms: first, by seasonally induced structural changes in dendritic morphology of neurons of the prepacemaker nucleus, thus leading to pronounced alterations in excitatory input. Second, by androgen-controlled changes in the innervation pattern of the prepacemaker nucleus by fibers expressing the neuropeptide substance P. In addition to these two dynamic processes, cells are generated continuously and at high number in the central posterior/prepacemaker nucleus during adulthood. This phenomenon may provide the basis for a “refreshment”, thus facilitating possible changes in the underlying neural network.

Apoptosis in the cerebellum of adult teleost fish, Apteronotus leptorhynchus

While involvement of programmed cell death (apoptosis) in embryogenesis is well established, only very little is known about this phenomenon in later stages of development. Based primarily on indirect evidence, it has been proposed that during postembryonic development of fish cell death does not occur. We have re-addressed this issue by examining the gymnotiform fish Apteronotus leptorhynchus. This teleost exhibits a high degree of proliferative activity in the brain during adulthood. Most of these cells are born in the cerebellum, where they differentiate, migrate into specific target regions, and are added to the population of already existing cerebellar cells. By applying morphological criteria and an in situ technique for the detection of DNA fragmentation (a feature characteristic of apoptotic cells), we show here that a large number of cerebellar cells undergo apoptosis. The density of apoptotic cells is significantly higher in the granule cell layers of the subdivisions of the cerebellum than in the corresponding molecular layers. This finding is consistent with previous observations indicating a drastic reduction in areal density of newborn cells within these granule cell layers in a period 4-7 weeks after their generation. In the granule cell layers of two cerebellar subdivisions, the corpus cerebelli and the valvula cerebelli pars medialis, the areal density of apoptotic cells displays a significant negative correlation with body weight, thus pointing to a decrease in the number of apoptotic events with age. The results of our investigation provide clear evidence for the existence of apoptosis during adulthood in fish and underline the significance of this process in the postembryonic development of the brain.

Neuronal regeneration in the cerebellum of adult teleost fish, Apteronotus leptorhynchus: guidance of migrating young cells by radial glia.

In contrast to mammals, adult fish exhibit an enormous potential to replace injured brain neurons by newly generated ones. In the present study, the role of radial glia, identified by immunostaining against fibrillary acidic protein (GFAP), was examined in this process of neuronal regeneration. Approximately 8 days after application of a mechanical lesion to the corpus cerebelli in the teleost fish Apteronotus leptorhynchus, the areal density of radial glial fibers increased markedly in the ipsilateral dorsal molecular layer compared to shorter survival times, or to the densities found in the intact brain or in the hemisphere contralateral to the lesion. This density remained elevated throughout the time period of up to 100 days examined. The increase in fiber density was followed approximately 2 days later by a rise in the areal density of young cells, characterized by labeling with the nuclear dye DAPI, in the ipsilateral dorsal molecular layer. Based on this remarkable spatio-temporal correlation, and the frequently observed close apposition of elongated young cells to radial glial fibers, we hypothesize that radial glia play an important role in the guidance of migrating young cells from their proliferation zones to the site of lesion where regeneration takes place.

Towards brain repair: Insights from teleost fish

In the adult mammalian brain, the ability to minimize secondary cell death after injury, and to repair nervous tissue through generation of new neurons, is severely compromised. By contrast, certain taxa of non-mammalian vertebrates possess an enormous potential for regeneration. Examination of one of these taxa, teleost fish, has revealed a close link between this phenomenon and constitutive adult neurogenesis. Key factors mediating successful regeneration appear to be: elimination of damaged cells by apoptosis, instead of necrosis; activation of mechanisms that prevent the occurrence of secondary cell death; increased production of new neurons that replace neurons lost to injury; and activation of developmental mechanisms that mediate directed migration of the new cells to the site of injury, the differentiation of the young cells, and their integration into the existing neural network. Comparative analysis has suggested that constitutive adult neurogenesis is a primitive vertebrate trait, the main function of which has been to ensure a numerical matching between muscle fibers/sensory receptor cells and central elements involved in motor control/processing of sensory information associated with these peripheral elements. It is hypothesized that, when in the course of the evolution of mammals a major shift in the growth pattern from hyperplasia to hypertrophy took place, the number of neurogenic brain regions and new neurons markedly decreased. As a consequence, the potential for neuronal regeneration was greatly reduced, but remnants of neurogenic areas have persisted in the adult mammalian brain in form of quiescent stem cells. It is likely that the study of regeneration-competent taxa will provide important information on how to activate intrinsic mechanisms for successful brain regeneration in humans.