Mitral Cell Dendritic Morphology in the Adult Zebrafish Olfactory Bulb following Growth, Injury and Recovery.

The role of afferent target interactions in dendritic plasticity within the adult brain remains poorly understood. There is a paucity of data regarding the effects of deafferentation and subsequent dendritic recovery in adult brain structures. Moreover, although adult zebrafish demonstrate ongoing growth, investigations into the impact of growth on mitral cell (MC) dendritic arbor structure and complexity are lacking. Leveraging the regenerative capabilities of the zebrafish olfactory system, we conducted a comprehensive study to address these gaps. Employing an eight-week reversible deafferentation injury model followed by retrograde labeling, we observed substantial morphological alterations in MC dendrites. Our hypothesis posited that cessation of injury would facilitate recovery of MC dendritic arbor structure and complexity, potentially influenced by growth dynamics. Statistical analyses revealed significant changes in MC dendritic morphology following growth and recovery periods, indicating that MC total dendritic branch length retained significance after 8 weeks of deafferentation injury when normalized to individual fish physical characteristics. This suggests that regeneration of branch length could potentially function relatively independently of growth-related changes. These findings underscore the remarkable plasticity of adult dendritic arbor structures in a sophisticated model organism and highlight the efficacy of zebrafish as a vital implement for studying neuroregenerative processes.

Diving into the streams and waves of constitutive and regenerative olfactory neurogenesis: insights from zebrafish.

The olfactory system is renowned for its functional and structural plasticity, with both peripheral and central structures displaying persistent neurogenesis throughout life and exhibiting remarkable capacity for regenerative neurogenesis after damage. In general, fish are known for their extensive neurogenic ability, and the zebrafish in particular presents an attractive model to study plasticity and adult neurogenesis in the olfactory system because of its conserved structure, relative simplicity, rapid cell turnover, and preponderance of neurogenic niches. In this review, we present an overview of the anatomy of zebrafish olfactory structures, with a focus on the neurogenic niches in the olfactory epithelium, olfactory bulb, and ventral telencephalon. Constitutive and regenerative neurogenesis in both the peripheral olfactory organ and central olfactory bulb of zebrafish is reviewed in detail, and a summary of current knowledge about the cellular origin and molecular signals involved in regulating these processes is presented. While some features of physiologic and injury-induced neurogenic responses are similar, there are differences that indicate that regeneration is not simply a reiteration of the constitutive proliferation process. We provide comparisons to mammalian neurogenesis that reveal similarities and differences between species. Finally, we present a number of open questions that remain to be answered.

Role of Macrophages and Microglia in Zebrafish Regeneration.

Currently, there is no treatment for recovery of human nerve function after damage to the central nervous system (CNS), and there are limited regenerative capabilities in the peripheral nervous system. Since fish are known for their regenerative abilities, understanding how these species modulate inflammatory processes following injury has potential translational importance for recovery from damage and disease. Many diseases and injuries involve the activation of innate immune cells to clear damaged cells. The resident immune cells of the CNS are microglia, the primary cells that respond to infection and injury, and their peripheral counterparts, macrophages. These cells serve as key modulators of development and plasticity and have been shown to be important in the repair and regeneration of structure and function after injury. Zebrafish are an emerging model for studying macrophages in regeneration after injury and microglia in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. These fish possess a high degree of neuroanatomical, neurochemical, and emotional/social behavioral resemblance with humans, serving as an ideal simulator for many pathologies. This review explores literature on macrophage and microglial involvement in facilitating regeneration. Understanding innate immune cell behavior following damage may help to develop novel methods for treating toxic and chronic inflammatory processes that are seen in trauma and disease.

Microglial response patterns following damage to the zebrafish olfactory bulb.

The inherent plasticity of the zebrafish olfactory system serves as a useful model for examining immune cell responses after injury. Microglia are the resident immune cells of the CNS that respond to damage by migrating to the site of injury and phagocytizing neuronal debris. While the olfactory system is renowned for its ability to recover from damage, the specific mechanisms of microglial involvement in olfactory system plasticity are unknown. To approach the potentially time-dependent effects of microglial activation after injury, we performed a time course analysis of microglial response profiles and patterns following different forms of damage: deafferentation by cautery ablation of the olfactory organ, deafferentation by chemical ablation of the olfactory epithelium, and direct lesioning of the olfactory bulb. Our aim was to demonstrate that immunocytochemistry and microscopy methods in zebrafish can be used to determine the timing of distinct microglial response patterns following various forms of injury. We found that permanent and temporary forms of damage to the olfactory bulb resulted in different microglial response profiles from 1 to 72 h after injury, suggesting that there may be critical timepoints in which microglia are activated that contribute to tissue and neuronal repair with a regenerative outcome versus a degenerative outcome. These distinctions between the different forms of damage suggest temporal changes relative to the potential for regeneration, since cautery deafferentation is permanent and unrecoverable while chemical ablation deafferentation and direct lesioning is reversible and can be used to observe the microglial relationship in neural regeneration and functional recovery in future studies.

The Olfactory System of Zebrafish as a Model for the Study of Neurotoxicity and Injury: Implications for Neuroplasticity and Disease.

The olfactory system, composed of the olfactory organs and the olfactory bulb, allows organisms to interact with their environment and through the detection of odor signals. Olfaction mediates behaviors pivotal for survival, such as feeding, mating, social behavior, and danger assessment. The olfactory organs are directly exposed to the milieu, and thus are particularly vulnerable to damage by environmental pollutants and toxicants, such as heavy metals, pesticides, and surfactants, among others. Given the widespread occurrence of olfactory toxicants, there is a pressing need to understand the effects of these harmful compounds on olfactory function. Zebrafish (Danio rerio) is a valuable model for studying human physiology, disease, and toxicity. Additionally, the anatomical components of the zebrafish olfactory system are similar to those of other vertebrates, and they present a remarkable degree of regeneration and neuroplasticity, making it an ideal model for the study of regeneration, reorganization and repair mechanisms following olfactory toxicant exposure. In this review, we focus on (1) the anatomical, morphological, and functional organization of the olfactory system of zebrafish; (2) the adverse effects of olfactory toxicants and injury to the olfactory organ; and (3) remodeling and repair neuroplasticity mechanisms following injury and degeneration by olfactory toxicant exposure.

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.

Reversible deafferentation of the zebrafish olfactory bulb with wax plug insertion.

BACKGROUND: Deafferentation of the zebrafish olfactory bulb allows investigation of neuroplasticity in a particularly dynamic brain region of a popular model animal known for its regenerative abilities. Current methods to remove sensory input to the zebrafish olfactory bulb differ in the extent of deafferentation and potential for recovery. NEW METHOD: We present a novel method of olfactory bulb deafferentation using continuous wax plug insertions into the nasal cavity of zebrafish. Wax plugs were placed in the nasal cavity and replaced if needed over 1wk or 3wk survival periods. Wax plugs were removed from fish after 1wk of occlusion to analyze the potential recovery of the olfactory organ and bulb. RESULTS: Wax plug insertions caused a dramatic reduction in olfactory organ size and structure and significantly reduced afferent input to the olfactory bulb after 1wk and 3wk. Removal of the wax plugs after 1wk allowed for recovery of the olfactory organ and subsequent reinnervation of the olfactory bulb. COMPARISONS WITH EXISTING METHODS: Chemical ablation with detergent causes partial, temporary deafferentation of the olfactory bulb. Cautery ablation causes complete, permanent deafferentation of the olfactory bulb. Wax plug insertions cause nearly complete, temporary deafferentation, allowing both significant deafferentation and the potential for reinnervation of the olfactory bulb. CONCLUSIONS: The wax plug insertion method of deafferentation described here is unique in that it destroys almost completely the structure of the olfactory organ and removes almost completely sensory input to the olfactory bulb, yet the organ returns to its typical morphology and afferent innervation returns.

Exposure to Zinc Sulfate Results in Differential Effects on Olfactory Sensory Neuron Subtypes in Adult Zebrafish.

Zinc sulfate is a known olfactory toxicant, although its specific effects on the olfactory epithelium of zebrafish are unknown. Olfactory organs of adult zebrafish were exposed to zinc sulfate and, after 2, 3, 5, 7, 10 or 14 days, fish were processed for histological, immunohistochemical, ultrastructural, and behavioral analyses. Severe morphological disruption of the olfactory organ was observed two days following zinc sulfate exposure, including fusion of lamellae, epithelial inflammation, and significant loss of anti-calretinin labeling. Scanning electron microscopy revealed the apical surface of the sensory region was absent of ciliated structures, but microvilli were still present. Behavioral analysis showed significant loss of the ability to perceive bile salts and some fish also had no response to amino acids. Over the next several days, olfactory organ morphology, epithelial structure, and anti-calretinin labeling returned to control-like conditions, although the ability to perceive bile salts remained lost until day 14. Thus, exposure to zinc sulfate results in rapid degeneration of the olfactory organ, followed by restoration of morphology and function within two weeks. Zinc sulfate appears to have a greater effect on ciliated olfactory sensory neurons than on microvillous olfactory sensory neurons, suggesting differential effects on sensory neuron subtypes.

Patterns of olfactory bulb neurogenesis in the adult zebrafish are altered following reversible deafferentation.

Adult brain plasticity can be investigated using reversible methods that remove afferent innervation but allow return of sensory input. Repeated intranasal irrigation with Triton X-100 in adult zebrafish diminishes innervation to the olfactory bulb, resulting in a number of alterations in bulb structure and function, and cessation of the treatment allows for reinnervation and recovery. Using bromodeoxyuridine, Hu, and caspase-3 immunoreactivity we examined cell proliferation, differentiation, migration, and survival under conditions of acute and chronic deafferentation and reafferentation. Cell proliferation within the olfactory bulb was not influenced by acute or chronic deafferentation or reafferentation, but cell fate (including differentiation, migration, and/or survival of newly formed cells) was affected. We found that chronic deafferentation caused a bilateral increase in the number of newly formed cells that migrated into the bulb, although the amount of cell death of these new cells was significantly increased compared to untreated fish. Reafferentation also increased the number of newly formed cells migrating into both bulbs, suggesting that the deafferentation effect on cell fate was maintained. Reafferentation resulted in a decrease in newly formed cells that became neurons and, although death of newly formed cells was not altered from control levels, survival was reduced in relation to that seen in chronically deafferented fish. The potential effect of age on cell genesis was also examined. While the amount of cell migration into the olfactory bulbs was not affected by fish age, more of the newly formed cells became neurons in older fish. Younger fish displayed more cell death under conditions of chronic deafferentation. In sum, our results show that reversible deafferentation affects several aspects of cell fate, including cell differentiation, migration, and survival, and age of the fish influences the response to deafferentation.

Reversible deafferentation of the adult zebrafish olfactory bulb affects glomerular distribution and olfactory-mediated behavior.

The olfactory system is a useful model for studying central nervous system recovery from damage due to its neuroplasticity. We recently developed a novel method of deafferentation by repeated exposure of Triton X-100 to the olfactory organ of adult zebrafish. This long-term, reversible method of deafferentation allows both degeneration and regeneration to be observed in the olfactory bulb. The aim of the present study is to examine olfactory bulb innervation, glomerular patterns, and olfactory-mediated behavior with repeated Triton X-100 treatment and the potential for recovery following cessation of treatment. Olfactory bulbs of control, chronic-treated, and recovery animals were examined for the presence or absence of glomeruli that have been identified in the zebrafish glomerular map. Following chronic treatment, the number of glomeruli was dramatically reduced; however, partial innervation remained in the lateral region of the bulb. When animals were given time to recover, complete glomerular distribution returned. A behavioral assay was developed to determine if innervation remaining correlated with behavior of the fish. Chronic-treated fish did not respond to odorants involved with social behavior but continued to react to odorants that mediate feeding behavior. Following recovery, responses to odorants involved with social behavior returned. The morphological and behavioral effects of chronic Triton X-100 treatment in the olfactory system suggest there may be differential susceptibility or resistance to external damage in a subset of sensory neurons. The results of this study demonstrate the remarkable regenerative ability of the olfactory system following extensive and long-term injury.

Peripheral sensory deafferentation affects olfactory bulb neurogenesis in zebrafish.

The potential effects of the removal of olfactory input on adult neurogenesis in the olfactory bulb were examined. Olfactory organs of adult zebrafish were permanently and completely ablated by cautery and animals were exposed to bromodeoxyuridine then examined following short (4-hour) or long (3-week) survival periods. Short survival times allowed analysis of cell proliferation in the olfactory bulb. Long survival times permitted investigation of survival of adult-formed cells. Deafferentation did not immediately affect the dividing cells in the bulb but did affect the number of adult-formed cells, some of which expressed a neuronal marker, present in the bulb 3 weeks later. Thus, afferent removal influenced the fate of newly formed cells by impacting subsequent divisions, maturation, or survival of those cells. One week of deafferentation altered the pattern of cell genesis, with a significant increase in the number of dividing cells located in the olfactory bulb and also in the ventral telencephalic proliferation zone. Sham surgery did not impact either proliferation or survival of adult-formed cells in the olfactory bulb, suggesting that the deafferentation effect is specific. Thus, afferent innervation is necessary for normal cell proliferation and maintenance of the olfactory bulb in adult zebrafish.