RESUMO
Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1-acting as master sex-determining gene-has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans-together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells-suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Oryzias/genética , Processos de Determinação Sexual/genética , Regiões 3' não Traduzidas/genética , Animais , Evolução Biológica , Feminino , Proteínas de Peixes/genética , Células Germinativas/metabolismo , Masculino , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vertebrados/metabolismoRESUMO
OBJECTIVE: Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff-person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood. METHODS: A cell-based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model. RESULTS: Glycine receptor function as assessed by glycine dose-response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N-terminal residues 29 A to 62 G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals. INTERPRETATION: Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff-person syndrome or progressive encephalitis with rigidity and myoclonus patients. ANN NEUROL 2020;88:544-561.
Assuntos
Autoanticorpos/imunologia , Encefalomielite/imunologia , Rigidez Muscular/imunologia , Receptores de Glicina/metabolismo , Rigidez Muscular Espasmódica/imunologia , Adulto , Idoso , Animais , Autoanticorpos/farmacologia , Autoantígenos/imunologia , Comportamento Animal/efeitos dos fármacos , Encefalomielite/metabolismo , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rigidez Muscular/metabolismo , Receptores de Glicina/imunologia , Rigidez Muscular Espasmódica/metabolismo , Peixe-ZebraRESUMO
Modulation of connectivity formation in the developing brain in response to external stimuli is poorly understood. Here, we show that the raphe nucleus and its serotonergic projections regulate pathfinding of commissural axons in zebrafish. We found that the raphe neurons extend projections toward midline-crossing axons and that when serotonergic signaling is blocked by pharmacological inhibition or by raphe neuron ablation, commissural pathfinding is disrupted. We demonstrate that the serotonin receptor htr2a is expressed on these commissural axons and that genetic knock-down of htr2a disrupts crossing. We further show that knock-down of htr2a or ablation of the raphe neurons increases ephrinB2a protein levels in commissural axons. An ephrinB2a mutant can rescue midline crossing when serotonergic signaling is blocked. Furthermore, we found that regulation of serotonin expression in the raphe neurons is modulated in response to the developmental environment. Hypoxia causes the raphe to decrease serotonin levels, leading to a reduction in midline crossing. Increasing serotonin in the setting of hypoxia restored midline crossing. Our findings demonstrate an instructive role for serotonin in axon guidance acting through ephrinB2a and reveal a novel mechanism for developmental interpretation of the environmental milieu in the generation of mature neural circuitry. SIGNIFICANCE STATEMENT: We show here that serotonin has a novel role in regulating connectivity in response to the developmental environment. We demonstrate that serotonergic projections from raphe neurons regulate pathfinding of crossing axons. The neurons modulate their serotonin levels, and thus alter crossing, in response to the developmental environment including hypoxia. The findings suggest that modification of the serotonergic system by early exposures may contribute to permanent CNS connectivity alterations. This has important ramifications because of the association between premature birth and accompanying hypoxia, and increased risk of autism and evidence associating in utero exposure to some antidepressants and neurodevelopmental disorders. Finally, this work demonstrates that the vertebrate CNS can modulate its connectivity in response to the external environment.
Assuntos
Axônios/metabolismo , Meio Ambiente , Efrina-B2/biossíntese , Rede Nervosa/embriologia , Rede Nervosa/metabolismo , Receptor 5-HT2A de Serotonina/deficiência , Animais , Animais Geneticamente Modificados , Feminino , Neurogênese/fisiologia , Gravidez , Núcleos da Rafe/embriologia , Núcleos da Rafe/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/deficiência , Serotonina/metabolismo , Peixe-ZebraRESUMO
Serotonin is a monoamine neurotransmitter that is involved in numerous physiological functions and its dysregulation is implicated in various psychiatric diseases. In all non-placental vertebrates, serotoninergic (5-HT) neurons are present in several regions of the brain, including the hypothalamus. In placental mammals, however, 5-HT neurons are located in the raphe nuclei only. In all species, though, 5-HT neurons constitute a functionally and molecularly heterogeneous population. How the non-raphe 5-HT populations are developmentally encoded is unknown. Using the zebrafish model we show that, in contrast to the raphe populations, hypothalamic 5-HT neurons are generated independently of the ETS-domain transcription factor Pet1 (Fev). By applying a combination of pharmacological tools and gene knockdown and/or overexpression experiments, we demonstrate that Fgf signalling acts via another ETS-domain transcription factor, Etv5b (Erm), to induce hypothalamic 5-HT neurons. We provide evidence that Etv5b exerts its effects by regulating cell cycle parameters in 5-HT progenitors. Our results highlight a novel role for Etv5b in neuronal development and provide support for the existence of a developmental heterogeneity among 5-HT neurons in their requirement for ETS-domain transcription factors.
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Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas c-ets/metabolismo , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo , Animais , Transporte Biológico , Ciclo Celular , Modelos Biológicos , Estrutura Terciária de Proteína , Núcleos da Rafe/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
First line pain relief medication during pregnancy relies nearly entirely on the over-the-counter analgesic acetaminophen, which is generally considered safe to use during gestation. However, recent epidemiological studies suggest a risk of developing attention-deficit/hyperactivity disorder (ADHD)-like symptoms in children if mothers use acetaminophen during pregnancy. Currently, there are no experimental proofs that prenatal acetaminophen exposure causes developmental brain alterations of progeny. Exposure to high acetaminophen concentrations causes liver toxicity, which is well investigated in different model organisms. However, sub-liver-toxic concentrations have not been experimentally investigated with respect to ADHD endophenotypes such as hyperactivity. We used zebrafish to investigate the potential impact of acetaminophen exposure on locomotor activity levels, and compared it to the established zebrafish Latrophilin 3 (Lphn3) ADHD-model. We determined the sub-liver-toxic concentration of acetaminophen in zebrafish larvae and treated wild-type and lphn3.1 knockdown larvae with increasing concentrations of acetaminophen. We were able to confirm that lphn3.1 knockdown alone causes hyperactivity, strengthening the implication of Lphn3 dysfunction as an ADHD risk factor. Neither acute nor chronic exposure to acetaminophen at sub-liver-toxic concentrations in wild-type or lphn3.1 knock-downs increases locomotor activity levels. Together our findings show that embryonic to larval exposure to acetaminophen does not cause hyperactivity in zebrafish larvae. Furthermore, there are no additive and/or synergistic effects of acetaminophen exposure in a susceptible background induced by knock-down of lphn3.1. Our experimental study suggests that there is, at least in zebrafish larvae, no direct link between embryonic acetaminophen exposure and hyperactivity. Further work is necessary to clarify this issue in humans.
Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Transtorno do Deficit de Atenção com Hiperatividade/induzido quimicamente , Fatores Etários , Análise de Variância , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Locomoção/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Peixe-ZebraRESUMO
Members of the family of metabotropic glutamate receptors are involved in the pathomechanism of several disorders of the nervous system. Besides the well-investigated function of dysfunctional glutamate receptor signaling in neurodegenerative diseases, neurodevelopmental disorders (NDD), like autism spectrum disorders (ASD) and attention-deficit and hyperactivity disorder (ADHD) might also be partly caused by disturbed glutamate signaling during development. However, the underlying mechanism of the type III metabotropic glutamate receptor 8 (mGluR8 or GRM8) involvement in neurodevelopment and disease mechanism is largely unknown. Here we show that the expression pattern of the two orthologs of human GRM8, grm8a and grm8b, have evolved partially distinct expression patterns in the brain of zebrafish (Danio rerio), especially at adult stages, suggesting sub-functionalization of these two genes during evolution. Using double in situ hybridization staining in the developing brain we demonstrate that grm8a is expressed in a subset of gad1a-positive cells, pointing towards glutamatergic modulation of GABAergic signaling. Building on this result we generated loss-of-function models of both genes using CRISPR/Cas9. Both mutant lines are viable and display no obvious gross morphological phenotypes making them suitable for further analysis. Initial behavioral characterization revealed distinct phenotypes in larvae. Whereas grm8a mutant animals display reduced swimming velocity, grm8b mutant animals show increased thigmotaxis behavior, suggesting an anxiety-like phenotype. We anticipate that our two novel metabotropic glutamate receptor 8 zebrafish models may contribute to a deeper understanding of its function in normal development and its role in the pathomechanism of disorders of the central nervous system.
RESUMO
Recent advances in the genetics of neurodevelopmental disorders (NDDs) have identified the transcription factor FOXP2 as one of numerous risk genes, e.g. in autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD). FOXP2 function is suggested to be involved in GABAergic signalling and numerous studies demonstrate that GABAergic function is altered in NDDs, thus disrupting the excitation/inhibition balance. Interestingly, GABAergic signalling components, including glutamate-decarboxylase 1 (Gad1) and GABA receptors, are putative transcriptional targets of FOXP2. However, the specific role of FOXP2 in the pathomechanism of NDDs remains elusive. Here we test the hypothesis that Foxp2 affects behavioural dimensions via GABAergic signalling using zebrafish as model organism. We demonstrate that foxp2 is expressed by a subset of GABAergic neurons located in brain regions involved in motor functions, including the subpallium, posterior tuberculum, thalamus and medulla oblongata. Using CRISPR/Cas9 gene-editing we generated a novel foxp2 zebrafish loss-of-function mutant that exhibits increased locomotor activity. Further, genetic and/or pharmacological disruption of Gad1 or GABA-A receptors causes increased locomotor activity, resembling the phenotype of foxp2 mutants. Application of muscimol, a GABA-A receptor agonist, rescues the hyperactive phenotype induced by the foxp2 loss-of-function. By reverse translation of the therapeutic effect on hyperactive behaviour exerted by methylphenidate, we note that application of methylphenidate evokes different responses in wildtype compared to foxp2 or gad1b loss-of-function animals. Together, our findings support the hypothesis that foxp2 regulates locomotor activity via GABAergic signalling. This provides one targetable mechanism, which may contribute to behavioural phenotypes commonly observed in NDDs.
Assuntos
Transtornos do Neurodesenvolvimento , Peixe-Zebra , Animais , Neurônios GABAérgicos , Locomoção , Ácido gama-AminobutíricoRESUMO
The glucose-fructose oxidoreductase domain containing gene family (GFOD) is small and contains only two members in human (GFOD1 and GFOD2). Information about its function is scarce. As the name implies the proteins contain an enzyme-defining domain, however, if this is functional and has enzymatic activity remains to be shown. A single nucleotide polymorphism situated in an intron of GFOD1 was found to be associated with inattentive symptomology in patients with attention-deficit/hyperactivity disorder. Further, in a large schizophrenia genome-wide association study the GFOD2 locus was found to be associated with the psychiatric condition. Until now, however, it is unclear what specific functions are associated with the two GFOD-family members, if they might be involved in neurodevelopment and how this may relate to the development of psychiatric disorders. In order to gain first insights into the hypothesis that GFOD-family members are involved in brain development and/or function we performed RNA in situ hybridization on zebrafish (Danio rerio) tissues at different developmental stages. We found that both family members are expressed in the central nervous system at embryonic, larvae and adult stages. We were able to define subtle differences of expression of the two gfod genes and we showed that a subset of GABAergic neurons express gfod1. Taken together, we conclude that both gfod gene family members are expressed in overlapping as well as in distinct regions in the zebrafish central nervous system. Our data suggest that gfod1 and gfod2 are relevant both for the developing and adult zebrafish brain. This study paves the way for further functional analyses of this yet unexplored gene family.
Assuntos
Encéfalo/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Encéfalo/embriologia , Embrião não Mamífero/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Humanos , Hibridização In SituRESUMO
Bioimage analysis of fluorescent labels is widely used in the life sciences. Recent advances in deep learning (DL) allow automating time-consuming manual image analysis processes based on annotated training data. However, manual annotation of fluorescent features with a low signal-to-noise ratio is somewhat subjective. Training DL models on subjective annotations may be instable or yield biased models. In turn, these models may be unable to reliably detect biological effects. An analysis pipeline integrating data annotation, ground truth estimation, and model training can mitigate this risk. To evaluate this integrated process, we compared different DL-based analysis approaches. With data from two model organisms (mice, zebrafish) and five laboratories, we show that ground truth estimation from multiple human annotators helps to establish objectivity in fluorescent feature annotations. Furthermore, ensembles of multiple models trained on the estimated ground truth establish reliability and validity. Our research provides guidelines for reproducible DL-based bioimage analyses.
Research in biology generates many image datasets, mostly from microscopy. These images have to be analyzed, and much of this analysis relies on a human expert looking at the images and manually annotating features. Image datasets are often large, and human annotation can be subjective, so automating image analysis is highly desirable. This is where machine learning algorithms, such as deep learning, have proven to be useful. In order for deep learning algorithms to work first they have to be 'trained'. Deep learning algorithms are trained by being given a training dataset that has been annotated by human experts. The algorithms extract the relevant features to look out for from this training dataset and can then look for these features in other image data. However, it is also worth noting that because these models try to mimic the annotation behavior presented to them during training as well as possible, they can sometimes also mimic an expert's subjectivity when annotating data. Segebarth, Griebel et al. asked whether this was the case, whether it had an impact on the outcome of the image data analysis, and whether it was possible to avoid this problem when using deep learning for imaging dataset analysis. For this research, Segebarth, Griebel et al. used microscopy images of mouse brain sections, where a protein called cFOS had been labeled with a fluorescent tag. This protein typically controls the rate at which DNA information is copied into RNA, leading to the production of proteins. Its activity can be influenced experimentally by testing the behaviors of mice. Thus, this experimental manipulation can be used to evaluate the results of deep learning-based image analyses. First, the fluorescent images were interpreted manually by a group of human experts. Then, their results were used to train a large variety of deep learning models. Models were trained either on the results of an individual expert or on the results pooled from all experts to come up with a consensus model, a deep learning model that learned from the personal annotation preferences of all experts. This made it possible to test whether training a model on multiple experts reduces the risk of subjectivity. As the training of deep learning models is random, Segebarth, Griebel et al. also tested whether combining the predictions from multiple models in a so-called model ensemble improves the consistency of the analyses. For evaluation, the annotations of the deep learning models were compared to those of the human experts, to ensure that the results were not influenced by the subjective behavior of one person. The results of all bioimage annotations were finally compared to the experimental results from analyzing the mice's behaviors in order to check whether the models were able to find the behavioral effect on cFOS. Segebarth, Griebel et al. concluded that combining the expert knowledge of multiple experts reduces the subjectivity of bioimage annotation by deep learning algorithms. Combining such consensus information in a group of deep learning models improves the quality of bioimage analysis, so that the results are reliable, transparent and less subjective.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Animais , Aprendizado Profundo , Medo , Corantes Fluorescentes , Masculino , Camundongos , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Peixe-ZebraRESUMO
In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the hindbrain serotonergic neurons, little is known about the development and function of these cells. Here, we identify fibroblast growth factor (Fgf)3 as the main Fgf ligand controlling the ontogeny of serotonergic CSF-c cells. We show that fgf3 positively regulates the number of serotonergic CSF-c cells, as well as a subset of dopaminergic and neuroendocrine cells in the posterior hypothalamus via control of proliferation and cell survival. Further, expression of the ETS-domain transcription factor etv5b is downregulated after fgf3 impairment. Previous findings identified etv5b as critical for the proliferation of serotonergic progenitors in the hypothalamus, and therefore we now suggest that Fgf3 acts via etv5b during early development to ultimately control the number of mature serotonergic CSF-c cells. Moreover, our analysis of the developing hypothalamic transcriptome shows that the expression of fgf3 is upregulated upon fgf3 loss-of-function, suggesting activation of a self-compensatory mechanism. Together, these results highlight Fgf3 in a novel context as part of a signalling pathway of critical importance for hypothalamic development.
RESUMO
The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington's and Alzheimer's diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders.
RESUMO
Serotonin (5HT) is a modulator of many vital processes in the spinal cord (SC), such as production of locomotion. In the larval zebrafish, intraspinal serotonergic neurons (ISNs) are a source of spinal 5HT that, despite the availability of numerous genetic and optical tools, has not yet been directly shown to affect the spinal locomotor network. In order to better understand the functions of ISNs, we used a combination of strategies to investigate ISN development, morphology, and function. ISNs were optically isolated from one another by photoconverting Kaede fluorescent protein in individual cells, permitting morphometric analysis as they developed in vivo. ISN neurite lengths and projection distances exhibited the greatest amount of change between 3 and 4 days post-fertilization (dpf) and appeared to stabilize by 5 dpf. Overall ISN innervation patterns were similar between cells and between SC regions. ISNs possessed rostrally-extending neurites resembling dendrites and a caudally-extending neurite resembling an axon, which terminated with an enlarged growth cone-like structure. Interestingly, these enlargements remained even after neurite extension had ceased. Functionally, application of exogenous 5HT reduced spinally-produced motor nerve bursting. A selective 5HT reuptake inhibitor and ISN activation with channelrhodopsin-2 each produced similar effects to 5HT, indicating that spinally-intrinsic 5HT originating from the ISNs has an inhibitory effect on the spinal locomotor network. Taken together this suggests that the ISNs are morphologically mature by 5 dpf and supports their involvement in modulating the activity of the spinal locomotor network. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018.
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A comparative study performed in mice investigating the action of DF302, a novel fluoride-containing gamma-carboline derivative, in comparison to the structurally similar neuroprotective drug dimebon. Drug effects on learning and memory, emotionality, hippocampal neurogenesis and mitochondrial functions, as well as AMPA-mediated currents and the 5-HT6 receptor are reported. In the step-down avoidance and fear-conditioning paradigms, bolus administration of drugs at doses of 10 or 40 mg/kg showed that only the higher dose of DF302 improved long-term memory while dimebon was ineffective at either dosage. Short-term memory and fear extinction remained unaltered across treatment groups. During the 5-day predation stress paradigm, oral drug treatment over a period of 2 weeks at the higher dosage regimen decreased anxiety-like behaviour. Both compounds supressed inter-male aggression in CD1 mice, the most eminent being the effects of DF302 in its highest dose. DF302 at the higher dose decreased floating behaviour in a 2-day swim test and after 21-day ultrasound stress. The density of Ki67-positive cells, a marker of adult neurogenesis, was reduced in the dentate gyrus of stressed dimebon-treated and non-treated mice, but not in DF302-treated mice. Non-stressed mice that received DF302 had a higher density of Ki67-positive cells than controls unlike dimebon-treated mice. Similar to dimebon, DF302 effectively potentiated AMPA receptor-mediated currents, bound to the 5-HT6 receptor, inhibited mitochondrial permeability transition and displayed cytoprotective properties in cellular models of neurodegeneration. Thus, DF302 exerts multi-target effects on the key mechanisms of neurodegenerative pathologies and can be considered as an optimized novel analogue of the neuroprotective agent dimebon.
Assuntos
Carbolinas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Compostos de Flúor/administração & dosagem , Memória/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Estresse Psicológico/tratamento farmacológico , Animais , Compostos de Flúor/metabolismo , Indóis/administração & dosagem , Indóis/química , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Ratos , Receptores de Serotonina/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologiaRESUMO
The tooth pulp has a dense sensory innervation which, upon stimulation, conveys sensory signals perceived as pain. This innervation, which originates from the trigeminal ganglion, is established through a series of regulated steps during development, and represents an interesting example of tissue targeting by pain-specific nerves. We have investigated various potentially neurotrophic and neurorepulsive influences during this process. The dental papilla/pulp appears to secrete neurite growth inhibitory molecular factors at early stages, which prevent nerve fibers from entering the tissue at what appears to be inappropriate timepoints. Later, a shift from repulsive to attractive factors apparently takes place, and nerve fibers then enter the tooth. When nerve fibers have invaded the dental mesenchyme, a complicated interplay of secreted and membrane-bound factors probably directs the nerve terminals to appropriate sites. Laminin-8 (alpha4beta1gamma1, Lm-411), which is produced by pulpal cells, emerges as an important candidate molecule in this context. Insights into the interactions between the dental pulp nerve fibers and their environment may become important in the search for novel ways to ameliorate pain in the tooth, as well as at other sites.
Assuntos
Polpa Dentária/inervação , Proteínas da Matriz Extracelular/fisiologia , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Nociceptores/crescimento & desenvolvimento , Animais , Polpa Dentária/metabolismo , Humanos , Terminações Nervosas/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Dente/inervação , Dente/metabolismoRESUMO
Septins are a highly conserved family of small GTPases that form cytoskeletal filaments. Their cellular functions, especially in the nervous system, still remain largely enigmatic, but there are accumulating lines of evidence that septins play important roles in neuronal physiology and pathology. In order to further dissect septin function in the nervous system a detailed temporal resolved analysis in the genetically well tractable model vertebrate zebrafish (Danio rerio) is crucially necessary. To close this knowledge gap we here provide a reference dataset describing the expression of selected septins (sept3, sept5a and sept5b) in the zebrafish central nervous system. Strikingly, proliferation zones are devoid of expression of all three septins investigated, suggesting that they have a role in post-mitotic neural cells. Our finding that three septins are mainly expressed in non-proliferative regions was further confirmed by double-stainings with a proliferative marker. Our RNA in situ hybridization (ISH) study, detecting sept3, sept5a and sept5b mRNAs, shows that all three septins are expressed in largely overlapping regions of the developing brain. However, the expression of sept5a is much more confined compared to sept3 and sept5b. In contrast, the expression of all the three analyzed septins is largely similar in the adult brain.
RESUMO
Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1-4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Septinas/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Animais , Sistema Nervoso Central/química , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/química , Brânquias/crescimento & desenvolvimento , Hibridização In Situ , Neurônios , Rombencéfalo/química , Rombencéfalo/crescimento & desenvolvimento , Inclusão do Tecido , Peixe-Zebra/genéticaRESUMO
Zebrafish intraspinal serotonergic neuron (ISN) morphology and distribution have been examined in detail at different ages; however, some aspects of the development of these cells remain unclear. Although antibodies to serotonin (5-HT) have detected ISNs in the ventral spinal cord of embryos, larvae, and adults, the only tryptophan hydroxylase (tph) transcript that has been described in the spinal cord is tph1a. Paradoxically, spinal tph1a is only expressed transiently in embryos, which brings the source of 5-HT in the ISNs of larvae and adults into question. Because the pet1 and tph2 promoters drive transgene expression in the spinal cord, we hypothesized that tph2 is expressed in spinal cords of zebrafish larvae. We confirmed this hypothesis through in situ hybridization. Next, we used 5-HT antibody labeling and transgenic markers of tph2-expressing neurons to identify a transient population of ISNs in embryos that was distinct from ISNs that appeared later in development. The existence of separate ISN populations may not have been recognized previously due to their shared location in the ventral spinal cord. Finally, we used transgenic markers and immunohistochemical labeling to identify the transient ISN population as GABAergic Kolmer-Agduhr double-prime (KAâ³) neurons. Altogether, this study revealed a novel developmental paradigm in which KAâ³ neurons are transiently serotonergic before the appearance of a stable population of tph2-expressing ISNs.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Serotonina/metabolismo , Medula Espinal/citologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva , Medula Espinal/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The serotonin (5-HT) system is generally considered as a single modulatory system, with broad and diffuse projections. However, accumulating evidence points to the existence of distinct cell groups in the raphe. Here, we review prior evidence for raphe cell heterogeneity, considering different properties of 5-HT neurons, from metabolism to anatomy, and neurochemistry to physiology. We then summarize more recent data in mice and zebrafish that support a genetic diversity of 5-HT neurons, based on differential transcription factor requirements for the acquisition of the 5-HT identity. In both species, PET1 plays a major role in the acquisition and maintenance of 5-HT identity in the hindbrain, although some 5-HT neurons do not require PET1 for their differentiation, indicating the existence of several transcriptional routes to become serotoninergic. In mice, both PET1-dependent and -independent 5-HT neurons are located in the raphe, but have distinct anatomical features, such as the morphology of axon terminals and projection patterns. In zebrafish, all raphe neurons express pet1, but Pet1-independent 5-HT cell groups are present in the forebrain. Overall, these observations support the view that there are a number of distinct 5-HT subsystems, including within the raphe nuclei, with unique genetic programming and functions.
Assuntos
Núcleos da Rafe/anatomia & histologia , Neurônios Serotoninérgicos/fisiologia , Serotonina/fisiologia , Animais , Axônios/fisiologia , Encéfalo/fisiologia , Fenômenos Eletrofisiológicos , Variação Genética , Camundongos , Camundongos Knockout , Núcleos da Rafe/citologia , Núcleos da Rafe/fisiologia , Neurônios Serotoninérgicos/citologia , Transmissão Sináptica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Neurons using serotonin (5-HT) as neurotransmitter and/or modulator have been identified in the central nervous system in representatives from all vertebrate clades, including jawless, cartilaginous and ray-finned fishes. The aim of this review is to summarize our current knowledge about the anatomical organization of the central serotonergic system in fishes. Furthermore, selected key functions of 5-HT will be described. The main focus will be the adult brain of teleosts, in particular zebrafish, which is increasingly used as a model organism. It is used to answer not only genetic and developmental biology questions, but also issues concerning physiology, behavior and the underlying neuronal networks. The many evolutionary conserved features of zebrafish combined with the ever increasing number of genetic tools and its practical advantages promise great possibilities to increase our understanding of the serotonergic system. Further, comparative studies including several vertebrate species will provide us with interesting insights into the evolution of this important neurotransmitter system.