Toward molecular genetic dissection of neural circuits for emotional and motivational behaviors

How does the brain process the emotional meaning of sensory stimuli and in turn drive behavior?Studies in the mammalian systems have identified various brain regions and neurotransmitter systems that are critical for emotional and motivational behaviors and have implicated their involvement in neuropsychiatric disorders including anxiety, depression, schizophrenia, and addiction. Despite these significant advancements, the precise neural circuitry underlying emotional and motivational behaviors remains to be understood at molecular and cellular levels. In this review, we discuss how the vertebrate model organism zebrafish can help us gain insights into the underlying circuitry. We first describe studies of several simple and relevant preference behaviors in this model organism, and then discuss approaches and technologies that can be used to uncover the development and function of neural circuits underlying these behaviors.     

幼年斑马鱼的视觉系统与捕食行为

神经环路的研究是揭示动物行为神经机制的关键。斑马鱼作为一种低等脊椎动物, 在神经环路的研究中有着独特优势。文章描述了斑马鱼视觉系统及其下游的神经环路, 重点讨论了它们在捕食行为中的可能作用。斑马鱼捕食行为主要依赖于视觉功能, 该过程涉及到视觉-运动通路各个层次的神经环路, 包括下游的网状脊髓命令神经元、脊髓内部的运动控制环路以及一些亟待研究的功能单元。随着在体记录和操纵神经元活动技术的成熟, 以及行为学范式的完善, 对斑马鱼捕食行为相关神经环路的研究将在未来数年内迅速发展, 同时也将推动神经科学相关研究的进步。     

Behavioral genetics in larval zebrafish: learning from the young

Deciphering the genetic code that determines how the vertebrate nervous system assembles into neural circuits that ultimately control behavior is a fascinating and challenging question in modern neurobiology. Because of the complexity of this problem, successful strategies require a simple yet focused experimental approach without limiting the scope of the discovery. Unbiased, large-scale forward genetic screens in invertebrate organisms have yielded great insight into the genetic regulation of neural circuit assembly and function. For many reasons, this highly successful approach has been difficult to recapitulate in the behavioral neuroscience field's classic vertebrate model organisms-rodents. Here, we discuss how larval zebrafish provide a promising model system to which we can apply the design of invertebrate behavior-based screens to reveal the genetic mechanisms critical for neural circuit assembly and function in vertebrates.     

Using transgenic zebrafish (Danio rerio) to study development of the craniofacial skeleton

Transgenic zebrafish provide amazing new tools for following and manipulating cells that form the skeleton. Transgenes that label distinct populations of embryonic cells, such as neural crest that forms most of the skull vault as well as the jaws and gills can be used to determine the embryonic origins of cartilages and bones. This provides a powerful model system for studies of the developmental basis for human birth defects and in a comparative context provides new insights into the developmental changes underlying morphological evolution. Targeting transgenes to nuclear or membrane compartments allows detailed tracking of cell shapes and movements. Here we review how such transgenic markers combined with mutants or tissue grafts to generate mosaic zebrafish embryos have already provided many new insights into skeletal development and disease. In the long run, transgenics designed to perturb gene expression hold great promise for studies of gene function.     

趋流行为下斑马鱼幼鱼的全脑成像

目的 趋流,意即在水中调整身体方向并逆流而上的能力,是一种在大多数鱼类与两栖类动物中存在的保守行为。虽然关于趋流的研究已有一段很长的历史,并且近年来斑马鱼幼鱼趋流行为的理论机制也被提出,但是分布式的神经环路是如何整合多感知信息、做出决策、并实现行为控制仍然是个未知数。对自由运动的斑马鱼进行全脑神经活动成像为理解这一困难的问题提供了特殊的机会。方法 本文开发了一种微流控装置精确控制环境水流并激发斑马鱼的趋流行为。将该微流控芯片与扩增视野光场显微镜以及追踪系统整合,从而记录自由行为下斑马鱼全脑的神经活动。结果 在整合的微流控装置中稳定观察到了斑马鱼在水流中保持自身位置不变、逆流而上等刻板的趋流行为现象。与此同时,实现了对斑马鱼幼鱼趋流行为过程中的全脑钙活动记录。本文初步发现了几个脑区的神经活动与趋流行为相关。结论 本研究第一次展示了在斑马鱼幼鱼趋流行为的同时记录全脑神经活动的技术。接下来对神经活动和行为数据的分析与建模将有助于更好地理解一种重要自然行为背后的感觉运动转换机制。     

Using imaging and genetics in zebrafish to study developing spinal circuits in vivo

Imaging and molecular approaches are perfectly suited to young, transparent zebrafish (Danio rerio), where they have allowed novel functional studies of neural circuits and their links to behavior. Here, we review cutting-edge optical and genetic techniques used to dissect neural circuits in vivo and discuss their application to future studies of developing spinal circuits using living zebrafish. We anticipate that these experiments will reveal general principles governing the assembly of neural circuits that control movements.     

From neuron to behavior: Sensory‐motor coordination of zebrafish turning behavior

Recent development of optogenetics brought non‐invasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models for this technique, which enables us to investigate neural circuits for behaviors based on a whole individual nervous system. In this article we review our recent finding that suggests sensory‐motor coordination in larval zebrafish escape behavior. When water vibration stimulates mechanosensory Rohon‐Beard (RB) neurons, intra‐spinal reflex circuit launches contralateral trunk muscle contraction that makes rapid body curvature for turning. In addition, positional information of the stimulus is conveyed to supra‐spinal circuits, and then regulates the curvature strength for appropriate escape pathway from the threat. Sensory‐motor coordination is a fundamental feature to adapt behaviors to environment, and zebrafish larvae would be an excellent model for elucidating its neural backbones.     

Linking genes to brain,behavior and neurological diseases: what can we learn from zebrafish?

How our brain is wired and subsequently generates functional output, ranging from sensing and locomotion to emotion, decision-making and learning and memory, remains poorly understood. Dys-regulation of these processes can lead to neurodegenerative, as well as neuro-psychiatric, disorders. Molecular genetic together with behavioral analyses in model organisms identify genes involved in the formation of neuronal circuits, the execution of behavior and mechanisms involved in neuro-pathogenesis. In this review I will discuss the current progress and future potential for study in a newly established vertebrate model organism for genetics, the zebrafish Danio rerio . Where available, schemes and results of genetic screens will be reviewed concerning the sensory, motor and neuromodulatory monoamine systems. Genetic analyses in zebrafish have the potential to provide important insights into the relationship between genes, neuronal circuits and behavior in normal as well as diseased states.     

Motion processing streams in Drosophila are behaviorally specialized

Motion vision is an ancient faculty, critical to many animals in a range of ethological contexts, the underlying algorithms of which provide central insights into neural computation. However, how motion cues guide behavior is poorly understood, as the neural circuits that implement these computations are largely unknown in any organism. We develop a systematic, forward genetic approach using high-throughput, quantitative behavioral analyses to identify the neural substrates of motion vision in Drosophila in an unbiased fashion. We then delimit the behavioral contributions of both known and novel circuit elements. Contrary to expectation from previous studies, we find that orienting responses to motion are shaped by at least two neural pathways. These pathways are sensitive to different visual features, diverge immediately postsynaptic to photoreceptors, and are coupled to distinct behavioral outputs. Thus, behavioral responses to complex stimuli can rely on surprising neural specialization from even the earliest sensory processing stages.     

Transgenic zebrafish expressing fluorescent proteins in central nervous system neurons

Zebrafish is a powerful model system for investigations of vertebrate neural development. The animal has also become an important model for studies of neuronal function. Both in developmental and functional studies, transgenic zebrafish expressing fluorescent proteins in central nervous system neurons have been playing important roles. We review here the methods for producing transgenic zebrafish. Recent advances in transposon- or bacterial artificial chromosome-based transgenesis greatly facilitate the creation of useful lines. We also present our study on alx -positive neurons to reveal how transgenic zebrafish expressing fluorescent proteins in a specific class of neurons can be used to investigate their development and function.     

The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

Genetic dissection of the zebrafish habenula, a possible switching board for selection of behavioral strategy to cope with fear and anxiety

The habenula is a part of an evolutionarily highly conserved conduction pathway within the limbic system that connects telencephalic nuclei to the brain stem nuclei such as interpeduncular nucleus(IPN), the ventral tegmental area (VTA), and the raphe.In mammals, the medial habenula receives inputs from the septohippocampal system, and relaying such information to the IPN. In contrast, the lateral habenula receives inputs from the ventral pallidum, a part of the basal ganglia. The physical adjunction of these two habenular nuclei suggests that the habenula may act as an intersection of the neural circuits for controlling emotion and behavior. We have recently elucidated that zebrafish has the equivalent structure as the mammalian habenula. The transgenic zebrafish, in which the neural signal transmission from the lateral subnucleus of the dorsal habenula to the dorsal IPN was selectively impaired, showed extremely enhanced levels of freezing response to presentation of the conditioned aversive stimulus. Our observation supports that the habenula may act as the multimodal switching board for controlling emotional behaviors and/or memory inexperience dependent manners.     

Zebrafish forebrain and temporal conditioning

The rise of zebrafish as a neuroscience research model organism, in conjunction with recent progress in single-cell resolution whole-brain imaging of larval zebrafish, opens a new window of opportunity for research on interval timing. In this article, we review zebrafish neuroanatomy and neuromodulatory systems, with particular focus on identifying homologies between the zebrafish forebrain and the mammalian forebrain. The neuroanatomical and neurochemical basis of interval timing is summarized with emphasis on the potential of using zebrafish to reveal the neural circuits for interval timing. The behavioural repertoire of larval zebrafish is reviewed and we demonstrate that larval zebrafish are capable of expecting a stimulus at a precise time point with minimal training. In conclusion, we propose that interval timing research using zebrafish and whole-brain calcium imaging at single-cell resolution will contribute to our understanding of how timing and time perception originate in the vertebrate brain from the level of single cells to circuits.     

Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain

How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.     

Molecular approaches to aggregation behavior and social attachment

In many animal species individuals aggregate to live in groups. A range of experimental approaches in different animals, including studies of social feeding in nematodes, maternal behavior in rats and sheep, and pair-bonding in voles, are providing insights into the neural bases for these behaviors. These studies are delineating multiple neural circuits and gene networks in the brain that interact in ways that are as yet poorly understood to coordinate social behavior.     

Activity-induced long-term potentiation of excitatory synapses in developing zebrafish retina in vivo

Neural activity-induced long-term potentiation (LTP) of synaptic transmission is believed to be one of the cellular mechanisms underlying experience-dependent developmental refinement of neural circuits. Although it is well established that visual experience and neural activity are critical for the refinement of retinal circuits, whether and how LTP occurs in the retina remain unknown. Using in?vivo perforated whole-cell recording and two-photon calcium imaging, we find that both repeated electrical and visual stimulations can induce LTP at excitatory synapses formed by bipolar cells on retinal ganglion cells in larval but not juvenile zebrafish. LTP induction requires the activation of postsynaptic N-methyl-D-aspartate receptors, and its expression involves arachidonic acid-dependent presynaptic changes in calcium dynamics and neurotransmitter release. Physiologically, both electrical and visual stimulation-induced LTP can enhance visual responses of retinal ganglion cells. Thus, LTP exists in developing retinae with a presynaptic locus and may serve for visual experience-dependent refinement of retinal circuits.     

Fear, anxiety, and control in the zebrafish

Emotional responses are triggered by environmental signals and involve profound changes at multiple levels, from molecular to behavior. Much has been learnt about two emotions, fear and anxiety, by studying mammalian models. In particular, neural circuits and the corresponding molecular mechanisms essential for the learning and retention of fear, as well as the activation of anxiety, are well known. In contrast, little is known about how these emotions are terminated. The zebrafish is a newcomer to the world of emotion research. A number of assays for fear and anxiety now exist, but the underlying neural circuitry is largely undefined. Recent experiments, however, appear to provide a hint as to how anxiety is downregulated. In particular, they point to an essential role for a circuit involving the posterior septum, medial habenula, and interpeduncular nucleus. This evolutionarily conserved circuit may fulfill a similar function in mammals.     

Genetic basis of male sexual behavior

Male sexual behavior is increasingly the focus of genetic study in a variety of animals. Genetic analysis in the soil roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster has lead to identification of genes and circuits that govern behaviors ranging from motivation and mate-searching to courtship and copulation. Some worm and fly genes have counterparts with related functions in higher animals and many more such correspondences can be expected. Analysis of mutations in mammals can potentially lead to insights into such issues as monogamous versus promiscuous sexual behavior and sexual orientation. Genetic analysis of sexual behavior has implications for understanding how the nervous system generates and controls a complex behavior. It can also help us to gain an appreciation of how behavior is encoded by genes and their regulatory sequences.