Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling.

A Large Field-of-view, Single-cell-resolution Two- and Three-Photon Microscope for Deep Imaging.

In vivo imaging of large-scale neuron activity plays a pivotal role in unraveling the function of the brain's network. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases.

Z-REX: shepherding reactive electrophiles to specific proteins expressed tissue specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.

This Protocol Extension describes the adaptation of an existing Protocol detailing the use of targetable reactive electrophiles and oxidants, an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of reactive electrophiles and oxidants technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments).

Whole-brain optical access in a small adult vertebrate with two- and three-photon microscopy.

Although optical microscopy has allowed scientists to study the entire brain in early developmental stages, access to the brains of live, adult vertebrates has been limited. , a genus of miniature, transparent fish closely related to zebrafish has been introduced as a neuroscience model to study the adult vertebrate brain. However, the extent of optically accessible depth in these animals has not been quantitatively characterized.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish.

Deep three-photon imaging of the brain in intact adult zebrafish.

Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model.

Features of the structure, development, and activity of the zebrafish noradrenergic system explored in new CRISPR transgenic lines.

The noradrenergic (NA) system of vertebrates is implicated in learning, memory, arousal, and neuroinflammatory responses, but is difficult to access experimentally. Small and optically transparent, larval zebrafish offer the prospect of exploration of NA structure and function in an intact animal. We made multiple transgenic zebrafish lines using the CRISPR/Cas9 system to insert fluorescent reporters upstream of slc6a2, the norepinephrine transporter gene.

Key Features of Structural and Functional Organization of Zebrafish Facial Motor Neurons Are Resilient to Disruption of Neuronal Migration.

The location of neurons early in development can be critical for their ability to differentiate and receive normal synaptic inputs. Indeed, disruptions in neuronal positioning lead to a variety of neurological disorders. Neurons have, however, shifted their positions across phylogeny, suggesting that changes in location do not always spell functional disaster.

In Vivo Measurement of Glycine Receptor Turnover and Synaptic Size Reveals Differences between Functional Classes of Motoneurons in Zebrafish.

The interplay between binding and unbinding of synaptic receptor proteins at synapses plays an important role in determining receptor concentration and synaptic strength, with known links between changes in binding kinetics and synaptic plasticity. The regulation of such kinetics may subserve the specific functional requirements of neurons in intact circuits. However, the majority of studies of synaptic turnover kinetics have been performed in cultured neurons outside the context of normal circuits, and synaptic receptor turnover has not been measured at individual synaptic sites in vivo.

A circuit motif in the zebrafish hindbrain for a two alternative behavioral choice to turn left or right.

Animals collect sensory information from the world and make adaptive choices about how to respond to it. Here, we reveal a network motif in the brain for one of the most fundamental behavioral choices made by bilaterally symmetric animals: whether to respond to a sensory stimulus by moving to the left or to the right. We define network connectivity in the hindbrain important for the lateralized escape behavior of zebrafish and then test the role of neurons by using laser ablations and behavioral studies.

TRP channel mediated neuronal activation and ablation in freely behaving zebrafish.

The zebrafish (Danio rerio) is a useful vertebrate model system in which to study neural circuits and behavior, but tools to modulate neurons in freely behaving animals are limited. As poikilotherms that live in water, zebrafish are amenable to thermal and pharmacological perturbations. We exploit these properties by using transient receptor potential (TRP) channels to activate or ablate specific neuronal populations using the chemical and thermal agonists of heterologously expressed TRPV1, TRPM8 and TRPA1.

Homeostatic regulation of dendritic dynamics in a motor map in vivo.

Neurons and circuits are remarkably dynamic. Their gross structure can change within minutes as neurons sprout and retract processes to form new synapses. Homeostatic processes acting to regulate neuronal activity contribute to these dynamics and predict that the dendritic dynamics within pools of neurons should vary systematically in accord with the activity levels of individual neurons in the pool during behaviour.

Chronic in vivo imaging in the mouse spinal cord using an implanted chamber.

Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation.

In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy.

Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy-a coherent, nonlinear, dye-free imaging modality-provides micrometer resolution imaging of myelin in the mouse CNS.

A structural and functional ground plan for neurons in the hindbrain of zebrafish.

The vertebrate hindbrain contains various sensory-motor networks controlling movements of the eyes, jaw, head, and body. Here we show that stripes of neurons with shared neurotransmitter phenotype that extend throughout the hindbrain of young zebrafish reflect a broad underlying structural and functional patterning. The neurotransmitter stripes contain cell types with shared gross morphologies and transcription factor markers.

Mapping a sensory-motor network onto a structural and functional ground plan in the hindbrain.

The hindbrain of larval zebrafish contains a relatively simple ground plan in which the neurons throughout it are arranged into stripes that represent broad neuronal classes that differ in transmitter identity, morphology, and transcription factor expression. Within the stripes, neurons are stacked continuously according to age as well as structural and functional properties, such as axonal extent, input resistance, and the speed at which they are recruited during movements. Here we address the question of how particular networks among the many different sensory-motor networks in hindbrain arise from such an orderly plan.

Movement, technology and discovery in the zebrafish.

Zebrafish provide unique opportunities for optogenetic studies of behavior. Here, we review the most recent work using optogenetic and imaging approaches to study the neuronal circuits controlling movements in the transparent zebrafish. Specifically, we focus on what we have learned from zebrafish about neuronal migration, network formation and behavioral control, and what the future may hold.

Some principles of organization of spinal neurons underlying locomotion in zebrafish and their implications.

Recent studies of the spinal motor system of zebrafish, along with work in other species, are leading to some principles that appear to underlie the organization and recruitment of motor networks in cord: (1) broad neuronal classes defined by a set of transcription factors, key morphological features, and transmitter phenotypes arise in an orderly way from different dorso-ventral zones in spinal cord; (2) motor behaviors and both motoneurons and interneurons differentiate in order from gross, often faster, movements and the neurons driving them to progressively slower movements and their underlying neurons; (3) recruitment order of motoneurons and interneurons is based upon time of differentiation; (4) different locomotor speeds involve some shifts in the set of active interneurons. Here we review these principles and some of their implications for other parts of the brain, other vertebrates, and limbed locomotion.

Spinal interneurons differentiate sequentially from those driving the fastest swimming movements in larval zebrafish to those driving the slowest ones.

Studies of neuronal networks have revealed few general principles that link patterns of development with later functional roles. While investigating the neural control of movements, we recently discovered a topographic map in the spinal cord of larval zebrafish that relates the position of motoneurons and interneurons to their order of recruitment during swimming. Here, we show that the map reflects an orderly pattern of differentiation of neurons driving different movements.

Shared versus specialized glycinergic spinal interneurons in axial motor circuits of larval zebrafish.

The neuronal networks in spinal cord can produce a diverse array of motor behaviors. In aquatic vertebrates such as fishes and tadpoles, these include escape behaviors, swimming across a range of speeds, and struggling. We addressed the question of whether these behaviors are accomplished by a shared set of spinal interneurons activated in different patterns or, instead, involve specialized spinal interneurons that may shape the motor output to produce particular behaviors.

Continuous shifts in the active set of spinal interneurons during changes in locomotor speed.

The classic 'size principle' of motor control describes how increasingly forceful movements arise by the recruitment of motoneurons of progressively larger size and force output into the active pool. We explored the activity of pools of spinal interneurons in larval zebrafish and found that increases in swimming speed were not associated with the simple addition of cells to the active pool. Instead, the recruitment of interneurons at faster speeds was accompanied by the silencing of those driving movements at slower speeds.

Synaptic homeostasis in a zebrafish glial glycine transporter mutant.

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects.

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.