Ethan K. Scott

Targeting neural circuitry in zebrafish using GAL4 enhancer trapping.

We present a pilot enhancer trap screen using GAL4 to drive expression of upstream activator sequence (UAS)-linked transgenes in expression patterns dictated by endogenous enhancers in zebrafish. The patterns presented include expression in small subsets of neurons throughout the larval brain, which in some cases persist into adult. Through targeted photoconversion of UAS-driven Kaede and variegated expression of UAS-driven GFP in single cells, we begin to characterize the cellular components of labeled circuits.

Optogenetic dissection of a behavioural module in the vertebrate spinal cord.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer–Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer–Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Remote Control of Neuronal Activity with a Light-Gated Glutamate Receptor

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

How do dendrites take their shape

Recent technical advances have made possible the visualization and genetic manipulation of individual dendritic trees. These studies have led to the identification and characterization of molecules that are important for different aspects of dendritic development. Although much remains to be learned, the existing knowledge has allowed us to take initial steps toward a comprehensive understanding of how complex dendritic trees are built. In this review, we describe recent advances in our understanding of the molecular mechanisms underlying dendritic morphogenesis, and discuss their cell-biological implications.

Filtering of visual information in the tectum by an identified neural circuit

Small Is Attractive The optic tectum of zebrafish larvae is required for the detection, tracking, and capture of small, highly motile prey. Del Bene et al. (p. 669) applied a combination of optical, genetic, and pharmacological tools to investigate how neural circuits in the optic tectum filter out low-frequency visual information. Most tectal neurons were tuned to respond selectively to small, moving objects in the fishs visual environment and responded very poorly to large stimuli. This spatial filtering mechanism depended on the activity of a small population of GABAergic, inhibitory interneurons at the tectal surface. Inactivation or destruction of these interneurons removed the size selectivity of deeper neurons and the zebrafish lost their ability to catch prey. A neural circuit in zebrafish is preferentially activated by small visual stimuli, facilitating the capture of prey. The optic tectum of zebrafish is involved in behavioral responses that require the detection of small objects. The superficial layers of the tectal neuropil receive input from retinal axons, while its deeper layers convey the processed information to premotor areas. Imaging with a genetically encoded calcium indicator revealed that the deep layers, as well as the dendrites of single tectal neurons, are preferentially activated by small visual stimuli. This spatial filtering relies on GABAergic interneurons (using the neurotransmitter γ-aminobutyric acid) that are located in the superficial input layer and respond only to large visual stimuli. Photo-ablation of these cells with KillerRed, or silencing of their synaptic transmission, eliminates the size tuning of deeper layers and impairs the capture of prey.

Neuronal activity biases axon selection for myelination in vivo

An essential feature of vertebrate neural development is ensheathment of axons with myelin, an insulating membrane formed by oligodendrocytes. Not all axons are myelinated, but mechanisms directing myelination of specific axons are unknown. Using zebrafish, we found that activity-dependent secretion stabilized myelin sheath formation on select axons. When VAMP2-dependent exocytosis was silenced in single axons, oligodendrocytes preferentially ensheathed neighboring axons. Nascent sheaths formed on silenced axons were shorter in length, but when activity of neighboring axons was also suppressed, inhibition of sheath growth was relieved. Using in vivo time-lapse microscopy, we found that only 25% of oligodendrocyte processes that initiated axon wrapping were stabilized during normal development and that initiation did not require activity. Instead, oligodendrocyte processes wrapping silenced axons retracted more frequently. We propose that axon selection for myelination results from excessive and indiscriminate initiation of wrapping followed by refinement that is biased by activity-dependent secretion from axons.

The cellular architecture of the larval zebrafish tectum, as revealed by gal4 enhancer trap lines.

We have carried out a Gal4 enhancer trap screen in zebrafish, and have generated 184 stable transgenic lines with interesting expression patterns throughout the nervous system. Of these, three display clear expression in the tectum, each with a distinguishable and stereotyped distribution of Gal4 expressing cells. Detailed morphological analysis of single cells, using a genetic “Golgi-like” labelling method, revealed four common cell types (superficial, periventricular, shallow periventricular, and radial glial), along with a range of other less common neurons. The shallow periventricular (PV) and a subset of the PV neurons are tectal efferent neurons that target various parts of the reticular formation. We find that it is specifically PV neurons with dendrites in the deep tectal neuropil that target the reticular formation. This indicates that these neurons receive the tectums highly processed visual information (which is fed from the superficial retinorecipient layers), and relay it to premotor regions. Our results show that the larval tectum, both broadly and at the single cell level, strongly resembles a miniature version of its adult counterpart, and that it has all of the necessary anatomical characteristics to inform motor responses based on sensory input. We also demonstrate that mosaic expression of GFP in Gal4 enhancer trap lines can be used to describe the types and abundance of cells in an expression pattern, including the architectures of individual neurons. Such detailed anatomical descriptions will be an important part of future efforts to describe the functions of discrete tectal circuits in the generation of behavior.

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis.

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains ∼20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.

Genetic and optical targeting of neural circuits and behavior — zebrafish in the spotlight

Methods to label neurons and to monitor their activity with genetically encoded fluorescent reporters have been a staple of neuroscience research for several years. The recent introduction of photoswitchable ion channels and pumps, such as channelrhodopsin (ChR2), halorhodopsin (NpHR), and light-gated glutamate receptor (LiGluR), is enabling remote optical manipulation of neuronal activity. The translucent brains of zebrafish offer superior experimental conditions for optogenetic approaches in vivo. Enhancer and gene trapping approaches have generated hundreds of Gal4 driver lines in which the expression of UAS-linked effectors can be targeted to subpopulations of neurons. Local photoactivation of genetically targeted LiGluR, ChR2, or NpHR has uncovered novel functions for specific areas and cell types in zebrafish behavior. Because the manipulation is restricted to times and places where genetics (cell types) and optics (beams of light) intersect, this method affords excellent resolving power for the functional analysis of neural circuitry.

Proneural gene-linked neurogenesis in zebrafish cerebellum

In mammals, cerebellar neurons are categorized as glutamatergic or GABAergic, and are derived from progenitors that express the proneural genes atoh1 or ptf1a, respectively. In zebrafish, three atoh1 genes, atoh1a, atoh1b, and atoh1c, are expressed in overlapping but distinct expression domains in the upper rhombic lip (URL): ptf1a is expressed exclusively in the ventricular zone (VZ). Using transgenic lines expressing fluorescent proteins under the control of the regulatory elements of atoh1a and ptf1a, we traced the lineages of the cerebellar neurons. The atoh1(+) progenitors gave rise not only to granule cells but also to neurons of the anteroventral rhombencephalon. The ptf1a(+) progenitors generated Purkinje cells. The olig2(+) eurydendroid cells, which are glutamatergic, were derived mostly from ptf1a(+) progenitors in the VZ but some originated from the atoh1(+) progenitors in the URL. In the adult cerebellum, atoh1a, atoh1b, and atoh1c are expressed in the molecular layer of the valvula cerebelli and of the medial corpus cerebelli, and ptf1a was detected in the VZ. The proneural gene expression patterns coincided with the sites of proliferating neuronal progenitors in the adult cerebellum. Our data indicate that proneural gene-linked neurogenesis is evolutionarily conserved in the cerebellum among vertebrates, and that the continuously generated neurons help remodel neural circuits in the adult zebrafish cerebellum.