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Neuronal Circuit Development

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Group leader : Filippo Del Bene
Neuronal Circuit Development

Keywords

Neuronal Circuit, Synaptogenesis, Optic Tectum, Axonal Transport, Zebrafish

Neuronal circuits represent the cellular substrate of complex behaviors. Understanding their development and function constitutes therefore a central question in neuroscience. We use the zebrafish visual system as a model to understand neuronal circuit formation taking advantage of its small size and  transparency. Our studies will provide crucial information to unravel the function of more complex brains such as ours, and perhaps to better understand the origin of many neurological disorders.

 

 

 

 

 

 

 

 

 

A major goal of modern neuroscience is the complete understanding of neuronal circuits development and function in an intact behaving animal. Our group examines the formation and function of the visual system of zebrafish larvae using in vivo time-lapse microscopy and state-of-the-art “connectomic” and “optogenetic” approaches to monitor and perturb neuronal activity. We apply complementary cellular and molecular analysis to dissect this circuit and identify the neuronal substrate of visual behaviors.

Neuronal circuit development in the zebrafish optic tectum

We focus our analysis on the zebrafish optic tectum. This midbrain structure has emerged as a potentially tractable visuomotor transformer, which integrates visual and other sensory inputs to produce a motor response. We apply anatomical and functional studies to a better understand of how behavior can be controlled by neuronal circuits. The tectum sits at the surface of the larval brain, located just under a single-layed epithelial tissue, and is  divided into two main areas, the stratum periventriculare (SPV), which contains the cell bodies of most tectal neurons (PVNs), and the synaptic neuropil area, which contains their dendrites and axons, as well as the axons of retinal afferents.
 


 Analysis of SIN connectivity and function

We are particularly interested in understanding the function, development and connectivity of a newly characterized class of inhibitory interneurons located in the superficial part of the tectal neuropil named SINs (superficial inhibitory interneurons). Our previous work based on functional imaging has placed SINs at the center of a tectal micro-circuit for size tuning of visual stimuli. We will dissect this working model by analyzing the physiological properties of SINs and their development and connectivity at the level of single synapses by imaging these cells in vivo using fluorescent reporters in transgenic animals.


Axonal transport function in retinotecal axonal development and synaptogenesis.


We are interested in investigating the effects of mutations in molecular motors on axonal branching and synaptogenesis. We focus our analysis to the retinotectal system to unravel the molecular processes that control its development. Using single cell loss-of-function experiments we want to perturb the expression or the activity of molecular motors and associated proteins. Given the links that have already been made between many of these proteins and human neurodegenerative and psychiatric disorders, our studies will provide novel insights into the underlying molecular mechanisms.

Zebrafish optic tectum Localization in the zebrafish head region (A) and anatomical subdivision (B)Zebrafish optic tectum Localization in the zebrafish head region (A) and anatomical subdivision (B)

SINs localization (white arrows) is revealed by GFP expression in a transgenic lineSINs localization (white arrows) is revealed by GFP expression in a transgenic line

 

 

 

 

Single retina ganglion cell labeled with GFP to reveal its axonal morphology, while the tectum neuropil is highlighted in red bySingle retina ganglion cell labeled with GFP to reveal its axonal morphology, while the tectum neuropil is highlighted in red by

 

Key publications

  • Year of publication : 2016

  • Escape behaviors deliver organisms away from imminent catastrophe. Here, we characterize behavioral responses of freely swimming larval zebrafish to looming visual stimuli simulating predators. We report that the visual system alone can recruit lateralized, rapid escape motor programs, similar to those elicited by mechanosensory modalities. Two-photon calcium imaging of retino-recipient midbrain regions isolated the optic tectum as an important center processing looming stimuli, with ensemble activity encoding the critical image size determining escape latency. Furthermore, we describe activity in retinal ganglion cell terminals and superficial inhibitory interneurons in the tectum during looming and propose a model for how temporal dynamics in tectal periventricular neurons might arise from computations between these two fundamental constituents. Finally, laser ablations of hindbrain circuitry confirmed that visual and mechanosensory modalities share the same premotor output network. We establish a circuit for the processing of aversive stimuli in the context of an innate visual behavior.
  • Year of publication : 2015

  • Development and function of highly polarized cells such as neurons depend on microtubule-associated intracellular transport, but little is known about contributions of specific molecular motors to the establishment of synaptic connections. In this study, we investigated the function of the Kinesin I heavy chain Kif5aa during retinotectal circuit formation in zebrafish. Targeted disruption of Kif5aa does not affect retinal ganglion cell differentiation, and retinal axons reach their topographically correct targets in the tectum, albeit with a delay. In vivo dynamic imaging showed that anterograde transport of mitochondria is impaired, as is synaptic transmission. Strikingly, disruption of presynaptic activity elicits upregulation of Neurotrophin-3 (Ntf3) in postsynaptic tectal cells. This in turn promotes exuberant branching of retinal axons by signaling through the TrkC receptor (Ntrk3). Thus, our study has uncovered an activity-dependent, retrograde signaling pathway that homeostatically controls axonal branching.
  • Year of publication : 2014

  • Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.
  • Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.
  • Year of publication : 2010

  • 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.
  • Year of publication : 2009

  • 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.
  • Year of publication : 2008

  • The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.