Estuardo Robles

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.

Src-Dependent Tyrosine Phosphorylation at the Tips of Growth Cone Filopodia Promotes Extension

Extracellular cues guide axon outgrowth by activating intracellular signaling cascades that control the growth cone cytoskeleton. However, the spatial and temporal coordination of signaling intermediates remains essentially unknown. Live imaging of tyrosine phosphorylation in growth cones revealed dynamic phospho-tyrosine (PY) signals in filopodia that directly correlate with filopodial behavior. Local PY signals are generated at distal tips of filopodia during extension and are lost during retraction. Active Src family kinases localize to the tips of filopodia, and Src activity regulates both filopodial dynamics and local PY signaling. Positive guidance cues stimulate filopodial motility by locally increasing tyrosine phosphorylation in a cell division cycle 42 (Cdc42)-dependent manner. Locally reduced Src activity on one side of the growth cone generates an asymmetry in filopodial motility and PY signaling that promotes repulsive turning, suggesting that local changes in filopodial PY levels may underlie growth cone pathfinding decisions. p21-activated kinase (PAK), a Cdc42 effector whose activity is regulated by Src phosphorylation, also localizes to the tips of extending filopodia and controls filopodial motility. Coordinated activation of cytoskeletal effector proteins by GTPase binding and Src-mediated tyrosine phosphorylation may function to produce specific growth cone behaviors in response to guidance cues.

Characterization of genetically targeted neuron types in the zebrafish optic tectum

The optically transparent larval zebrafish is ideally suited for in vivo analyses of neural circuitry controlling visually guided behaviors. However, there is a lack of information regarding specific cell types in the major retinorecipient brain region of the fish, the optic tectum. Here we report the characterization of three previously unidentified tectal cell types that are specifically labeled by dlx5/6 enhancer elements. In vivo laser-scanning microscopy in conjunction with ex vivo array tomography revealed that these neurons differ in their morphologies, synaptic connectivity, and neurotransmitter phenotypes. The first type is an excitatory bistratified periventricular interneuron that forms a dendritic arbor in the retinorecipient stratum fibrosum et griseum superficiale (SFGS) and an axonal arbor in the stratum griseum centrale (SGC). The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS. The third type is a GABAergic periventricular projection neuron that extends a dendritic arbor into the SGC and a long axon to the torus semicircularis, medulla oblongata, and anterior hindbrain. Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale. This approach revealed several novel aspects of tectal circuitry, including: (1) a glutamatergic mode of transmission from the superficial, retinorecipient neuropil layers to the deeper, output layers, (2) the presence of interneurons with mixed dendrite/axon arbors likely involved in local processing, and (3) a heretofore unknown GABAergic tectofugal projection to midbrain and hindbrain. These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system.

Assembly of Lamina-Specific Neuronal Connections by Slit Bound to Type IV Collagen

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.

Focal adhesion kinase modulates Cdc42 activity downstream of positive and negative axon guidance cues.

Summary There is biochemical, imaging and functional evidence that Rho GTPase signaling is a crucial regulator of actin-based structures such as lamellipodia and filopodia. However, although Rho GTPases are believed to serve similar functions in growth cones, the spatiotemporal dynamics of Rho GTPase signaling has not been examined in living growth cones in response to known axon guidance cues. Here we provide the first measurements of Cdc42 activity in living growth cones acutely stimulated with both growth-promoting and growth-inhibiting axon-guidance cues. Interestingly, we find that both permissive and repulsive factors can work by modulating Cdc42 activity, but in opposite directions. We find that the growth-promoting factors laminin and BDNF activate Cdc42, whereas the inhibitor Slit2 reduces Cdc42 activity in growth cones. Remarkably, we find that regulation of focal adhesion kinase (FAK) activity is a common upstream modulator of Cdc42 by BDNF, laminin and Slit. These findings suggest that rapid modulation of Cdc42 signaling through FAK by receptor activation underlies changes in growth cone motility in response to permissive and repulsive guidance cues.

Precise lamination of retinal axons generates multiple parallel input pathways in the tectum

The axons of retinal ganglion cells (RGCs) form topographic connections in the optic tectum, recreating a two-dimensional map of the visual field in the midbrain. RGC axons are also targeted to specific positions along the laminar axis of the tectum. Understanding the sensory transformations performed by the tectum requires identification of the rules that control the formation of synaptic laminae by RGC axons. However, there is little information regarding the spatial relationships between multiple axons as they establish laminar and retinotopic arborization fields within the same region of neuropil. Moreover, the contribution of RGC axon lamination to the processing of visual information is unknown. We used Brainbow genetic labeling to visualize groups of individually identifiable axons during the assembly of a precise laminar map in the larval zebrafish tectum. Live imaging of multiple RGCs revealed that axons target specific sublaminar positions during initial innervation and maintain their relative laminar positions throughout early larval development, ruling out a model for lamina selection based on iterative refinements. During this period of laminar stability, RGC arbors undergo structural rearrangements that shift their relative retinotopic positions. Analysis of cell-type-specific lamination patterns revealed that distinct combinations of RGCs converge to form each sublamina, and this input heterogeneity correlates with different functional responses to visual stimuli. These findings suggest that lamina-specific sorting of retinal inputs provides an anatomical blueprint for the integration of visual features in the tectum.

Assembly of synaptic laminae by axon guidance molecules

A prominent feature of nervous systems is the organization of synapses into discrete layers, or laminae. Such laminae are essential for the spatial segregation of synaptic connections conveying different types of information. A prime example of this is the inner plexiform layer (IPL) of the vertebrate retina, which is subdivided into at least ten sublaminae. Another example gaining prominence is the layered neuropil of the zebrafish optic tectum. Three recent papers have shed light on the extracellular signals that control the precise stratification of pre- and postsynaptic neuronal processes in these two areas. The new studies implicate well-known axon guidance cues, including class 5 and 6 semaphorins in the retina, as well as Slit in the optic tectum. Remarkably, the short-range action of Slit, which is required for neurite stratification, appears to be achieved by anchoring the secreted guidance factor to the basement membrane at the surface of the tectum.

Imaging calcium dynamics in developing neurons.

Here we describe the techniques developed to image Ca2+ signals in motile nerve growth cones both in culture and in the developing Xenopus spinal cord. We have used these methods to identify two spatially and temporally distinct classes of Ca2+ transients in growth cones. Imaging Ca2+ in morphologically complex migratory cells allows for analysis and correlation of discrete signals with a wide variety of cellular behaviors. For example, we find that localized Ca2+ changes at the tips of individual filopodia correlate with reduced filopodial motility. Further, rapid fixation after Ca2+ imaging made it possible to determine that transients occur at integrin receptor clusters that may generate and in turn be regulated by these local signals. We describe the use of caged-Ca2+ to locally impose Ca2+ transients in individual filopodia and find this treatment sufficient to repel neurite outgrowth. Calcium signals across broad spatial and temporal dimensions are universal regulators of numerous complex and varied cellular functions. The imaging methods we describe here begin to view growth cones over a range of spatial resolutions and temporal frequencies necessary to detect different types of Ca2+ transients, however it is clear that not all dimensions have been examined. In particular, imaging cells more rapidly and at higher magnification may one day allow us to detect more elemental events such as single-channel openings, as has been achieved in nonneuronal cells. We also describe techniques used to examine Ca2+ signals in growth cones migrating within the spinal cord. These types of studies are ultimately necessary to confirm the relevance of in vitro findings. Although designed for the Xenopus spinal cord, the methods we outline should be applicable to other tissues and organisms. Finally, we use caged Ca2+ as a tool to reproduce very precise changes in cytosolic Ca2+ levels. This is a powerful means to test the function of different types of Ca2+ transients and assess the downstream regulators of those signals. These types of manipulations can also be used with other types of caged compounds, many of which are commercially available (Molecular Probes) or readily synthesized.