Functional Integration of Newborn Neurons in the Zebrafish Optic Tectum

Neurogenesis persists during adulthood in restricted parts of the vertebrate brain. In the optic tectum (OT) of the zebrafish larva, newborn neurons are continuously added and contribute to visual information processing. Recent studies have started to describe the functional development and fate of newborn neurons in the OT. Like the mammalian brain, newborn neurons in the OT require sensory inputs for their integration into local networks and survival. Recent findings suggest that the functional development of newborn neurons requires both activity-dependent and hard-wired mechanisms for proper circuit integration. Here, we review these findings and argue that the study of neurogenesis in non-mammalian species will help elucidate the general mechanisms of circuit assembly following neurogenesis

Sensorimotor Transformations in the Zebrafish Auditory System

Organisms use their sensory systems to acquire information from their environment and integrate this information to produce relevant behaviors. Nevertheless, how sensory information is converted into adequate motor patterns in the brain remains an open question. Here, we addressed this question using two-photon and light-sheet calcium imaging in intact, behaving zebrafish larvae. We monitored neural activity elicited by auditory stimuli while simultaneously recording tail movements. We observed a spatial organization of neural activity according to four different response profiles (frequency tuning curves), suggesting a low-dimensional representation of frequency information, maintained throughout the development of the larvae. Low frequencies (150–450 Hz) were locally processed in the hindbrain and elicited motor behaviors. In contrast, higher frequencies (900–1,000 Hz) rarely induced motor behaviors and were also represented in the midbrain. Finally, we found that the sensorimotor transformations in the zebrafish auditory system are a continuous and gradual process that involves the temporal integration of the sensory response in order to generate a motor behavior.Fil: Privat, Martin. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Romano, Sebastián Alejo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Pietri, Thomas. Centre National de la Recherche Scientifique; Francia. Inserm; FranciaFil: Jouary, Adrien. Champalimaud Centre For The Unknown; Portugal. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Boulanger Weill, Jonathan. Centre National de la Recherche Scientifique; Francia. Inserm; FranciaFil: Elbaz, Nicolas. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Duchemin, Auriane. Centre National de la Recherche Scientifique; Francia. Inserm; FranciaFil: Soares, Daphne. New Jersey Institute of Technology; Estados UnidosFil: Sumbre, Germán. Centre National de la Recherche Scientifique; Francia. Inserm; Franci

An integrated calcium imaging processing toolbox for the analysis of neuronal population dynamics

The development of new imaging and optogenetics techniques to study the dynamics of large neuronal circuits is generating datasets of unprecedented volume and complexity, demanding the development of appropriate analysis tools. We present a comprehensive computational workflow for the analysis of neuronal population calcium dynamics. The toolbox includes newly developed algorithms and interactive tools for image pre-processing and segmentation, estimation of significant single-neuron single-trial signals, mapping event-related neuronal responses, detection of activity-correlated neuronal clusters, exploration of population dynamics, and analysis of clusters' features against surrogate control datasets. The modules are integrated in a modular and versatile processing pipeline, adaptable to different needs. The clustering module is capable of detecting flexible, dynamically activated neuronal assemblies, consistent with the distributed population coding of the brain. We demonstrate the suitability of the toolbox for a variety of calcium imaging datasets. The toolbox open-source code, a step-by-step tutorial and a case study dataset are available at https://github.com/zebrain-lab/Toolbox-Romano-et-al

Intégration fonctionnelle des neurones nouveaux-nés dans des circuits déjà établis dans le système visuel de la larve de poisson zèbre

In the vertebrate brain, mechanisms leading to the incorporation of newborn neurons into already functional networks still remain poorly understood. Indeed, since most of the studies have been performed at the single-cell level, a detailed description of the circuit dynamics is lacking. To investigate this phenomenon, I have developed a pioneer methodology using the zebrafish larva as an experimental model and a multidisciplinary approach combining genetics, two-photon microscopy and optogenetics to monitor the developing activity of genetically targeted newborn neurons and the surrounding matured networks, in an intact and non-anesthetized vertebrate. Using this technique I have described for the first time, and in the time course of several days, the developmental dynamics of the functional properties of newborn neurons before and during their incorporation into the mature tectal circuit, the zebrafish most complex layered structure and highest visual center. Overall, these results suggest a developmental sequence of events during which newborn neurons capable of generating intrinsic activity dynamics first connect to their pre-synaptic sensory organ (the retina). At a second stage, the newborn neurons gradually incorporate into the tectal mature circuit showing sparse correlations with mature neurons. At a third stage, the spatial organization of the correlation between the newborn and the mature neurons is refined, becoming denser. I thus suggest that the newborn neurons first connect to a large population of sparsely located mature neurons and subsequently distant connections are pruned, permitting the newborn-labeled neuron to acquire a stable and robust functional signature (e.g. sharp receptive fields). In the recent years, treatments based on the transplantation of neural tissue have been developed to target neurodegenerative diseases such as Parkinson's disease. Because these therapies face the problem of poor survival and long-term functional incorporation, this study may provide better understanding of neuronal circuits formation and might pave the way to improve the efficiency of stem-cells-based treatments for human-brain reparation.Au cours du développement cérébral des vertébrés, le processus permettant à des neurones nouveaux-nés de s'incorporer dans des réseaux déjà établis est mal compris. En effet, la majorité des études ayant été réalisées à l'échelle de la cellule, une description détaillée de la dynamique des circuits au cours de ce phénomène est manquante. Pour l'étudier, j'ai développé une méthode innovante utilisant la larve de poisson zèbre comme modèle expérimental et une approche pluridisciplinaire combinant la génétique, la microscopie bi-photonique et l'optogénétique pour suivre le développement de l'activité de neurones nouveaux-nés et des réseaux matures voisins dans un vertébré intacte et non-anesthésié. En utilisant cette technique j'ai décrit pour la première fois, pendant plusieurs jours consécutifs, le développement des propriétés fonctionnelles de neurones nouveaux nés avant et pendant leur incorporation dans les circuits du toit optique, la structure cérébrale la plus complexe du poisson zèbre permettant l'intégration l'information visuelle. Les résultats obtenus suggèrent une séquence de développement durant laquelle les neurones morphologiquement immatures spontanément actifs se connectent en premier à la rétine. Dans un second temps, ces neurones s'incorporant graduellement au circuit mature en montrant des corrélations avec des neurones matures éparses. Troisièmement, l'organisation spatiale des corrélations entre les neurones nouveaux-nés est raffinée et devient plus dense. Ces résultats suggèrent que les neurones nouveaux-nés se connectent dans un premier temps a une population éparse de neurones matures avant que les connections a longue distance disparaissent permettant aux neurones en développement d'obtenir une signature fonctionnelle robuste (ex. réponses restreintes spatialement). Récemment, des traitements basés sur la transplantation des tissues neuronaux ont été développées pour certaines maladies neuro-dégénératives (ex. maladie de Parkinson). Cependant ces thérapies sont actuellement limitées par le faible taux de survie et l'incorporation des neurones injectés. Ces travaux apportent une meilleure compréhension des mécanismes à l’œuvre lors de la formation de circuits neuronaux et pourront peut-être permettre d'améliorer l'efficacité des traitements utilisant des cellules souches pour réparer le cerveau humain

Intégration fonctionnelle des neurones nouveaux-nés dans des circuits déjà établis dans le système visuel de la larve de poisson zèbre

In the vertebrate brain, mechanisms leading to the incorporation of newborn neurons into already functional networks still remain poorly understood. Indeed, since most of the studies have been performed at the single-cell level, a detailed description of the circuit dynamics is lacking. To investigate this phenomenon, I have developed a pioneer methodology using the zebrafish larva as an experimental model and a multidisciplinary approach combining genetics, two-photon microscopy and optogenetics to monitor the developing activity of genetically targeted newborn neurons and the surrounding matured networks, in an intact and non-anesthetized vertebrate. Using this technique I have described for the first time, and in the time course of several days, the developmental dynamics of the functional properties of newborn neurons before and during their incorporation into the mature tectal circuit, the zebrafish most complex layered structure and highest visual center. Overall, these results suggest a developmental sequence of events during which newborn neurons capable of generating intrinsic activity dynamics first connect to their pre-synaptic sensory organ (the retina). At a second stage, the newborn neurons gradually incorporate into the tectal mature circuit showing sparse correlations with mature neurons. At a third stage, the spatial organization of the correlation between the newborn and the mature neurons is refined, becoming denser. I thus suggest that the newborn neurons first connect to a large population of sparsely located mature neurons and subsequently distant connections are pruned, permitting the newborn-labeled neuron to acquire a stable and robust functional signature (e.g. sharp receptive fields). In the recent years, treatments based on the transplantation of neural tissue have been developed to target neurodegenerative diseases such as Parkinson's disease. Because these therapies face the problem of poor survival and long-term functional incorporation, this study may provide better understanding of neuronal circuits formation and might pave the way to improve the efficiency of stem-cells-based treatments for human-brain reparation.Au cours du développement cérébral des vertébrés, le processus permettant à des neurones nouveaux-nés de s'incorporer dans des réseaux déjà établis est mal compris. En effet, la majorité des études ayant été réalisées à l'échelle de la cellule, une description détaillée de la dynamique des circuits au cours de ce phénomène est manquante. Pour l'étudier, j'ai développé une méthode innovante utilisant la larve de poisson zèbre comme modèle expérimental et une approche pluridisciplinaire combinant la génétique, la microscopie bi-photonique et l'optogénétique pour suivre le développement de l'activité de neurones nouveaux-nés et des réseaux matures voisins dans un vertébré intacte et non-anesthésié. En utilisant cette technique j'ai décrit pour la première fois, pendant plusieurs jours consécutifs, le développement des propriétés fonctionnelles de neurones nouveaux nés avant et pendant leur incorporation dans les circuits du toit optique, la structure cérébrale la plus complexe du poisson zèbre permettant l'intégration l'information visuelle. Les résultats obtenus suggèrent une séquence de développement durant laquelle les neurones morphologiquement immatures spontanément actifs se connectent en premier à la rétine. Dans un second temps, ces neurones s'incorporant graduellement au circuit mature en montrant des corrélations avec des neurones matures éparses. Troisièmement, l'organisation spatiale des corrélations entre les neurones nouveaux-nés est raffinée et devient plus dense. Ces résultats suggèrent que les neurones nouveaux-nés se connectent dans un premier temps a une population éparse de neurones matures avant que les connections a longue distance disparaissent permettant aux neurones en développement d'obtenir une signature fonctionnelle robuste (ex. réponses restreintes spatialement). Récemment, des traitements basés sur la transplantation des tissues neuronaux ont été développées pour certaines maladies neuro-dégénératives (ex. maladie de Parkinson). Cependant ces thérapies sont actuellement limitées par le faible taux de survie et l'incorporation des neurones injectés. Ces travaux apportent une meilleure compréhension des mécanismes à l’œuvre lors de la formation de circuits neuronaux et pourront peut-être permettre d'améliorer l'efficacité des traitements utilisant des cellules souches pour réparer le cerveau humain

Functional Interactions between Newborn and Mature Neurons Leading to Integration into Established Neuronal Circuits

From development up to adulthood, the vertebrate brain is continuously supplied with newborn neurons that integrate into established mature circuits. However, how this process is coordinated during development remains unclear. Using two-photon imaging, GCaMP5 transgenic zebrafish larvae, and sparse electroporation in the larva's optic tectum, we monitored spontaneous and induced activity of large neuronal populations containing newborn and functionally mature neurons. We observed that the maturation of newborn neurons is a 4-day process. Initially, newborn neurons showed undeveloped dendritic arbors, no neurotransmitter identity, and were unresponsive to visual stimulation, although they displayed spontaneous calcium transients. Later on, newborn-labeled neurons began to respond to visual stimuli but in a very variable manner. At the end of the maturation period, newborn-labeled neurons exhibited visual tuning curves (spatial receptive fields and direction selectivity) and spontaneous correlated activity with neighboring functionally mature neurons. At this developmental stage, newborn-labeled neurons presented complex dendritic arbors and neurotransmitter identity (excitatory or inhibitory). Removal of retinal inputs significantly perturbed the integration of newborn neurons into the functionally mature tectal network. Our results provide a comprehensive description of the maturation of newborn neurons during development and shed light on potential mechanisms underlying their integration into a functionally mature neuronal circuit.Fil: Boulanger Weill, Jonathan. Ecole Normale Supérieure; FranciaFil: Coste, Virginie. Ecole Normale Supérieure; FranciaFil: Jouary, Adrien. Ecole Normale Supérieure; FranciaFil: Romano, Sebastián Alejo. Ecole Normale Supérieure; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Pérez Schuster, Verónica. Ecole Normale Supérieure; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Sumbre, Germán. Ecole Normale Supérieure; Franci

The Emergence of the Spatial Structure of Tectal Spontaneous Activity Is Independent of Visual Inputs

International audienceGraphical Abstract Highlights d Development of tectal circuitry is influenced by the onset of retinal inputs d Enucleations impact the development of the tectum's spontaneous activity correlations d Enucleations only delay the topography of the correlated activity d In the absence of retinal inputs, the tectal circuitry is capable of predicting behavior In Brief The influence of retinal inputs on the development of the spontaneous neuronal activity of the tectal circuit is unknown. Pietri et al. show that retinal inputs are dispensable for the development of the spatial structure of spontaneous tectal activity, suggesting that the tectal circuit is preconfigured for its functional role

Overview of the toolbox workflow.

<p>Colored boxes indicate the processing modules (color code in top left corner). Dashed boxes correspond to optional procedures. Tutorial step numbers related to each procedure are indicated in the bottom-right corner of each box. Single-headed thin and thick arrows respectively depict the processing-pipeline flow, and the option to import pre-analyzed data from other methods into the stand-alone modules of the pipeline.</p

Detection of neuronal assemblies for the case study.

<p>(<b>A</b>) Screenshot for the selection of the zMax threshold to determine the neuronal composition of the assemblies. After setting the smooth parameter with the slider (top-right), the threshold is chosen with a mouse click on the graph of the density distribution (red arrow). (<b>B</b>) Screenshot showing the topography of 3 representative assemblies (out of 42). ROIs that belong to each assembly are labeled in yellow. (<b>C</b>) Screenshot of two user-defined anatomical axes (see tutorial) over which assemblies will be spatially organized. Each curve is automatically colored so that the combined curves reproduce the hue gradient used in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005526#pcbi.1005526.g005" target="_blank">Fig 5A and 5C</a>. The chosen curves span the rostro-caudal retinotopic axis of each tectal hemisphere. (<b>D</b>) Screenshot of the figure obtained displaying the spatial organization of the assemblies along the selected axes. Assemblies' ROIs are colored according to the defined axis (i.e., the position of the assemblies’ spatial centroid with respect to the defined axis). The comparison with <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005526#pcbi.1005526.g005" target="_blank">Fig 5C</a> confirms that assemblies reproduce the tectal retinotopic functional map.</p

Toolbox performance in inferring neuronal activity from calcium imaging data.

<p>(<b>A</b>) Two examples with different signal-to-noise ratios (SNRs) from different neurons in the cai-1 ground-truth dataset[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005526#pcbi.1005526.ref028" target="_blank">28</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005526#pcbi.1005526.ref039" target="_blank">39</a>]. For each example, we show: top, GCaMP6f ΔF/F0 traces (black) and the significant fluorescent transients detected by the module (red); bottom, ticks representing the simultaneously recorded spikes (those associated with a significant calcium event are highlighted in red; asterisks mark single spikes); middle, spiking rate of the neuron calculated by temporally convolving spikes with a Gaussian filter of σ = 20 ms. Imaging was performed at 60 Hz. A spike was considered as associated with a significant calcium event if it was followed by an event of significant fluorescence within 40 ms (GCaMP6f rise time τ_peak = 45± 4 ms, for 1 spike[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005526#pcbi.1005526.ref028" target="_blank">28</a>]). Significant transients were calculated with default toolbox parameters in Dynamic threshold mode, with a GCaMP6f τ_decay = 250 ms[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005526#pcbi.1005526.ref028" target="_blank">28</a>]. (<b>B</b>) Boxplot summary of performance for all recordings (n = 37). Top: coefficients obtained when correlating the spiking rates with the raw fluorescence traces or with the ΔF/F0 traces of significant transients (where non-significant fluctuations were set to 0). Bottom: Percentage of “detected” spikes (i.e., those associated with a significant calcium event), for all recorded spikes or for single spikes only.</p