Schooling in Xenopus laevis Tadpoles as a Way to Assess Their Neural Development.

Escape behaviors, orienting reflexes, and social behaviors in Xenopus laevis tadpoles have been well-documented in the literature. Schooling behavior experiments allow for the observation of tadpole social interactions and in the past have been used to characterize behavioral deficits in models of neurodevelopmental disorders. Unlike other species of frogs, Xenopus tadpoles show polarized schooling. Not only do tadpoles aggregate, they also swim in the same direction. Quantifying both aggregation and relative swim angle can give us an important measure of social behavior and sensory integration. Past iterations of these experiments have required the continued presence of an experimenter throughout the duration of each trial and relied on expensive software for subsequent data analysis. The instrument configuration and analysis protocol outlined here provide an automated method to assess schooling by delivering a series of timed vibratory stimuli to a group of tadpoles to induce swimming behavior and then controlling a camera to document their positions via still images. Both stimulus delivery and image acquisition are automated using the Python programming language. Analysis is done using ImageJ and custom Python scripts, which are provided in this protocol. The specific equipment configuration and scripts shown here provide one solution, but other equipment and custom scripts can be substituted.

Analysis of Visual Collision Avoidance in Xenopus Tadpoles.

In teaching, the best exam questions are those that seem simple at first but can lead to deep and nuanced conversations. Similarly, to probe brain development, we should look for behaviors that are easy to evoke and quantify, but that are demanding, malleable, and inherently variable. Visual collision avoidance is an example of such a behavior; it is ecologically relevant, robust, and easy to record, but also nuanced and shaped by the sensory history of the animal. Here we describe how to set up a visual avoidance assay and how to use it to test sensory processing and sensorimotor transformations in the vertebrate brain.

Intrinsic temporal tuning of neurons in the optic tectum is shaped by multisensory experience.

For a biological neural network to be functional, its neurons need to be connected with synapses of appropriate strength, and each neuron needs to appropriately respond to its synaptic inputs. This second aspect of network tuning is maintained by intrinsic plasticity; yet it is often considered secondary to changes in connectivity and mostly limited to adjustments of overall excitability of each neuron. Here we argue that even nonoscillatory neurons can be tuned to inputs of different temporal dynamics and that they can routinely adjust this tuning to match the statistics of their synaptic activation. Using the dynamic clamp technique, we show that, in the tectum of Xenopus tadpole, neurons become selective for faster inputs when animals are exposed to fast visual stimuli but remain responsive to longer inputs in animals exposed to slower, looming, or multisensory stimulation. We also report a homeostatic cotuning between synaptic and intrinsic temporal properties of individual tectal cells. These results expand our understanding of intrinsic plasticity in the brain and suggest that there may exist an additional dimension of network tuning that has been so far overlooked.NEW & NOTEWORTHY We use dynamic clamp to show that individual neurons in the tectum of Xenopus tadpoles are selectively tuned to either shorter (more synchronous) or longer (less synchronous) synaptic inputs. We also demonstrate that this intrinsic temporal tuning is strongly shaped by sensory experiences. This new phenomenon, which is likely to be mediated by changes in sodium channel inactivation, is bound to have important consequences for signal processing and the development of local recurrent connections.

A cellular mechanism for inverse effectiveness in multisensory integration.

To build a coherent view of the external world, an organism needs to integrate multiple types of sensory information from different sources, a process known as multisensory integration (MSI). Previously, we showed that the temporal dependence of MSI in the optic tectum of Xenopus laevis tadpoles is mediated by the network dynamics of the recruitment of local inhibition by sensory input (Felch et al., 2016). This was one of the first cellular-level mechanisms described for MSI. Here, we expand this cellular level view of MSI by focusing on the principle of inverse effectiveness, another central feature of MSI stating that the amount of multisensory enhancement observed inversely depends on the size of unisensory responses. We show that non-linear summation of crossmodal synaptic responses, mediated by NMDA-type glutamate receptor (NMDARs) activation, form the cellular basis for inverse effectiveness, both at the cellular and behavioral levels.

Emergence of Selectivity to Looming Stimuli in a Spiking Network Model of the Optic Tectum.

The neural circuits in the optic tectum of Xenopus tadpoles are selectively responsive to looming visual stimuli that resemble objects approaching the animal at a collision trajectory. This selectivity is required for adaptive collision avoidance behavior in this species, but its underlying mechanisms are not known. In particular, it is still unclear how the balance between the recurrent spontaneous network activity and the newly arriving sensory flow is set in this structure, and to what degree this balance is important for collision detection. Also, despite the clear indication for the presence of strong recurrent excitation and spontaneous activity, the exact topology of recurrent feedback circuits in the tectum remains elusive. In this study we take advantage of recently published detailed cell-level data from tadpole tectum to build an informed computational model of it, and investigate whether dynamic activation in excitatory recurrent retinotopic networks may on its own underlie collision detection. We consider several possible recurrent connectivity configurations and compare their performance for collision detection under different levels of spontaneous neural activity. We show that even in the absence of inhibition, a retinotopic network of quickly inactivating spiking neurons is naturally selective for looming stimuli, but this selectivity is not robust to neuronal noise, and is sensitive to the balance between direct and recurrent inputs. We also describe how homeostatic modulation of intrinsic properties of individual tectal cells can change selectivity thresholds in this network, and qualitatively verify our predictions in a behavioral experiment in freely swimming tadpoles.

Multisensory integration in the developing tectum is constrained by the balance of excitation and inhibition.

Multisensory integration (MSI) is the process that allows the brain to bind together spatiotemporally congruent inputs from different sensory modalities to produce single salient representations. While the phenomenology of MSI in vertebrate brains is well described, relatively little is known about cellular and synaptic mechanisms underlying this phenomenon. Here we use an isolated brain preparation to describe cellular mechanisms underlying development of MSI between visual and mechanosensory inputs in the optic tectum of Xenopus tadpoles. We find MSI is highly dependent on the temporal interval between crossmodal stimulus pairs. Over a key developmental period, the temporal window for MSI significantly narrows and is selectively tuned to specific interstimulus intervals. These changes in MSI correlate with developmental increases in evoked synaptic inhibition, and inhibitory blockade reverses observed developmental changes in MSI. We propose a model in which development of recurrent inhibition mediates development of temporal aspects of MSI in the tectum.

Multivariate analysis of electrophysiological diversity of Xenopus visual neurons during development and plasticity.

Biophysical properties of neurons become increasingly diverse over development, but mechanisms underlying and constraining this diversity are not fully understood. Here we investigate electrophysiological characteristics of Xenopus tadpole midbrain neurons across development and during homeostatic plasticity induced by patterned visual stimulation. We show that in development tectal neuron properties not only change on average, but also become increasingly diverse. After sensory stimulation, both electrophysiological diversity and functional differentiation of cells are reduced. At the same time, the amount of cross-correlations between cell properties increase after patterned stimulation as a result of homeostatic plasticity. We show that tectal neurons with similar spiking profiles often have strikingly different electrophysiological properties, and demonstrate that changes in intrinsic excitability during development and in response to sensory stimulation are mediated by different underlying mechanisms. Overall, this analysis and the accompanying dataset provide a unique framework for further studies of network maturation in Xenopus tadpoles.

Valproate-induced neurodevelopmental deficits in Xenopus laevis tadpoles.

Autism spectrum disorder (ASD) is increasingly thought to result from low-level deficits in synaptic development and neural circuit formation that cascade into more complex cognitive symptoms. However, the link between synaptic dysfunction and behavior is not well understood. By comparing the effects of abnormal circuit formation and behavioral outcomes across different species, it should be possible to pinpoint the conserved fundamental processes that result in disease. Here we use a novel model for neurodevelopmental disorders in which we expose Xenopus laevis tadpoles to valproic acid (VPA) during a critical time point in brain development at which neurogenesis and neural circuit formation required for sensory processing are occurring. VPA is a commonly prescribed antiepileptic drug with known teratogenic effects. In utero exposure to VPA in humans or rodents results in a higher incidence of ASD or ASD-like behavior later in life. We find that tadpoles exposed to VPA have abnormal sensorimotor and schooling behavior that is accompanied by hyperconnected neural networks in the optic tectum, increased excitatory and inhibitory synaptic drive, elevated levels of spontaneous synaptic activity, and decreased neuronal intrinsic excitability. Consistent with these findings, VPA-treated tadpoles also have increased seizure susceptibility and decreased acoustic startle habituation. These findings indicate that the effects of VPA are remarkably conserved across vertebrate species and that changes in neural circuitry resulting from abnormal developmental pruning can cascade into higher-level behavioral deficits.

Excitation and inhibition in recurrent networks mediate collision avoidance in Xenopus tadpoles.

Information processing in the vertebrate brain is thought to be mediated through distributed neural networks, but it is still unclear how sensory stimuli are encoded and detected by these networks, and what role synaptic inhibition plays in this process. Here we used a collision avoidance behavior in Xenopus tadpoles as a model for stimulus discrimination and recognition. We showed that the visual system of the tadpole is selective for behaviorally relevant looming stimuli, and that the detection of these stimuli first occurs in the optic tectum. By comparing visually guided behavior, optic nerve recordings, excitatory and inhibitory synaptic currents, and the spike output of tectal neurons, we showed that collision detection in the tadpole relies on the emergent properties of distributed recurrent networks within the tectum. We found that synaptic inhibition was temporally correlated with excitation, and did not actively sculpt stimulus selectivity, but rather it regulated the amount of integration between direct inputs from the retina and recurrent inputs from the tectum. Both pharmacological suppression and enhancement of synaptic inhibition disrupted emergent selectivity for looming stimuli. Taken together these findings suggested that, by regulating the amount of network activity, inhibition plays a critical role in maintaining selective sensitivity to behaviorally-relevant visual stimuli.

Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets.

The Xenopus tadpole model offers many advantages for studying the molecular, cellular and network mechanisms underlying neurodevelopmental disorders. Essentially every stage of normal neural circuit development, from axon outgrowth and guidance to activity-dependent homeostasis and refinement, has been studied in the frog tadpole, making it an ideal model to determine what happens when any of these stages are compromised. Recently, the tadpole model has been used to explore the mechanisms of epilepsy and autism, and there is mounting evidence to suggest that diseases of the nervous system involve deficits in the most fundamental aspects of nervous system function and development. In this Review, we provide an update on how tadpole models are being used to study three distinct types of neurodevelopmental disorders: diseases caused by exposure to environmental toxicants, epilepsy and seizure disorders, and autism.

GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles.

In the developing mammalian brain, gamma-aminobutyric acid (GABA) is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl(-) in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF) may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl(-) concentrations, and therefore a more depolarized GABA receptor (GABAR) reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.

Visual experience-dependent maturation of correlated neuronal activity patterns in a developing visual system.

The functional properties of neural circuits become increasingly robust over development. This allows circuits to optimize their output in response to a variety of input. However, it is not clear whether this optimization is a function of hardwired circuit elements, or whether it requires neural experience to develop. We performed rapid in vivo imaging of calcium signals from bulk-labeled neurons in the Xenopus laevis optic tectum to resolve the rapid spatiotemporal response properties of populations of developing tectal neurons in response to visual stimuli. We found that during a critical time in tectal development, network activity becomes increasingly robust, more correlated, and more synchronous. These developmental changes require normal visual input during development and are disrupted by NMDAR blockade. Our data show that visual activity and NMDAR activation are critical for the maturation of tectal network dynamics during visual system development.

Questioning the depolarizing effects of GABA during early brain development.

During early brain development, γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the adult brain, has been thought to be an important source of excitatory neurotransmission. This view, however, was recently challenged by a series of studies that claim that the excitatory effect of GABA is due to non-physiological in vitro experimental conditions. In this article, we aim to summarize results that support and challenge the traditional point of view, and indicate some strong and weak points of both positions.