Characterization of Na+ currents regulating intrinsic excitability of optic tectal neurons.

Developing neurons adapt their intrinsic excitability to maintain stable output despite changing synaptic input. The mechanisms behind this process remain unclear. In this study, we examined Xenopus optic tectal neurons and found that the expressions of Nav1.1 and Nav1.6 voltage-gated Na+ channels are regulated during changes in intrinsic excitability, both during development and becsuse of changes in visual experience. Using whole-cell electrophysiology, we demonstrate the existence of distinct, fast, persistent, and resurgent Na+ currents in the tectum, and show that these Na+ currents are co-regulated with changes in Nav channel expression. Using antisense RNA to suppress the expression of specific Nav subunits, we found that up-regulation of Nav1.6 expression, but not Nav1.1, was necessary for experience-dependent increases in Na+ currents and intrinsic excitability. Furthermore, this regulation was also necessary for normal development of sensory guided behaviors. These data suggest that the regulation of Na+ currents through the modulation of Nav1.6 expression, and to a lesser extent Nav1.1, plays a crucial role in controlling the intrinsic excitability of tectal neurons and guiding normal development of the tectal circuitry.

Role of matrix metalloproteinase-9 in neurodevelopmental deficits and experience-dependent plasticity in Xenopus laevis.

Matrix metalloproteinase-9 (MMP-9) is a secreted endopeptidase targeting extracellular matrix proteins, creating permissive environments for neuronal development and plasticity. Developmental dysregulation of MMP-9 may also lead to neurodevelopmental disorders (ND). Here, we test the hypothesis that chronically elevated MMP-9 activity during early neurodevelopment is responsible for neural circuit hyperconnectivity observed in Xenopus tadpoles after early exposure to valproic acid (VPA), a known teratogen associated with ND in humans. In Xenopus tadpoles, VPA exposure results in excess local synaptic connectivity, disrupted social behavior and increased seizure susceptibility. We found that overexpressing MMP-9 in the brain copies effects of VPA on synaptic connectivity, and blocking MMP-9 activity pharmacologically or genetically reverses effects of VPA on physiology and behavior. We further show that during normal neurodevelopment MMP-9 levels are tightly regulated by neuronal activity and required for structural plasticity. These studies show a critical role for MMP-9 in both normal and abnormal development.

Early Developmental Exposure to Fluoxetine and Citalopram Results in Different Neurodevelopmental Outcomes.

Although selective serotonin reuptake inhibitors are commonly prescribed for prenatal depression, there exists controversy over adverse effects of SSRI use on fetal development. Few studies have adequately isolated outcomes due to SSRI exposure and those due to maternal psychiatric conditions. Here, we directly investigated outcomes of exposure to widely-used SSRIs Fluoxetine and Citalopram on the developing nervous system of Xenopus laevis tadpoles, using an integrative experimental approach. We exposed tadpoles to low doses of Citalopram and Fluoxetine during a critical developmental period and found that different experimental groups displayed opposing behavioral effects. While both groups showed reduced schooling behavior, the Fluoxetine group showed increased seizure susceptibility and reduced startle habituation. In contrast, Citalopram treated tadpoles had decreased seizure susceptibility and increased habituation. Both groups had abnormal dendritic morphology in the optic tectum, a brain area important for behaviors tested. Whole-cell electrophysiological recordings of tectal neurons showed no differences in synaptic function; however, tectal cells from Fluoxetine-treated tadpoles had decreased voltage gated K+ currents while cells in the Citalopram group had increased K+ currents. Both behavioral and electrophysiological findings indicate that cells and circuits in the Fluoxetine treated optic tecta are hyperexcitable, while the Citalopram group exhibits decreased excitability. Taken together, these results show that early developmental exposure to SSRIs is sufficient to induce neurodevelopmental effects, however these effects can be complex and vary depending on the SSRI. This may explain some discrepancies across human studies, and further underscores the importance of serotonergic signaling for the developing nervous system.

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.

Fragile X mental retardation protein knockdown in the developing Xenopus tadpole optic tectum results in enhanced feedforward inhibition and behavioral deficits.

BACKGROUND: Fragile X Syndrome is the leading monogenetic cause of autism and most common form of intellectual disability. Previous studies have implicated changes in dendritic spine architecture as the primary result of loss of Fragile X Mental Retardation Protein (FMRP), but recent work has shown that neural proliferation is decreased and cell death is increased with either loss of FMRP or overexpression of FMRP. The purpose of this study was to investigate the effects of loss of FMRP on behavior and cellular activity. METHODS: We knocked down FMRP expression using morpholino oligos in the optic tectum of Xenopus laevis tadpoles and performed a series of behavioral and electrophysiological assays. We investigated visually guided collision avoidance, schooling, and seizure propensity. Using single cell electrophysiology, we assessed intrinsic excitability and synaptic connectivity of tectal neurons. RESULTS: We found that FMRP knockdown results in decreased swimming speed, reduced schooling behavior and decreased seizure severity. In single cells, we found increased inhibition relative to excitation in response to sensory input. CONCLUSIONS: Our results indicate that the electrophysiological development of single cells in the absence of FMRP is largely unaffected despite the large neural proliferation defect. The changes in behavior are consistent with an increase in inhibition, which could be due to either changes in cell number or altered inhibitory drive, and indicate that FMRP can play a significant role in neural development much earlier than previously thought.

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.

A population of gap junction-coupled neurons drives recurrent network activity in a developing visual circuit.

In many regions of the vertebrate brain, microcircuits generate local recurrent activity that aids in the processing and encoding of incoming afferent inputs. Local recurrent activity can amplify, filter, and temporally and spatially parse out incoming input. Determining how these microcircuits function is of great interest because it provides glimpses into fundamental processes underlying brain computation. Within the Xenopus tadpole optic tectum, deep layer neurons display robust recurrent activity. Although the development and plasticity of this local recurrent activity has been well described, the underlying microcircuitry is not well understood. Here, using a whole brain preparation that allows for whole cell recording from neurons of the superficial tectal layers, we identified a physiologically distinct population of excitatory neurons that are gap junctionally coupled and through this coupling gate local recurrent network activity. Our findings provide a novel role for neuronal coupling among excitatory interneurons in the temporal processing of visual stimuli.

Neurobiology: Setting the Set Point for Neural Homeostasis.

Neural homeostasis allows neural networks to maintain a dynamic range around a given set point. How this set point is determined remains unknown. New evidence shows that alterations of activity during a critical developmental period can alter the homeostatic set point, resulting in epilepsy-like activity.

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.

A competition-based mechanism mediates developmental refinement of tectal neuron receptive fields.

Neural activity plays an important role in development and maturation of visual circuits in the brain. Activity can be instructive in refining visual projections by directly mediating formation and elimination of specific synaptic contacts through competition-based mechanisms. Alternatively, activity could be permissive-regulating production of factors that create a favorable environment for circuit refinement. Here we used the Xenopus laevis tadpole visual system to test whether activity is instructive or permissive for shaping development of the retinotectal circuit. In vivo spike output was dampened in a small subgroup of tectal neurons, starting from developmental stages 44-46, by overexpressing Shaker-like Xenopus Kv1.1 potassium channels using electroporation. Tadpoles were then reared until stage 49, a time period when significant refinement of the retinotectal map occurs. Kv1.1-expressing neurons had significantly decreased spike output in response to both current injection and visual stimuli compared to untransfected controls, with spiking occurring during a more limited time interval. We found that Kv1.1-expressing neurons had larger visual receptive fields, decreased receptive field sharpness, and more persistent recurrent excitation than control neurons, all of which are characteristics of immature neurons. Transfected cells, however, had normal spontaneous excitatory synaptic currents and dendritic arbors. These results suggest that spike output of a tectal neuron plays an important instructive role in development of its receptive field properties and refinement of local circuits. However, other activity-dependent processes, such as synaptogenesis and dendritic growth, remain unaffected due to the permissive environment created by otherwise normal network activity.

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.

A neuroprotective role for polyamines in a Xenopus tadpole model of epilepsy.

Polyamines are endogenous molecules involved in cell damage following neurological insults, although it is unclear whether polyamines reduce or exacerbate this damage. We used a developmental seizure model in which we exposed Xenopus laevis tadpoles to pentylenetetrazole (PTZ), a known convulsant. We found that, after an initial PTZ exposure, seizure onset times were delayed in response to a second PTZ exposure 4 h later. This protective effect was a result of activity-dependent increases in synthesis of putrescine, the simplest polyamine. Unlike more complex polyamines that directly modulate ion channels, putrescine exerted its effect by altering the balance of excitation to inhibition. Tectal neuron recordings, 4 h after the initial seizure, revealed an elevated frequency of GABAergic spontaneous inhibitory postsynaptic currents. Our data suggest that this effect is mediated by an atypical pathway that converts putrescine into GABA, which then activates presynaptic GABA(B) receptors. Our data suggest that polyamines have a previously unknown neuroprotective role in the developing brain.

Sensory modality-specific homeostatic plasticity in the developing optic tectum.

We found a previously unknown form of homeostatic synaptic plasticity in multisensory neurons in the optic tectum of Xenopus laevis tadpoles. Individual tectal neurons are known to receive converging inputs from multiple sensory modalities. We observed that long-term alterations in either visual or mechanosensory activity in vivo resulted in homeostatic changes specific to each sensory modality. In contrast with typical forms of homeostatic synaptic plasticity, such as synaptic scaling, we found that this type of plasticity occurred in a pathway-specific manner that is more reminiscent of hebbian-type plasticity.

Learning to see: patterned visual activity and the development of visual function.

To successfully interact with their environments, developing organisms need to correctly process sensory information and generate motor outputs appropriate to their size and structure. Patterned sensory experience has long been known to induce various forms of developmental plasticity that ultimately shape mature neural circuits. These same types of plasticity also allow developing organisms to respond appropriately to the external world by dynamically adapting neural circuit function to ongoing changes in brain circuitry and sensory input. Recent work on the visual systems of frogs and fish has provided an unprecedented view into how visual experience dynamically affects circuit function at many levels, ranging from gene expression to network function, ultimately leading to system-wide functional adaptations.

Neurodevelopmental effects of chronic exposure to elevated levels of pro-inflammatory cytokines in a developing visual system.

BACKGROUND: Imbalances in the regulation of pro-inflammatory cytokines have been increasingly correlated with a number of severe and prevalent neurodevelopmental disorders, including autism spectrum disorder, schizophrenia and Down syndrome. Although several studies have shown that cytokines have potent effects on neural function, their role in neural development is still poorly understood. In this study, we investigated the link between abnormal cytokine levels and neural development using the Xenopus laevis tadpole visual system, a model frequently used to examine the anatomical and functional development of neural circuits. RESULTS: Using a test for a visually guided behavior that requires normal visual system development, we examined the long-term effects of prolonged developmental exposure to three pro-inflammatory cytokines with known neural functions: interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. We found that all cytokines affected the development of normal visually guided behavior. Neuroanatomical imaging of the visual projection showed that none of the cytokines caused any gross abnormalities in the anatomical organization of this projection, suggesting that they may be acting at the level of neuronal microcircuits. We further tested the effects of TNF-alpha on the electrophysiological properties of the retinotectal circuit and found that long-term developmental exposure to TNF-alpha resulted in enhanced spontaneous excitatory synaptic transmission in tectal neurons, increased AMPA/NMDA ratios of retinotectal synapses, and a decrease in the number of immature synapses containing only NMDA receptors, consistent with premature maturation and stabilization of these synapses. Local interconnectivity within the tectum also appeared to remain widespread, as shown by increased recurrent polysynaptic activity, and was similar to what is seen in more immature, less refined tectal circuits. TNF-alpha treatment also enhanced the overall growth of tectal cell dendrites. Finally, we found that TNF-alpha-reared tadpoles had increased susceptibility to pentylenetetrazol-induced seizures. CONCLUSIONS: Taken together our data are consistent with a model in which TNF-alpha causes premature stabilization of developing synapses within the tectum, therefore preventing normal refinement and synapse elimination that occurs during development, leading to increased local connectivity and epilepsy. This experimental model also provides an integrative approach to understanding the effects of cytokines on the development of neural circuits and may provide novel insights into the etiology underlying some neurodevelopmental disorders.