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1. Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2

   https://doi.org/10.1002/dneu.22791  

   Terzi, Aslihan ; Roeder, Haley ; Weaver, Cory J. ; Suter, Daniel M. ( December 2020 , Developmental Neurobiology)

   NADPH oxidases (Nox) are membrane‐bound multi‐subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox‐derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin‐1, slit2, and brain‐derived neurotrophic factor (BDNF). To test the role of Nox in cue‐mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2‐mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin‐1 on growth rates, growth in the presence of netrin‐1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin‐1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo.astray/nox2double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons.

    

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2. Microscale Neuronal Activity Collectively Drives Chaotic and Inflexible Dynamics at the Macroscale in Seizures

   https://doi.org/10.1523/JNEUROSCI.0171-22.2023  

   Burrows, Dominic R. ; Diana, Giovanni ; Pimpel, Birgit ; Moeller, Friederike ; Richardson, Mark P. ; Bassett, Dani S. ; Meyer, Martin P. ; Rosch, Richard E. ( May 2023 , The Journal of Neuroscience)

   Neuronal activity propagates through the network during seizures, engaging brain dynamics at multiple scales. Such propagating events can be described through the avalanches framework, which can relate spatiotemporal activity at the microscale with global network properties. Interestingly, propagating avalanches in healthy networks are indicative of critical dynamics, where the network is organized to a phase transition, which optimizes certain computational properties. Some have hypothesized that the pathologic brain dynamics of epileptic seizures are an emergent property of microscale neuronal networks collectively driving the brain away from criticality. Demonstrating this would provide a unifying mechanism linking microscale spatiotemporal activity with emergent brain dysfunction during seizures. Here, we investigated the effect of drug-induced seizures on critical avalanche dynamics, usingin vivowhole-brain two-photon imaging of GCaMP6s larval zebrafish (males and females) at single neuron resolution. We demonstrate that single neuron activity across the whole brain exhibits a loss of critical statistics during seizures, suggesting that microscale activity collectively drives macroscale dynamics away from criticality. We also construct spiking network models at the scale of the larval zebrafish brain, to demonstrate that only densely connected networks can drive brain-wide seizure dynamics away from criticality. Importantly, such dense networks also disrupt the optimal computational capacities of critical networks, leading to chaotic dynamics, impaired network response properties and sticky states, thus helping to explain functional impairments during seizures. This study bridges the gap between microscale neuronal activity and emergent macroscale dynamics and cognitive dysfunction during seizures.

   SIGNIFICANCE STATEMENTEpileptic seizures are debilitating and impair normal brain function. It is unclear how the coordinated behavior of neurons collectively impairs brain function during seizures. To investigate this we perform fluorescence microscopy in larval zebrafish, which allows for the recording of whole-brain activity at single-neuron resolution. Using techniques from physics, we show that neuronal activity during seizures drives the brain away from criticality, a regime that enables both high and low activity states, into an inflexible regime that drives high activity states. Importantly, this change is caused by more connections in the network, which we show disrupts the ability of the brain to respond appropriately to its environment. Therefore, we identify key neuronal network mechanisms driving seizures and concurrent cognitive dysfunction.

    

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3. In toto imaging of early enteric nervous system development reveals that gut colonization is tied to proliferation downstream of Ret

   https://doi.org/10.1242/dev.200668  

   Baker, Phillip A. ; Ibarra-García-Padilla, Rodrigo ; Venkatesh, Akshaya ; Singleton, Eileen W. ; Uribe, Rosa. A. ( November 2022 , Development)

   ABSTRACT The enteric nervous system is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and these are pertinent subjects for study due to their relevance to the human congenital disease Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in wild type and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs localized along the gut, and these ENCCs failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation-dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs. These results suggest that Ret signaling may regulate maintenance of a stem state in ENCCs. 

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4. Sparse decomposition light-field microscopy for high speed imaging of neuronal activity

   https://doi.org/10.1364/OPTICA.392805  

   Yoon, Young-Gyu ; Wang, Zeguan ; Pak, Nikita ; Park, Demian ; Dai, Peilun ; Kang, Jeong Seuk ; Suk, Ho-Jun ; Symvoulidis, Panagiotis ; Guner-Ataman, Burcu ; Wang, Kai ; et al ( October 2020 , Optica)

   One of the major challenges in large scale optical imaging of neuronal activity is to simultaneously achieve sufficient temporal and spatial resolution across a large volume. Here, we introduce sparse decomposition light-field microscopy (SDLFM), a computational imaging technique based on light-field microscopy (LFM) that takes algorithmic advantage of the high temporal resolution of LFM and the inherent temporal sparsity of spikes to improve effective spatial resolution and signal-to-noise ratios (SNRs). With increased effective spatial resolution and SNRs, neuronal activity at the single-cell level can be recovered over a large volume. We demonstrate the single-cell imaging capability of SDLFM within vivoimaging of neuronal activity of whole brains of larval zebrafish with estimated lateral and axial resolutions of$\sim <\#comment/>3.5\text{µ<\#comment/>}\mathrm{m}$and$\sim <\#comment/>7.4\text{µ<\#comment/>}\mathrm{m}$, respectively, acquired at volumetric imaging rates up to 50 Hz. We also show that SDLFM increases the quality of neural imaging in adult fruit flies.

    

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5. Imaging Spontaneous Neuronal Activity with Voltage‐Sensitive Dyes

   https://doi.org/10.1002/cpz1.48  

   Raliski, Benjamin K. ; Kirk, Molly J. ; Miller, Evan W. ( March 2021 , Current Protocols)

   Accurately mapping changes in cellular membrane potential across large groups of neurons is crucial for understanding the organization and maintenance of neural circuits. Measuring cellular voltage changes by optical means allows greater spatial resolution than traditional electrophysiology methods and is adaptable to high‐throughput imaging experiments. VoltageFluors, a class of voltage‐sensitive dyes, have recently been used to optically study the spontaneous activity of many neurons simultaneously in dissociated culture. VoltageFluors are particularly useful for experiments investigating differences in excitability and connectivity between neurons at different stages of development and in different disease models. The protocols in this article describe general procedures for preparing dissociated cultures, imaging spontaneous activity in dissociated cultures with VoltageFluors, and analyzing optical spontaneous activity data. © 2021 Wiley Periodicals LLC.

   This article was corrected on 20 July 2022. See the end of the full text for details.

   Basic Protocol 1: Preparation of dissociated rat hippocampal or cortical cultures

   Alternate Protocol: Preparation of microisland dissociated cultures

   Basic Protocol 2: Imaging of spontaneous activity in dissociated cultures using voltage‐sensitive dyes

   Basic Protocol 3: Analysis of spontaneous activity imaging data

    

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