Optical mapping of neuronal activity during seizures in zebrafish

Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acoustooptic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License

Optical method for neuronal activity mapping during seizures in zebrafish

Conventionally, epileptic seizures are detected and characterized in zebrafish either as behavioral alterations [1, 2, 3] or by means of direct electrographic recordings [1, 2, 4]. To overcome the inherent limitations of these conventional approaches (namely limited sensitivity of behavioural assays and lack of spatial resolution of electrographic recordings), we implemented the real time optical mapping of neuronal activity during the onset and propagation of epileptic seizures. We employed a transgenic zebrafish line with pan-neuronal expression of the genetically encoded calcium indicator GCaMP6s5 to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetetrazole (PTZ). With this approach, we simultaneously measured the neuronal activity in different regions of the larva brain, showing the high sensitivity of this method to detect different levels of alteration, as induced by increasing PTZ concentrations. We also performed simultaneous recording of brain and locomotor activity, demonstrating that GCaMP measurements can be much more sensitive than behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on hypomorphic human muta- tions. Moreover, taking advantage of the strong fluorescence signal of GCaMP reporter, we developed a novel and comprehensive high-throughput drug screening method able to measure neuronal activity and behavioural parameters on 60 individual larvae, at the same time. The methodological approach described here could provide a deeper comprehension of the mecha- nisms underlying epilepsy and improve the development of new anticonvulsant drugs. Further- more, this method can also be easily applied to the study of many human neuropathologies modelled in zebrafish, enabling a simple but detailed investigation of brain activity alterations associated with the pathological phenotype. References 1. Barabanetal.Pentylenetetrazoleinducedchangesinzebrafishbehavior,neuralactivityandc-FoSexpression.2005 Neuroscience 131, 759\u2013768. 2. Afrikanova et al. Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs. 2013 PloS one 8, e54166. 3. Winteretal.Validationofalarvalzebrafishlocomotorassayforassessingtheseizureliabilityofearly-stagedevelop- ment drugs. 2008 J Pharmacol Toxicol Methods 57, 176\u2013187. 4. Zdebik et al. Epilepsy in kcnj10 morphant zebrafish assessed with a novel method for long-term EEG recordings. 2013 PloS one 8, e79765. 5. Vladimirov et al. Light-sheet functional imaging in fictively behaving zebrafish. 2014 N Meth 11, 883\u2013884

Two-photon high-speed light-sheet volumetric imaging of brain activity during sleep in zebrafish larvae

Although it is well known that zebrafish display the behavioural signature of sleep, the neuronal correlates of this state are not yet completely understood, due to the complexity of the measurements required. For example, when performed with visible excitation light, functional imaging can disrupt the day/night cycle due to the induced visual stimulation. To addreß this ißue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not visible to the larva) for excitation, we are able to record wholebrain neuronal activity with high temporal- and spatial-resolution without affecting the sleep state. In two-photon light-sheet microscopy the maximum achievable frame rate is limited by the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarisation, and we implemented remote scanning of the focal plane to record without disturbing the sample. Using this setup, as a preliminary result, we characterized the intensity spectra of neuronal calcium traces of 4 days post fertilisation larvae during the day/night phases. We aim to extend these results to multiple brain regions and frequency bands

Two-photon light-sheet microscopy for high-speed whole-brain functional imaging of zebrafish neuronal physiology and pathology

We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a visual response, that otherwise would affect the neuronal activity. In particular, we were able to record whole-brain neuronal activity of the larva with high temporal- and spatial-resolution during the nocturnal period without affecting the circadian rhythm. Analyzing the spatially resolved power spectra of GCaMP signal, we found significant differences for several frequency bands between the day/night phases in various brain regions. Moreover, we studied the fast dynamics that characterize the acutely induced pathological epileptic activity of the larvae, identifying the brain structures that are more susceptible to the action of the epileptogenic drug. In conclusion, the high speed two-photon light-sheet microscope that we developed is proving to be an important tool to study both the physiological and the pathological activity of the zebrafish larval brain without undesired visual stimulation