Liu baraban investigated spatiotemporal patterns of seizure-like activity in the brain of zebrafish ( danio rerio) larvae by combining high-speed imaging of calcium indicator fluorescence with electrophysiological recording of histiocytoma surgery cost local field potentials (lfps). Whereas mammalian studies of epilepsy are sometimes restricted to electrophysiological histiocytoma surgery cost recordings of in vitro slice preparations or focal brain regions histiocytoma surgery cost in vivo, the small brain size of zebrafish permits a broad view histiocytoma surgery cost of all brain regions affected by seizures. This brain-wide view is important as seizures involve highly synchronous activity histiocytoma surgery cost across macro-neuronal networks. In their eneuro publication, liu baraban demonstrate distinct patterns in calcium signaling following drug-induced epileptiform activity or during a non-seizure hyperexcitable state in the brain of zebrafish larvae. These findings have implications for the understanding of how seizure histiocytoma surgery cost activity propagates from sites of seizure initiation to other brain histiocytoma surgery cost regions.
As a model organism, zebrafish, including its larval stages, exhibit several characteristics that are advantageous for this type of histiocytoma surgery cost investigation. The zebrafish nervous system is simple, well-characterized, and displays epileptiform-like seizure activity following exposure to convulsant drugs or genetic histiocytoma surgery cost manipulations. Direct absorption of drugs through the skin of larvae at histiocytoma surgery cost these early stages of development enables rapid drug-induced manipulation of neural activity. Zebrafish larvae are transparent facilitating optical imaging of the central histiocytoma surgery cost nervous system. Lastly, genetic modifications can be introduced relatively rapidly, as compared to mammals, into the zebrafish genome to create stable transgenic or knockout histiocytoma surgery cost lines.
Here, the authors used the tg(neurod1:gcamp6f) zebrafish line in which expression of a fluorescent, genetically encoded calcium indicator (gcamp) is driven by a neuronal promoter. Experiments were performed on larvae at 5–6 days postfertilization. Pancuronium was added to the embryo media in the recording histiocytoma surgery cost chamber to maintain paralysis and eliminate artifacts caused by body histiocytoma surgery cost movements, throughout all experiments. Two independent experimental groups were exposed to common convulsant drugs: (1) pentylenetetrazole (PTZ), a GABA A receptor antagonist that disinhibits network activity causing histiocytoma surgery cost spontaneous seizures or (2) 4-aminopyridine (4-AP), a potassium channel blocker that enhances neuronal firing activity, inducing a non-seizure hyperexcitable state at the concentrations used in the study. Imaging was started ∼ 40 minutes after the addition of convulsant drugs to the histiocytoma surgery cost media; high-speed confocal scanning (20–30 frames per second) using 5× and 20× objectives enabled whole brain and subsequent single neuron level imaging, respectively. Figure 1 shows a schematic illustration of the experimental timeline histiocytoma surgery cost and setup and representative images of zebrafish brain anatomy and histiocytoma surgery cost fluorescent expression of gcamp driven by the neurod1 promoter.
Figure 1. High-speed confocal imaging and LFP recording in the forebrain of histiocytoma surgery cost gcamp-expressing zebrafish larvae. (A) timeline of confocal imaging and LFP recording relative to immobilization histiocytoma surgery cost of larvae in agar and exposure to convulsant drugs. High-speed confocal recordings were obtained ∼40 minutes after drug exposure, with 5× and 20× objectives used for whole-brain and single neuron imaging, respectively. (B) simultaneous high-speed confocal imaging and LFP recording in agar-embedded larvae. (C) schematic illustration depicting sub-regions of the larval zebrafish brain. (D) representative 5× image of tg(neurod1:gcamp6f) zebrafish at 5–6 days postfertilization. (adapted from figure 1 in liu baraban et al., 2019.)
Whole-brain changes in calcium fluorescence revealed distinct neural activity patterns histiocytoma surgery cost during seizures or a non-seizure state of hyperexcitability. Compared to control larvae that exhibited basal levels of neural histiocytoma surgery cost activity and only very small changes in calcium fluorescence during histiocytoma surgery cost imaging, 4-AP-treated larvae showed high frequency, short duration LFP spikes and low-amplitude calcium transients, both indicative of a non-seizure hyperexcitable state. However, PTZ exposure resulted in high-amplitude, low frequency, long-duration LFP spikes and more significant changes in calcium fluorescence histiocytoma surgery cost compared to control or 4-AP-treated larvae. Analysis of changes in calcium fluorescence between brain regions showed histiocytoma surgery cost that calcium signaling was propagated in an anterior-to-posterior direction from the site of seizure initiation. These observations highlight the importance of understanding the integration of histiocytoma surgery cost different neuronal circuits in seizure activity.
Liu baraban also used high-resolution fast confocal calcium imaging to determine the effects of histiocytoma surgery cost convulsant drugs on ensembles of coactive neurons within one microcircuit, the optic tectum. In control larvae, calcium imaging showed that the incidence of coactive neurons was histiocytoma surgery cost rare (movie 1), whereas dispersed ensembles of coactive neurons were observed in 4-AP-treated larvae (movie 2). Interestingly, in larvae undergoing ictal-like seizure activity induced by PTZ exposure, many more coactive neurons were found in a spatially localized histiocytoma surgery cost activity pattern (movie 3).
Overall, these results show that the brain network dynamics underlying seizures histiocytoma surgery cost or a hyperexcitable state have characteristic patterns of neural activity histiocytoma surgery cost propagation and distinct involvement of coactive neuron ensembles. P rofessor baraban’s (university of california san francisco, CA) current research focuses on genetic editing to create transgenic zebrafish histiocytoma surgery cost lines that recapitulate known genetic causes of epilepsy in humans. In combination with drug screens, genetic editing, and fast confocal calcium imaging, this line of research will provide a basic understanding of histiocytoma surgery cost the cellular contributions to seizure activity and guide therapeutic interventions. Future studies incorporating chemogenetics or dreadds ( designer receptors exclusively activated by designer drugs) in novel transgenic zebrafish lines representing human forms of epilepsy histiocytoma surgery cost will also improve knowledge of the neural mechanisms underlying this histiocytoma surgery cost neurological disorder.
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