Zijun Sun1, Thomas C Südhof2. 1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: sunzijun@stanford.edu. 2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Abstract
BACKGROUND: Ca2+-imaging is a powerful tool to measure neuronal dynamics and network activity. To monitor network-level changes in cultured neurons, neuronal activity is often evoked by electrical or optogenetic stimulation and assessed using multi-electrode arrays or sophisticated imaging. Although such approaches allow detailed network analyses, multi-electrode arrays lack single-cell precision, whereas optical physiology generally requires advanced instrumentation that may not be universally available. NEW METHOD: Here we developed a simple, stimulation-free protocol with associated Matlab algorithms that enables scalable analyses of spontaneous network activity in cultured human and mouse neurons. The approach allows analysis of the overall network activity and of single-neuron dynamics, and is amenable to screening purposes. RESULTS: We validated the new protocol by assessing human neurons with a heterozygous conditional deletion of Munc18-1, and mouse neurons with a homozygous conditional deletion of neurexins. The approach described enabled identification of differential changes in these mutant neurons, allowing quantifications of the synchronous firing rate at the network level and of the amplitude and frequency of Ca2+-spikes at the single-neuron level. These results demonstrate the utility of the approach. COMPARISION WITH EXISTING METHODS: Compared with current imaging platforms, our method is simple, scalable, accessible, and easy to implement. It enables quantification of more detailed parameters than multi-electrode arrays, but does not have the resolution and depth of more sophisticated yet labour-intensive methods, such as patch-clamp electrophysiology. CONCLUSION: The method reported here is scalable for a rapid direct assessment of neuronal function in culture, and can be applied to both human and mouse neurons. Thus, the method can serve as a basis for phenotypical analysis of mutations and for drug discovery efforts.
BACKGROUND: Ca2+-imaging is a powerful tool to measure neuronal dynamics and network activity. To monitor network-level changes in cultured neurons, neuronal activity is often evoked by electrical or optogenetic stimulation and assessed using multi-electrode arrays or sophisticated imaging. Although such approaches allow detailed network analyses, multi-electrode arrays lack single-cell precision, whereas optical physiology generally requires advanced instrumentation that may not be universally available. NEW METHOD: Here we developed a simple, stimulation-free protocol with associated Matlab algorithms that enables scalable analyses of spontaneous network activity in cultured human and mouse neurons. The approach allows analysis of the overall network activity and of single-neuron dynamics, and is amenable to screening purposes. RESULTS: We validated the new protocol by assessing human neurons with a heterozygous conditional deletion of Munc18-1, and mouse neurons with a homozygous conditional deletion of neurexins. The approach described enabled identification of differential changes in these mutant neurons, allowing quantifications of the synchronous firing rate at the network level and of the amplitude and frequency of Ca2+-spikes at the single-neuron level. These results demonstrate the utility of the approach. COMPARISION WITH EXISTING METHODS: Compared with current imaging platforms, our method is simple, scalable, accessible, and easy to implement. It enables quantification of more detailed parameters than multi-electrode arrays, but does not have the resolution and depth of more sophisticated yet labour-intensive methods, such as patch-clamp electrophysiology. CONCLUSION: The method reported here is scalable for a rapid direct assessment of neuronal function in culture, and can be applied to both human and mouse neurons. Thus, the method can serve as a basis for phenotypical analysis of mutations and for drug discovery efforts.
Authors: Christopher Patzke; Yan Han; Jason Covy; Fei Yi; Stephan Maxeiner; Marius Wernig; Thomas C Südhof Journal: J Clin Invest Date: 2015-08-17 Impact factor: 14.808
Authors: Eftychios A Pnevmatikakis; Daniel Soudry; Yuanjun Gao; Timothy A Machado; Josh Merel; David Pfau; Thomas Reardon; Yu Mu; Clay Lacefield; Weijian Yang; Misha Ahrens; Randy Bruno; Thomas M Jessell; Darcy S Peterka; Rafael Yuste; Liam Paninski Journal: Neuron Date: 2016-01-07 Impact factor: 17.173
Authors: Trevor J Wardill; Tsai-Wen Chen; Eric R Schreiter; Jeremy P Hasseman; Getahun Tsegaye; Benjamin F Fosque; Reza Behnam; Brenda C Shields; Melissa Ramirez; Bruce E Kimmel; Rex A Kerr; Vivek Jayaraman; Loren L Looger; Karel Svoboda; Douglas S Kim Journal: PLoS One Date: 2013-10-14 Impact factor: 3.240
Authors: Peter Verstraelen; Michiel Van Dyck; Marlies Verschuuren; Nachiket D Kashikar; Rony Nuydens; Jean-Pierre Timmermans; Winnok H De Vos Journal: Front Neurosci Date: 2018-06-26 Impact factor: 4.677
Authors: Jasmeet K Virdee; Gabriella Saro; Antoine Fouillet; Jeremy Findlay; Filipa Ferreira; Sarah Eversden; Michael J O'Neill; Joanna Wolak; Daniel Ursu Journal: Sci Rep Date: 2017-11-03 Impact factor: 4.379
Authors: Jie Wang; Yi Miao; Rebecca Wicklein; Zijun Sun; Jinzhao Wang; Kevin M Jude; Ricardo A Fernandes; Sean A Merrill; Marius Wernig; K Christopher Garcia; Thomas C Südhof Journal: Cell Date: 2021-11-09 Impact factor: 41.582