Mohammad I K Hamad1, Solieman Daoud2, Petya Petrova2, Obada Rabaya2, Abdalrahim Jbara2, Nesrine Melliti3, Sarah Stichmann2, Gebhard Reiss3, Joachim Herz4, Eckart Förster2. 1. Institute for Anatomy and Clinical Morphology, School of Medicine, Faculty of Health, University of Witten/Herdecke, Witten, Germany; Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany. Electronic address: mohammad.hamad@uni-wh.de. 2. Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany. 3. Institute for Anatomy and Clinical Morphology, School of Medicine, Faculty of Health, University of Witten/Herdecke, Witten, Germany. 4. Departments of Molecular Genetics, Neuroscience, Neurology and Neurotherapeutics, Center for Translational Neurodegeneration Research, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Abstract
BACKGROUND: Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. NEW METHOD: We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. RESULTS: Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating. COMPARISON WITH EXISTING METHODS: The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. CONCLUSION: The method is optimal for short-time investigation of calcium signals in acute slices.
BACKGROUND: Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. NEW METHOD: We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. RESULTS: Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating. COMPARISON WITH EXISTING METHODS: The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. CONCLUSION: The method is optimal for short-time investigation of calcium signals in acute slices.
Authors: Zhan-Lu Ma; Markus Werner; Christoph Körber; Illah Joshi; Mohammad Hamad; Petra Wahle; Michael Hollmann Journal: J Neurosci Res Date: 2007-01 Impact factor: 4.164
Authors: Mohammad I K Hamad; Petya Petrova; Solieman Daoud; Obada Rabaya; Abdalrahim Jbara; Nesrine Melliti; Jennifer Leifeld; Igor Jakovčevski; Gebhard Reiss; Joachim Herz; Eckart Förster Journal: Development Date: 2021-09-06 Impact factor: 6.862