title: Single-cell juxtacellular transfection and recording technique.
abstract: Genetic modifications and pharmacological studies enable the analysis of protein function in living cells. While many of these studies investigate the effect of proteins by bulk administration or withdrawal of the protein in complex cellular networks, understanding the more subtle mechanisms of protein function requires fine-tuned changes on a single-cell level without affecting the balance of the system. In order to analyse the consequences of protein modification at the single-cell level, we have developed a single-cell transfection method in the loose patch configuration, which allows juxtacellular recordings of neuronal cells prior to juxtacellular transfection. CA1 pyramidal neurons were selected based on morphological and electrophysiological criteria. Using a patch clamp amplifier which allows sensitive recordings of action currents in the loose seal mode as well as electroporation with high-voltage electrical stimulation the identified neurons were transfected with a combination of specific nucleotides, e.g. siRNA and a plasmid coding for GFP for later cell retrieval. Two days after transfection, whole-cell patch clamp recordings of transfected cells were performed to analyse electrophysiological properties. Action potential firing and synaptic transmission of single electroporated CA1 pyramidal cells were comparable to untransfected cells. Our study presents a method which enables identification of neurons by juxtacellular recording prior to single-cell juxtacellular transfection, allowing subsequent analysis of morphological and electrophysiological parameters several days after the genetic modification.
authors.affiliation: Institute of Physiology, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Daniel
authors.forename: Hans Reiner
authors.initials: HR
authors.lastname: Polder
authors.forename: Volkmar
authors.initials: V
authors.lastname: Lessmann
authors.forename: Tanja
authors.initials: T
authors.lastname: Brigadski
mesh_terms: D000200:Action Potentials; D000818:Animals; D056547:CA1 Region, Hippocampal / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques / Q000379:methods*; D017966:Pyramidal Cells / Q000502:physiology*; D014162:Transfection / Q000379:methods*