title: Hybrid voltage sensor imaging of eGFP-F expressing neurons in chicken midbrain slices.
abstract: BACKGROUND
  Dendritic computation is essential for understanding information processing in single neurons and brain circuits. Optical methods are suited best to investigate function and biophysical properties of cellular compartments at high spatial and temporal resolution. Promising approaches include the use of voltage sensitive dyes, genetically encoded voltage sensors, or hybrid voltage sensors (hVoS) consisting of fluorescent proteins and voltage-dependent quenchers that, so far, are not available in avian neuroscience.
  
  
  METHODS
  We have adapted a hVoS system for a chicken midbrain slice preparation by combining genetically expressed farnesylated eGFP with dipicrylamine (DPA). Depending on the cellular potential, DPA is shifted in the membrane, resulting in quenching of eGFP fluorescence linearly to the membrane potential by Förster resonance electron transfer.
  
  
  RESULTS
  In ovo electroporation resulted in labelled neurons throughout the midbrain with a high level of fine structural detail. After application of DPA, we were able to optically record electrically evoked action potentials with high signal-to-noise ratio and high spatio-temporal resolution.
  
  
  METHODS
  Standard methods available for avian neuroscience such as whole-cell patch clamp yield insufficient data for the analysis of dendritic computation in single neurons. The high spatial and temporal resolution of hVoS data overcomes this limitation. The results obtained by our method are comparable to hVoS data published for mammals.
  
  
  CONCLUSIONS
  With the protocol presented here, it is possible to optically record information processing in single avian neurons at such high spatial and temporal resolution, that cellular and subcellular events can be analysed.
authors.affiliation: Technische Universität München, Chair of Zoology, Liesel-Beckmann Straße 4, 85354 Freising, Germany. Electronic address: stefan.weigel@wzw.tum.de.
authors.forename: S
authors.initials: S
authors.lastname: Weigel
authors.affiliation: Technische Universität München, Chair of Livestock Biotechnology, Liesel-Beckmann Straße 1, 85354 Freising, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Flisikowska
authors.affiliation: Technische Universität München, Chair of Livestock Biotechnology, Liesel-Beckmann Straße 1, 85354 Freising, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schnieke
authors.affiliation: Technische Universität München, Chair of Zoology, Liesel-Beckmann Straße 4, 85354 Freising, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Luksch
mesh_terms: D000818:Animals; D002645:Chickens; D005240:Feasibility Studies; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D016014:Linear Models; D008564:Membrane Potentials / Q000502:physiology; D008636:Mesencephalon / Q000166:cytology / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D055096:Optical Devices; D055095:Optics and Photonics / Q000379:methods*; D010851:Picrates / Q000494:pharmacology; D013997:Time Factors; D046509:Tissue Culture Techniques; D014162:Transfection
chemical_list: D010851:Picrates; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C011454:dipicrylamine
keywords: Chicken; Dendritic computation; Golgi staining; In ovo electroporation; Optical imaging; Optogenetic