Preparation of <em>Drosophila</em> Central Neurons for <em>in situ</em> Patch Clamping
- Publikationstyp:
- Zeitschriftenaufsatz
- Metadaten:
-
- Autoren
- Stefanie Ryglewski
- Carsten Duch
- DOI
- 10.3791/4264
- eISSN
- 1940-087X
- Ausgabe der Veröffentlichung
- 68
- Zeitschrift
- Journal of Visualized Experiments
- Sprache
- en
- Online publication date
- 2012
- Status
- Published online
- Herausgeber
- MyJove Corporation
- Herausgeber URL
- http://dx.doi.org/10.3791/4264
- Datum der Datenerfassung
- 2023
- Titel
- Preparation of <em>Drosophila</em> Central Neurons for <em>in situ</em> Patch Clamping
Datenquelle: Crossref
- Andere Metadatenquellen:
-
- Abstract
- Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker(1,2). Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain(3,4) and ventral nerve cord of embryonic(5,6), larval(7,8,9,10), and adult Drosophila(11,12,13,14). A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5(15)), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.
- Addresses
- School of Life Sciences, Arizona State University, AZ, USA. ryglewsk@uni-mainz.de
- Autoren
- Stefanie Ryglewski
- Carsten Duch
- DOI
- 10.3791/4264
- eISSN
- 1940-087X
- Externe Identifier
- PubMed Identifier: 23092999
- PubMed Central ID: PMC3490319
- Funding acknowledgements
- NINDS NIH HHS: R01 NS072128
- Open access
- false
- ISSN
- 1940-087X
- Ausgabe der Veröffentlichung
- 68
- Zeitschrift
- Journal of visualized experiments : JoVE
- Schlüsselwörter
- Motor Neurons
- Animals
- Drosophila melanogaster
- Green Fluorescent Proteins
- Dissection
- Patch-Clamp Techniques
- Sprache
- eng
- Medium
- Electronic
- Online publication date
- 2012
- Paginierung
- 4264
- Datum der Veröffentlichung
- 2012
- Status
- Published
- Datum der Datenerfassung
- 2012
- Titel
- Preparation of Drosophila central neurons for in situ patch clamping.
- Sub types
- Video-Audio Media
- research-article
- Journal Article
Files
https://europepmc.org/articles/PMC3490319?pdf=render
Datenquelle: Europe PubMed Central
- Abstract
- Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker(1,2). Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain(3,4) and ventral nerve cord of embryonic(5,6), larval(7,8,9,10), and adult Drosophila(11,12,13,14). A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5(15)), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.
- Autoren
- Stefanie Ryglewski
- Carsten Duch
- Autoren-URL
- https://www.ncbi.nlm.nih.gov/pubmed/23092999
- DOI
- 10.3791/4264
- eISSN
- 1940-087X
- Externe Identifier
- PubMed Central ID: PMC3490319
- Funding acknowledgements
- NINDS NIH HHS: R01 NS072128
- Ausgabe der Veröffentlichung
- 68
- Zeitschrift
- J Vis Exp
- Schlüsselwörter
- Animals
- Dissection
- Drosophila melanogaster
- Green Fluorescent Proteins
- Motor Neurons
- Patch-Clamp Techniques
- Sprache
- eng
- Country
- United States
- PII
- 4264
- Datum der Veröffentlichung
- 2012
- Status
- Published online
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2013
- Titel
- Preparation of Drosophila central neurons for in situ patch clamping.
- Sub types
- Journal Article
- Video-Audio Media
Datenquelle: PubMed
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- Eigentum von