A method for the three-dimensional reconstruction of Neurobiotin(TM)-filled neurons and the location of their synaptic inputs

Fogarty, Matthew J., Hammond, Luke A., Kanjhan, Refik, Bellingham, Mark C. and Noakes, Peter G. (2013) A method for the three-dimensional reconstruction of Neurobiotin(TM)-filled neurons and the location of their synaptic inputs. Frontiers in Neural Circuits, 7 OCT: 1-18. doi:10.3389/fncir.2013.00153

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Author Fogarty, Matthew J.
Hammond, Luke A.
Kanjhan, Refik
Bellingham, Mark C.
Noakes, Peter G.
Title A method for the three-dimensional reconstruction of Neurobiotin(TM)-filled neurons and the location of their synaptic inputs
Formatted title
A method for the three-dimensional reconstruction of Neurobiotin™-filled neurons and the location of their synaptic inputs
Journal name Frontiers in Neural Circuits   Check publisher's open access policy
ISSN 1662-5110
Publication date 2013-10-01
Year available 2013
Sub-type Article (original research)
DOI 10.3389/fncir.2013.00153
Open Access Status DOI
Volume 7
Issue OCT
Start page 1
End page 18
Total pages 18
Editor Eve Marder
Place of publication Lausanne, Switzerland
Publisher Frontiers Research Foundation
Language eng
Formatted abstract
Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit α1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses.
Keyword Motor neuron
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Article # 153

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Brain Institute Publications
Official 2014 Collection
School of Biomedical Sciences Publications
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Created: Tue, 03 Dec 2013, 22:34:39 EST by Dr Mark Bellingham on behalf of School of Biomedical Sciences