Computer-assisted estimation in the CNS of 3D multimarker 'overlap' or 'touch' at the level of individual nerve endings: a confocal laser scanning microscope application

Floris G Wouterlood, Amber J Boekel, Gerrit A Meijer, Jeroen A M Beliën

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Presynaptic boutons and associated postsynaptic structures in the CNS express markers that are highly synapse type-specific. In multilabel immunofluorescence imaging, coexpression of such markers appears as overlap of signals in the same structures whereas closely related yet segregated markers, e.g., located pre-and postsynaptically, translate into signals that touch. 'Overlap' and 'touch' occur in three dimensions (3D). The instrument of choice to study overlap vs. touch of small objects in tissue volumes is the confocal laser scanning microscope (CSLM). To quantify overlap and touch we used two paradigms. Overlap was studied in rat brain sections triple-immunostained with antibodies against markers predominantly located presynaptically: glutamic acid decarboxylase, vesicular glutamate transporter 2, and calretinin. Touch was studied in rat temporal cortex where afferent, tracer-labeled entorhinohippocampal fibers in the subiculum were imaged together with possible postsynaptic target neurons immunostained with an antibody against the calcium binding protein, parvalbumin. Z-series of CLSM images were obtained in multiple channels. After post-acquisition deconvolution we further processed the images via software written in the C/C++ SCIL Image computer programming environment. The software receives parameters via scripts, after which it first identifies 3D objects through establishing isodensity envelopes around pixels representing small biologic structures (in our case: boutons) and then compares associated Z-series in which it determines whether there is overlap or touch between recognized 3D objects. Finally, graphic and numeric output is produced. With this script-commanded software we feel equipped to accurately and objectively quantify overlap and touch.

Original languageEnglish
Pages (from-to)1215-28
Number of pages14
JournalJournal of Neuroscience Research
Volume85
Issue number6
DOIs
Publication statusPublished - 1 May 2007

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