Abstract
Analyzing cell morphology is a key component to understand neuronal function. Several staining techniques have been developed to facilitate the morphological analysis of neurons, including the use of fluorescent markers, such as DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate). DiI is a carbocyanine membrane dye that exhibits enhanced fluorescence upon insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and prominent fluorescence of the dye serves as an effective means of illuminating cellular architecture in individual neurons, including detailed dendritic arborizations and spines in cell culture and tissue sections. Here, we specifically optimized a simple and reliable method to fluorescently label and visualize dissociated hippocampal neurons using DiI and high-resolution confocal microscopic imaging. With high efficacy, this method accurately labels neuronal and synaptic morphology to permit quantitative analysis of dendritic spines. Accurate imaging techniques of these fine neuronal specializations are vital to the study of their morphology and can help delineate structure-function relationships in the central nervous system.
Highlights
Dendritic spines are small protrusions from the dendritic shaft of various types of neurons that act as the postsynaptic compartments of most excitatory synapses in the central nervous system (CNS)
OF FINDINGS The results obtained through this protocol demonstrated that DiI staining in cells prepared with a lower percentage
of fixative yielded the highest quality of images
Summary
Dendritic spines are small protrusions from the dendritic shaft of various types of neurons that act as the postsynaptic compartments of most excitatory synapses in the central nervous system (CNS). They are known to play a significant role in neuronal plasticity and synaptic integration through their ability to undergo structural rearrangements during development (Rochefort and Konnerth, 2012). Structural classifications of spines with accurate labeling and imaging techniques can spawn vital information on neuronal function, and in turn offer insight into the etiology of neurological diseases (Lin and Koleske, 2010; Penzes et al, 2011)
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