Background

Neurodegenerative diseases share a common predisposing factor, the aging of the brain. So, a greater understanding of the normal aging brain may be necessary before we can fully understand the causes of pathological aging and cognitive decline (Yankner et al., 2008).

The existence of a complete neuronal connectivity map and genetic tractability of C. elegans make this animal model useful for studying human neurological diseases. Analysis of multiple genetic databases shows that a considerable number of human genes associated with different diseases have a significant homology to C. elegans genes, and the genetic tools available for this nematode have allowed the construction of predictive models for studying the molecular mechanism of these diseases (Alexander et al., 2014; Griffin et al., 2017).

In the aging C. elegans nervous system, synapses appear physically less robust than in younger animals. The average synapse in an elderly animal is smaller in its presynaptic density and includes fewer synaptic vesicles compared to young adults; this is similar to what occurs in the human brain, where loss of synaptic integrity contributes to its deterioration and dysfunction. Away from the synapse, typical changes in aging neurons include reduced axon caliber, poor axonal transport, smaller nuclei, and reduced cytoplasmic volume. In addition, part of the loss of mobility has an origin in defects of the nervous system (Toth et al., 2012). In recent years, numerous protocols have emerged to facilitate the visualization of C. elegans neurons, taking advantage of the transparent nature of this nematode. DiI is a long-chain dialkylcarbocyanine dye that uniformly labels neurons via lateral diffusion in the plasma membrane (Godement et al., 1987; Hofmann and Bleckmann, 1999). Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks. DiI specifically labels amphid (ADL, ASH, ASI, ASJ, ASK, and AWB), and phasmid (PHA and PHB) IL1 and IL2 neurons, as well as IL sheath and socket cells to highlight the chemosensory structures of live nematodes (Wormatlas.org [Reference 12]). These dyes do not appear to affect the survival, development, or basic physiological properties of neurons, and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane (Honig and Hume, 1986). Labeling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology (Honig and Hume, 1989). The aim of this protocol is to provide a modified technique for Dil staining, to allow the study of neurodegeneration in dementia disease models of C. elegans.