In actio optophysiological analyses reveal functional diversification of dopaminergic neurons in the nematode C. elegans

Abstract

Many neuronal groups such as dopamine-releasing (dopaminergic) neurons are functionally divergent, although the details of such divergence are not well understood. Dopamine in the nematode Caenorhabditis elegans modulates various neural functions and is released from four left-right pairs of neurons. The terminal identities of these dopaminergic neurons are regulated by the same genetic program, and previous studies have suggested that they are functionally redundant. In this study, however, we show functional divergence within the dopaminergic neurons of C. elegans. Because dopaminergic neurons of the animals were supposedly activated by mechanical stimulus upon entry into a lawn of their food bacteria, we developed a novel integrated microscope system that can auto-track a freely-moving (in actio) C. elegans to individually monitor and stimulate the neuronal activities of multiple neurons. We found that only head-dorsal pair of dopaminergic neurons (CEPD), but not head-ventral or posterior pairs, were preferentially activated upon food entry. In addition, the optogenetic activation of CEPD neurons alone exhibited effects similar to those observed upon food entry. Thus, our results demonstrated functional divergence in the genetically similar dopaminergic neurons, which may provide a new entry point toward understanding functional diversity of neurons beyond genetic terminal identification.

Figure 1. DAergic neurons mediate food-dependent slowing behaviour.

(a) A schematic diagram of arrangement of the four DAergic neuron pairs. Note that this is the top view of an animal on an agar surface. (b) An arrangement of patches of bacterial lawns on an agar surface. Similar experimental condition was recently reported by Hardaway et al.. (c) Food-dependent slowing response of wild-type and mutant animals. Well-fed animals (black bars) or animals starved for 30 min (grey bars) were transferred to the centre position of the assay plate shown in b and the bending numbers were scored for 20 s after the food entry. The numbers of animals used in each condition are shown at the bottom.

Figure 2. Schematic drawing of the OSaCaBeN system.

Setups for calcium imaging (a) and optogenetic analysis (b) are shown. The motorized stage is controlled to lock-on a part of the animal’s body at the centre of the view field. In (a), blue light is illuminated to the full view field to excite GCaMP6f and mCherry in all of the DAergic neurons. In (b), green light for mCherry is illuminated to the full view field to monitor the positions of all of the DAergic neurons, and blue light is illuminated to one of the DAergic neurons to stimulate ChR2 when necessary. The position of the blue light illumination is updated in real time because the target neuron moves in the view field due to the limitation of tracking accuracy. An IR light was used to acquire bright field images for pattern matching and tracking.

Figure 3. CEPD and CEPV neuron pairs were asymmetrically activated upon food entry.

(a,b) Responses of CEPD (top) and CEPV (middle) as well as speed (bottom) of well-fed (a) or starved (b) wild-type animals upon food entry are shown. The time when an animal entered a bacterial lawn was determined as t = 0. The R/R₀ values (average ± SEM) are shown. The responses of CEPD and CEPV were monitored from the same animals. Twenty-eight and 11 animals were analysed for well-fed and starved conditions, respectively. When the average R/R₀ value was lower than 1.1 in both of CEPD and CEPV in an animal, that animal was regarded as “not responding”, and those data were excluded. (c) Scatter plot comparing averaged R/R₀ values between CEPD and CEPV of well-fed (left) or starved (right) wild-type animals upon food entry. A two-tailed Mann-Whitney test was used for the statistical analysis.

Figure 4. Activation of CEPD neuron pairs is mainly responsible for the slowing behaviour.

(a) Expressions of mCherry and ChR2::GFP were at similar levels between CEPD and CEPV but was lower in ADE. (b) Comparison of the effects of optogenetic stimulation on slowing. When all of the dopaminergic neurons or only CEPD were illuminated, but not CEPV or PDE, significant slowing occurred. A Mann-Whitney test was used to compare the normalized locomotion speeds before (−) and during (+) the blue light illumination within each targeted cell type, while a Kruskal-Wallis test with a post-hoc Steel-Dwass test was used to compare cell types during illumination. Details of the statistical analyses are shown in Supplementary Table S4.