Habituation as an adaptive shift in response strategy mediated by neuropeptides

Abstract

Habituation is a non-associative form of learning characterized by a decremented response to repeated stimulation. It is typically framed as a process of selective attention, allowing animals to ignore irrelevant stimuli in order to free up limited cognitive resources. However, habituation can also occur to threatening and toxic stimuli, suggesting that habituation may serve other functions. Here we took advantage of a high-throughput Caenorhabditis elegans learning assay to investigate habituation to noxious stimuli. Using real-time computer vision software for automated behavioral tracking and optogenetics for controlled activation of a polymodal nociceptor, ASH, we found that neuropeptides mediated habituation and performed an RNAi screen to identify candidate receptors. Through subsequent mutant analysis and cell-type-specific gene expression, we found that pigment-dispersing factor (PDF) neuropeptides function redundantly to promote habituation via PDFR-1-mediated cAMP signaling in both neurons and muscles. Behavioral analysis during learning acquisition suggests that response habituation and sensitization of locomotion are parts of a shifting behavioral strategy orchestrated by pigment dispersing factor signaling to promote dispersal away from repeated aversive stimuli.

Fig. 1

Habituation of glutamate transmission mutants reveals a glr-1 mutant phenotype. a Representative raster plots depicting the behavioral state at the beginning (left) and end (right) of training. Pixels are color coded for speed with negative values corresponding to backward locomotion. Black bars indicate 2 s of whole-plate illumination with blue light at 250 μW/mm². b, c Proportion of the population reversing to each of thirty 2 s light pulses administered at 0.1 Hz. b The glr-1-independent response did not persist across the assay, c but could be rescued with a GLR-1 expressing transgene (GLR-1::GFP) or suppressed by loss of egl-3. Mean ± SEM. ‘#’, ‘¥’, and ‘&’ denote groups that are statistically different based on the likelihood of responding to the final stimulus. N = 6 plates/strain

Fig. 2

GPCR RNAi screen. a Proportion of the population reversing to each stimulus for animals fed RNAi clones that increased the probability of a reversal to the final stimulus. Mean ±SEM. ‘#’ and ‘&’ denote significantly different groups based on the likelihood of responding to the final stimulus. b Proportion of animals reversing to the final stimulus for populations fed RNAi clones to knockdown egl-3 or one of 57 GPCRs. Knockdown was done in a background sensitized to neuronal RNAi by feeding: glr-1; lin-15b; sid-1; unc-119p::sid-1. Each circle is the mean of three plates, with multiple replicates for the control and egl-3 targeting vector. Dashed lines mark upper and lower critical values (values corresponding to |z-score| > 4.46; P < (0.05/57 =) 0.0008)

Fig. 3

PDF signaling promotes habituation. Proportion of the population reversing (a), reaction time (b), and response duration (c) to each of thirty 2 s light pulses administered at 0.1 Hz. d–f Effects of restoring PDFR-1 expression to neurons or muscles. Mean ± SEM. ‘#’ denotes groups significantly different from control and ‘&’ denotes groups significantly different from both control and the pdfr-1 mutant based on the response to the final stimulus. N = 2–4 plates/strain

Fig. 4

PDFR-1 signals through cAMP to promote habituation. Proportion of the population reversing (a), reaction time (b), and response duration (c) to each of 30 2 s light pulses administered at 0.1 Hz. Mean  ±  SEM. ‘#’ and ‘&’ denote significantly different groups based on the response to the final stimulus. N = 3 plates/strain

Fig. 5

PDF signaling promotes dispersal during habituation training. a Representative trajectories during a 20 s interval at the beginning (0–20 s), middle (140–160 s), and end (280–300 s) of the habituation assay with thirty 2 s light pulses delivered at 0.1 Hz. Individual tracks were randomly assigned colors and set to start from the same point. N = 4 plates/strain. Scale bar = 1 mm b Displacement (shortest distance between the start and endpoint) over the first and final 30 s of the assay for the PDF signaling mutants. c Displacement over the first and final 30 s of the assay for PDF signaling mutant rescue lines. ‘#’ denotes a significant increase in displacement (one-tailed, P < 0.01, with Bonferroni correction for multiple comparisons) over the assay. Circles are plate means, crosses are population means  ±  SEM

Fig. 6

Shift in locomotion between stimuli. Proportion of the population’s time spent moving forward, backward, or not at all during the 3 s interval immediately preceding each stimulus delivered at 0.1 Hz for control (a) and pdfr-1 (b) and pdf-;pdf-2 (c) mutants. d Speed of worms moving forward during the same 3 s intervals. Mean ± SEM. N = 6 plates/strain