A behavioral and genetic dissection of two forms of olfactory plasticity in Caenorhabditis elegans: adaptation and habituation

Abstract

Continuous presentation of an olfactory stimulus causes a decrement of the chemotaxis response in the nematode Caenorhabditis elegans. However, the differences between the learning process of habituation (a readily reversible decrease in behavioral response) and other types of olfactory plasticity such as adaptation (a decrement in response due to sensory fatigue, which cannot be dishabituated) have not been addressed. The volatile odorant diacetyl (DA) was used within a single paradigm to assess the distinct processes of olfactory adaptation and habituation. Preexposing and testing worms to 100% DA vapors caused a chemotaxis decrement that was not reversible despite the presentation of potentially dishabituating stimuli. This DA adaptation was abolished in worms with an odr-10 mutation (encoding a high-affinity DA receptor on the AWA neuron), even though naive chemotaxis remained unaffected. Conversely, DA adaptation remained intact in odr-1 mutants (defective in AWC neuron-mediated olfactory behavior), even though naive chemotaxis to DA decreased. Surprisingly, exposure to vapors of intermediate concentrations of DA (0.01% and 25%) did not cause worms to exhibit any response decrement. In contrast to preexposure to high DA concentrations, preexposure to low DA concentrations (0.001%) produced habituation of the chemotaxis response (a dishabituating stimulus could reverse the response decrement back to baseline levels). The distinct behavioral effects produced by DA preexposure highlight a concentration-dependent dissociation between two decremental olfactory processes: adaptation at high DA concentrations versus habituation at low DA concentrations.

Figure 1

Habituation/adaptation apparatus and dose response to DA. (A) Conditioning agar plates were used for exposing worms to the various concentrations of diacetyl (DA) during training. Circles with hatched bars represent agar plugs, each saturated with 1 μl of DA, and short wavy lines represent aggregates of worms. Following treatment, hundreds of worms were placed at the origin point on a test plate with a 1 μl control spot (ethanol, EtOH) and test spot (DA) placed on either end. After a 60 min test period, worms were placed at 4°C, and later counted to establish a chemotaxis index (CI) per plate. Each plate consisting of hundreds of worms was considered an n = 1 each treatment group had an average of n  = 4 per experiment. (B) Worms chemotax to a broad range of DA concentrations. Approach was elicited to a variety of different concentrations of DA, with the highest approach to DA seen at the highest dilutions. n = 4 for all groups.

Figure 2

Increasing exposure time, but not volume, affects degree of approach decrement after preexposure to 100% DA. (A) Worms preexposed to 5 μl of 100% DA for 30 min to 2 hr had significantly lower mean chemotaxis to 100% DA. The unstarved naive group and the naive group that was starved for 2 hr were not different from each other. After 1 hr of DA exposure, worms showed a significant decrease in approach, but the effect was most pronounced after the 2 hr exposure. n  = 4 for all groups. (B) A 2 hr preexposure time to DA with varying volumes of odorant shows that the approach decrement decreased to ∼40% of naive levels for all groups, regardless of preexposure volume. n  = 4 for all groups.

Figure 3

Lack of dishabituation after preexposure to 100% DA. (A) Preexposure to 3 μl of 100% DA for 2 hr caused worms to exhibit a response decrement to 100% DA which could not be reversed despite being presented with dishabituating stimuli ranging from 100–1000g spins. The naive group was given a 1000g dishabituation treatment without preexposure to DA. All groups preexposed and given dishabituation stimuli were significantly different from the naive group that was not preexposed, but not different from the preexposed group without dishabituation stimuli. 100g = a 1 min spin at 100g followed by a 2 min spin at 100g; 300g = a 1 min spin at 300g followed by a 2 min spin at 300g; 500g = a 1 min spin at 500g followed by a 2 min spin at 500g; 1000g = a 1 min spin at 1000g followed by a 2 min spin at 1000g. n  = 4 for all groups, except n = 3 for naive. (B) Worms preexposed to 5 μl of 100% DA for 1 hr do not show a dishabituated response to 100% DA following different dishabituating treatments. Naive group given dishabituating stimulus without DA preexposure. Preexpose = preexposed without dishabituating treatments; Group 1 = preexposure + 2 × 2000g spins (3 min total) + 30 sec vortex; Group 2 = preexposure + cold shock treatment of 4°C for 15 min, followed by a 1 min spin at 100g; Spont. Recovery = spontaneous recovery after 3 hr. n  = 20, 18, 4, 8, and 4, respectively, from left to right.

Figure 3

Lack of dishabituation after preexposure to 100% DA. (A) Preexposure to 3 μl of 100% DA for 2 hr caused worms to exhibit a response decrement to 100% DA which could not be reversed despite being presented with dishabituating stimuli ranging from 100–1000g spins. The naive group was given a 1000g dishabituation treatment without preexposure to DA. All groups preexposed and given dishabituation stimuli were significantly different from the naive group that was not preexposed, but not different from the preexposed group without dishabituation stimuli. 100g = a 1 min spin at 100g followed by a 2 min spin at 100g; 300g = a 1 min spin at 300g followed by a 2 min spin at 300g; 500g = a 1 min spin at 500g followed by a 2 min spin at 500g; 1000g = a 1 min spin at 1000g followed by a 2 min spin at 1000g. n  = 4 for all groups, except n = 3 for naive. (B) Worms preexposed to 5 μl of 100% DA for 1 hr do not show a dishabituated response to 100% DA following different dishabituating treatments. Naive group given dishabituating stimulus without DA preexposure. Preexpose = preexposed without dishabituating treatments; Group 1 = preexposure + 2 × 2000g spins (3 min total) + 30 sec vortex; Group 2 = preexposure + cold shock treatment of 4°C for 15 min, followed by a 1 min spin at 100g; Spont. Recovery = spontaneous recovery after 3 hr. n  = 20, 18, 4, 8, and 4, respectively, from left to right.

Figure 4

No response decrement seen after exposure to intermediate DA concentrations (25%, 0.01%). (A) Preexposures to 2 μl of 25% DA for 90 min or 3 μl of 25% DA for 2 hr were not sufficient to elicit a response decrement to 25% DA in wild-type animals. The dishabituating stimulus was a 1 min centrifugation at 500g, followed by a 2 min 500g spin. n  = 4 for all, except n  = 3 for naive 2 μl/90 min exposure group. (B) A 5 μl preexposure to 0.01% DA did not cause a decreased chemotaxis approach to 0.01% DA despite varying the time of exposure from 15–60 min. The dishabituating stimulus was a 1 min centrifugation at 500g, followed by a 2 min 500g spin. n  = 4 for all 15 min groups, and n  = 3 for all 60 min groups.

Figure 5

Nonassociative learning occurs after preexposure to low concentrations of DA (0.001%). Preexposure to 0.001% DA leads to habituation. Worms preexposed for 15 min to 5 μl of 0.001% DA showed a 25% response decrement when tested to 0.001% DA compared with naive values. Preexposed + spin groups centrifuged twice for a total of 3 min at 250g exhibited a recovery of chemotaxic approach to 0.001% DA to the levels seen in the naive or spontaneous recovery groups. All naive worms (Naive) were given the dishabituating treatment (spin) without DA preexposure. n  = 100, 106, 103, and 10, respectively, from left to right.

Figure 6

Adaptation is odr-10 dependent, but odr-1 independent. Wild-type, odr-10, and odr-1 worms were preexposed to 5 μl of 100% DA for 60 min and tested to 100% DA, 0.1% DA, or 1% BZ. Wild-type worms displayed a 50% decrease in CI to 100% DA and a 75% decrease in CI to 0.1% DA (compared with their respective naive values), but the CI to BZ did not show a similar decrement after 100% DA preexposure. Preexposed group = preexposure with a gentle wash and settled in a tube; Preexposed + Spin = preexposed and spun at 500g for 1 min followed by another 500g spin for 2 min. Within each strain, there was no difference between preexposed or preexposed + spin treatments. odr-10 animals, which showed a nearly zero response to 0.1% DA, did not demonstrate DA adaptation after 100% DA preexposure and testing, and did not show any difference in BZ approach compared to wild-type. odr-1 animals displayed lower baseline CIs to 100% DA and 0.1% DA and no response to BZ, yet still demonstrated DA adaptation to 100% and 0.1% DA after 100% DA preexposure. Respectively from left to right: for Wild-type 100% DA, n  = 17, 16, 15; 0.1% DA, n  = 17, 18, 15; 1% BZ, n  = 12, 11, 14. For odr-10 100% DA, n  = 9, 9, 8; 0.1% DA, n  = 9, 9, 10; 1% BZ n  = 9 for all groups. For odr-1 100% DA, n  = 7, 6, 8; 0.1% DA, n  = 11, 12, 12; 1% BZ, n  = 11, 9, 8.

Figure 7

Adaptation and habituation are separate processes that affect the behavioral response after preexposure to DA. (A) Under baseline conditions, the putative low-affinity DA receptor on AWC mediates a portion of the naive approach to 100% DA via an odr-1-dependent pathway. Although the ODR-10 receptor on AWA is not necessary for naive approach to DA, it can account for a portion of the response to 100% DA in the absence of AWC function. Preexposure to 100% DA causes excessive activation of the high-affinity odr-10 receptor on AWA which leads to a down regulation of the AWC mediated approach pathway causing adaptation. This interaction between AWC and AWA may be via a common downstream neuronal target. Lack of odr-1 in the AWC neuron partially eliminates baseline approach to 100% DA, but does not prevent the odr-10-mediated adaptation. Any sensitization that may be occurring in AWA is overshadowed by the adaptation. (B) Preexposure to intermediate DA concentrations causes less stimulation of the low-affinity DA receptor on AWC and strong stimulation of the ODR-10 DA receptor (but less stimulation than with 100% DA so that adaptation is less and/or sensitization is greater). This leads to the processes of adaptation and sensitization to be equivalently favored and the competition between these two opposing processes results in no net behavioral decrement in DA response. (C) Preexposure to low DA concentrations causes stimulation of only the ODR-10 receptor on AWA (and no activation of the low-affinity DA receptor on AWC), eliminating any possible response adaptation. This results in the nonassociative learning processes of habituation, dependent on AWA function. Gray = inactive neurons; blue = activated AWA neuron; red = activated AWC neuron.