An automated olfactory learning assay using individual animals
displays olfactory preference on a solid substrate by chemotaxis, using both a “pirouette strategy” and a “weathervane strategy”. In the “pirouette strategy”, locomotion is characterized by periods of forward movements that are interrupted by reorienting maneuvers “pirouettes”, including reversals and Ω turns (sharp turns in which animal’s body shape resembles the Greek letter omega Ω). When an animal experiences improving conditions, such as a positive gradient of attractive chemical cues, it reduces the frequency of reorienting maneuvers; when an animal encounters declining conditions, it increases the frequency of reversals and turns. This behavioral strategy resembles the biased random walk that bacteria exhibit during chemotaxis (Berg and Brown, 1972
; Chalasani et al., 2007
; Pierce-Shimomura et al., 1999
). In the “weathervane strategy”, animals gradually steer themselves during periods of forward movement to move towards an attractant (Iino and Yoshida, 2009
). Swimming C. elegans
also exhibits chemotaxis and displays olfactory preference by regulating the frequency of omega turns. Attractive odorant molecules suppress turns and their removal evokes turns (Luo et al., 2008
; Pierce-Shimomura et al., 2008
). Therefore, the frequency of omega turns during swimming is negatively correlated with an animal’s preference for an olfactory stimulus.
To carry out systematic laser ablation analysis of olfactory learning, we employed a microdroplet assay to automate the analysis of olfactory behaviors using individual animals (). Previously, it was shown that adult animals exhibit similar levels of olfactory learning whether exposed to pathogenic bacteria only as adults or exposed throughout their lifetimes (Zhang et al., 2005
). We raised animals under standard conditions until adulthood and then transferred half of the animals onto a training plate containing a fresh bacterial lawn of a pathogenic bacterium P. aeruginosa
PA14 and the other half onto a control plate containing a fresh lawn of a benign bacterium E. coli
OP50 (so they would remain naïve to the smell of PA14). After four to six hours, we analyzed trained and naïve animals side by side in the microdroplet assay by subjecting animals, each freely swimming in a microdroplet of buffer, to 12 cycles of alternating airstreams that were odorized with either PA14 or OP50. Switching between airstreams was under computer control (). We analyzed video records of swimming animals with a machine-vision algorithm that automatically detected omega turns exhibited by each animal. We quantified olfactory preference between the two odorized airstreams with a choice index based on the number of omega turns detected during exposure to each airstream for each assay ().
C. elegans displays aversive olfactory learning on the pathogenic bacterium PA14 in the microdroplet assay
Because the frequency of omega turns is negatively correlated with the preference for an olfactory stimulus, a positive choice index indicates an olfactory preference for pathogen PA14 and a negative choice index indicates a preference for OP50. We defined the learning index as the difference in choice indexes exhibited by naïve and trained animals (). We found that naive animals turned much less frequently when exposed to the smell of PA14 and generated a positive choice index, indicating that the naïve olfactory preference is to PA14. However, after training, animals generated similar numbers of turns when exposed either to the smell of PA14 or to the smell of OP50 and produced a choice index close to zero. Therefore, a positive learning index indicates that trained animals learn to decrease their attraction to the smell of PA14 after exposure to PA14 ().
exhibits spontaneous omega turns even when subjected to clean air that carries no olfactory stimulus (). Thus, olfactory stimuli do not directly generate omega turns, but instead modulate the frequency of spontaneous turns. When exposed to the alternating airstreams odorized with buffer and bacteria culture of either OP50 or PA14, both naïve and trained animals exhibited a lower turning rate to the smell of either bacterial culture than to buffer, indicating that they are attracted to the smell of food (Supplemental Figure 1A
). Thus, learning specifically modulates the olfactory preference between benign and pathogenic bacteria without abolishing the general attraction of food smell.
This form of aversive olfactory learning occurs and reverses rapidly. Adult animals trained on PA14 exhibited a significant amount of learning with two hours of training and became fully trained after 4 hours. When fully-trained adult animals were transferred back to a bacterial lawn of OP50, the learned olfactory preference gradually diminished over 2 hours (Supplemental Figure 1B, 1C
), indicating that the training process does not generate permanent alteration to the nervous system. Thus, the fully-developed nervous system of adult C. elegans
is capable of experience-dependent modulations to avoid the smell of pathogenic bacteria.
Although the aversive olfactory learning analyzed by the microdroplet assay reverses rapidly, three observations suggest that it is distinct from adaptation. First, no alteration in olfactory preference was detected in this assay when adult animals were trained with a series of non-pathogenic bacterial strains (Supplemental Figure 1D
), suggesting that the aversive learning is contingent on the pathogenesis of the training pathogen. Second, two adaptation mutants, egl-4
, both displayed normal learning ability in the microdroplet assay (Supplemental Figure 1E
). Third, aversive olfactory learning of PA14 requires the negative reinforcing effect that directly results from ingestion of the pathogen. To show this, we put adult animals on a lawn of OP50 while exposing them for 6 hours to the smell of a PA14 lawn, which was grown on the lid of the plate. In this experiment, trained animals were exposed to the smell of PA14, but were fed on OP50. These trained animals exhibited olfactory preference comparable to that of the control animals that fed on OP50 without exposure to the smell of PA14 (Supplemental Figure 1F
Previously, we used a two-choice assay that quantified the overall movements of populations of crawling worms to elucidate the role of serotonergic neurotransmission in aversive olfactory learning (Zhang et al., 2005
). Importantly, the automated microdroplet assay that we utilized in this study recapitulates the phenotypes that were obtained using the two-choice assay and supports the role of serotonin in aversive olfactory learning. The cat-1
mutation, which disrupts both dopamine and serotonin neurotransmission (Duerr et al., 1999
), greatly reduced olfactory learning quantified using the microdroplet assay while the cat-2
mutation, which specifically disrupts dopamine production (Lints and Emmons, 1999
), had no effect on learning (). The tph-1(mg280)
mutant, which is deficient in the only C. elegans
tryptophan hydroxylase required for biosynthesis of serotonin (Sze et al., 2000
), was completely defective in olfactory learning in the microdroplet assay (). In addition, the mod-1(ok103)
mutant, which is defective in a serotonin-gated chloride channel (Ranganathan et al., 2000
), also showed greatly reduced learning in the microdroplet assay (). Thus, the microdroplet assay for swimming animals assigns phenotypes that are consistent with the two-choice assay that we previously used. The important advantage of the microdroplet assay is that it allows us to quantify olfactory preference with small numbers of animals.
Aversive olfactory learning requires the AWB and AWC olfactory sensory neurons
To characterize the neuronal network that regulates the switch of olfactory preference, we began by identifying chemosensory neurons required for olfactory plasticity. We first tested an osm-6
mutant, which is defective in development and sensory function of all ciliated chemosensory neurons (Collet et al., 1998
). The osm-6
mutant showed significantly reduced learning to avoid the smell of PA14 (). By comparing choice indexes before and after training, we found that the osm-6
mutant was unable to reduce its olfactory preference for the smell of PA14 after training (Supplemental Figure 2A
). These results indicate a requirement for the function of chemosensory neurons in generating a learned preference. The residual learning ability of the osm-6
mutant likely results from its residual olfactory sensory ability in the microdroplet assay (Supplemental Figure 2B
). The osm-6
mutant exhibited lower turning frequencies than wild-type animals, especially towards the smell of PA14 (Supplemental Figure 2C
), consistent with other studies in which osm-6
mutant exhibited lower omega turn frequencies in crawling and swimming assays (Gray et al., 2005
; Srivastava et al., 2009
). We also measured the innate immune response of C. elegans
to PA14 infection following an established procedure (Tan et al., 1999
) and found that the osm-6
mutant displayed the same immune response to PA14 infection as wild type animals (Supplemental Figure 2D
), suggesting that its learning defect did not result from an altered immune response.
Aversive olfactory learning requires the AWB and AWC olfactory sensory neurons
is expressed in all ciliated chemosensory neurons, we tested other chemosensory mutants to identify the set of chemosensory neurons that are necessary for learning. An osm-3
mutant, which is defective in function of most chemosensory neurons except olfactory neurons (Tabish et al., 1995
), showed learning ability and turning rate comparable to wild type animals. A che-1
mutant, which is impaired in development of the major gustatory neurons ASE (Uchida et al., 2003
), also displayed normal learning ability and turning rate (, Supplemental Figure 2A and 2C
). However, a mutation in ceh-36
, which compromises development of ASE and olfactory sensory neurons AWC (Lanjuin et al., 2003
), caused a defective learning and exhibited a much reduced naïve olfactory preference for the smell of bacteria (, Supplemental Figure 2A and 2C
). Together, these results suggest that the AWC olfactory neurons are required to detect the smell of bacteria and olfactory sensory inputs are required to generate aversive olfactory learning in the microdroplet assay.
AWA, AWB and AWC represent three pairs of olfactory sensory neurons in the C. elegans
nervous system. AWC and AWB sensory neurons mediate attractive and repulsive olfactory responses, respectively, to a variety of odorants (Bargmann et al., 1993
; Troemel et al., 1997
). It was shown that AWB mediate behavioral response to stay off the lawns of pathogenic bacteria (Pradel et al., 2007
). We found that the expression of osm-6
complementary DNA in the olfactory sensory neurons AWB and AWC together fully rescued the learning defect of the osm-6
mutant, while expression of osm-6
cDNA in AWC alone or in the gustatory neurons ASE did not rescue ( and Supplemental Figure 2E
), suggesting that the combined function of AWB and AWC regulates aversive olfactory learning.
Since different Pseudomonas
bacteria strains secrete 2-butanone, iso-amyl alcohol, 2,3-pentanedione and 2,4,5-trimethylthiazole, which are detected by the sensory neurons AWC and AWA as attractive odorants (Bargmann et al., 1993
; Zechman and Labows, 1985
), one possibility is that training with Pseudomonas aeruginosa
PA14 lowers the animal's attraction towards these molecules, or even inverts the animal's response, turning them into repellents. Using the microdroplet assay, we quantified the strength of olfactory responses to this set of chemicals in both naïve animals and animals trained with PA14 and found no significant difference (Supplemental Figure 3
). One possibility is that C. elegans
recognizes the smell of PA14 as a coherent percept corresponding to a particular mix of different odorant molecules. Another possibility is that the smell of PA14 is recognized through an as yet unidentified odorant molecule that is unique to it.
Two different neural circuits regulate naïve and learned olfactory preferences
Next, we asked which neurons operate downstream of AWB and AWC to display naïve and learned olfactory preferences. Serial-section electron micrography has revealed the complete wiring diagram of the C. elegans
nervous system (Chen et al., 2006
; White et al., 1986
). Potential functional circuits can be identified as groups of neurons that are heavily interconnected by large numbers of synapses. This straightforward method of counting synapses allowed previous investigators, for example, to map pathways that regulate the ability of crawling worms to generate spontaneous omega turns and reversals (Gray et al., 2005
). We applied this method to identify integrated circuits downstream of AWB and AWC based on strong chemical synaptic connections (Supplemental Table 1
and Supplemental Experimental Procedures). This analysis uncovered a multilayered neural network composed of sensory neurons (AWB, AWC, and ADF), interneurons (AIB, AIY, AIZ, RIA, and RIB), and motor neurons (RIM, RIV, RMD, SAA, and SMD) ( and Supplemental Table 2
). Most neurons in this candidate network represent pairs of bilaterally symmetric neurons except that SMD and SAA are groups of four neurons and RMD are six neurons. This network downstream of the AWB and AWC olfactory sensory neurons overlaps partly with a previously mapped network that regulates the frequency of reversals and omega turns of crawling worms (Gray et al., 2005
; Tsalik and Hobert, 2003
). For this study, the network downstream of AWB and AWC provides candidate pathways for understanding how olfactory preference and plasticity are generated in the C. elegans
To characterize how the neuronal function of the olfactory network in allows animals to display different olfactory preferences before and after learning, we performed a systematic laser ablation analysis of each neuronal type in the network. We conducted laser ablations with a femtosecond laser microbeam (Chung et al., 2006
) on L2 larvae and cultivated the operated animals under standard conditions until the adult stage. At this point, we transferred half of the operated animals onto a fresh lawn of OP50 as naïve controls and the other half onto a fresh lawn of PA14 to induce aversive olfactory learning (). After six hours, we measured turning frequency of these naïve and trained animals towards alternating airstreams odorized with OP50 or PA14 ( and ), which allowed us to analyze choice indexes to quantify olfactory preference (). We quantified effects of neuronal ablation by comparing the choice indexes of naïve and trained ablated animals with those of matched mock controls. Mock controls went through laser surgery, post-surgery cultivation, and olfactory training and analysis procedures in the same way as the ablated animals but without the neurons being ablated. These comparisons allowed us to define the contribution of each neuronal type in the network to the generation of naïve and learned olfactory preferences.
Olfactory sensory neurons in the AWB-AWC sensorimotor circuit mediate naïve olfactory preference
Modulations to signal transduction downstream of sensory neurons generate the learned preference
We found that the naïve olfactory preference for PA14 is disrupted by laser ablation of a specific group of neurons. For example, AWB-ablated animals exhibited no naïve olfactory preference for PA14 and trained AWB-ablated animals did not exhibit any olfactory preference either, producing a learning index that was close to zero (). It is important to note that all choice indexes that we measured as being close to zero in this study were due to similar turning rates during the OP50 and the PA14 airstreams, and not due to inability to swim or generate Ω turns. This notion is evidenced by the analyses on turning rates in and for all the ablation results.
Two different neural circuits in an olfactory network regulate the naïve and learned olfactory preferences
Individually ablating AWC or AIY produced an effect similar to, albeit smaller than, that of ablating AWB. Ablating AIZ or AIY and AIB together generated the same effects on the naïve preference as ablating AWB (). Ablating the ADF serotonergic neurons also moderately reduced the naïve choice index, indicating that ADF might have a small sensory contribution to the naïve olfactory preference for PA14. Ablating any other neurons in the network did not significantly alter naïve olfactory preference (). Thus, AWB, AWC, AIY and AIB, AIZ and possibly ADF play essential roles in generating the naïve olfactory preference between the smells of OP50 and PA14. These neurons are strongly interconnected with chemical synapses. The similar effects caused by ablating these neuronal types suggest that these neurons constitute a functional circuit (an AWB-AWC sensorimotor circuit) that allows C. elegans to encode and display its naïve olfactory preference for PA14 (blue symbols in ).
Within the AWB-AWC sensorimotor circuit, the functions of different neurons are diverse. Animals lacking AWB or AWC or AIY and AIB together are not only defective in their naïve preference, but also deficient in generating any clear preference after training and, thus, produce low learning indexes (). The low learning indexes of these animals could be caused either by defects in sensing or distinguishing between the smells of different bacteria, defects in learning, or both. Although the severe defects in the naïve preference caused by ablating AWB, AWC or AIY and AIB together clearly points to their role in producing the naïve preference, their contribution to producing the learned preference cannot be excluded and deserves further examination. In contrast, AIZ-ablated animals exhibited a strong olfactory aversion to the smell of PA14 after training, despite showing no naïve olfactory preference between OP50 and PA14 (). Both naïve and trained AIZ-ablated animals exhibited lower turning rates when exposed to the smell of OP50 than mock control animals, with a greater decrease in turning rate after training ( and ), implicating the specific function of AIZ in generating the naïve olfactory preference for PA14. The difference in naïve and trained choice indexes of AIZ-ablated animals yielded a learning index comparable to wild type animals (), indicating that ablating AIZ did not affect olfactory learning ability. The distinct effects of ablating AIZ on olfactory preference and plasticity point to different cellular mechanisms for generating naïve olfactory preference and learning.
We next sought to identify neurons that might regulate olfactory plasticity without affecting naïve olfactory preference. Further laser ablation analysis uncovered such a group of neurons. For example, ablating the RIA interneurons had no effect on the naïve olfactory preference for PA14, but completely abolished the ability to shift olfactory preference away from PA14 after training. Animals without RIA continued to exhibit an olfactory preference for PA14 after training, leading to a low learning index (). Similarly, killing ADF or RIM or SMD significantly changed the learned preference and disrupted learning ability without substantially altering naïve olfactory preference (). Except for the mild effect of killing RIB, ablating any other neuronal types in the network did not generate comparable defects (). The RIA interneurons connect with ADF sensory neurons and SMD motor neurons with large numbers of synapses, and the RIM motor neurons send out a few synapses to SMD. Ablating any neurons in this circuit - RIA, ADF, SMD or RIM - abolished olfactory plasticity without affecting the naïve olfactory preference for PA14. Thus, this circuit (the ADF modulatory circuit) is specifically required to generate experience-dependent plasticity after training with PA14 (pink symbols in ). Previously, we found that the serotonergic neurons ADF play an essential role in regulating aversive olfactory learning on pathogenic bacteria (Zhang et al., 2005
). Here, by analyzing the function of neurons that are strongly connected to ADF, we identified the pathway downstream of ADF that causes worms to shift their olfactory preference away from PA14 after training.
In summary, two different neural circuits – the AWB-AWC sensorimotor circuit and the ADF modulatory circuit -- allow C. elegans to display the naïve olfactory preference and to change olfactory preference after experience. The ADF neurons contribute to both the naïve olfactory preference and the change in olfactory preference after experience ().
Correspondence between the microdroplet assay for swimming worms and the two-choice assay for crawling worms
Next, we sought to verify that phenotypes of neuronal ablation that were quantified using individual swimming worms in the microdroplet assay could also be obtained using crawling worms in the two-choice assay that we established earlier (Zhang et al., 2005
). Since the two-choice assay requires a large number of animals for each experiment, we were able to do this with the RIA interneurons by ectopically expressing a cell-death molecule caspase with strong cell specific promoter (Brockie et al., 2001
; Chelur and Chalfie, 2007
). In the microdroplet assay, laser ablation of RIA did not alter the naïve olfactory preference for PA14, but generated a significant deficiency in changing olfactory preference away from PA14 after training (). Similarly, we found that RIA-genetically-killed animals exhibited a naïve olfactory preference comparable to wild type animals and non-transgenic siblings, but exhibited no ability to shift olfactory preference away from PA14 after training, resulting in a complete loss of learning ability (). Thus, the results of both assays are consistent in identifying a specific role for RIA in generating olfactory plasticity.
Correspondence between the microdroplet assay and the two-choice assay
We also compared phenotypes obtained in the microdroplet assay and the two-choice assay using osm-6 mutants and transgenic animals in which function of osm-6 is rescued in olfactory neurons AWB and AWC. We found that in both the microdroplet assay and the two-choice assay the trained choice indexes of osm-6 mutants were significantly different from that of wild type animals and expression of osm-6 cDNA in AWB and AWC neurons fully rescued the learning defect (). Thus, the microdroplet assay is as reliable as the two-choice assay in defining phenotypes for olfactory preference and learning. Unlike the two-choice assay, however, the microdroplet assay can be combined with systematic laser ablation analysis of any neuron within the circuit.
Olfactory sensory neurons mediate the naïve olfactory preference
As shown above, naïve animals prefer the smell of PA14, evidenced by an increase in their turning rate when airstreams switch from the smell of PA14 to the smell of OP50. In contrast, animals that have been trained by exposure to PA14 display similar turning rates towards the smells of PA14 and OP50, producing a comparatively lower olfactory preference for PA14. We next asked how the neurons for the naïve and learned olfactory preferences regulate turning rate to exhibit olfactory preference.
We first analyzed the AWB-AWC sensorimotor circuit for the naïve olfactory preference (blue symbols in ). AWB and AWC mediate repulsive and attractive olfactory responses, respectively. To characterize their function in determining naïve preference, we measured neuronal activity within these sensory neurons upon exposure to the smells of OP50 or PA14 using intracellular calcium imaging. First, we studied transgenic animals expressing the genetically encoded calcium sensitive fluorescent protein G-CaMP in the AWCON
cell, one of the two AWC neurons. It was previously shown that the two AWC neurons, AWCON
, generate similar calcium responses to the odorants that they both detect (Chalasani et al., 2007
). Removal of attractive odorants stimulates AWC calcium response while exposure to attractants suppresses it (Chalasani et al., 2007
). We subjected naïve transgenic animals to alternating streams of PA14-conditioned medium and OP50-conditioned medium in the order of OP50-PA14-OP50, and found that OP50-conditioned medium stimulated calcium transients in AWCON
while PA14-conditioned medium suppressed the activation (). We also did control experiments by exposing transgenic animals to the two stimuli in the order of PA14-OP50-PA14 and found similar results (Supplemental Figure 4A
). Next, we subjected naïve transgenic animals to alternating streams of clean buffer and streams conditioned with either OP50 or PA14, and found that AWCON
calcium transients were suppressed by either type of bacterial conditioned medium (). Together, these results indicate that the AWC neurons in naïve animals respond to both the smell of PA14 and OP50 as attractants, but respond to the smell of PA14 as a more attractive stimulus than the smell of OP50. Thus, the response properties of the AWC neuron match the olfactory preference of the naïve behaving animal.
We next examined transgenic animals that express G-CaMP in the AWB olfactory sensory neurons, which mediate repulsive olfactory behavioral response to repellants including 2-nonanone (Troemel et al., 1997
). We found that removal of 2-nonanone stimulated AWB calcium transients and exposure to 2-nonanone suppressed AWB (Supplemental Figure 4E
). This result suggests that the switch from a repellent to the removal of the repellent activates AWB. We subjected these naïve transgenic animals to alternating streams of OP50 and PA14-conditioned mediums in the order of either OP50-PA14-OP50 or PA14-OP50-PA14. We found that the switch from OP50-conditioned medium to PA14-conditioned medium activated AWB calcium transients ( and Supplemental Figure 4C
). When we alternated streams of clean buffer with streams conditioned by either OP50 or PA14, calcium transients in AWB neurons were activated by switching either type of bacterial conditioned medium to buffer (). Taken together, these results indicate that both OP50 and PA14-conditioned mediums contain repellents that are detected by AWB and in naïve animals AWB respond to OP50 as a more repulsive stimulus than PA14. Thus, the neuronal response of AWB is consistent with the olfactory preference of naïve animals towards PA14 at the level of behavior.
Next, we asked how the olfactory sensitivities of AWC and AWB are transduced into olfactory behavioral preference by the regulation of turning rate exhibited by swimming worms in response to the smells of OP50 and PA14. To do this, we examined the effects of neuronal ablation on the turning rate of naïve animals. Ablating the AWC sensory neurons, AIB or AIZ interneurons, or SMD motor neurons significantly decreased the turning rate to the smell of OP50, suggesting that the smell of OP50 promotes turns through these neurons. In contrast, ablating both AIY and AIB or ablating the RIM motor neurons increased the turning rate towards the smells of both OP50 and PA14, indicating that these neurons inhibit turns in response to olfactory inputs (). Individually ablating any other neurons did not affect turning rate in naive animals (). The AIB and AIY interneurons are postsynaptic to AWC, and regulate turns during odor sensation in crawling animals (Chalasani et al., 2007
). RIM, which receive inputs from both AIY and AIB, synapse onto the four SMD motor neurons. Taken together, these results indicate that these strongly connected interneurons and motor neurons regulate turning rates downstream of AWB and AWC (). When naïve worms are subjected to alternating smells of OP50 and PA14, the smell of OP50 activates AWC neurons and raises turning rate, whereas the smell of PA14 inactivates AWC and lowers turning rates (). Thus, the differential responses of AWC to the smells of PA14 and OP50 could regulate downstream neurons to generate stimulus-specific turning rates that are displayed as the olfactory preference for PA14 in naïve animals ().
Although the activity of the AWB neurons also reflects the naïve olfactory preference for PA14 (), ablating AWB eliminated naïve olfactory preference without significantly changing turning rates. It is possible that AWB might regulate AIZ directly and/or indirectly through ADF (). Ablating AIZ interneurons specifically lowered the turning rate upon exposure to the smell of OP50 in both naïve and trained animals, eliminating the naïve olfactory preference for PA14 without affecting olfactory learning (, and ). Although AIB or RIM or SMD also contribute to the generation of different turning rates upon exposure to the smell of OP50 and PA14 in naïve animals (), the ablation effects were not specific (RIM) or prominent enough (AIB or SMD) to significantly change the naïve olfactory preference for PA14 (). Together, our results indicate that in naïve animals AWB and AWC exhibit stimulus-specific patterns of activity. Differential response of AWC to the smells of OP50 and PA14 regulates downstream circuit to display olfactory preference through the control of turning rate. AIZ contribute to naïve olfactory preference by regulating the response to the smell of OP50 ().
Modulations to signal transduction to the network downstream of olfactory sensory neurons generate the learned preference
Finally, we investigated how this network is changed by training with PA14 to generate learned olfactory preference. First, we studied intracellular calcium responses in the AWB and AWC olfactory neurons upon exposure to the smells of OP50 and PA14 after training. Surprisingly, while AWCON
neuronal responses are strongly correlated with the behavioral preference for PA14 over OP50 in naïve animals, AWCON
neuronal responses in trained animals did not reflect the shift in olfactory preference away from the smell of PA14. As we did with naïve animals, we subjected trained worms to alternating streams conditioned with either OP50 or PA14. We found that OP50-conditioned medium stimulated AWCON
calcium response and PA14-conditioned medium suppressed AWC when animals were exposed to the two stimuli either in the order of OP50-PA14-OP50 or in the order of PA14-OP50-PA14 ( and Supplemental Figure 4B
). And the switch from buffer to medium conditioned with either OP50 or PA14 also suppressed AWCON
calcium transients in trained animals (). These patterns of AWCON
calcium dynamics in trained animals indicate that AWCON
continues to respond to PA14 as the more attractive stimulus than OP50, just as in naïve animals. Calcium dynamics in the AWB olfactory sensory neurons were also unchanged by training, continuing to respond to the smell of OP50 as more repulsive than the smell of PA14 ( and Supplemental Figure 4D
). Thus, the behavioral shift of olfactory preference away from PA14 is not generated by the patterns of AWC or AWB sensory response, pointing to experience-dependent changes to the signal transduction to downstream neurons.
To identify the changes to the network that are caused by training, we examined the turning rates exhibited by trained animals in response to the smells of OP50 and PA14. We found that ablating RIA, the interneurons that are specifically required to shift olfactory preference away from PA14 after training, specifically decreased the PA14-induced turning rate without affecting the OP50-induced turning rate (). Ablating SMD, four motor neurons connecting with RIA, reduced the turning rates towards the smells of both OP50 and PA14, but with a stronger reduction towards the smell of PA14 (). These results suggest that RIA and SMD function downstream of the neural network to increase turning rate towards the smell of PA14 in trained animals. Although ablating AWC or AIB or RIB also altered turning rates of trained animals, the effects on turning rate were similar towards either the smell of OP50 or PA14, resulting in little change on olfactory preference after training ( and ). These results suggest that olfactory learning to shift olfactory preference away from the smell of PA14 after exposure to PA14 works by modulating the turning rate response upon exposure to the smells of OP50 and PA14, not by modifying the neuronal responses of AWC or AWB sensory neurons to the smell of either bacterium. It was previously shown that the ADF serotonergic neurons, major presynaptic partners of RIA, are essential for aversive olfactory learning in crawling animals. Long term exposure of C. elegans
to PA14 increases the serotonin content of ADF, suggesting that serotonin might represent the negative-reinforcing cue (Zhang et al., 2005
). Our results suggest that ADF function together with their downstream RIA interneurons and SMD motor neurons to drive aversive olfactory learning. RIA connect with SMD through a large number of reciprocal synapses. Thus, RIA may regulate aversive olfactory learning by integrating the negatively reinforcing serotonergic signal with locomotory response to the olfactory sensory input. This regulation can modulate motor outputs of the olfactory network during training, resulting in an increased turning rate, and thus a decreased olfactory preference towards PA14 in trained animals.