Part 3:Identification Of Dopaminergic Neurons That Can Both Establish Associative Memory And Acutely Terminate Its Behavioral Expression
Mar 19, 2022
Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791
test, we introduced a waiting period. During this period, the larvae were either kept in darkness or exposed to blue light for 3 min for 864-DAN activation (Fig. 7A). When the recall test was conducted in darkness, a significant memory-based search was observed regardless of 864-DAN activation during the waiting phase (Fig. 7A, left and middle box plot). This suggests that 864- DAN activation during the waiting period does not promote forgetting.
Indirect activation of DANs, via two-step cross-compart- mental feedback from MBONs, has recently been discovered to promote extinction in adult flies (Felsenberg et al., 2018). Extinction refers to the learning that takes place when the odor, but no external reinforcement, is presented. To see whether 864-DAN activation would promote this process, we repeated the experiment, but this time presented the previously trained odor during the waiting period (without external reinforcement), either without or including 864-DAN activation. Again, we observed robust memory-based search in the recall test in both cases (Fig. 7B, left, and middle box plot). 864-DAN activation fully terminated learned search in both experiments (Fig. 7A, B).
Together, these results suggest that activation of 864-DAN, reliably covering only the DAN-i1 neuron, does not promote for- getting or extinction. Rather, 864-DAN activation can confer both a reward signal for memory formation during training and an acute search termination signal during the recall test.
Given that the 58E02- and 864-DAN neurons, just like natural rewards, can mediate both rewards and search termination signals, we decided to investigate further the behavioral relation between the signals carried by these neurons and natural reward.
Relation of 58E02- and 864-DAN activation to sugar reward To test for the relationship between the signals carried by 58E02- and 864-DANs and a natural sugar reward, we took advantage of the nature of memory-based behavior as a search. Regarding natural rewards, we have previously shown that a memory-based search for a reward is terminated by a reward only if the sought-for reward matches the reward that is present (Schleyer et al., 2011, 2013, 2015a). Thus, if the activation of these neurons and sugar reward were plainly to “mean the same thing” to the larvae, then the search for one should be fully and mutually terminated by the presence of the other. However, in the case of 58E02-DAN, this effect is partial and not mutual; and in the case of 864-DAN, it is full yet not mutual, either (Fig. 8):
• Memory-based search induced by 58E02-DAN activation during training was tuned down to about half in the presence of sugar during the test (Fig. 8A). Using a two-odor, differential conditioning paradigm, the same result was observed (Fig. 8E). In contrast, sugar did fully terminate memory-based search when the more specific 864-DAN activation was used for training (Fig. 8B; for two repetitions of this experiment, see Fig. 8F, G). These results can be explained by a representation of sugar reward that partially covers the 58E02-DANs, and fully covers the 864-days. Whether these DANs are necessary for the observed effects of sugar on search behavior is not known.
• Second, we asked whether the memory-based search for sugar can in turn be terminated by activating the 58E02- DANs or 864-DANs, that is, whether activation of these DANs would be sufficient to terminate the search for sugar. This was not the case in the present type of assay (Fig. 8C, D). Thus, with respect to its behavioral significance, there are major aspects of the sugar reward representation that are covered neither by the 58E02-DANs nor the 864-DANs, with the result that the search for sugar continues despite the search termination signal conveyed by these neurons.
With respect to the behavioral relevance in the present type of assay, it is thus a plausible working hypothesis that the representation of sugar reward partially overlaps with 58E02-DAN, and fully encompasses 864-DAN (Fig. 8H). In order to lend further plausibility to this scenario, we investigated whether 864-DAN activation would share additional basic behavioral features with a sugar reward. First, we asked whether 864-DAN activation, like sugar, can induce memories of opposite valence in a contingency-dependent manner.
864-DAN activation induces memories of opposite valence in a contingency-dependent manner
After paired training with odor and sugar, odor preferences during the test are increased relative to baseline, whereas after presenting odor and sugar in an unpaired manner (i.e., during separate trials), the odor preferences are decreased (Fig. 9A) (Saumweber et al., 2011; Schleyer et al., 2011, 2015b; Paisios et al., 2017; for review, see Schleyer et al., 2018). This conforms to widely applied learning theory (Rescorla and Wagner, 1972; Sutton and Barto, 1981; Malaka, 1999) and makes intuitive sense as well: after paired odor-sugar training, the odor predicts where sugar can be found; whereas after presentations of odor separately from sugar, the odor predicts precisely where sugar cannot be found. In both cases, the animals’ behavior reflects a memory-based search for sugar, yet their memory guides them in opposite directions relative to the odor. Optogenetic activation of 864- DAN establishes both these types of memory (Fig. 9B),
Figure 8. Relation of 58E02- and 864-DAN activation to sugar reward. A Larvae were trained with 58E02-DAN activation as in Figure 6. Memory-based search is expressed when the recall test is conducted in the absence of sugar but is tuned down in its presence. Sample sizes: 31, 21. B, Same as in A, but with 864-DAN activation. Memory-based search is observed when the recall test is conducted in the absence of sugar but is terminated in its presence. Sample sizes: 24 each. C, Larvae were trained such that an odor was presented either paired or unpaired with sugar. A memory-based search for sugar was observed regardless of whether or not 58E02-DAN was activated by blue light during the recall test. Sample sizes: 19, 17. D, Same as in C, but with 864-DAN activation. A memory-based search for sugar was observed regardless of whether or not 864-DAN was activated by blue light during the recall test. Sample sizes: 18, 18. E, The experiment shown in A, using 58E02-DAN, was replicated using a differential, two-odor version of the experiment with n-amyl acetate diluted 1:50 and undiluted 1-octanol. Each training trial lasted for 5min, and training and test were performed on a Petri dish of 9cm inner diameter. The larvae showed memory-based search in the absence of the sugar reward. When sugar was presented during the recall test, however, a memory-based search was tuned down. Sample sizes: 14, 13. F, In a replication of the experiment shown in B, using 864-DAN, both training and test were performed on a Petri dish of 9cm inner diameter. The larvae showed memory-based search in the absence but not in the presence of the sugar reward. Sample sizes: 10, 11. G, In a further replication of the experiment shown in B, the same result was observed. Sample sizes: 19, 18. H, The results from A–G suggest, as a working hypothesis, that the representation of sugar reward partially overlaps with 58E02-DAN, and fully encompasses 864-DAN. Different lettering above the box plots indicates pairwise significance (Mann–Whitney U test, p , 0.05, corrected according to Bonferroni-Holm). *Significance from zero (one-sample sign test, p , 0.05, corrected according to Bonferroni-Holm). All statistical tests and their results are reported along with the source data in Extended Data Figure 1-1.
suggesting that, as for sugar, the reward signal carried by 864- DAN can induce memories of opposite valence depending on the contingency of its occurrence with the odor presentation. Strikingly, the resulting behavioral tendencies, memory-based odor approach and aversion, are both terminated by 864-DAN activation during the test (Fig. 9C). Thus, like sugar, activating 864-DAN provides a reward signal that can induce memories of opposite valence during training, and a signal to terminate the search behavior based on either kind of memory during the test. We next asked whether more specifically 864-DAN activation results in the same “micro behavioral” modulations of search behavior as sugar.
Figure 9. 864-DAN activation induces memories of opposite valence in a contingency-dependent manner. A, After paired odor-sugar training, the larvae approached the odor when tested on plain agarose; after unpaired training, they avoided the odor. This difference was abolished in the presence of sugar, meaning that the larvae behaved independently of their odor-sugar associative memory. Therefore, the data from this condition can be combined as a baseline against which the effects of associative memory can be assessed (stippled line). This shows that paired and unpaired training with a natural reward establishes memories of opposite valence, leading to increased or decreased preference compared with baseline, respectively, during the recall test (for review, see Schleyer et al., 2018). Sample sizes: 29, 29, 28, 28. These data underlie the PI scores presented in Figure 1A. B, After paired training with 864-DAN activation, too, the odor preference is increased, whereas, after unpaired 864-DAN training, it is decreased, compared with the baseline preference when the recall test is performed in the presence of sugar. This demonstrates that the reward signal carried by 864-DAN can establish memories of opposite valence, depending on the contingency with the odor. Sample sizes: 43, 42, 42, 43. These data are pooled from the experiments presented in Figure 8B, G. C, Just like sugar, activation of 864-DAN during the recall test terminates both the memory-based odor approach after paired training and the memory-based odor avoidance after unpaired training. Sample sizes: 47 each. These data are pooled from the data presented in Figure 6B, D. Different lettering above the box plots indicates pairwise significance (Mann–Whitney U test, p, 0.05, corrected according to Bonferroni-Holm). All statistical tests and their results are reported along with the source data in Extended Data Figure 1-1.
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864-DAN activation affects the same aspects of search behavior as sugar Larval behavior in an odor gradient can be characterized by relatively straight runs, interrupted by lateral HCs that are followed by changes of direction (Fig. 10A, B). We find that search based on an 864-DAN memory, just like memories for sugar (Schleyer et al., 2015b; Paisios et al., 2017; Thane et al., 2019), can be characterized by modulations of both these two aspects of behavior, namely, the rate of HCs, and their direction (Fig. 10C, D) (Saumweber et al., 2018):
• After paired training with odor and 864-DAN activation, larvae made more HCs while heading away from the odor than while heading toward it (Fig. 10C, leftmost box plot showing HC rate modulation . 0). Such modulation of their HC rate brings the animals closer to the odor source. After unpaired training with odor and 864-DAN activation, the opposite was observed (Fig. 10C, second box plot from the left showing HC rate modulation, 0).
• In addition, after paired training, the animals directed their HCs more toward the odor source than after unpaired training (Fig. 10D, black-filled box plots).
We next wondered whether 864-DAN activation during the test can also terminate both these behavioral effects, as has been reported for sugar (Schleyer et al., 2015b; Paisios et al., 2017), and indeed found this to be the case (Fig. 10C,D, blue-filled box plots). Thus, the reward signal carried by 864-DAN causes the same modulations of memory-based search as sugar, and the search termination signal carried by 864-DAN, just like sugar, terminates both these behavioral modulations.
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Discussion
The current study shows that the optogenetic activation of DANs can have two effects: it can confer a reward signal during training such that associated odors are learned and can later be
used by the animal to direct its search for the reward. And during the recall test, the activation of the same DANs can confer a signal to acutely terminate that very search. Before we discuss which of the synaptic partners of the DANs might be receiving these signals, we compare the effects of DAN activation to those of sugar as a natural reward.
The relationship between natural rewards and optogenetic DAN activation
Optogenetic 864-DAN activation, just like sugar as a natural reward (Fig. 9A) (Saumweber et al., 2011; Schleyer et al., 2011, 2015b; Paisios et al., 2017; for review, see Schleyer et al., 2018), can establish memories of opposite behavioral valence in larvae, depending on the contingency with the odor (Fig. 9B). Thus, 864-DAN activation as such does not signal valence. Rather, as for natural rewards, the valence of 864-DAN activation arises only on convergence with olfactory processing. Extrapolating from what has been established in adult D. melanogaster, this might correspond at the physiological level to the depression of the KC!MBON synapse was observed after paired odor-DAN training, and its potentiation after their unpaired presentation (Cohn et al., 2015; see also Yarali et al., 2012; Handler et al., 2019). Importantly, memories from both paired and unpaired 864-DAN training can be prevented from behavioral expression by optogenetically activating 864-DAN during the recall test (Fig. 9C), as is the case for sugar (Fig. 9A)(Schleyer et al., 2018). This shows that the search termination signal likewise pertains to memories establishedby864-DAN, regardless of their behavior avalanche.
Olfactory memories from 864-DAN activation of either valence further resemble sugar memories in the specific modulations of memory-based search behavior (Fig. 10) (Paisios et al., 2017; Saumweber et al., 2018). Moreover, both 864-DAN activation and sugar can also terminate the opposing modulations of behavior underlying learned search behavior after paired and unpaired training (Fig. 10).
Figure 10. 864-DAN activation affects the same aspects of search behavior as sugar. A, Sample track from a video recording of a larva with runs and HCs. An HC is detected whenever the angular speed of the head exceeds 635°/s (for details, see Paisios et al., 2017). B, When the recall test is performed in darkness, larvae typically approached the odor after paired training with odor and 864-DAN activation, and avoided the odor after unpaired training (black-filled circles). When tested in light (i.e., while 864-DAN was activated), the animals’ preference after paired and unpaired training was indistinguishable (blue-filled circles). Four example tracks for each condition are shown. Arrowheads indicate the start and the end of the tracks. C, After paired training with odor and 864-DAN activation, larvae modulated their HC rate such that they made relatively more HCs while heading away from the odor source than while heading to- ward it. After unpaired training, the opposite modulation was observed. When 864-DAN was activated during the recall test, larvae behaved independently of their previous training. D, After paired training with odor and 864-DAN activation, larvae directed their HCs more toward the odor source than after unpaired training. When 864-DAN was activated during the recall test, larvae behaved independently of their training. For this analysis, data from the experiments displayed in Figures 6B, D were used. Sample sizes from left to right: 46, 48, 48, 45. Different lettering above the box plots indicates pairwise significance (Mann–Whitney U test, p, 0.05, corrected according to Bonferroni-Holm). All statistical tests and their results are reported along with the source data in Extended Data Figure 1-1.
Furthermore, the search termination signal from 864-DAN activation, like that conferred by sugar (Fig. 1) (Schleyer et al., 2011, 2015a,b), affects only memory-based olfactory search, but not innate odor preference (Fig. 6). This specific effect of 864- DAN activation on only learned behavior contrasts with the recently reported DAN-mediated enhancement of innate food exploitation behavior in C. elegans (Oranth et al., 2018).
Finally, the presence of sugar terminates memory-based search after 864-DAN training, suggesting that, after such training, larvae may be searching for a sugar-like reward (Fig. 8B).
However, the search termination signal conveyed by 864- DAN activation is sufficient to terminate search after 864-DAN training (Figs. 6, 7) but not after sugar training (Fig. 8D), consistent with 864-DANs being only a subset of the neurons represent- ing sugar reward (Fig. 8H). Moreover, whether in an experiment the activation of the 864-DANs is necessary for sugar to terminate search is not known. Thus, our results suggest that in its behavioral relevance 864-DAN activation bears some resemblance to a sugar reward but certainly does not equal it.
Who receives the DAN signals?
Given the two effects of DAN activation (i.e., to confer a reward and a search termination signal) and given that DANs has two main targets (i.e., the KCs and the MBONs), the question arises what the effects of receiving a DAN signal are in KCs and MBONs, respectively.
Concerning the KCs, it is established that during training the coincidence of odor activation and dopaminergic input is detected by the type I adenylate cyclase and turned into presynaptic depression of the KC!MBON synapse (for larvae: for review, see Widmann et al., 2018; Thum and Gerber, 2019; adults: Cognigni et al., 2018; Tumkaya et al., 2018). For DANs carrying a reward signal, this would lead to less drive toward avoidance-promoting MBONs and hence to odor attraction based on the residual, intact drive from KCs to approach-promoting MBONs in other compartments (adults: Aso et al., 2014b; Hige et al., 2015; Oswald et al., 2015; Felsenberg et al., 2018). Presenting the odor unpaired from DAN activation can lead to potentiation of the KC!MBON synapse (Cohn et al., 2015), is a process that is not yet understood at the molecular level. Thus, during training, signaling from DANs can have opposite effects on the KC depending on the activity state of the KC (see also Aso et al., 2019; Handler et al., 2019). In the present case, such depression/potentiation of the KC!MBON synapse could support the odor approach/avoidance we observe after odor presentation paired/unpaired from 864-DAN activation, respectively (Fig. 9B). In summary, the reward signal generated by optogenetic 864-DAN activation is very likely delivered via the DAN!KC synapses.
Much less is known about the MBONs, almost exclusively from adult flies. However, the fact that they are required for the expression of learned behavior (larvae: Saumweber et al., 2018; adults: Sejourne et al., 2011; Places et al., 2013; Bouzaiane et al., 2015; Oswald et al., 2015; Shyu et al., 2017; Wu et al., 2017)makes them plausible candidates to receive a search termination signal from the DANs during the recall test. Driving adult DANs has a monosynaptic, excitatory effect on MBONs that is mediated by dopamine (Takemura et al., 2017), and adult MBONs express all four types of D. melanogaster dopamine receptors (Perisse et al., 2016; Crocker et al., 2016; Takemura et al., 2017; Aso et al., 2019). Whether any of these receptors mediates a search termination signal remains unknown, however. Indeed, DAN presynapses can harbor both dense-core and clear vesicles, suggesting that they can use an additional neurotransmitter (adults: Takemura et al., 2017; larvae: Eichler et al., 2017), and some DANs in adults can signal by nitric oxide (Aso et al., 2019). In any case, a plausible working hypothesis is that after paired training the activation of 864-DAN during the test would increase the reduced MBON activity back to baseline levels (Takemura et al., 2017), such that the balance between avoidance- and approach-promoting MBONs was restored and learned behavior was terminated. After unpaired training, according to such a scenario, the enhanced MBON activity would be decreased by DAN activation. In other words, very similar to what was discussed above for the effect of DAN activation on KCs, DAN activation might also have opposite effects on the MBONs, in this case depending on the activity state of the MBON. We would like to add that an alternative would be to use the DAN signal to short-circuit avoidance- and approach-promoting MBONs, such that the net output of the MBON network would be neutral. Indeed, adult MBONs express innexin genes (Aso et al., 2019), and there is a precedent for dopamine-dependent electrical coupling in cardiac motor neurons in crabs, Mauthner cells in fish, and the mammalian retina (Cachope and Pereda, 2012; Lane et al., 2018; Roy and Field, 2019). In summary, although it is a plausible working hypothesis that the search termination signal from the DANs operates through the MBONs, direct evidence is still lacking.
A general principle?
In Pavlovian terminology, the odor in our paradigm corresponds to a conditioned stimulus, sugar to an unconditioned stimulus (US), and optogenetic DAN activation to a US’s reinforcing capacity. Furthermore, learned behavior toward the odor in our paradigm corresponds to the conditioned response, whereas behavior toward sugar would correspond to the unconditioned response. Terminating learned search in the presence of the US in Pavlovian terms would thus correspond to a “dominance” of the unconditioned response over the conditioned response.
Although this seems to be generally adaptive because USs are of intrinsic value whereas conditioned stimuli are not, it is unknown how generally such a rule applies. Indeed, testing for the applicability of this rule requires testing for the conditioned response in the presence of the US, thereby defying what might be called Clause One of Pavlovian practice. In the present case, doing so has allowed us to reveal that in the brain of larval D. melanogaster the same DANs can meditate on the one hand a reward signal during training to establish associative memory and, on the other hand, a signal that can terminate its behavioral expression. Given the role of DANs in mediating reinforcement signals across animals and humans, we wonder whether this reflects a principle of DAN function.
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