Part 2:Identification Of Dopaminergic Neurons That Can Both Establish Associative Memory And Acutely Terminate Its Behavioral Expression
Mar 19, 2022
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Dendrogram analysis. The neuron dendrograms are 2D sketches of the 3D neuron reconstructions from Eichler et al. (2017). As “linearized”, flattened versions of morphologically complex neurons, the dendrograms are compact visualizations that serve to display the spatial distribution of synapses. Details of the dendrogram drawing algorithm were described by Strauch et al. (2018). Briefly, the dendrograms are topologically correct neuron sketches with a dominant primary branch drawn as a straight line, and shorter side branches deflected at stereotypical angles and pointing upward and downward in an alternating fashion to avoid overlap. They are drawn such that cable length distances and synapse positions are approximately correct. Slight modifications to improve readability involve an automatic rearrangement of synapse symbols to resolve clutter and overlap (Strauch et al., 2018).
For the analysis of the synaptic organization, we computed a (synapse) distance matrix containing geodesic distances (i.e., the distance as “cable length” along the neuron) between the locations of all synapses on the neuron. The geodesic distances were computed as Euclidean distances in the 3D coordinates of the reconstructed neuron by traversing along the neuron’s branches. We clustered the synapse locations with divisive analysis clustering (DIANA) (Kaufman, 2005) operating on the geodesic distance matrix. Based on silhouette score analysis, we set the number of clusters per neuron to 10. Clusters that did not contain all four synapse classes of interest (KC!DAN-i1, DAN-i1!KC, DAN- i1!MBON-i1 left, DAN-i1!MBON-i1 right) were removed from the analysis. For each cluster, a central point was defined as the most central synapse of the cluster as determined by partitioning around medoids clustering (PAM with k=1 cluster) (Kaufman, 2005) based on the geodesic synapse distances within the cluster. Finally, we determined the geodesic distances between all synapses and the cluster’s central point.
Experimental design and statistical analysis. Two-tailed nonparametric tests were used, and statistical assumptions for these tests were met throughout. When multiple comparisons were performed within one analysis, a Bonferroni-Holm correction was applied to keep the experiment-wide error rate to,5% (Holm, 1979). All the performed statistical tests and their results are reported in Extended Data Fig. 1-1 along with the source data.
For all behavioral experiments, values were compared across multiple groups with Kruskal–Wallis tests. In case of significance, subsequent pairwise comparisons used Mann–Whitney U tests. To test whether the values of a given group differed from zero, one-sample sign tests were used.
The experiments in Figures 1, 6C–F, and 8 followed a two-group- design, comparing two testing conditions for the experimental genotype. Both groups were compared with a Mann–Whitney U test, and their significance from zero was tested by one-sample sign tests.
The experiments in Figures 6A, B followed a six-group design, with three genotypes (experimental genotype expressing ChR2-XXL, driver, and an effector control) and two test conditions. First, a Kruskal–Wallis test was performed across all groups. In case of significance, we performed pairwise Mann–Whitney U-tests between the experimental genotype and each of the genetic controls of the same test condition, as well as between the test conditions for the experimental genotype (total five Mann–Whitney U tests). Each group was tested for its significance from zero by one-sample sign tests.
The experiments in Figure 7 followed a three-group design with animals of the experimental genotype expressing ChR2-XXL. After an initial Kruskal–Wallis test across all groups, we performed pairwise Mann– Whitney U tests for the group without blue light exposure after training and both the other groups. Significance from zero was tested for each group (one-sample sign tests).
The experiments in Figures 9 and 10 followed a four-group design with animals of the experimental genotype expressing ChR2-XXL. After an initial Kruskal–Wallis test across all groups, we performed pairwise Mann–Whitney U tests between groups that had received paired and unpaired training within the same test condition, and between test conditions for a given kind of training (total four Mann–Whitney U tests). No one-sample sign tests were performed.
We present behavioral data as box plots with the median as the middle line and 25%/75% and 10%/90% as box boundaries and whiskers, respectively. Outliers are not displayed. Experimenters were blind to genotype and regarding the absence or presence of sugar reward during the recall test, if applicable. Sample sizes (biological replications) were chosen based on previous studies revealing moderate to mild effect sizes (Paisios et al., 2017; Saumweber et al., 2018) and are displayed in the figure legends. A sample size of n =1 included;20 animals of both sexes for all innate preference experiments, and 2 20 animals (20 for each reciprocally trained group) of both sexes for all learning experiments.
To compare synapse numbers between the ipsilateral and contralateral sides of the MBON-i1 neurons (see Fig. 3A), we tested for their distribution using x2 tests, separately for KC!MBON-i1 synapses and DAN-i1!MBON-i1 synapses. To compare the distances of synapses to the center of their respective center-surround structure on the DAN-i1 neuron (see Fig. 4C,D), we performed Kruskal–Wallis tests across all synapse types, as well as pairwise Mann–Whitney U tests between all synapse types (total 6 Mann–Whitney U-tests), separately for the left and the right DAN-i1 neuron.
Results
The spatial arrangement of synapses in a DAN-KC-MBON matrix
The recent electron microscope reconstruction of a complete larval mushroom body circuit has provided a connectome of the DANs, KCs, and MBONs based on the number of chemical synapses between them (Eichler et al., 2017). The axonal branches of
Figure 3. Organization of synaptic inputs to the MBON-i1 neuron. A Number of KC!MBON-i1 and DAN-i1!MBON-i1 synapses to each MBON-i1 neuron, separated by hemisphere. Both MBON-i1 neurons have more synapses with those KCs and that DAN-i1 is located in the hemisphere contralateral to their cell body. *Significant x2 tests (p, 0.05, corrected according to Bonferroni-Holm). Data are from Eichler et al. (2017). B, Semischematic overview of the distribution of synapses from the ipsilateral and contralateral hemisphere to MBON-i1. KCs and both DAN-i1 neurons form more synapses with the contralateral part of MBON-i1 than with its ipsilateral part. C, Distribution of the number of mature KCs forming the indicated number of synapses with the left or right MBON-i1 neuron. KCs from the left hemisphere form fewer synapses with the left than the right MBON-i1 neuron (left). KCs from the right hemisphere, conversely, form more synapses with the left than the right MBON-i1 (right). Given that KCs do not cross the midline, all synapses that, e.g., the right MBON-i1 formed with KCs from the left hemisphere are located on the part of MBON-i1 contralateral to its cell body. D, Despite the fact that the MBON-i1 neurons receive less input from the KCs of the hemisphere ipsilateral to their cell bodies than from the contralateral hemisphere, both MBON-i1 neurons receive information about the full space of KC inputs, in either hemisphere. To determine the relationship between MBON-i1 and the sensory input projected onto the KCs, the total input that MBON-i1 receives from KCs that in turn receive their input from uniglomerular olfactory projection neurons (PNs) (white and dark gray), multiglomerular olfactory PNs (light gray), and nonolfactory PNs (black) were taken into account to calculate the matrix product of the respective PN!KC and KC!MBON-i1 synapses. This was done separately for MBON-i1 left and its connections to the right (i.e., contralateral) KCs (top left), and those to the left (i.e., ipsilateral) KCs (top right), and accordingly for MBON-i1 right (bottom). Data are based on Eichler et al. (2017). All statistical tests and their results are reported along with the source data in Extended Data Figure 1-1.
individual DANs and the dendritic branches of individual MBONs are each confined to, and overlap within, specific compartments along with the KC axon bundles (larvae: Pauls et al., 2010;
Eichler et al., 2017; Saumweber et al., 2018; Eschbach et al., 2020; adults: Aso et al., 2014a; Takemura et al., 2017). DAN-i1 innervates the i-compartment of the mushroom body medial lobe (sometimes called the upper toe of the medial lobe). The reconstruction revealed that it not only signals to KCs but actually has two main targets: the KCs and MBON-i1 (Fig. 2A, B) (Eichler et al., 2017). Correspondingly, MBON-i1 has two main upstream partners: the KCs and DAN-i1. A similar organization of DANs and MBONs is found across mushroom body compartments (larvae: Eichler et al., 2017; adults: Takemura et al., 2017). Specifically, for DAN-i1, 75% and 21% of its output synapses in the mushroom body connect to KCs and MBON-i1, respectively, and MBON-i1 receives;74% and 14% of its input synapses in the mushroom body from KCs and DAN-i1, respectively (Eichler et al., 2017; Saumweber et al., 2018). Although such analyses are informative in many ways, they do not consider the spatial arrangement of synapses along the neurons. To shed light on this arrangement in the i-compartment, we use dendrograms that allow us to display the complex, 3D Gestalt of a neuron as a 2D sketch that captures its branching structure, preserves branch lengths, and identifies synapse location along the branches (Strauch et al., 2018). Such detailed analyses surpass the resolution by which neurons can presently be manipulated in freely behaving animals (Aso and Rubin, 2020). Still, they offer a glimpse of the actual complexity of the neurons under study, and of the computational implications of the circuit motifs they are part of (see also Bilz et al., 2020). From our analyses, we highlight the following points (high-resolution images of the DAN-i1 and MBON-i1 neurons from both hemispheres well as of all mature KCsare available in Extended Data Figs. 2-1, 2-2, and 2-3, respectively):
• Both DAN-i1 and MBON-i1 harbor massive interhemispheric connections. This is remarkable given how little interhemispheric crosstalk is otherwise observed along ascending pathways, including the KCs (Fig. 2A) (Berck et al., 2016; Eichler et al., 2017; Saumweber et al., 2018; Thum and Gerber, 2019). Specifically, both DAN-i1 neurons are reciprocally connected with most mature KCs, in both hemispheres. Moreover, the DAN-i1 neurons provide output to both MBON-i1 neurons, both ipsilaterally and contralaterally (Fig. 2E; for high-resolution images of both DAN-i1 neurons, see Extended Data Fig. 2-1). This implies that each MBON-i1 neuron, in turn, receives input from both DAN-i1 neurons, both ipsilaterally and contralaterally (Fig. 2F; for high-resolution images of both MBON-i1 neurons, see Extended Data Fig. 2-2). Likewise, the MBON-i1 neurons receive input from most mature KCs, in both hemispheres (Eichler et al., 2017), and provide output outside the mushroom body, also in both hemispheres. The dendrograms more specifically reveal that the outputs from DAN-i1 onto the MBON-i1 neuron of the ipsilateral and contralateral hemisphere are intermingled, as are the inputs to MBON-i1 from the DAN-i1 neuron of the ipsilateral and the contralateral hemisphere (Fig. 2E,F). Notably, MBON-i1 is not hemispherically symmetrical in terms of the number of synapses it receives in the mushroom body: both MBON-i1 neurons receive more synapses in the hemisphere contralateral to their cell bodies (Figs. 2F, 3A, B); nevertheless, both MBON- i1 neurons receive information of the complete sensory input space in either hemisphere (Fig. 3C,D).
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• The branching patterns of both DAN-i1 and MBON-i1 are structured into a few major regions. For DAN-i1, three such major regions can be identified. Starting from the cell body, in the first region, DAN-i1 receives input from its partners from outside the mushroom body (marked with 1 in Fig. 2C–E) (Eschbach et al., 2020), whereas the second and third regions coincide with the i-compartment of the ipsilateral and contralateral mushroom body, respectively (marked with 2 and 3 in Fig. 2C–F). In the mushroom body, DAN-i1 is reciprocally connected to KCs and provides output toward MBON-i1. The primary neurite of MBON-i1 crosses the midline, and the neuron then innervates the i-compartment of the mushroom body contralateral to its cell body (region marked with 3 in Fig. 2C–F); here, it mainly receives input from KCs and DAN-i1. Then, MBON-i1 crosses back over the midline and likewise innervates the mushroom body ipsilateral to its cell body (region marked with 2 in Fig. 2C–F). In addition, MBON-i1 features several small branches in both hemispheres that connect to partners close to, but im- mediately outside, the mushroom body.
• Within these regions, compartment-like divisions that are recognizable in the DAN-i1 and in the MBON-i1 neurons of both hemispheres cannot be identified (Fig. 2E,F; Extended Data Figs. 2-1, 2-2). However, for the DAN-i1 neurons, multiple, laterally “idiosyncratic” synapse-rich center-surround structures are apparent (Fig. 4). Toward their center, all the above-mentioned types of the synapse of the DAN-KC-MBON matrix are found, whereas their surround features almost exclusively KC!DAN synapses (Fig. 4). Notably, any given KC usually establishes all its synapses in only one or two of those center-surround structures (Extended Data Fig. 1-1).
• Regarding mature KCs, 86% and 88% of them establish synapses with the left and right DAN-i1 neuron, respectively, and 78% and 74% with the left and right MBON-i1 neuron, respectively. Individual KCs have medians of 4 KC!DAN- i1, 4 DAN-i1!KC, and 5 KC!MBON-i1 synapses (Eichler et al., 2017; for more details on DAN-i1, see Saumweber et al., 2018). According to the dendrograms of all the individual KCs, the KC!DAN-i1, DAN-i1!KC, and KC! MBON-i1 synapses appear to be intermingled with no apparent divisions across the array of KCs, as do the synapses with theleftandtherightDAN-i1neuron, and the left and the right MBON-i1neuron(ExtendedDataFig.2-3).
Together, these analyses reveal the detailed spatial organization of the DAN-KC-MBON matrix of the i-compartment. Of note is that the synapses of DAN-i1 with its two main targets, the KCs and MBON-i1, are locally intermingled within the multiple synapse-rich structures along the axonal branches of DAN-i1, rather than being separated out between them (Figs. 2E, 4; Extended Data Fig. 2-1). Such architecture might imply that DAN-i1 delivers correlated, rather than independently modulated, signals to the KCs and MBON-i1 within these synapse-rich structures.
Activation of the same neurons can establish memory and terminate its recall
We were intrigued by the fact that DAN-i1 has two main targets, namely KCs and MBON-i1; that its synapses with these two targets are so closely intermingled; and that natural rewards have two functions, namely, to establish memory in training and to acutely terminate the behavioral expression of this memory during the recall test (Fig. 1A) (Gerber and
Figure 4. Organization of synapses on the DAN-i1 neuron. Cluster analysis revealed that synapses from and to the left DAN-i1 neuron in regions 2 and 3 (see Fig. 2) are arranged in six clusters (labeled as 29,299, etc.). These clusters appear to have a center-surround organization: toward their center, DAN!KC, KC!DAN, and DAN!MBON synapses are found, whereas their surround features are almost exclusively KC!DAN synapses. B, Same as in A, but for the right DAN-i1 neuron. Seven center-surround structures can be identified. C, To quantify this organization of synapses, we determined the distance (in cable length along the neuron) of different types of synapses to the center of their respective cluster, and found that KC!DAN synapses are further from the center than all other types of synapses. This confirms the organization of the clusters as center-surround structures. D, Same as in C, but for the right DAN-i1 neuron. E, Schematic sketch of the center-surround structure (top) and a magnification of the connections in the center (bottom). 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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Hendel, 2006; Schleyer et al., 2011, 2013, 2015a,b, 2018; Paisios et al., 2017). We, therefore, wondered whether optogenetic activation of DANs in training and during the test could confer these two functions. We started out using the 58E02- Gal4 driver strain that covers DAN-i1 plus two DANs innervating other compartments of the medial lobe (Fig. 5A, B) (Rohwedder et al., 2016), and UAS-ChR2-XXL as the effector strain (Dawydow et al., 2014). The 58E02-DANs were activated either paired or unpaired with odor in training, and the difference in odor preference in a subsequent recall test was used to quantify associative memory through the PI. We observed positive PI scores indicating appetitive associative memory in the experimental genotype, whereas genetic controls heterozygous for either only the transgenic effector or the driver construct did not show memory (Fig. 6A, top, black-filled box plots) (Rohwedder et al., 2016). Thus, 58E02-DAN activation during training can provide a reward signal for associative memory formation.
Next, we repeated the experiment but activated 58E02-DAN during the recall test, too. Doing so reduced PI values (i.e., reduced memory-based search) to approximately half (Fig. 6A, top, blue-filled box plots; for a replication of this result, see Fig. 6C). Critically, just as is the case for natural rewards (Fig. 1B) (Schleyer et al., 2011, 2015a,b), 58E02-DAN activation did not affect innate olfactory behavior (Fig. 6A, bottom). Thus, 58E02- DAN activation can also provide an acute search termination signal during the recall test.
The previous experiments demonstrate that the three 58E02- DANs can confer the same two signals as a natural reward (i.e.,
Figure 5. Transgene expression pattern of the 58E02-DAN and 864-DAN driver strains. A, B, A previous flip-out-based analysis of the 58E02-DAN driver strain identified the neurons DAN-h1, DAN-i1, and DAN-j1 (also called pPAM1, pPAM3, and pPAM4), as well as one additional neuron outside of the mushroom body (Rohwedder et al., 2016). This pattern was confirmed: the driver strain was crossed to either (A1-A5) pJFRC-10xUAS-IVS-mCD8::GFP or (B1-B4) UAS-ChR2-XXL. A1, B1, Signal of the a-GFP or a-ChR antibody, respectively. A2, B2, Signal of the background staining via the a-FASII or a-HRP antibody, respectively. A3, B3, Merge of the respective signals. A4, B4, A partial z projection reveals strong innervation of the medial lobe of the mushroom body. A5, No innervation of the lateral appendix is detected (arrow). C, D, The 864-DAN driver strain has been reported to express strongly in DAN-i1 (Saumweber et al., 2018). Here, we crossed it to either (C1-C5) pJFRC-10xUAS-IVS-mCD8::GFP or (D1-D4) UAS-ChR2-XXL. C1, D1, Signal of the a-GFP or a-ChR antibody, respectively. C2, D2, Signal of the background staining via the a-FASII or a-HRP antibody, respectively. C3, D3, Merge of the respective signals. C4, D4, A partial z projection reveals strong innervation of the i-compartment in both hemispheres (arrows), confirming expression in DAN-i1. C5, As previously reported (Saumweber et al., 2018), the driver strain occasionally expresses in additional neurons, but if so, typically in only one brain hemisphere, and in neurons differing in identity between preparations. Relatively most frequently, we observed the MBON-d1 neuron, innervating the lateral appendix in one hemisphere (arrow). Across the multiple animals used for our group-based assay, such stochastic additional expression is unlikely to cause systematic effects.
the reward signal during the training and the search termination signal during the recall test). However, it remains unclear whether each of the three DANs confers both signals, or whether there is a division of labor, with different DANs being responsible for the reward signal and the search termination signal. We, therefore, chose the split-Gal4 driver strain 864, which supports strong and reliable transgene expression in only one of the DANs covered by 58E02-DAN, the DAN-i1 neuron, in both hemispheres (Fig. 5C, D) (Saumweber et al., 2018). Associative 864-DAN activation in training established positive performance indices (i.e., memory-based search; Fig. 6B, top, black-filled box plots) (Saumweber et al., 2018). Activating 864-DAN also during the recall test terminated this memory-based search(Fig.6B, top,blue-filled box plots). Three independent datasets confirm the observation of search being tuned down (Fig. 6D–F). Innate olfactory behavior wasnotaffectedby864-DANactivation(Fig.6B, bottom).
The activity of DANs between training and the recall test (i.e., during the retention period) has previously been found to pro-mote forgetting in adult D. melanogaster (Berry et al., 2012, 2015; Shuai et al., 2015; Aso and Rubin, 2016). We, therefore, wondered whether, in our paradigm, the 864-DAN activity would promote forgetting, as opposed to acutely terminating memory-based search and leaving memory intact. To test for this possibility, larvae were trained as before, but before the recall
Figure 6. Activation of the same neurons can establish memory and terminate its recall. A Top, Larvae were trained with odor presented either paired or unpaired with 58E02-DAN activation by blue light. In the recall test, positive PI values in the experimental genotype, but not in the genetic controls, indicate learned search behavior based on associative memory for the odor (leftmost box plot). Thus, during training, 58E02-DAN activation can mediate a reward signal. The memory-based search of the experimental genotype was tuned down when 58E02-DAN was activated by blue light during the recall test as well (leftmost blue-filled box plot). Thus, 58E02 activation can also confer a search termination signal. Sample sizes: 16, 15, 17, 18, 15, 18. Bottom, Larvae were tested for innate odor preference, either in the absence or the presence of blue light. All genotypes preferred the odor equally strongly under both conditions, suggesting that activation of 58E02-DAN had no effect on innate odor preference. Sample sizes: 24 each. B, Same as in A, but with 864-DAN activation by blue light. Top, A positive PI, and thus memory-based search, was observed only in the experimental genotype and only when the recall test was performed in darkness. Sample sizes: 19, 19, 19, 19, 19, 20. Bottom, 864-DAN activation had no effect on innate odor preference. Sample sizes: 16 each. C, In a replication of the experiment shown in A (top) for the experimental genotype, the larvae showed memory-based search in darkness. When 58E02-DAN was activated by blue light during the recall test, this search was tuned down. Sample sizes: 15, 16. D, In a replication of the experiment shown in B (top) for the experimental genotype, the larvae exhibited memory-based search in darkness. When 864-DAN was activated during the recall test, this search was terminated. Sample sizes: 28, 28. E, In a further replication of D, the test was performed on a Petri dish of 9cm inner diameter instead of 15cm. Memory-based search during the recall test was observed in darkness and, in this case, was partially tuned down through 864-DAN activation. Sample sizes: 22, 22. F, Larvae were trained in a differential, two-odor version of the experiment, using n-amyl acetate diluted 1:50, and undiluted 1-octanol. The recall test was performed on a Petri dish of 9cm inner diameter. The memory-based search was observed in darkness and was tuned down through 864-DAN activation. Sample sizes: 26, 26. 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). ns: Nonsignificance across all experimental conditions (Kruskal–Wallis-test, p . 0.05). All statistical tests and their results are reported along with the source data in Extended Data Figure 1-1.
Figure 7. Activation of 864-DAN between training and the recall test does not enhance forgetting or extinction. A Group of larvae was trained as in Figure 6. Then, the larvae were kept either in darkness or blue light for 3 min (Wait) and were subsequently tested. When that recall test was conducted in darkness, a memory-based search was observed independent of the light condition, and thus independent of 864-DAN activity, during the waiting phase (left and middle box plot). We confirmed that also after such a waiting period 864- DAN activation during the recall test terminates memory-based search (right box plot). Sample sizes: 25, 24, 19. B, Same as in A, but this time with the odor present during the waiting period. Sample sizes: 28 each. Different lettering above the box plots indicates pair-wise 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.
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