Visualization of postsynaptic calcium in mushroom body output neurons
Acquisition and formation of an associative short-term memory is localized to the KC presynapses of the γ-lobe of the MB–. Thus, when looking for a readout of the synaptic plasticity underlying this form of memory, we focused on the population of compartment-specific MBONs that lie directly downstream of the γ-type KCs. These neurons each receive synaptic input from KCs in their respective compartment, the weight of which is modulated by input from DANs and comprises the canonical site of learning-induced plasticity within this circuit (Fig. 1a-c),–. In addition, MBONs receive direct modulatory input from DANs as well (Fig. 1c). Therefore, we utilized a postsynaptically localized tool, dHomer-fused GCaMP3, that allowed for the monitoring of odor-evoked activity precisely at the site of MBON input. To first verify the localization of dHomer-GCaMP to the postsynaptic compartments of the MBONs, we removed the brains of flies expressing this construct in individual MBONs of the mushroom body γ-lobe and subjected them to immunohistochemistry and confocal microscopy. When compared to the cytosolic GCaMP6f, dHomer-GCaMP results in a spatially restricted, overall more punctuated fluorescence that is localized primarily in the dendritic compartments of neurons (Fig. 1d). This difference was also observed when neurons were visualized in vivo using two-photon microscopy (Fig. 1e and f), with the large neurites that predominate the GCaMP6f fluorescence being absent in dHomer-GCaMP-expressing flies (the γ1 compartment innervation by MBON-γ1pedc > α/β is shown here as an example). To further verify that the latter can also be used to detect postsynaptic odor-evoked activity in MBONs, flies were presented with MCH and 3-Oct and changes in fluorescence were quantified (Fig. 1e and f). Indeed, reflective of the less intense overall fluorescence, postsynaptic odor responses monitored using dHomer-GCaMP were lower in magnitude than the gross calcium signal observed using GCaMP6f, although still robust enough to be reliably detected and analyzed. With this, we validate dHomer-GCaMP as a viable tool for studying localized odor representations at the level of the MBON postsynapse.
γ-lobe MBONs receive heterogeneous odor-evoked inputs
Congruent with their receipt of inputs from large populations of KCs, previous studies have shown that MBONs innervating the MB lobes have broad odor response profiles. In this vein, we first sought to analyze the postsynaptic responsiveness of our γ-MBONs of interest to the experimental odors, MCH and 3-Oct. To do so, we carried out in vivo functional imaging of individual female flies expressing dHomer-GCaMP in each of the MBONs and quantified the odor-evoked changes in postsynaptic calcium (Fig. 2; individual and mean response traces shown in Supplementary Fig. 1).
This analysis revealed that, indeed, in the case of the MBONs innervating the γ1, γ3, and γ4 compartments, robust responses to both odors were observable in the majority of flies measured (Fig. 2a, c, d). Surprisingly, the γ2-innervating MBON (γ2α’1) only responded to 3-Oct in a small number of flies, in contrast to MCH (Fig. 2b). It has previously been reported that the dendritic arbors of MBONs in the γ5 compartment (MBON-γ5β’2a, MBON-β’2mp) show little or no response to olfactory stimuli. We also observed this result, with responses being detected in only ~17% of flies measured (Fig. 2e, Supplementary Fig. 1). Differences in the magnitude of responses are also notable, with those measured at the MBON-γ3/MBON- γ3β’1 postsynapse sometimes being 4-5x greater than those measured in, for example, MBON-γ1pedc > α/β. These differences in magnitude were also be observed between odors within animals, with MCH eliciting stronger responses than 3-Oct in most cases. These findings indicate that, first, olfactory responses between the γ-lobe MBONs and, thus, the efficiency of KC-to-MBON connections in different γ-lobe compartments, are not homogenous. Second, within MBON types, responses to different odors are not homogenous across individuals. Therefore, we confirm that the neurons in this MBON population possess distinct and individualized odor response properties that could indeed influence and/or result from individual experience such as olfactory learning, as already reported using a cytosolic calcium sensor.
Associative conditioning gives rise to compartment-specific plasticity
To investigate if and how these odor representations are influenced by associative learning, we subjected the same flies expressing dHomer-GCaMP in the γ-lobe MBONs to an aversive conditioning protocol under the microscope and monitored odor-evoked postsynaptic calcium. Flies were placed in a custom-built imaging chamber in which they could be exposed to both odor and electric shock stimuli during functional imaging, allowing for the visualization of odor-evoked postsynaptic activity before and after an aversive training in which flies learned to associate a given odor with punishment.
Based on the hypothesis that the combinatorial activity of the γ-lobe MBONs holds behavior-instructive information about learned odor valence, we first hypothesized that aversive associative conditioning would lead to bidirectional modulation of MBONs such that approach-mediating MBONs would be suppressed in response to the aversively paired odor and avoidance-mediating MBONs would be potentiated. Indeed, such effects have been reported when measuring with cytosolic calcium indicators or using electrophysiology–. In the following sections, however, we demonstrate that modulation directly at the MBON postsynapse is a highly specialized occurrence, localized to a singular compartment of the γ-lobe.
Pairing of an odor with electric shock led to a significant reduction in the postsynaptic calcium response elicited by the trained odor (CS+) in MBON-γ1pedc > α/β (Wilcoxon signed rank test, Z=2.01399, p=0.03906) (Fig. 3c, left). This is consistent with the identification of this neuron in the signaling of positive stimulus valence . Neither the CS- odor (that was explicitly not paired with electric shock) nor the control odor (1-Oct) elicited statistically significant changes in response after conditioning (CS-: Wilcoxon signed rank test, Z=0, p=1; control odor: Wilcoxon signed rank test, Z=0.82929, p=0.42578) (Fig. 3c, center and right). This was also the case in the control groups for odor presentation, but without any electric shock (‘CS only’ control; Fig. 3d). This finding demonstrates association-specific modulation of the odor-driven inputs to MBON-γ1pedc > α/β, dependent on CS-US contiguity. Interestingly, the stimulation with electric shock, but without any odor presentation (‘US only control’; Fig. 3e) induced a slight, but not statistically significant increase in MBON-γ1pedc > α/β response at the given sample size. However, when data for both odorants that were also used for associative training, MCH and 3-Oct, were pooled, a statistically significant increase was detected (Supplementary Fig. 3) (Wilcoxon signed rank test, Z= -2.55729; p=0.00831). This illustrates that the postsynaptic calcium response in this particular MBON can be bidirectionally modulated; it decreases in response to an odor associated with punishment, and it increases in response to an odor if the punishment does not occur in temporal coincidence with it.
Conversely, no association-induced changes were observable in MBONs of the remaining γ-lobe compartments (Fig. 4). Rather, a strong decrease in odor-evoked calcium activity occurred between the pre- and post-training odor response measurements in MBONs innervating the γ3 and γ4 compartments, but both to the CS+ and CS- odor (Fig. 4a, c, e, g). The γ5-innervating MBONs represent an exception in that, in most cases, they showed no responses to odors throughout experiments (Fig. 4g). In MBONs innervating the γ2 compartment, this effect was not statistically significant, perhaps because of relatively weak odor-evoked calcium activity before training in this group of animals (Fig. 4a). The relatively high variability in odor-evoked calcium activity across individuals is in accordance with previous reports that suggest highly individualized, perhaps experience-dependent responsiveness in MBONs innervating the lobes. Flies that received the ‘odor only’ control procedure also displayed strong reductions in responses (Fig. 4b, d, f). Strikingly, the previously high amplitude responses in the γ3-innverating MBON are almost entirely lost through either of these protocols (Fig. 4c, d). In most cases, this adaptation is not odor-identity specific and is generalized to the third odor, 1-Oct, that is not presented during training (Supplementary Fig. 2). We conclude that this adaptation is likely caused by the prolonged odor exposure that occurs during both the conditioned (paired) and the ‘odor only’ control protocols, as adaptation is much weaker in flies that received the ‘shock only’ control procedure (Supplementary Fig. 3).
These results go beyond confirmation that the MBONs of the γ-lobe show differential naïve odor responses, and that the manner in which those odor responses are modulated by dopamine is diverse across different MBONs. Our data indicate that experience-dependent changes in odor-evoked, postsynaptic calcium activity occur in MBONs γ1 – γ4 MBONs. However, a differential modulation resulting from CS-US coincidence is restricted to the γ1 compartment. Therefore, the synaptic DAN-KC-MBON microcircuitry that mediates the CS-US association process during aversive olfactory conditioning (i.e., the memory trace in a strict sense) is confined to a single γ-lobe compartment and not distributed across different compartments.
MBON responses are indicative of whether an odor has been aversively trained or not.
Given this finding, we sought to test in an unbiased manner whether these observed changes in postsynaptic calcium responses are actually indicative of whether the odor-evoked calcium transients have been aversively trained or not. To do so, we applied a machine learning approach, and used pseudo-populations of γ1-γ5 MBON responses to train a classifier. Over multiple training sessions, the classifier was provided with the pre- or post-training odor responses of such pseudo populations of γ-lobe MBONs and was then asked to predict whether the response was from one of two conditions, CS+ or CS-. On average, the conditioned odor (CS+) was distinguished with significantly greater accuracy than any other condition (one sample Wilcoxon sign rank test against level of chance (50%), Z=16.78943, p<0.0001). (Fig. 5a) Indeed, all other experimental conditions (pre-training, ‘odor only,’ or ‘shock only’ conditions), resulted in an approximate 50% success rate (one sample Wilcoxon sign rank test against level of chance (50%), pre-training: Z= 1.7353, p=0.08269, ‘odor only’ control: Z=0.19191, p=0.84782, ‘shock only’ control: Z=1.15577, p=0.24778), indicating that the odor representations that we observed at the MBON postsynapse are in fact indicators of whether the odor has been trained as aversive or not. We then compared the accuracy with which the classifier could distinguish between CS- and CS+ odors after training in a situation in which one of the five γ-lobe MBON types was removed from the training data sets. Only removing the MBON innervating the γ1 compartment decreased the accuracy of differentiating CS+ from CS- to the level of chance (Fig. 5b) (one sample Wilcoxon signed rank test against level of chance (50%), Z= 1.77029, p= 0.07668). Excluding any other MBON type did not significantly affect the accuracy of discriminability (Fig. 5b) (one sample Wilcoxon signed rank test against level of chance (50%), γ2 excluded: Z=16.39812, p<0.0001, γ3 excluded: Z=9.87016, p<0.0001, γ4 excluded: Z=10.72866, p<0.0001, γ5 excluded: Z=13.54734, p<0.0001), corroborating the finding of the γ1 compartment as the site of differential synaptic plasticity underlying aversive discrimination learning.