Figure 6.

PD2a1 and PD2b1 Receive Input from Appetitive PNs and Are Broadly Tuned

(A) Summary heatmap of antennal lobe glomeruli with uniglomerular, excitatory PN connectivity to individual PD2a1 and PD2b1 neurons as determined by EM reconstruction. The connectivity heatmap is separated by neuropil location: PD2a1 and PD2b1 LH dendrites, PD2b1 MB calyx dendrites and total across all PD2a1 and PD2b1 dendrites. Cell numbers represent the number of synapses, and heatmap coloring represents the synapse count normalized by the total number of postsynapses in that neuropil. Uniglomerular PNs with no connectivity are not shown. Uniglomerular PNs from connected glomeruli or MBON-ɑ2sc are ordered by connection strength. PN names are colored by their behavioral significance based on published studies.

(A’) The number of synaptic inputs for all PD2a1 and PD2b1 dendrites traced in this study. Input is either undefined (gray), uniglomerular PN (oranges), or MBON-ɑ2sc (purple).

(B) Reconstruction of all presynaptic partners to PD2a1#1 in the EM volume. Shown is the PD2a1#1 EM-reconstructed skeleton with dendritic postsynapses highlighted in blue.

(B’) Right: stacked bar chart showing the percentages of postsynapses contributed by different types of input neurons (different colors). Left: histogram showing the number of upstream postsynpatic partners against their synaptic weight (number of synapses onto PD2a1#1). The gray box highlights that 50% of PD2a1#1’s postsynapses are spent on neurons that only input PD2a1#1 by less than 10 synaptic connections. MBON-ɑ2sc is indicated by purple arrowheads.

(C) Electrophysiological recording raster plot from a representative PD2a1 neuron. The responses of each cell to the different odors are stacked, black squares represent action potentials, and there are 4 presentations of each odor. The red block represents the odor stimulation period.

(C’) Tuning curve of PD2a1 and PD2b1 neurons. Responses are shown in hertz. Data are mean ± SEM; n = 7 cells, consisting of one PD2b1, one PD2a1 or PD2b1, and five PD2a1 neurons. Odors in the text are shown in cyan.

(D) Schematic for imaging experiments with MBON-ɑ2sc silencing. Flies express Shi^ts in MBON-ɑ2sc and GCaMP3 in PD2a1 and PD2b1 neurons for calcium imaging. At the permissive temperature (left), there is no effect on MBON-ɑ2sc neurotransmission, and PD2a1 and PD2b1 neurons receive input from both MBON-ɑ2sc and directly from the antennal lobe. MBON-ɑ2sc is silenced at the restrictive temperature (right), although the PD2a1 and PD2b1 neurons still receive input from the antennal lobe.

(E) Response of PD2a1 and PD2b1 axons to Oct with or without MBON-ɑ2sc silencing. Left: time traces of normalized GCaMP3 fluorescence (STAR Methods) are shown at permissive (blue) and restrictive (red) temperature in response to Oct stimulation (light blue bar). Right: the integral of the absolute odor responses for each fly at the permissive (blue) and restrictive (red) temperatures are plotted, which revealed decreased response to Oct after MBON-ɑ2sc silencing (n = 6, paired t test = 0.044).

(F) Response of PD2a1 and PD2b1 axons to Mch with or without MBON-ɑ2sc silencing. The layout of the data is the same as in (E). This revealed a decreased response to Mch after MBON-ɑ2sc silencing (n = 6, paired t test = 0.0015).

(G) Response of PD2a1 and PD2b1 axons to vinegar with or without MBON-ɑ2sc silencing. The layout of the data is the same as in (E). There was no change in response to vinegar after MBON-ɑ2sc silencing (n = 6, paired t test = 0.67).

(H) Response of PD2a1 and PD2b1 axons to ethyl acetate with or without MBON-ɑ2sc silencing. The layout of the data is the same as in (E). This revealed a decreased response to ethyl acetate after MBON-ɑ2sc silencing (n = 6, paired t test = 0.039).

(I) Response of PD2a1 and PD2b1 axons to isoamyl acetate with or without MBON-ɑ2sc silencing. This revealed a decreased response to isoamyl acetate after MBON-ɑ2sc silencing (n = 6, paired t test = 0.012).

^∗p < 0.05, ^∗∗p < 0.01. See also Figure S10.