Extended Data Fig. 5: PFL3 neurons receive inputs from heading-sensitive neurons in the protocerebral bridge and FC2 neurons represent a columnar-neuron class with a large number of synaptic inputs to PFL3 neurons.

All data in this figure were extracted from the hemibrain connectome, neuPrint v1.2¹³. a, PFL3 neurons receive inputs from two sets of heading-sensitive neurons in the protocerebral bridge: EPG neurons (14% of all PFL3 bridge inputs) and Δ7 neurons (77% of all PFL3 bridge inputs). b, A single EPG neuron innervates one wedge of the ellipsoid body and projects to one glomerulus in the bridge (top). c, If one assumes that the ellipsoid body circle represents 360° of azimuthal space around the fly, consistent with physiological observations^2,34, then each bridge glomerulus can be assigned an angle based on the wedge in the ellipsoid body from which the EPG cells that innervate that glomerulus originate (bottom). The angles thus assigned to the bridge yield 45° azimuthal spacing between bridge glomeruli, except the inner two inner glomeruli, which are separated by only 22.5° (see ref. ⁴). d, A single Δ7 neuron receives dendritic inputs (thin neurites in image) from EPG neurons across multiple glomeruli in the protocerebral bridge and expresses axonal terminals in 2-3 bridge glomeruli, typically spaced eight glomeruli apart^4,12,31. Two axon terminals are visible in the example ∆7 cell shown. e, Based on the anatomy of ∆7 neurons, one can index the glomeruli of the bridge with angles that repeat every 8 glomeruli, creating a 45° spacing between adjacent glomeruli⁴. Given that individual ∆7 axons are offset from the peak density of their dendritic arbors by ~180°, the angular assignments to their axon terminals in specific bridge glomeruli could be expected to be ~180° offset from the EPG assignments to those glomeruli. However, because ∆7 cells are glutamatergic⁶⁷ and appear to act in sign-inverting/inhibitory fashion on most of their downstream targets, their influence is expected to be roughly aligned with that of EPG cells, with a slight offset. Therefore, the resulting ∆7 angles have a + 11.25° and –11.25° offset relative to EPG angles for the right and left bridge respectively. f, Three different ∆7 neurons. Each ∆7 cell is assigned an angle (grey arrows) based on the glomeruli in which it has its outputs using the mapping shown in e. Note that ∆7 L4R6 (middle) has outputs that are nine glomeruli apart instead of the usual eight. In this case, the cell is assigned the same angle as ∆7 L3R6 (top), since its dendritic arborization pattern across the bridge is more similar than that of ∆7 L4R5 (bottom). Likewise, ∆7 L6R4 can be assigned the same angle as ∆7 L6R3 and ∆7 L7R3 can be assigned the same angle as L7R2. g, Δ7 to PFL3 connectivity matrix. Each row represents a different ∆7 cell (42 total). Each column represents a postsynaptic PFL3 neuron (24 total, each labeled by the glomerulus or glomeruli it innervates). The heatmap depicts the total number of synapses between each ∆7-PFL3 pair. The arrows at the bottom of the heatmap are the angles assigned to each PFL3 neuron based on the angle of the ∆7 class from which it receives the most of its inputs. We used these angles as the value for H_pref in our full PFL3 neuron model. These angles are the same as one would obtain from assigning each PFL3 neuron its angle based on which bridge glomerulus it innervates and the mapping shown in e, except for the two PFL3 neurons that innervate two glomeruli (PFL3 L3/L4 and PFL3 R3/R4). Within the L4 and R4 glomeruli, these PFL3 cells receive inputs from ∆7 L4R6 and ∆7 L6R4 respectively, and are therefore assigned angles corresponding to the more inner glomeruli that they innervate. h, The top 50 cell classes with synaptic inputs to PFL3 neurons in the fan-shaped body. These cell classes constitute 94% of all PFL3 inputs in the fan-shaped body. Each bar shows the total number of synapses between a presynaptic cell type and PFL3 neurons. FC2 neurons (purple) are a population of columnar neurons composed of three subtypes: FC2A, FC2B and FC2C. Together they constitute a third of columnar-cell synapses onto PFL3 cells in the fan-shaped body. Other columnar cell classes, such hDeltaA, hDeltaI, and hDeltaM cells could also provide goal information to PFL3 neurons during menotaxis or other goal-directed behaviours. Unlike columnar neurons, tangential cells have neurites that cut across all the columns of the fan-shaped body. These cells are likely to serve a role in modulating and impacting columnar goal information to the PFL3 cells, but their anatomy makes it less likely that they communicate column-specific information independent of their interaction with columnar neurons. i, Skeletons of FC2A, FC2B and FC2C populations. j, FC2 to PFL3 connectivity matrix. Each column represents an individual PFL3 neuron, sorted by its column in the fan-shaped body (C1 to C12) and whether it innervates the left (L) or right (R) LAL. C1 is on the very left of the fan-shaped body and C12 on the very right. Each row represents an individual FC2 neuron. k, Pairwise Pearson correlation matrix between individual PFL3 neurons based on their FC2 neuron inputs. The synaptic connections from all FC2 neurons to a given PFL3 neuron are treated as a vector and the correlation between each vector is computed. This analysis highlights that left and right PFL3 neurons innervating the same column receive highly similar inputs. PFL3 neurons can be viewed as forming nine functional columns instead of twelve¹². In this view, the four PFL3 neurons innervating the anatomical columns C3 and C4 (in the 12-column numbering scheme) would form a single functional column. The same would be true for C6 and C7, and C9 and C10. The cell groupings of the 9 and 12-column schemes are illustrated by the dendrograms in the margins. One justification for the 9-column scheme is that the PFL3 neurons which would be combined to form a single functional column, and innervate the same side of the LAL, share the same angles (see Fig. 4b). However, given that PFL3 neurons innervating C6 and C7, for example, receive different FC2 inputs, physiological evidence demonstrating that these FC2 inputs are in fact functionally identical would be required, we believe, to justify merging two anatomical columns into a single functional column and employing a 9-column fan-shaped body functional scheme instead of the 12-column scheme used herein.