A correlative approach to analyze the ultrastructure of identified olfactory glomeruli

A-B: Two-photon laser scans of the antennal lobe in Orco-Gal4; UAS-GCaMP6s flies where Orco-positive olfactory sensory neurons (OSNs) in the glomerular neuropil were labeled by GCaMP (green fluorescence). Glomeruli DA2 (A) and DL5 (B) are encircled. Schematics show their relative position in the antennal lobe. Once the glomerulus of interest was identified, its boundaries were delineated using fiducial marks (white triangles) via laser branding, which enabled their identification at the ultrastructural level. C-D: Representative images of the same glomeruli (DA2 in C and DL5 in D) obtained with focused-ion-beam electron microscopy (FIB-SEM), showing their ultrastructure. Asterisks indicate the main neurite of uniglomerular projection neurons entering the glomerulus. White triangle shows a 2-photon laser mark (see also A and B). E: FIB-SEM image of a polyadic synapse: the presynaptic site (red arrowhead) is composed of a T-bar shaped presynaptic density, surrounded by small vesicles, and is apposed to several postsynaptic profiles (cyan dots). Scheme of a tetrad synapse: a presynaptic site with its T-bar (red arrowhead) forms four output connections (arrows) with four postsynaptic input sites (cyan dots). F: A skeleton-based reconstruction of an OSN axon terminal (green line) with presynaptic (red dots) and postsynaptic sites (cyan dots). The dark grey shading surrounding the OSN trace represents the volume-based reconstruction of the same neuron. Tracing and reconstruction were performed within the FIB-SEM dataset (light grey area).

Neuron classification and neuronal composition of the DA2 and DL5 glomeruli

A: Representative examples of each neuron class in glomeruli DA2 and DL5. Shown are FIB-SEM images (left column), volumetric neuronal reconstructions (middle column), and skeleton-based neuron traces (right column) for olfactory sensory neurons (OSNs, green), uniglomerular projection neurons (uPNs, magenta), and multiglomerular neurons (MGNs, blue). Key ultrastructural features including T-bars (black triangle), mitochondria (asterisks) and spinules (white triangle) are indicated on the FIB-SEM images. Volumetric reconstructions (middle column) depict the general morphology of each neuron class. On the skeleton traces, presynaptic and postsynaptic sites are marked with red and cyan dots, respectively (right column). B: Average branching intensity (branching points per µm of neuronal-fiber length) for each neuron class OSNs, uPNs and MGNs in DA2 and DL5. Data represent mean+ standard deviation (error bars). Data points represent single values. Means were compared using Wilcoxon two-sample test. No significant differences of branching points/µm were observed for OSNs or MGNs between glomeruli (significance was not tested for uPNs due to the presence of a single uPN in DL5). C: Schematic summary, for each glomerulus, showing its volume (in µm3), the number of neurons per class (excluding MGNs), the total fiber length of all neurons for each neuron class and the total number of individual synaptic contacts.

Glomerular innervation and synaptic composition

Quantitative comparison of neuronal and synaptic parameters between glomeruli DA2 and DL5 for each neuron class-OSNs (green), uPNs (magenta) and MGNs (blue) – as well as the combined totals. Row 1: Total length of all neurons per neuron class and the overall total length for all neurons for each glomerulus. Row 2-4: Synapse counts: number of input sites (inputs), output sites (outputs) and T-bars. Row 5: Innervation density: calculated as total neuron length (µm; row 1)/glomerular volume (µm3); glomerular volumes: DA2=1500 µm3 and DL5=2600 µm3 (see Figure 1C). Row 6-8: Synaptic density per unit glomerular volume (µm3): total number of all input sites (inputs), output sites (outputs) and T-bars for each neuron class and all neurons divided by glomerular volume. Row 9-11: Average synaptic density along neuronal fibers (see also Figure 3 – supplement 1): number of inputs, outputs or T-bars per µm of neuron length. Row 12-13: Average synaptic ratios: T-bars-to-inputs or outputs-to-inputs. Row 14: Polyadicity: the average number of postsynaptic sites at each T-bar in DA2 and DL5. The ratios in rows 12-14 were calculated based on synaptic counts normalized to neuron length (rows 9-11). The color shading highlights values with a relative difference greater than 20% between DA2 and DL5 (see relative differences Table S1). Dark shades highlight values greater in DA2 than DL5 (green = OSNs), magenta = uPNs), blue = MGNs); light shades indicate values lower in DA2 than in DL5.

Innervation density and synaptic density in DA2 and DL5

A-E: Quantification of average glomerular innervation density of OSNs (A), uPNs (B), MGNs (C) and all glomerular neurons combined (D); and synaptic density of input sites (inputs), output sites (outputs), T-bars and average polyadicity. Innervation density: total length (µm) of each neuronal fiber normalized to one µm3 of glomerular (glom.) volume. Synaptic density: number of input sites, output sites, or T-bars for each neuronal fiber normalized to one µm3 of glomerular volume. Polyadicity: average number of single output sites per T-bar for each neuronal fiber. Data for DA2 shown in dark colors and for DL5 in light colors. Number of neurons in DA2: OSNs (green) n= 44; uPNs (magenta) n= 7; MGNs (blue) n=180; all neurons n=231, in DL5: OSNs n=46; uPN n=1; MGNs n=221; all neurons n=268. Data are presented as mean with standard deviation (error bars). Data points represent single neuron values. Means were compared using either Student’s t-test (OSNs) or Wilcoxon two-sample test (MGNs and all neurons). uPNs were not compared to the single uPN of th DL5 glomerulus. Significance value: p>0.05 (not significant, no star), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***). Values are provided at data availability; polyadicity values are listed in Table 1, row 14.

Lateralization of OSN terminals in the antennal lobes

A: Illustration of an ipsilateral (dark green) and a contralateral (light green) OSN with dendrites in the corresponding antennae and their axonal projections to the ipsilateral olfactory glomerulus in the antennal lobe (AL) (dashed rectangle). B: Exemplary skeleton traces of an ipsilateral (dark green) and a contralateral (light green) OSN terminal inside glomerulus DA2. The ipsilateral OSN axons reach the glomerulus via the ipsilateral antennal nerve (arrow down) and leave the glomerulus towards the AL commissure (arrow up) while OSN axons originating at the contralateral antenna reach the glomerulus via the AL commissure. Red dots: presynaptic sites; blue dots: postsynaptic sites. C: Boxplots showing the fraction of synaptic output to uPNs (in magenta), - to OSNs (in green) or - to MGNs (in blue), for the ipsilateral OSNs (dark green boxplot) and contralateral OSNs (light green), respectively, in the DA2, DL5 and VA1v glomeruli. D: Boxplots showing the fraction of synaptic input of the same ipsilateral and contralateral OSNs that they receive from OSNs and MGNs. Connection polarity is indicated by arrows in the schematic neuronal drawings on the left of each plot. Dots represent single values. Means were compared using Student’s T-test. Significance value: p>0.05 (not significant, no star)), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***). Mean and Median values are provided at data availability. The data for glomerulus VA1v was extracted from (Horne et al., 2018).

Strength of synaptic connections between neuron classes in the circuitry of DA2, DL5 and VA1v.

A: Schematic representation of the principal connection motifs among the neuron classes OSNs (green), uPNs (magenta) and MGNs (blue). Synaptic connections directed towards uPNs are defined as feedforward and those directed towards OSNs or from uPNs to MGNs are classified as feedback connections (indicated by arrows). B-D: Alluvial diagrams of the glomerular circuitry in DA2 (B), DL5 (C) and VA1v (D). Each diagram shows the relative synaptic strength calculated as the proportion of 1:1 single synaptic contact between each neuron class in relation to the total number of synaptic contacts in their respective glomerulus. The synaptic strength between each neuron class, given as percentage, is indicated by the thickness of the lines. The proportions (as percentage) of output (left side) or input (right side) are illustrated by colored rectangles to the left or right of each alluvial diagram. The total number of synaptic contacts is indicated below the diagrams. Percentages of the relative synaptic strength and synaptic counts are listed in the supplementary Table S1. E: Stacked bar charts depict output (E’) and input (E’’) fractions (given as percentages) of each neuron class: OSNs (green), uPNs (magenta), MGNs (blue), schematically illustrated next to the bar charts respectively, to each of the other neuron classes for glomeruli DA2, DL5 and VA1v. Fractions are color-coded according to the neuron class of the respective connecting partner.

Differences in connectivity strength in glomeruli DA2, DL5 and VA1v

A: Schematic representation of synaptic connection motifs (arrows) between OSNs (green), uPNs (magenta), and MGNs (blue) in glomeruli DA2, DL5 and VA1v. The number of neurons of each class - or truncated neuronal fibers (indicated in brackets) - is shown within the corresponding circle. B: Diagrams of connection motifs (left) that are consistently stronger or weaker in DA2 and VA1v compared to DL5. Relative differences (expressed as percentages) between DA2 and DL5, and between VA1v and DL5 are illustrated as upward (stronger) or downward (weaker) arrows, with arrow intensity indicating the magnitude of the difference (see legend at the bottom) from the perspective of the target glomerulus (as defined in the table header). All relative differences values are provided in Table S2.

Distribution of pre- and postsynaptic partners of autapses in the uPN dendrite of the DL5

A: Distribution of autaptic presynaptic (red dots) and postsynaptic (cyan dots) sites mapped onto a dendrogram of the single uPN dendrite in glomerulus DL5. The basal root node (black dot) marks the entry site of the uPN dendrite into the glomerulus (i.e., the point closest to its soma). Clusters of autaptic input sites along specific branches are encircled. B: Simplified dendrogram of the uPN illustrating distinct strahler orders, with distal branches (orders 1-4) and at basal branches (orders 5-8); see legend on the right. C: Distribution of autaptic presynaptic (left) and postsynaptic (right) input sites along the dendrite, shown as proportions at each corresponding strahler order (color coded). Note that autaptic postsynaptic sites are located almost exclusively at the most distal dendritic branches. D: Dendrogram of the DL5-uPN showing the locations of presynaptic sites (triangles) and postsynaptic sites (circles) for selected autapses (color-matched pairs). Autapses with large geodesic distance between their components are labeled with numbers; those with short distances are encircled and labeled with letters. E: Schemes of the dendrogram illustrating the location of the presynaptic (red dot) and postsynaptic (cyan dot) sites of individual autapses, the geodesic distance between them (measured along the dendrite in µm), and the number of branching points (orange dots) separating the pre- and postsynaptic components. F: Histogram showing the number of autapses grouped by geodesic distance between their pre- and postsynaptic sites (as illustrated in E). G: the number of autapses categorized by the number of branch points between their pre- and postsynapses along the uPN dendrite (as illustrated in E).

Graphic summary illustrating genera l differences between narrowly and broadly tuned olfactory circuits.

Graphic summary of key circuit features that distinguish the two narrowly tuned olfactory glomeruli studied here (dark grey circle) from a broadly tuned on (light grey circle). Olfactory glomeruli are the first relay station where olfactory information is processed before being transmitted to higher brain centers, including the lateral horn (LH) and mushroom body calyces (MBc). The model is based on a comparative analysis of the narrowly tuned circuits of DA2 and VA1v, and the broadly tuned circuit of DL5. Circuit components include uniglomerular projection neurons (uPNs, magenta), local interneurons (LNs, blue), and olfactory sensory neurons (OSNs, green) from both ipsilateral and contralateral brain hemispheres. In the narrowly tuned circuits, multiple uPNs are present, whereas in the broadly tuned circuit LNs are more numerous, as indicated by the number of circles. Connectivity strength between neuronal types is inferred from synaptic counts (1:1 presynaptic-postsynaptic sites) and is represented by the size of the connecting triangles. Differences in neuron number are indicated by the number of circles. In narrowly tuned circuits, OSNs exhibit stronger synaptic output (1) and form stronger reciprocal connections with sister OSNs (2) and uPNs (3). The lateralization of OSN connectivity is reduced compared to broadly tuned circuits, where ipsilateral output to uPNs and contralateral output to the LNs dominates (4). Feedback synapses from uPNs to LNs (5) and from LNs to OSNs (6) are weaker in narrowly tuned circuits. In contrast, the broadly tuned circuit DL5 exhibits weaker OSN output and stronger lateralization of OSN inputs. Additionally, autapses are observed in the single uPN of DL5, whereas reciprocal uPN connections are a feature of narrowly tuned circuits. The model attributes to LNs the features quantified for a larger group of neurons (multiglomerular neurons, MGNs), of which the great majority are LNs, as discussed in the main text.

Neuronal volume and polyadicity

A: Ratio between neuronal fiber volume and length in glomerulus DA2. Data represent mean + standard deviation (error bars) (OSNs n=30; uPNs n =5; MGNs n = 15). B-D: Frequency of T-bars associated with a number of postsynaptic contacts (Polyadicity) in OSNs (B), uPNs (C) and MGNs (D) in DA2 (dark shade) and DL5 (light shade)

Relative differences of innervation and synaptic composition between glomeruli DA2 and DL5

The table lists the relative differences between DA2 and DL5 (see Methods for calculations). Values that are at least 20% greater in DA2 than in DL5 are highlighted in dark shades and values that are at least 20% less in DA2 than in DL5 are highlighted in light shades.

Synaptic density along neuronal fibers in DA2 and DL5

Counts of synaptic inputs, synaptic outputs and T-bars normalized to 1 µm of neuronal length along OSN, uPN or MGN fibers and collectively sampled for all neurons within the glomeruli DA2 (dark colors) and DL5 (light colors). DA2: OSNs (green) n= 44; uPNs (magenta) n= 7; MGNs (blue) n=180; all neurons n=231. DL5: OSNs n=46; uPN n=1; MGNs n=221; all neurons n=268. Data represent mean + standard deviation (error bars). Data points represent single values. Means are compared using either Student’s T-test (in OSNs) or Wilcoxon two-sample test (in MGNs and all neurons). The uPNs of the DA2 are not compared to the single uPN of the DL5. Significance value: p>0.05 (not significant, no star), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***). Values are listed in Table 1, row 9-11.

Properties of ipsi- and contralateral OSNs.

A: Boxplots depicting the total neuronal-fiber length and synaptic density (measured as the number of inputs, outputs, T-bars per unit of neuronal fiber length) for ipsilateral (dark green) and contralateral OSN terminals (light green). Dots represent single values. Means were compared using Student’s T-test. Significance value: p≤0.05 (*), p≤0.01 (**), p≤0.001 (***).

Synaptic connectivity and relative differences between DA2, DL5 and VA1v

Synapse counts and synaptic strength of each connection type in the DA2, DL5 and VA1v glomerulus. Three pairwise comparisons are shown: DA2 vs. DL5 (top table), VA1v vs. DL5 (middle table), and VA1v vs. DA2 (bottom table). The relative synaptic strength (rel syn strength) of each connection type (listed on the left) and the relative differences (rel differences) (listed on the right) are color coded.

Connectivity of single neurons in DA2

Connectivity of single neurons in DL5

Distribution of synapses and autapses along the DL5 uPN dendrite in DL5

A: 3D-reconstruction of the dendritic tree of the single uPN in glomerulus DL5, shown as a skeleton trace with artificial thickness scaled according to Strahler order. Autapses are indicated as cyan dots. The entry site of the uPN main dendrite into the glomerulus (the point closest to the soma) is defined as the basal root node. B: Number of autaptic presynaptic (magenta) and postsynaptic (cyan) sites plotted against their geodesic distance from the basal root node (indicated by a black circle in A). C: Proportional distribution of all presynaptic and postsynaptic sies (excluding autaptic connections) across Strahler orders in the DL5 uPN dendritic tree(see legend inset). Note the high proportion of postsynaptic sites located on the most distal dendritic branches.