A correlative approach to analyze the ultrastructure of identified olfactory glomeruli

A-B: Two-photon laser scans of the antennal lobes in Orco-Gal4; UAS-GCaMP6s flies where Orco-positive olfactory sensory neurons (OSNs) in the glomerular neuropils 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 glomeruli of interest were identified, glomerular borders were marked with 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 opposed by 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: Example FIB-SEM images (left column), volumetric neuronal reconstructions (middle column), and skeleton-based neuron traces (right column) of a representative example of each neuron class: OSNs (green), uniglomerular projection neurons (uPNs, red) and multiglomerular neurons (MGNs, blue). The ultrastructure of neurons, including T-bars (black triangle), mitochondria (asterisks) and spinules (white triangle) are indicated. Exemplar volumetric reconstructions (middle column) show the general morphology of each neuron class. Presynapses and postsynapses are indicated with red and cyan dots on the skeleton traces (right column). B: Average branching intensity (branching points per µm of neuronal-fiber length) of 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 in OSNs or MGNs between glomeruli were found (significance was not tested for uPNs due to the presence of a single uPN in DL5). C: Schematic summary indicating, for each glomerulus, its volume (in µm3), the number of neurons of each class (MGNs were not counted), the total fiber length of all neurons for each neuron class and the total number of single synaptic contacts for each glomerulus.

Glomerular innervation and synaptic composition

Quantitative neuronal data comparing glomeruli DA2 and DL5, detailing glomerular innervation and synaptic properties for each neuronal class: OSNs (green), uPNs (red) and MGNs (blue) and the sum of all of them. Row 1: Total length of all neurons of each neuron class and total length for all neurons in each glomerulus. Row 2-4: Synaptic counts: input sites (inputs), output sites (outputs) and T-bars (T-bars). Row 5: Innervation density: total neuron length (µm; row 1)/glomerular volume (µm3); glomerular volume: DA2=1500 µm3 and DL5=2600 µm3 (see Figure 1C). Row 6-8: Total synaptic density per unit of glomerular volume (µm3): sum of all input sites (inputs), output sites (outputs) and T-bars of each neuron class or of all neurons/glomerular volume. Row 9-11: Average synaptic density along neuronal fibers (illustrated also in Figure 3 – supplement 1): number of inputs, outputs or T-bars/neuron length (µm). Row 12-13: Average synaptic ratios: the ratio of 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 that have a relative difference greater than 20% (see relative differences Table S1) between DA2 and DL5. Dark shades highlights values that are greater in DA2 than in DL5 (green (OSNs), red (uPNs), blue (MGNs)) and light colors highlight values that are less in DA2 than in DL5.

Innervation density and synaptic density in DA2 and DL5

A-E: The average glomerular innervation density of OSNs (A), uPNs (B), MGNs (C) and collectively of all glomerular neurons (D); the average synaptic density of input sites (inputs), output sites (outputs) and T-bars and the average polyadicity. Innervation density: 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 of each neuronal fiber normalized to one µm3 of glomerular volume. Polyadicity: average number of single output sites per T-bar in 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 (red) 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 represent mean + standard deviation (error bars). Data points represent single values. Means were compared using either Student’s t-test (OSNs) or Wilcoxon two-sample test (MGNs and all neurons). uPNs were not compared, since the DL5 has only one. 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: presynapses; blue dots: postsynapses. C: Boxplots showing the fraction of synaptic output to uPNs (in red), - to OSNs (in green) or - toMGNs (in blue), for the ipsilateral OSNs (dark green boxplot) and contralateral OSNs (light green), respectively, in the DA2, DL5 and VA1v glomeruli (Horne et al., 2018). 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.

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

A: Schematic representation of principal connection motifs between the neuron classes OSNs (green), uPNs (red) and MGNs (blue). The synaptic flow directed towards uPNs is a feedforward and that directed towards OSNs or from uPNs to MGNs defined as a feedback connection (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 contacts 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 (red), 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 (red), and MGNs (blue) in glomeruli DA2, DL5 and VA1v. The number of neurons of each class or truncated neuronal fibers (in brackets) is noted in the corresponding circle. B: Schematics of connection motifs (left) that are jointly stronger or weaker in DA2 and VA1v than in DL5. The relative differences (as percentage) between DA2 and DL5 as well as VA1v and DL5 are illustrated as arrows up (stronger) or arrows down (weaker) according to their intensity (see legend at the bottom) from the perspective of the target glomerulus (defined in the table header). The values of relative differences are listed in the 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 sites (cyan dots) mapped in a dendrogram of the dendrite of the single uPN in glomerulus DL5. The basal root node (black dot) represents the entry site of the uPN dendrite into the glomerulus (closest point to its soma). Clustering of autaptic input sites along some branches are encircled. B: Simplified representation of the uPN’s dendrogram illustrating the distinct strahler orders, at distal branches (1-4) and at basal branches (5-8); see legend on the right). C: Distribution of autaptic presynaptic (left) and postsynaptic input sites (right) along the dendrite, 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 distribution of presynaptic sites (triangles) and postsynaptic sites (circles) of selected autapses (indicated by same color). Distant pairs of pre- and postsynapses (long geodesic distance) are indicated by numbers whereas closely attached synaptic sites (short geodesic distance) are encircled and labelled with letters. E: Schematic of the dendrogram illustrating the location of the presynaptic (red dot) and postsynaptic (cyan dot) sites of a single autapse, the geodesic distance between them, i.e. the distance along the dendrite (µm), and the number of branching points (orange dots) between the pre- and postsynaptic components of the same autapse. F: Number of autapses with distinct geodesic distances between their pre- and postsynapses (illustrated in E). G: Number of autapses with the number of branch points between their pre- and postsynapses counted along the uPN dendrite (illustrated in E).

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 for all neurons within glomeruli DA2 (dark colors) and DL5 (light colors). DA2: OSNs (green) n= 44; uPNs (red) 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 for total neuronal-fiber length and synaptic density (inputs, outputs, T-bars per unit of neuronal fiber length) of 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 DA2, DL5 and VA1v. Three comparisons are shown: DA2 compared with DL5 (top table), VA1v with DL5 (middle) and VA1v with DA2 (bottom). The relative synaptic strength (rel syn strength) of each connection type is listed on the left side and the relative differences (rel differences) are listed on the right side.

Connectivity of single neurons in DA2

(see extra file)

Connectivity of single neurons in DL5

(see extra file)

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 skeleton trace with artificial thickness according to Strahler order. Autapses are shown as cyan dots. The entry site of the uPN main dendrite into the glomerulus (point closest to the soma) is the basal root node. B: Number of autaptic presynaptic (red) and postsynaptic sites (cyan) according to their geodesic distance to the basal root node point (indicated with a black circle in A). C: Proportional distribution of all presynapses and postsynapses (excluding autaptic connections) in the DL5 uPN at each strahler order (see legend inset). Note the high proportion of postsynaptic sites on most distal dendritic branches.