The Wiring Logic of an Identified Serotonergic Neuron That Spans Sensory Networks

Abstract

Serotonergic neurons project widely throughout the brain to modulate diverse physiological and behavioral processes. However, a single-cell resolution understanding of the connectivity of serotonergic neurons is currently lacking. Using a whole-brain EM dataset of a female Drosophila, we comprehensively determine the wiring logic of a broadly projecting serotonergic neuron (the CSDn) that spans several olfactory regions. Within the antennal lobe, the CSDn differentially innervates each glomerulus, yet surprisingly, this variability reflects a diverse set of presynaptic partners, rather than glomerulus-specific differences in synaptic output, which is predominately to local interneurons. Moreover, the CSDn has distinct connectivity relationships with specific local interneuron subtypes, suggesting that the CSDn influences distinct aspects of local network processing. Across olfactory regions, the CSDn has different patterns of connectivity, even having different connectivity with individual projection neurons that also span these regions. Whereas the CSDn targets inhibitory local neurons in the antennal lobe, the CSDn has more distributed connectivity in the LH, preferentially synapsing with principal neuron types based on transmitter content. Last, we identify individual novel synaptic partners associated with other sensory domains that provide strong, top-down input to the CSDn. Together, our study reveals the complex connectivity of serotonergic neurons, which combine the integration of local and extrinsic synaptic input in a nuanced, region-specific manner.SIGNIFICANCE STATEMENT All sensory systems receive serotonergic modulatory input. However, a comprehensive understanding of the synaptic connectivity of individual serotonergic neurons is lacking. In this study, we use a whole-brain EM microscopy dataset to comprehensively determine the wiring logic of a broadly projecting serotonergic neuron in the olfactory system of Drosophila Collectively, our study demonstrates, at a single-cell level, the complex connectivity of serotonergic neurons within their target networks, identifies specific cell classes heavily targeted for serotonergic modulation in the olfactory system, and reveals novel extrinsic neurons that provide strong input to this serotonergic system outside of the context of olfaction. Elucidating the connectivity logic of individual modulatory neurons provides a ground plan for the seemingly heterogeneous effects of modulatory systems.

Keywords: Drosophila; connectomics; olfaction; serotonin.

Figure 1.

CSDn EM reconstruction and distribution of synaptic sites. A, MultiColor FlpOut (green) highlights the arborization pattern of a single CSDn. N-Cadherin labeling delineates neuropil (blue). B, Reconstruction of the left-hand CSDn (soma in the fly's left hemisphere) from the FAFB dataset. C–F, Example images of the diversity of small clear vesicles and larger dense core-like vesicles found within the CSDn branches (blue) in the AL (C–E) and LH (F). Scale bars, 500 nm. G, The CSDn has a mix of input and output sites across most olfactory neuropil. Shading based on the input/output index of the CSDn was calculated as the # inputs/(# inputs + # outputs) where 0 = output only and 1 = input only. H, Expression of Brp-short puncta (yellow) labels CSDn active zones in the LH. GFP (cyan) labels the CSDn's arborizations and NC82 delineates neuropil (magenta). Scale bar, 50 μm. I, Heat maps of the distribution of Brp-short puncta (i.e., CSDn active zones) in the LH. Puncta density is higher in the mediodorsal and posterior regions of LH (top and bottom heat maps, respectively).

Figure 2.

Glomerulus-specific innervation by the CSDn. A, Glomeruli rank-ordered by the total number of CSDn synaptic sites. The number of CSDn presynaptic sites (red bars) and postsynaptic sites (blue bars) are correlated to total CSDn cable length in each glomerulus (black diamonds) (R² = 0.8231). B, Input/output index of the CSDn in each glomerulus is fairly consistent across glomeruli (mean = 0.4359, SEM = 0.01, coefficient of variation = 22.96%). CSDn branch length in glomeruli is highly correlated to (C) CSDn presynaptic sites (R² = 0.924, p < 0.0001) and (D) number of CSDn postsynaptic sites (R² = 0.861, p < 0.0001). E, CSDn presynaptic density (red) (# sites/nm branch length) and postsynaptic density (blue) are fairly consistent across glomeruli (coefficient of variation = 26.26% and 30.85%, respectively), although postsynaptic is more distributed (p < 0.005, Levene's test for homogeneity of variance). F, CSDn branch length per glomerulus weakly correlates to glomerular volume (R² = 0.201, p = 0.001).

Figure 3.

The CSDn has distinct connectivity across glomeruli. A, Rendering of the AL highlighting glomeruli in which CSDn synaptic partners were reconstructed. B, Percent of input from the CSDn onto all postsynaptic partners reconstructed in the 9 glomeruli. The CSDn predominantly targets LNs across all glomeruli. Glomerulus order based on rank-ordered amount of input from the CSDn to LNs. C, Percent of input to the CSDn from all presynaptic partners reconstructed in 9 glomeruli. Composition of presynaptic targets within each glomerulus is more diverse than for CSDn output. D, Synapse fractions segregate into 3 clusters in PC space, based on whether the fraction is for upstream partners (magenta) or downstream partners (green and blue) based on k-means clustering. E, The mean distance between downstream points in the PCA (D) is significantly different from the mean distance between upstream points (***, p = 0.0007, Student s t test). F, Fraction of input from the CSDn onto LNs is inversely correlated to the percent of input from the CSDn onto ORNs.

Figure 4.

Classification of LNs with which the CSDn synapses. Examples of LNs with connectivity to the CSDn are as follows: A, Dense ABAF LNs innervate ∼50 glomeruli. B, The sparse ABAF also innervates ∼50 glomeruli but has far less branchpoints compared with the dense ABAF. C, Patchy LNs' characteristic looping structure within the glomeruli that they innervate. D, vLNs have their soma ventral to the AL and are likely glutamatergic. E, AL-projecting SEZ LNs (LN_SEZ) have their somata ventral and project bilaterally to both ALs. These LNs resemble the keystone LNs reconstructed in a larval EM dataset (Berck et al., 2016). F, Distribution of KNN analyses showing the distance of the nearest neighboring branchpoints of different subclasses of LNs. Left, The distributions of the two dense ABAFs are consistent. Thus, they belong to the same morphologic class. Middle, KNN showing that sparse ABAF and dense ABAF LNs belong to two different morphologic subclasses. The patchy LN is included as an outgroup. Right, KNN distribution showing that two patchy LNs belong to the same morphologic class. Plots show distance from the first (top), third (middle), and eighth (bottom), nearest neighboring branchpoints.

Figure 5.

The CSDn has distinct connectivity with LN subpopulations. A, Percent of synaptic input from the CSDn onto LN subtypes. The CSDn provides input to most LN subtypes across all 9 glomeruli, except for LN_SEZs. Dense ABAFs and patchy LNs appear to be the main LN type the CSDn targets regardless of glomerulus identity. B, The CSDn receives far more of its LN synaptic input from patchy LNs across 9 glomeruli. C, The CSDn is overall most strongly connected to two dense ABAF LNs as well as patchy LNs. “Other LNs” include LN_SEZs, vLNs, and otherwise uncategorized LNs. #, number of neurons within a population. Synaptic connections derive from the 9 completely reconstructed glomeruli in Figure 3 and synaptic connections from other glomeruli found in the course of reconstructing the LN.

Figure 6.

CSDn connectivity with PNs in the AL and LH. EM reconstructions of PNs with connectivity to the CSDn (A) mALT uPNs, (B) mlALT mPNs, and (C) mALT mPNs. Number of synaptic connections of PN subtypes with the CSDn in the AL (D) and LH (D′). E, uPNs have varied connectivity with the CSDn across the AL and LH. Number indicates number of neurons, rather than synapse counts.

Figure 7.

CSDn connectivity with LH neurons. EM reconstruction of (A) LHONs, (B) LHLNs, and (C) LHINs that have connectivity with both CSDns. Colorization based on transmitter content (see E). D, The CSDn reciprocally connectivity to each class of LH neurons. E, Percent of input to and from the CSDns onto populations of LH neurons. The CSDns have the most connectivity to and from cholinergic LHONs followed by glutamatergic and GABAergic LHLNs. F, Connectivity of LH neuron types that are strongly connected based on transmitter content.

Figure 8.

WPN_Bs provide top-down input to the CSDn. A, WPN_Bs (light gray) provide input to the CSDn (dark gray) in the antler. Cyan represents CSDn postsynaptic site markers. B, EM reconstructions of 12 WPN_Bs that provide a top-down input to the CSDn in the antler and portions of the protocerebrum. EM reconstructions of a Tier 1 WPN_B (C; green), Tier 2 WPN_B (D; magenta), and Tier 3 WPN_B (E; blue). F, The WPN_Bs provide input to the CSDns in a three-tiered, feedforward network where the Tier 1 WPN_Bs (green) are morphologically distinct from Tier 2 (blue) and Tier 3 (magenta). G, Tier 3 WPN_Bs provide strong input to the Tier 1 and Tier 2 WPN_Bs as well as the CSDn. H, R25C01-driven expression of GFP (green) includes populations of WPN_Bs. I, MultiColor FlpOut of R25C01-GAl4 highlights the expression of the two Tier 1 WPN_Bs (blue) and a Tier 2/3 WPN_B (green, arrow). N-Cadherin delineates neuropil (magenta). J, Some WPN_Bs' somata (green; R25C01-GAL4) colocalize with ChAT (magenta) Trojan-LexA::QFAD protein trap but not vGLUT (K; magenta) Trojan-LexA::QFAD protein trap. Scale bars: A, 25 μm; H, I, 50 μm; J, K, 20 μm.

Figure 9.

Extrinsic input from a protocerebral neuron. EM reconstruction of a previously undescribed protocerebral neuron that provides strong input (at least 90 synapses) to the CSDns in the superior medial protocerebrum, superior lateral protocerebrum, and antler.

Figure 10.

Novel extrinsic input from the SIMPAL neurons. A, EM reconstruction of the four SIMPAL neurons, which provide strong input (>10 synapses) to the CSDns in food odor-associated glomeruli. B, DC1, DP1l, DP1m, VA2, VC4, and VL2p. C, Connectivity of individual SIMPAL neurons to the CSDn is predominantly nonreciprocal. D, The CSDns, dense ABAF LNs, and SIMPAL neurons form a feedback loop, suggesting that the CSDn may influence the SIMPAL neurons polysynaptically. E, The CSDn provides strong input to the ABAF LNs in the 6 food odor-associated glomeruli in which the SIMPAL neurons synapse on the CSDn.

Figure 11.

The wiring logic of the CSDn. Schematic depicting the broad connectivity of the CSDn in the AL, LH, and the superior protocerebrum (SP). Color coding for each cell type matches earlier figures. Arrow weight indicates the number of synaptic connections. Top left, In the LH, CSDn connectivity to LHNs varies with LHN transmitter content. Top right, Within the SP, the CSDns receive input from a feedforward network of WPN_Bs and from the SIMPAL neurons, which originate in the SP yet synapse on the CSDns in select AL glomeruli. Within the AL (bottom left), the majority of CSDn output is directed toward LNs, although it also synapses on the three different PN types. Whereas CSDn output is relatively uniform, input to the CSDn is glomerulus-specific (bottom right).