Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain

Abstract

Animals exhibit innate and learned preferences for temperature and humidity-conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior.

Keywords: Drosophila; antennal lobe; circadian clock; connectomics; hygrosensation; lateral accessory calyx; lateral horn; mushroom body; projection neuron; thermosensation.

Graphical abstract

Figure 1

Sensory Neurons Define Seven Distinct Glomeruli in the Ventroposterior Antennal Lobe (A) Frontal view illustration of the VP AL summarizing the sensory modalities, locations, and numbers of thermo- and hygrosensory neurons reported in previous studies. D: dorsal; L: lateral; M: medial; V: ventral. (B and B’) Glomerular meshes generated from sensory neurons reconstructed in the right hemisphere of FAFB. Dashed circle labeled ANT: right antennal nerve. (B) Posterior meshes: VP2, VP3, VP4, and VP5. (B’) Anterior meshes: VP1d, VP1l, and VP1m. (C–I) ALs with glomerular meshes enclosing reconstructions of individual VP sensory neurons (in black, with presynaptic sites in red and postsynaptic sites in cyan, and number of neurons indicated in parentheses). Dashed line: the midline. (C) VP1d, (D) VP1l, (E) VP1m, (F) VP2, (G) VP3, (H) VP4, (I) VP5. (J–L) Frontal views of receptor expression data in the AL. White dashed line: the midline. (J and J’) VP1m and VP5 sensory neurons express Ir68a (green) but not Ir40a (magenta). (K and K’) VP1l and VP3 sensory neurons express Ir21a (green) but not Ir40a (magenta). (L) VP2 sensory neurons express Gr28b.d (magenta), but Ir21a+ VP1l sensory neurons (green) do not. (M) Chamber I and II sensory neurons express Ir68a (green). Inserts show bright field overlay with GFP of selected frames. (N) Chamber I sensory neurons express Ir21a (green). Inserts show bright field overlay with GFP of selected frames. Arrowhead: Ir21a+ neurons in the arista. (O) Dorsal view of VP4 RNs. Arrows: distinct axon bundles from each antennal nerve. (P) A model of the organization of thermo- and hygrosensory neurons in the sacculus and arista, with the specific receptors they express and the VP AL glomeruli they innervate. See also Figures S1 and S2.

Figure 2

Projection Neurons Relay Information from Individual VP Glomeruli to Higher Brain Centers (A) Frontal view of all VP PNs, color-coded by primary glomerulus. (B) Summary diagram of the tracts and neuroblast lineages of VP PNs. (C) Summary diagram of the target brain neuropils of VP PNs, color-coded to correspond with all other figure panels. AL: antennal lobe; AMMC: antennal mechanosensory and motor center; CA: mushroom body calyx; ICL: inferior clamp; IPS: inferior posterior slope; lACA: lateral accessory calyx; LH: lateral horn; PLP: posterior lateral protocerebrum; PVLP: posterior ventral protocerebrum; SCL: superior clamp; SEZ: subesophageal zone; SLP: superior lateral protocerebrum; SMP: superior medial protocerebrum; WED: wedge. (D) Morphological hierarchical clustering based on NBLAST scores for VP PNs in each hemisphere, cut at height 0.8 (dashed line), which groups the PNs by type. Dark cyan: RHS. Dark red: LHS. Square brackets: unique VP PNs matched across hemispheres. For each type with multiple examples, numbers (#1, etc.) reflect order of appearance in the SKID_R or SKID_L columns in Table 1. (E–Q’) Frontal views of reconstructions (black) of candidate thermo- and hygrosensory PNs that innervate one primary glomerulus (color-coded as in Figures 1B and 1B’, non-VP glomeruli in gray). Presynaptic sites in red and postsynaptic sites in cyan. Neuropils color-coded as in (C). Asterisks: primarily olfactory PNs; black outline: candidate novel thermosensory PNs; dashed outline: PNs previously described but not recognized as VP or thermosensory. (E) VP1d il2PN, (E') VP1d il2PN, (F) VP1l+ lvPN1#1, (G) VP2+ adPN, (H) VP3+ l2PN1#1, (I) VP4 vPN, (J) VP4+ vPN, (J') VP4+ vPN, (K) VP5 l2PN, (L) VP1m l2PN, (M) VP2 l2PN, (N) VC5+ adPN*, (O) VP2 adPN, (P) VP3 vPN, (Q) VP3+ l2PN2#1, (Q') VP3+ l2PN2#1.

Figure 3

Projection Neurons Relay Information from Multiple VP Glomeruli and Other Modalities to Higher Brain Centers (A–F’’’) Frontal views of reconstructions (black) of candidate thermo- and hygrosensory PNs that innervate more than one VP glomerulus. (A) VP1m+VP5 ilPN, (A') VP1m+VP5 ilPN, (B) VP1l+VP3 ilPN, (B') VP1l+VP3 ilPN, (C) VP3+VP1l ivPN, (C') VP3+VP1l ivPN, (D) VP1d+VP4 l2PN1, (D') VP1d+VP4 l2PN1, (E) VP1d+VP4 l2PN2, (F) VP1m+VP2 lvPN1#1, (F') VP1m+VP2 lvPN1#2, (F'') VP1m+VP2 lvPN1#3. (G and H) Proportion of inputs of each type (G) and best fit line to linear regression model plotted in R with the lm() function showing correlation of cable length in each glomerulus with RN input for two RHS multiglomerular VP PNs, VP1m+VP2 lvPN1#1 and VP1l+VP3 ilPN (H). (I–L) Frontal views of reconstructions (black) of candidate thermo- and hygrosensory PNs that innervate the subesophageal ganglion (red) as well as VP glomeruli. (I) VP1m+VP2 lvPN2, (J) VP1m+ lvPN, (K) VP2+SEZ lvPN1#1, (L) VP5+SEZ adPN. (M–O’) Frontal views of reconstructions (black) of candidate thermo- and hygrosensory PNs that innervate non-VP glomeruli. Presynaptic sites in red and postsynaptic sites in cyan. Brain neuropils color-coded as in Figure 2C. VP glomeruli color-coded as in Figures 1B and 1B’; non-VP glomeruli in gray. Black outline: candidate novel thermosensory PNs; dashed outline: PNs previously described but not recognized as VP or thermosensory, or associated with the wrong glomeruli. (M) VP4+VP2+VL1 l2PN, (M') VP4+VP2+VL1 l2PN, (N) VP2+VC5 l2PN, (N') VP2+VC5 l2PN, (O) VC5++ l2PN1#1•, (O') VC5++ l2PN1#2*.

Figure 4

Both Dedicated and Mixed Modality Kenyon Cells Receive Thermosensory Input from the Lateral Accessory Calyx (A and A’) Frontal (A) and dorsal (A’) views of VP PN presynapses (red) versus olfactory PN presynapses (gray). CA: mushroom body main calyx; LH: lateral horn; PLP: posterior lateral protocerebrum; PS: posterior slope. (B–D) Workflow for randomized downstream sampling from key VP PNs in the lACA. (B) Frontal view of reconstructed VP PNs used for sampling from the lACA in the right hemisphere of FAFB. (C) Annotation of presynapses and associated postsynapses from these two VP PNs in the lACA. Orange: presynapse; cyan arrow: edge to associated postsynapse. (D) Frontal view of an example target neuron reconstructed during the sampling process. (E) Partial circuit diagram illustrating connectivity between lACA-associated KCs receiving at least five inputs in the lACA and their VP and olfactory inputs. Circular nodes represent individual neurons; hexagonal nodes represent pooled neurons, with the number pooled after the hashtag. Synapse numbers correlate with line thicknesses. lACA: PNs targeting KCs in the lACA; VP3: VP3 vPN; VP2: VP2 adPN; 1m2: VP1m+VP2 lvPN1#1-2; VC5: VC5+ adPN; 1m5: VP1m+VP5 ilPN; CA: olfactory PNs (O), lACA PNs (LP), and other VP PNs (VP) targeting KC dendrites in the main calyx. (F–J) Frontal views of lACA-associated KCs. Green: whole mushroom body; purple: dACA (J only). (F) α: KCα'β', (G) γ: KCγ, (H) t: tKCα'β', (I) s: KCγ-s2, (J) KCγ-s1. (K) lACA-associated KCs (black) derive from one (green) of four mushroom body neuroblast lineages (green/cyan/red/violet). (L) Expected (circles) versus observed (bars) numbers of lACA KC dendritic claws receiving input from olfactory and VP PN partners in the CA (color-coded by valence, key on right). PN inputs labeled as aversive (red), attractive (green), or unknown (black). (M) Summary of expected (circles) versus observed (bars) numbers of lACA KC dendritic claws receiving input of various valences (key on right) from olfactory and VP PN partners in the CA. See also Figure S5 and Table S1.

Figure 5

The Lateral Accessory Calyx Is a Thermo- and Hygrosensory Hub with Outputs to Multiple Neuropils (A–K, except D’’) Frontal views of reconstructions (black) of non-KC lACA target neurons. Brain neuropils have been color-coded to correspond with all other figure panels. (A) All identified targets with at least five connections to lACA PNs within the lACA. (B–K) Strong downstream targets of lACA PNs. Presynaptic sites in red and postsynaptic sites in cyan. (B) LHAV3q1#1, (C) AV3r1#1, (D) DN1a#1, (D') DN1a#2, (E) DN1-l, (F) LHPV4c1#1, (F') LHPV4c1#2, (G) LHAV3f1#1, (H) PD5g1#1, (I) LHPD5a1#1, (J) LHAV2d1#1, (K) PV6r1#1. AME: accessory medulla. (D’’) Gal4 driver line labeling DN1a neurons (source: Fly Light). (L–M) Circuit diagrams depicting (L) within-lACA and (M) total connectivity between lACA PNs and their strong downstream targets. Letters correspond to those of panels in this figure (except for elements carried over from Figure 3 and dark red VP2: VP2+ adPN). Circular nodes represent individual neurons; hexagonal nodes represent pooled neurons, with the number pooled after the hashtag. Synapse numbers correlate with line thicknesses, with dashed arrows in (M) representing inputs from (L) of <10 connections. CA PNs: olfactory PNs (O) and VP PNs (VP) targeting KC dendrites in the main calyx. lACA output neurons: neurons receiving input from lACA PNs that have <10 outputs to other lACA circuit neurons. See also Figure S6 and Table S2.

Figure 6

VP PNs Relay Information to Diverse Targets, Including a Descending Neuron (A–F) Frontal views of selected strong downstream targets of RHS and bilaterally symmetric VP PNs, with brain neuropils color coded as in Figure 2C. Reconstructed neurons in black, with presynaptic sites in red and postsynaptic sites in cyan. (A) Seventeen strong downstream targets of VP PNs. (B) Local LH neuron LHPV6g1#1. (C) VM1++ l2PN. (D) VP1m++ smPN^∗. (E) LH and SEZ interneuron LHPV10c1#1. (F) Descending neuron DNp44. (G) Heatmap showing contributions by VP PN classes to 17 downstream targets, normalized with total VP PN input = 1. Cell numbers denote raw connectivity. Heatmap is thresholded >2 synapses. Source labels are color coded by top glomerulus. See also Table S3. (H) Summary stacked bar graph showing proportion of inputs from VP, VP + SEZ, gustatory, olfactory, and visual PNs, and lACA target neurons onto each of the 17 targets, normalized with total identified inputs = 1. Total inputs and total identified inputs are shown on the right. For both (G) and (H), only the dendrites of VM1++ l2PN and KCγ-s2R and the axon of VP1m++ smPN were considered. See also Table S3.