Most insect species, including agricultural pests and disease vectors, rely on olfaction to guide critical behaviors including foraging, feeding, mating, egg-laying, and aggression (Kim et al., 2017). Odor detection begins with the olfactory organs, the antenna and maxillary palp, which are covered with sensory hairs, or sensilla, that encapsulate the dendrites of olfactory receptor neurons (ORNs). In Drosophila melanogaster, olfactory sensilla fall into four morphological classes—basiconic, coeloconic, intermediate, and trichoid—which are distinguished by their length, size, shape, and cuticular pore arrangement (Shanbhag et al., 1999). Notably, ORNs housed in different sensillum classes have distinct dendritic branching patterns and odor tuning properties. The outer dendritic segments of basiconic and intermediate ORNs display multiple branches, whereas the outer dendrites of coeloconic and trichoid neurons are typically unbranched (Shanbhag et al., 1999). While basiconic ORNs respond to a wide range of odorants, trichoid neurons respond predominantly to pheromones, and most coeloconic neurons are selectively tuned to amines and carboxylic acids (Dweck et al., 2015; Hallem and Carlson, 2006; Lin et al., 2016; Silbering et al., 2011; van der Goes van Naters and Carlson, 2007; Yao et al., 2005). The link between sensillum class, dendritic branching pattern, and odor tuning property suggests that the morphological features of sensilla and ORNs likely influence olfactory function. However, testing this possibility requires a systematic survey of these morphological features at nanoscales for neurons whose receptors have been identified and whose odor response profiles have been characterized.

The majority of antennal sensilla house multiple ORNs. Each neuron is assigned a designation indicating the host sensillum and its relative extracellular spike amplitude among grouped neurons. Typically two and up to four ORNs are compartmentalized in a sensillum in stereotyped combinations—whereby a neuron expressing a particular receptor is always paired with a neighboring neuron that expresses another specific receptor (Benton et al., 2009; Couto et al., 2005; Fishilevich and Vosshall, 2005; Hallem and Carlson, 2006; Hallem et al., 2004). For example, the Or59b receptor is expressed in the ab2A neuron, which is located in the antennal basiconic sensillum of type 2, while the ‘A’ indicates that its spike amplitude is greater than that of the neighboring ab2B neuron, which invariably expresses the Or85a receptor (Hallem et al., 2004). Interestingly, deleting or substituting the tuning receptor for an ORN does not alter its characteristic spike amplitude (Dobritsa, van der Goes van Naters and Carlson, 2007; Hallem et al., 2004), suggesting that spike amplitude is a feature independent of receptor identity. The genetically predetermined pairing of neurons implies functional significance for this arrangement.

Indeed, ORNs housed in the same sensillum can inhibit each other by means of direct electrical interaction, termed ephaptic coupling, which can also modulate fruitfly behavior in response to odor mixtures (Su et al., 2012; Zhang et al., 2019). Strikingly, in most sensillum types, lateral inhibition is asymmetric between compartmentalized ORNs: the large-spike neuron is not only capable of exerting greater ephaptic influence but is also less susceptible to ephaptic inhibition by its small-spike neighbors. Mechanistically, this functional disparity arises from the size difference between grouped neurons. The large-spike ORN has a larger soma than its small-spike neighbor(s); this feature is translated into a smaller input resistance for the ‘A’ neuron, thus accounting for its dominance in ephaptic interaction (Zhang et al., 2019). These findings indicate a causal relationship between an ORN’s morphometric features and its circuit function. Taken together, these observations highlight the necessity and value of a systematic morphometric survey of genetically identified ORNs.

Pioneering transmission electron microscopy (TEM) studies of high-pressure frozen and freeze-substituted (i.e., cryofixed) antennal tissues have provided a high-quality morphological atlas of Drosophila ORNs (Shanbhag et al., 1999; Shanbhag et al., 2000). However, for morphometric analysis, the utility of this atlas is limited because the 3D structure and molecular identity of ORNs are undefined. Taking advantage of the CryoChem method, which we have previously developed to permit high-quality ultrastructural preservation of cryofixed and genetically labeled samples for volume EM (Tsang et al., 2018), we have acquired serial block-face scanning electron microscopy (SBEM) images of antennal tissues in which select ORNs expressed a membrane-tethered EM marker (APEX2-mCD8GFP or APEX2-ORCO) (Tsang et al., 2018; Zhang et al., 2019). In a previous study, we focused our analysis on the soma of labeled ORNs and their co-compartmentalized neuronal partners in five sensillum types (Zhang et al., 2019). Yet, in addition to the APEX2-labeled hairs, each SBEM data set contains many other sensilla, whose identity can be determined by cross-referencing the sensillum morphology and the number of its compartmentalized ORNs (Benton et al., 2009; Couto et al., 2005; Fishilevich and Vosshall, 2005; Grabe et al., 2016; Lin and Potter, 2015; Shanbhag et al., 1999, Shanbhag et al., 2000). For example, among the three large basiconic sensillum types, ab1 can be identified without genetic labeling because the sensillum houses four neurons, whereas ab2 and ab3 both house two ORNs. Moreover, within a known sensillum, the ‘A’ or ‘B’ ORN can be distinguished according to the soma size. That is, the spike amplitude ratio between grouped neurons reflects their soma size differential (Zhang et al., 2019). Thus, the cellular identities of ORNs can be determined for many unlabeled neurons in our SBEM data sets (see Table 1—source data 1 for details).

Here, we conduct a systematic morphological and morphometric analysis of identified Drosophila ORNs. By segmenting SBEM images, we reconstruct 3D structures for neurons housed in 13 of the 19 genetically defined antennal sensillum types (Benton et al., 2009; Lin and Potter, 2015), covering all four morphological classes: the basiconic (ab1–5), coeloconic (ac1–4), intermediate (ai2 and ai3), and trichoid sensilla (at1 and at4) from a total of 158 reconstructed 3D sensillum models. Furthermore, based on 122 reconstructed 3D ORN models, we perform morphometric analysis for 33 of the 40 identified antennal ORN types (Task and Lin, 2021), as well as for 4 novel neuronal types. Our high-resolution survey uncovers multiple novel features, including extracellular vacuoles in the sensillum lumen, mitochondria-enriched inner dendritic enlargement, empty sensilla which house no ORNs, and two hitherto uncharacterized basiconic sensillum types. We also show that the outer dendritic branching pattern is much more intricate than previously appreciated. The information provided here defines new questions about how the size and shape of sensory neurons influence their responses, sensitivity, and circuit function, and sets the stage for future investigations into the molecular and cellular mechanisms underlying diverse ORN morphologies.

We began with a detailed survey of the SBEM images we had previously generated using female antennal tissues (Zhang et al., 2019). Individual data sets, acquired from different flies and named on the basis of the APEX2-expressing ORNs, included numerous sensory hairs in addition to the sensilla that housed labeled neurons (Figure 1A). In agreement with the characteristic topographical distribution of sensilla on the antenna (de Bruyne et al., 2001; Grabe et al., 2016; Shanbhag et al., 1999), the four morphological sensillum classes were unevenly represented in our eight SBEM data sets (Figure 1B,C). A total of 541 sensilla were sampled from these data sets, comparable to the total number of sensilla on an antenna (Shanbhag et al., 1999). Among the whole sensilla which were sampled in their entirety, we identified 37 basiconic, 30 coeloconic, 40 intermediate, and 51 trichoid sensilla based on their distinctive morphological features (Shanbhag et al., 1999). Briefly, the cuticle of basiconic, intermediate, and trichoid sensilla is decorated with numerous pores which serve as passages for odorants. The pores of basiconic sensilla are arranged in characteristic arrays, whereas those of trichoid and intermediate sensilla appear to scatter irregularly on the cuticular surface. The trichoid and intermediate sensilla are further recognized by their cylindrical, thickened bases, termed the ‘basal drum’ (Figure 1D). In contrast, the cuticle of the coeloconic sensilla does not contain any pores but instead consists of multiple cuticular fingers (Figure 1D), between which numerous spoke channels allow odorants access to ORNs (Shanbhag et al., 1999).

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For the completely sampled sensilla, we reconstructed their 3D models and performed morphometric analysis. Within a morphological class, sensillum identity was determined by the number of enclosed neurons and by genetic labeling when this information was available. Of note, the distribution of individual sensillum types on the antenna follows a characteristic topographical pattern (de Bruyne et al., 2001; Grabe et al., 2016; Shanbhag et al., 1999). Therefore, for an unlabeled sensillum, its proximity to the labeled sensillum type may further aid its identification (see Large basiconic sensillum section below). For basiconic sensilla, we identified all three large basiconic sensilla (ab1, ab2, and ab3), one thin basiconic (ab4), and one small basiconic sensillum (ab5). Unexpectedly, we also found two novel types of basiconic sensilla: one large basiconic housing three neurons, which we termed abx(3), and the other, a small basiconic housing only one neuron, which we termed abx(1). For the other three morphological sensillum classes, all of the reported subtypes were identified, including four coeloconic, two intermediate, and three trichoid sensilla (Figure 1D).

We further measured the length, surface area, cuticular, and lumen volumes of these identified sensilla (Table 1). The coeloconic sensilla are the shortest (5–8 μm), followed by the ab5 small basiconic (8–10 μm). The intermediate, thin, and large basiconic sensilla are of similar length (10–14 μm), while the trichoid sensilla are the longest (19–24 μm) (Figure 1E). In the remainder of this article, we focus on individual sensillum types, describe their identification, and provide morphological and morphometric information for their neurons (see Source data files for detailed information).

Trichoid sensilla

T1 sensillum

Distinctly among trichoids, the T1 sensillum houses only a single neuron expressing the Or67d receptor that responds to the male fly pheromone, cVA (Couto et al., 2005; Kurtovic et al., 2007; Shanbhag et al., 1999; Ha and Smith, 2006; van der Goes van Naters and Carlson, 2007). The ORN soma is located approximately 8–16 μm beneath the antennal surface (Figure 2A and Figure 2—figure supplement 1), with an average size of 47 μm³ (Table 2). The dendrite is divided into the inner and outer segments by the ciliary constriction (Figure 2A; Shanbhag et al., 1999). Situated entirely beneath the antennal surface, the inner dendritic segment is ensheathed by the thecogen cell, which is itself enveloped by the trichogen processes and then further surrounded by the tormogen cell (Figures 2A–7 and 8). The inner dendrite exhibits a wider diameter (0.75–1.5 μm) than the outer dendrite (~0.3 μm), which extends into the sensillum lumen with a slight taper (Figure 2A) or with a markedly flattened distal segment (Figure 2—figure supplement 1A and Video 1). Although the T1 outer dendrite is believed to be typically unbranched (Shanbhag et al., 1999), we observed that among the four segmented T1 ORNs, one exhibits three branches and another six (Figure 2—figure supplement 1B,C and Video 1). In both cases, the dendrites began branching approximately halfway through the dendritic length inside the lumen, with secondary branching occurring further along distally (Figure 2—figure supplement 1B,C). The outer dendrite typically extends to the tip of the sensillum (Figure 2—figure supplement 1), but some terminate before reaching the endpoint (Figure 2A).

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Table 2

ORN identity		Soma		Inner dendrite			Outer dendrite
		Volume (μm3)	Surface area (μm2)	Volume (μm3)	Surface area (μm2)	Mitochondria number	Volume (μm3)	Surface area (μm2)	Branch number
T1 (n=4)	A (Or67d) (DA1)	47.44±0.95	79.21±2.25	5.08±0.20	31.80±2.14	NA	2.58±0.21	36.70±3.93	2.75±1.18
T2 (at4) (n=4)	A (Or47b) (VA1v)	63.42±5.41	92.70±4.54	6.50±0.20	37.62±1.42	NA	2.40±0.21	26.98±1.57	1.00±0.00
	B (Or88a) (VA1d)	34.90±1.04	65.07±1.44	4.50±0.61	29.70±2.44	NA	1.00±0.05	19.99±1.27	2.00±0.41
T3 (at4) (n=8)	A (Or47b) (VA1v)	67.10±3.75	100.19±4.09	7.28±0.46	39.04±2.84	NA	2.75±0.38	30.55±2.85	1.57±0.27
	B (Or65a) (DL3)	26.15±1.18	54.01±1.65	3.57±0.60	26.53±2.72	NA	1.02±0.33	13.46±1.64	1.25±0.25
	C (Or88a) (VA1d)	44.35±3.63	83.73±4.77	6.10±0.57	37.23±2.92	NA	1.59±0.44	23.47±2.85	1.50±0.38
ai2 (n=3)	A (Or83c) (DC3)	47.96±2.95	78.43±2.85	7.23±0.30	29.28±1.77	NA	2.92±0.17	41.85±2.44	7.67±1.76
	B (Or23a) (DA3)	30.22±2.06	62.92±0.68	3.07±0.08	19.69±1.85	NA	0.64±0.07	13.18±1.63	3.33±0.33
ai3 (n=2)	A (Or19a) (DC1)	64.05±2.45	108.63±6.88	8.78±0.84	31.71±3.96	NA	2.51±0.12	35.85±2.15	16.00±2.00
	B (Or2a) (DA4m)	43.65± 1.30	78.26±3.49	6.26±1.00	31.16±5.55	NA	1.46±0.09	29.08±2.29	15.5±0.50
	C (Or43a) (DA4l)	36.72±5.21	71.48±5.45	4.44±1.16	22.66±5.21	NA	1.03±0.14	19.08±2.29	9.00±0.00
ab1 (n=2)	A(Or42b) (DM1)	69.96±4.14	117.76±1.55	19.39±4.63	63.22±7.02	123.50±14.50	NA	NA	NA
	B (Or92a) (VA2)	60.96±4.11	98.22±3.89	21.15±0.83	70.57±11.40	81.50±21.50	NA	NA	NA
	C (Gr21a/63a) (V)	52.10±1.00	105.23±0.48	6.73±0.63	29.84±1.20	26.00±2.00	NA	NA	NA
	D (Or10a) (DL1)	28.03±1.69	61.00±1.17	2.83±0.01	19.92±1.53	9.00±3.00	NA	NA	NA
abx(3) (n=3)	A	59.20±5.98	125.35±5.38	17.49±6.17	74.81±9.04	NA	NA	NA	NA
	B	53.79±2.83	126.34±8.41	18.41±2.68	89.24±23.85	NA	NA	NA	NA
	C	43.71±0.88	109.37±13.37	5.90±0.02	43.71±3.72	NA	NA	NA	NA
ab2 (n=1)	A (Or59b) (DM4)	93.69	131.28	20.01	63.01	125	9.8	275.42	312
	B (Or85a) (DM5)	34.40	68.89	3.32	26.73	6	0.49	13.78	1
ab3 (n=5)	A (Or22a) (DM2)	50.35±2.42	98.27±3.74	13.42±1.17	65.64±5.33	75*	6.15*	144.73*	83*
	B (Or85b) (VM5d)	32.97±1.49	87.30±6.68	3.69±0.42	26.92±3.46	14*	1.95*	39.55*	19*
ab4 (n=3)	A (Or7a) (VM5v)	73.44±3.38	101.94±3.10	9.44±0.63	32.60±2.91	62.33±6.01	3.90±0.42	79.89±18.34	46.67±20.79
	B (Or56a) (DA2)	35.22±1.12	67.72±4.01	3.63±0.45	18.89±2.49	17.67±4.48	1.65±0.40	30.79±3.74	12.67±4.10
ab5 (n=4)	A (Or82a) (VA6)	31.21±1.16	61.53±3.02	3.77±0.34	19.79±2.05	NA	0.98±0.05	27.25±2.18	39.25±8.78
	B (Or47a) (DM3)	41.11±3.70	72.45±4.73	1.77±0.45	14.70±2.17	NA	0.82±0.16	23.77±4.77	44.75±12.38
abx(1) (n=3)	A	38.42*	89.88*	7.00±0.74	36.09±3.85	38.50±1.50	1.28±0.02	20.94±2.75	6.00±3.00
ac1# (n=4)	A (Rh50/Amt) (VM6)	23.20±0.55	46.98±0.92	1.38±0.08	9.96±0.87	4.75±0.48	0.62±0.04	7.95±0.43	1.00±0.00
	B (Ir92a) (VM1)	11.56±0.40	32.02±1.29	0.84±0.01	8.47±0.27	3.50±0.96	0.13±0.01	3.72±0.15	1.00±0.00
	C (Ir31a or 75d) (VL2p or VL1)	9.62±0.33	26.03±0.62	0.54±0.03	5.29±0.20	1.50±0.50	0.13±0.01	3.83±0.11	1.00±0.00
	D (Ir31a or 75d) (VL2p or VL1)	7.92±0.35	25.08±1.50	0.67±0.04	8.54±0.92	2.25±0.63	0.20±0.01	4.65±0.13	1.00±0.00
ac2 (n=3)	A (Ir75a) (DP1l)	26.69±1.48	64.46±4.81	3.08±1.00	20.41±5.89	NA	0.36±0.02	7.36±0.32	1.00±0.00
	B (Ir75d or Ir41a) (VL1 or VC5)	24.05±0.81	54.33±1.48	2.07± 0.06	14.87±0.85	NA	0.48±0.05	8.59±0.34	1.00±0.00
	C (Ir75d or Ir41a) (VL1 or VC5)	15.42±0.58	40.32±4.12	1.55±0.22	11.54±1.36	NA	0.26±0.05	6.41±0.73	1.00±0.00
ac3II (n=4)	A (Ir75c) (DL2)	41.27±5.65	99.52±6.22	3.75±0.36	26.53±1.84	NA	0.91±0.07	12.18±0.49	1.25±0.25
	B (Or35a) (VC3)	23.49±1.30	60.44±3.70	3.02±0.43	24.96±2.47	NA	0.59±0.11	11.01±1.15	3.00±0.71
ac4 (n=1)	A (Ir84a) (VL2a)	38.30	64.62	1.99	11.76	NA	0.24	5.17	1.00
	B (Ir75d) (VL1)	31.99	59.07	2.72	15.89	NA	0.47	7.44	1.00
	C (76a) (VM4)	17.92	39.60	1.62	10.65	NA	0.25	5.01	1.00

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Video 1

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Besides the dendritic branches, we observed free, extracellular membrane-bound structures within the lumen in 4 of the 19 analyzed T1 sensilla (21%) (see Table 1—source data 1 for details). These membranous structures, which we referred to as vacuoles, were predominantly found in the proximal region of the sensillum (Figure 2A-4-6). They vary considerably in size (0.2–2.0 μm in diameter) and do not appear to have a regular shape (Figure 2A). In some cases, the vacuole occupied nearly an entire segment of the lumen, reducing the ORN dendrite to the margin (Figures 2A–4). Although their functional significance remains to be determined, the extracellular vacuoles are likely derived from the tormogen auxiliary cell. In one T1 sensillum, we found that its tormogen cell extended a bulbous protrusion into the lumen, resembling the process of membrane budding (Figure 2B). Immediately adjacent to the bulbous protrusion was a small vacuole (Figure 2B). The close proximity between these two structures suggests that the free vacuoles within the lumen may have originated from the tormogen cell.

T2 and T3 sensilla

In addition to T1, we identified the other two trichoid sensillum types—T2 and T3—containing two and three neurons, respectively (Shanbhag et al., 1999; Figure 1). All trichoid sensilla are similar in length (19–24 μm), but T1 is distinctively narrower than T2 or T3 with smaller sensillum surface area and volume (Figure 1C,D and Table 1). In contrast, T2 and T3 cannot be distinguished externally from their morphological or morphometric features (Figure 1C,D and Table 1). On the basis of recent molecular and functional studies, T3 likely corresponds to the at4 sensillum, which houses three pheromone-sensing neurons: the Or47b (at4A), Or65a (at4B), and Or88a ORNs (at4C) (Couto et al., 2005; Dweck et al., 2015; Lin et al., 2016; van der Goes van Naters and Carlson, 2007). However, the molecular identity for T2 neurons remains hitherto unknown. Unexpectedly, in the data sets in which Or47b ORNs were genetically marked (Or47b-GAL4>10XUAS-myc-APEX2-Orco), we identified labeled neurons in both T2 and T3 sensilla (Figure 3A,B). Similarly, in the Or88a data set (Or88a-GAL4>10XUAS-myc-APEX2-Orco), labeled neurons were found in both T2 and T3 sensilla (Figure 3—figure supplement 1). Taken together, these results indicate that Or47b and Or88a ORNs are the shared constituents for T2 and T3.

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In both sensillum types, the at4A/Or47b ORN is the largest cell among compartmentalized neurons, measuring approximately 65 μm³ in soma volume. In comparison, the at4C/Or88a soma is smaller, averaging 35 μm³ in T2 and 44 μm³ in T3. Although the Or65a ORN was previously designated as at4B without clear evidence for its intermediate spike amplitude, its soma is in fact the smallest among the three at4 ORNs, averaging only 26 μm³ (Figure 3C and Table 2). As with soma size, the average inner and outer dendritic volumes are both greatest for at4A/Or47b neurons, followed by at4C/Or88a, and then at4B/Or65a ORNs (Figure 3D,E). However, compared to soma size, a greater variability was observed for dendritic volumes. For example, in one T3 sensillum, the at4B/Or65a outer dendrite was larger than that of the neighboring at4C/Or88a neuron; in other instances, the at4C/Or88a dendritic volume was greater than that of at4A/Or47b (Figure 3E).

Similar to the T1 neurons, the inner dendritic segments of T2 or T3 ORNs are insulated from one another by thecogen processes (Figures 3A-3 and 4 and Figures 3B-3 and 4). The outer dendrites of some T2 and T3 ORNs also exhibit a small number of branches: among 12 pairs of segmented neurons, three Or47b ORNs display two or three dendritic branches, and five Or88a neurons have two to four branches. In comparison, only one of the eight segmented Or65a ORNs exhibit branched outer dendrites (Table 2 and Table 2—source data 1 for details). Also similar to T1 neurons, the outer dendritic morphology of T2 and T3 neurons are typically cylindrical with a gradual taper toward the tip, while some neurons have portions of their outer dendritic segments flattened like a rippled ribbon (Figure 3—figure supplement 2A,B and Video 2). Notably, we observed ring-shaped outer dendritic profiles in the cross-section images of some unbranched Or47b ORNs; 3D reconstruction reveals that these profiles correspond to dendritic regions with a hollow center (Figure 3—figure supplement 2C,D). In one neuron, the hollowed region was short, observed in the mid-section of the outer dendrite (Figure 3—figure supplement 2C). In another neuron, the hollowed section was considerably longer, exceeding 1/3 of the outer dendritic length, and was found in the distal segment of the dendrite (Figure 3—figure supplement 2D). Their variable position and length suggest that this dendritic structure is dynamic, possibly reflecting the process of dendritic branching. Finally, as with T1 sensilla, we observed extracellular vacuoles in the proximal regions of the sensillum lumen in 27% of the analyzed T2 sensilla and in 29% of T3 sensilla (see Table 1—source data 1 for details).

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Coeloconic sensilla

Conical in shape, the coeloconic sensilla are the shortest antennal sensilla, ranging from 4.8 to 7.6 μm long (Figure 1C,D). Unlike other olfactory sensilla, the cuticle of the coeloconic sensilla contains no pores and instead consists of closely apposed cuticular fingers, between which longitudinal grooves permit odorants to enter the inner sensillum lumen through interspersed spoke channels (Shanbhag et al., 1999). The number of cuticular fingers ranges from 5 to 9, and does not correlate with sensillum types (see Table 1—source data 1 for details). The cuticle is double-walled, wherein the inner cuticular cylinder lines the inner lumen, the volume of which is the smallest among antennal sensilla (0.3–1.0 μm³) (Table 1). Extracellular vacuoles were not typically observed within the small luminal volumes, as vacuoles were found only in 6 of the 90 analyzed coeloconic sensilla (7%) (see Table 1—source data 1 for details). The inner sensillum lumen encapsulates only a short distal segment of the outer dendrite (2–3 μm), while approximately 75% of the outer dendritic length is beneath the antennal surface. In addition, the coeloconic outer dendrites typically terminate before reaching the sensillum tip (Figure 4 and Video 3).

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Intermediate sensilla

The intermediate sensilla share similar cuticular morphology with trichoid sensilla (Figure 1A,B), but their length is shorter than that of trichoids (Figure 1D and Table 1). The intermediate sensilla also display the basal drum at the sensillum base which, although not as prominent as the trichoid counterpart, can be recognized in isosurface reconstructions (Figure 1C) or by the thick cuticular walls in SBEM sections (Figure 5A). Another distinctive feature for intermediate sensilla is the darkly stained lumen which contrasts with the light luminal staining in the basiconic and trichoid sensilla from the same SBEM data set (Figure 5A).

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In agreement with an earlier TEM study (Shanbhag et al., 1999), we also identified two populations of intermediate sensilla that house either two or three neurons, likely corresponding to the ai2 and ai3 sensilla, respectively. The ai2 ORNs express the Or83c (ai2A) and Or23a (ai2B) receptors, while the ai3 neurons express the Or19a (ai3A), Or2a (ai3B), and Or43a receptors (ai3C) (Dweck et al., 2013; Lin and Potter, 2015; Ronderos et al., 2014). On average, ai2 is ~3 μm longer than ai3 (Figure 1D and Table 1). For the ai2 sensillum, the A neuron soma is approximately 50% larger than the neighboring B neuron, and the size differential is greater for the inner dendritic segment, and still greater for the outer dendritic segment (Figure 5B-D and Table 2). All segmented ai2 neurons exhibit branched outer dendrites (Figure 5B, Figure 5—figure supplement 1, and Video 4) with branches numbering from 5 to 11 for ai2A and 3 to 4 for ai2B (Table 2). Of note, striking morphological variability was observed for ai2A dendrites. While the majority of branches are cylindrical with a gradual taper toward the tip, we found one that was flattened like a ribbon which spiraled upwards (Figure 5—figure supplement 1A). In another ai2A, the flattened region was observed in the unbranched segment of outer dendrite, which then gave rise to multiple cylindrical and some flattened sub-branches (Figure 5—figure supplement 1B). As for the ai3 sensilla, a similar size difference was observed for their grouped neurons: the ‘A’ neuron exhibits the largest soma, inner, and outer dendrites, and is followed by the neighboring ai3B and then ai3C neurons (Figure 5E-G). As with ai2, all analyzed ai3 neurons have branched outer dendrites (Table 2 and Video 4). Finally, extracellular vacuoles were observed in the lumen among 21% and 56% of the analyzed ai2 and ai3 sensilla, respectively.

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Large basiconic sensilla ab1 sensillum

Characteristic of this basiconic subtype is the large, club-shaped cuticular apparatus which has a rounded tip and a slight narrowing at the base (Figure 1C). Large basiconic sensilla range from 10 to 13 μm in length (Figure 1D and Table 1). Among large basiconics, ab1 is unique because it houses not two but four neurons (Figure 6A,B), which express the Or42b (ab1A), Or92a (ab1B), Gr21a/Gr63a (ab1C), and Or10 receptors (ab1D) (Couto et al., 2005; de Bruyne et al., 2001; Hallem et al., 2004; Jones et al., 2007; Kwon et al., 2007). Compartmentalized ab1 neurons have distinctive sizes, with the soma volumes in the ratio of 2.5:2.2:1.9:1, following the rank order A≥B>C>D (Figure 6C). Interestingly, ab1C, which senses CO₂ (de Bruyne et al., 2001; Jones et al., 2007; Kwon et al., 2007), exhibits multiple membranous protrusions that resemble neuronal processes extending from the basolateral side of the soma (Figure 6B, arrows). Because of this unique feature, ab1C soma has the second largest surface area among the four grouped neurons (A>C>B>D), despite its relatively small soma volume (Table 2).

Figure 6

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More interestingly still, both ab1A and ab1B display markedly enlarged inner dendritic segments such that the size differential of ab1 neurons is even greater for this region—with a volumetric ratio of 6.8:7.5:2.4:1 (Figure 6B,C). As with the atypical ac1A (Figure 4—figure supplement 1), these inner dendritic enlargements contain numerous mitochondria (Figure 6A,B), with an average number of 124 for ab1A and 82 for ab1B (Figure 6D and Table 2). In comparison, the neighboring small-spike ‘C’ and ‘D’ neurons contain an average of 26 and 9 mitochondria, respectively (Figure 6D, Table 2 and Video 5). The disparity in mitochondrial number is also reflected in total mitochondrial volume (Figure 6D). We note that in vertebrate ORNs, mitochondria play a direct role in regulating cytosolic Ca²⁺ response profiles and thereby ensure a broad dynamic range for the neurons’ spike responses (Fluegge et al., 2012). Although it is unclear whether mitochondria play a similar role in insect olfactory signaling, a recent study shows that odor-induced Ca²⁺ signals in Drosophila ORNs are shaped by mitochondria (Lucke et al., 2020). Therefore, it will be interesting to investigate the functional significance of this striking mitochondrial disparity between grouped ORNs in future research.

Video 5

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ab2 and ab3 sensilla

Among the two large basiconic sensilla that house two neurons, we distinguished ab2 from ab3 by its lack of DAB staining in the Or22a data set, in which ab3A was genetically labeled by APEX2. Apart from the Or22a data set, an ab2 sensillum was identified in the Or7a data set on the basis of its proximity to the labeled ab4 sensilla, because ab3 is not found in the same topographical region as ab4 (de Bruyne et al., 2001). The ab2 and ab3 receptors are Or59b (ab2A), Or85a (ab2B), Or22a (ab3A), and Or85b (ab3B) (Dobritsa et al., 2003; Hallem et al., 2004). On average, the cuticular volume of ab2 (~32 μm³) is smaller than that of the genetically identified ab3 (~39 μm³) (Table 1). Among the four ab2 sensilla found in our data sets, we were able to segment a pair of ab2 ORNs in their entirety (Figure 6E,F and Video 5). In agreement with ab2’s large A/B spike amplitude ratio (Zhang et al., 2019), the soma size differential is notably high for the two neurons (2.7:1) (Figure 6G, top left panel). Of note, ab2A but not ab2B displays a bulbous inner dendritic enlargement and numerous long membranous protrusions (Figure 6E inset), which further widens the size disparity between the two neurons for this region (6:1) (Figure 6G, top right panel). As with the large-spike ab1 neurons, the ab2A inner dendritic enlargement contains over 100 mitochondria, markedly more numerous and with a greater total volume than ab2B (Figure 6G, middle panels, and Table 2). Moreover, an even greater disparity was observed in the outer dendritic segments: ab2A exhibits 312 dendritic branches while ab2B has only one (Figure 6G, bottom panels).

With respect to the ab3 large basiconic sensilla, four pairs of ORNs were identified and partially segmented from the Or22a-labeled data set in which the ab3A neurons were marked genetically. Additionally, we segmented a pair of ab3 neurons in their entirety from another data set (Figure 7A,B and Video 5), in which the ab3 sensillum was identified on the basis of its characteristic morphometric features (Table 1). Morphometric analysis of the soma and inner dendrite revealed a marked size difference between ab3A and ab3B (Figure 7C), as described in our previous work (Zhang et al., 2019). As with ab2A, the inner dendrite of ab3A also contains numerous mitochondria and displays several long membranous protrusions (Figure 7A,B). Similar to the other large basiconic neurons, the inner dendritic size disparity for grouped ab3 neurons (3.6:1) is greater than the somatic size difference (1.5:1) (Figure 7C). The outer dendritic disparity for ab3 neurons is less striking when compared to ab2 ORNs; the completely segmented ab3A and ab3B exhibit 83 and 19 dendritic branches, respectively (Table 2).

Figure 7

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Thin and small basiconic sensilla ab4 sensillum

All seven molecularly defined thin and small basiconic sensillum types (ab4–ab10) house two ORNs (Couto et al., 2005). We therefore focused our analysis on the genetically labeled ab4 and ab5 sensilla, which express Or7a (ab4A), Or56a (ab4B), Or82a (ab5A), and Or47a (ab5B) as odor-tuning receptors (Hallem et al., 2004; Stensmyr et al., 2012). Matching the description of a thin basiconic (Shanbhag et al., 1999), the ab4 sensillum is similar to the large basiconics in length (~11 μm) but with a more slender profile (Figure 1C,D and Table 1). Three pairs of ab4 neurons, including their elaborate dendritic branches, were completely segmented (Figure 8A-C and Video 6). Compartmentalized ab4 ORNs also have distinctive sizes, with the soma volume in the ratio of 2.1:1 (Figure 8D). Although the inner dendrite of ab4A is similarly enlarged and enriched with mitochondria (Figure 8B), the enlargement is less pronounced: ab4A’s inner dendritic volume is ~9 μm³ as opposed to ~20 μm³ for ab1A or ab2A (Table 2). As such, the inner dendritic volume ratio for ab4A and ab4B is 2.6:1, which is close to the somatic size differential (Figure 8D). A similar volume difference was also observed in the outer dendrites (2.4:1) (Figure 8D).

Figure 8 with 1 supplement see all

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Video 6

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In addition to morphometric analysis, we examined the branching pattern of ab4 outer dendrites and found it to be strikingly complex, contrary to the simple paintbrush model proposed on the basis of TEM longitudinal-section images of a sensillum, whereby all basiconic dendritic branches appear to share a common diverging point (Shanbhag et al., 1999). Instead in ab4A, we observed multiple branching points. A primary branch can bifurcate several times, giving rise to numerous secondary branches that in turn can give rise to tertiary, quaternary, or even higher-order branches. Moreover, some high-order branches can converge with others to form lateral connections (Figure 8C, top panel). In other instances, a branch can diverge into two sub-branches which then merge back into one (Video 7). In comparison to ab4A, the dendritic branching pattern of ab4B is less elaborate, with a smaller number of branches and a lower degree of branching (Figure 8C, bottom panel). Despite their varying complexity, the ab4A and ab4B outer dendrites start branching at similar positions near the sensillum base (Figure 8A-C).

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Empty sensilla

Among the 541 antennal sensilla sampled in this study, we were surprised to find two hairs that did not contain any ORNs. Such ‘empty sensilla’ have never been reported before. One is a trichoid sensillum identified from an Or47b-labeled data set (Figure 9A). Based on its cuticular morphometric features, the empty trichoid—named T(0)—likely belonged to the T1 subtype (Table 1 and Figure 1D). Devoid of any neurons, the empty trichoid also did not contain any intact auxiliary cells; only remnants of what appeared to be tormogen processes were observed near the sensillum base (Figure 9A). The second empty sensillum is a small basiconic found in the Or7a-labeled data set (designated as abx(0), Figure 9B and Table 1). Interestingly, and unlike the empty trichoid, all three auxiliary cells were present and morphologically indistinguishable from those found in adjacent sensilla containing ORNs (Figure 9C). While the biological significance of empty sensilla remains to be determined, it is possible that they represent a small fraction of antennal olfactory hairs whose neurons have undergone apoptosis in the process of neuronal turnover (Fernández-Hernández and Hu, 2020).

Figure 9

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Olfactory auxiliary cells

In addition to the sensilla and ORNs described in previous sections, we segmented two groups of auxiliary cells and reconstructed their 3D models. Auxiliary cells support olfactory function by insulating ORN soma and inner dendrites, maintaining transepithelial potential, expressing ion transporters, and secreting odorant-binding proteins and odorant degrading enzymes into the sensillum lymph (Kaissling, 1986; Larter et al., 2016; Leal, 2013; Menuz et al., 2014; Shanbhag et al., 2001; Shanbhag et al., 2000; Sun et al., 2018). We note that the ultrastructure of these cells has been described in detail through high-quality TEM images (Shanbhag et al., 2000). Therefore, our objective was to illustrate their positions in 3D space in relation to other auxiliary cells, their associated ORNs, and the sensillum apparatus. Most olfactory sensilla contain three auxiliary cells, except for the coeloconic class which has four (Shanbhag et al., 2000). In the following sections, we featured the auxiliary cells for ab4 and ac3 to represent these two possible cellular arrangements.

Auxiliary trio

The three auxiliary cell types are the thecogen, trichogen, and tormogen cells (Shanbhag et al., 2000). Among them, the thecogen is situated most deeply beneath the antennal surface, and forms a sleeve enveloping parts of the ORN soma, inner dendrite, and proximal portion of the outer dendrite (Figure 10A-C and Video 8). The plasma membrane of thecogen cell is typically smooth but can form microvilli or microlamellae in regions where the auxiliary cell ensheathes the inner dendrites (e.g., Figure 10C; Figures 3B-4 and Figures 6A-1). The nucleus of the auxiliary cell is at a similar level as the ORN soma such that thecogen cells are often found adjacent to the cell body of neurons (e.g., Figure 10A-C; Figures 2A-9, Figures 3A-5 and B-5).

Figure 10

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Video 8

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The distal region of the thecogen cell—where it envelops the inner dendrites—is in turn enwrapped by the trichogen cell. The apical surface of the trichogen cell forms extensive, elaborate microvilli and microlamellae that border the sensillum-lymph cavity (Figure 10A,B and D). Also characteristic of the thecogen cell are its abundant mitochondria and numerous tubular membrane invaginations (Figure 10A,B and D). The nucleus of the trichogen cell is located slightly distal to that of the thecogen cell at the level of proximal inner dendrite (Figure 10A and Video 8).

Moving toward the antennal surface, a small portion of the apical thecogen cell is further enveloped by the tormogen cell, which occupies the most distal and peripheral position of the auxiliary trio. Similar to the trichogen cell, the apical surface of the tormogen cell forms microvilli and microlamellae bordering the sensillum lymph cavity (Figure 10A,B and E). However, the tormogen processes are less numerous and elaborate. In turn, these processes surround the subcuticular segment of outer dendrites and terminate more distally near the sensillum base or occasionally into the sensillum lumen (e.g., Figure 10A,B; Figures 2B and 5E). Moreover, in lieu of tubular invaginations, the tormogen cell displays an abundance of large, interconnected vesicles which open into the extracellular space (Figure 10E). The tormogen nucleus is situated at the level of dendritic ciliary constriction. The basal region of the tormogen cell is distinctive in that it extends a long, stalk-like protrusion that terminates below the level of ORN soma (Figure 10E and Video 8).

Auxiliary quartet

Focusing on ac3, we segmented its four auxiliary cells, which have been reported to include one thecogen, one trichogen, and two tormogen cells (Shanbhag et al., 2000). Similar to the cellular arrangement described above, the coeloconic thecogen is the innermost cell located at the bottom of the quartet, ensheathing parts of ORN soma, inner dendrite, and the proximal portion of outer dendrite (Figure 11A-C and Video 8). The apical thecogen region is likewise enveloped by trichogen processes, whose elaborate microvilli and microlamellae surround the proximal outer dendrites. Compared to the basiconic trichogen cell, the invaginations of coeloconic trichogen appear less tubular but are rather vesicle-like (Figure 11D). In addition, the coeloconic trichogen nucleus is located more distally, at or above the level of ciliary constriction. Interestingly, we identified two trichogen cells instead of one based on their similar morphological features, including elaborate microlamellae, the position of their apical surface, and the lack of tormogen’s stalk-like protrusion (Figure 11B,D). This observation indicates that the coeloconic auxiliary quartet is in fact composed of one thecogen, one tormogen, and two trichogen cells. Finally, the coeloconic tormogen cell bears a strong resemblance to its basiconic counterpart. Both surround the subcuticular portion of outer dendrites with their apical processes, terminate their processes near the sensillum base, and extend a long, stalk-like basal protrusion (Figure 11E and Video 8). In future research, it will be interesting to determine whether the two coeloconic trichogen cells are functionally distinct, and how the morphological characteristics of individual auxiliary cells subserve function.

Figure 11

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All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Table 1, Table 2 and Individual datasets. The SBEM image volumes are available in the Cell Image Library (http://www.cellimagelibrary.org/home). The accession numbers are CIL:54606 (Or22a/ab3A-labeled volume); CIL:54614 (Or7a/ab4A-labeled volume); CIL:54610 (Or56a/ab4B-labeled volume); CIL:54611 (Ir75c/ac3AII-labeled volume); CIL:54612 (Or47a/ab5B-labeled volume); CIL:54607 and CIL:54608 (Or47b/at4A-labeled volumes); CIL:54609 (Or88a/at4C-labeled volume).

1. 1. Tsang TK
   2. Bushong EA
   3. Ellisman MH
   4. Su C-Y

   (2021) Cell Image Library

   ID CIL:54609. Or88a-GAL4>10XUAS-myc-APEX2-Orco.

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2. 1. Tsang TK
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   ID CIL:54608. Or47b-GAL4>10XUAS-myc-APEX2-Orco.

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   ID CIL:54607. Or47b-GAL4>10XUAS-myc-APEX2-Orco.

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Author details

1. Cesar Nava Gonzales

   Neurobiology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology

   Contributed equally with

   Quintyn McKaughan

   Competing interests

   No competing interests declared

2. Quintyn McKaughan

   Neurobiology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Cesar Nava Gonzales

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6611-6530

3. Eric A Bushong

   National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, San Diego, United States

   Contribution

   Software, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Kalyani Cauwenberghs

   Neurobiology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States

   Contribution

   Software, Formal analysis, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

5. Renny Ng

   Neurobiology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States

   Contribution

   Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

6. Matthew Madany

   National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, San Diego, United States

   Contribution

   Software, Methodology

   Competing interests

   No competing interests declared

7. Mark H Ellisman

   National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, San Diego, United States

   Contribution

   Resources, Funding acquisition

   Competing interests

   No competing interests declared

8. Chih-Ying Su

   Neurobiology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   c8su@ucsd.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0005-1890

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