Description

The odorant receptor co-receptor Orco forms ligand-gated ion channels with odorant receptors (ORs) tuned to specific odors; Orco is required for trafficking of these complexes to the dendrites of olfactory neurons (Larsson et. al., 2004). Mutation of the Orco gene not only abolishes olfactory neuron activity but has also revealed potential new roles for this gene in the development and maintenance of olfactory systems in a variety of insects. In ants and honeybees, Orco mutation leads to a loss of olfactory sensory neurons in the periphery, as well as severe defects in the morphology of the antennal lobe, the first olfactory processing center in the insect brain (Trible et al., 2017; Yan et al., 2017; Chen et al., 2021). These deficits are likely developmental as they are apparent at adult eclosion. Orco mutant hawkmoth males show more modest losses in antennal lobe volume associated with pheromone-sensing olfactory neurons (Fandino et al., 2019). In contrast, in Anopheles coluzzii and Aedes aegypti mosquitoes Orco mutation does not appear to lead to olfactory neuron loss or gross deficits in antennal lobe anatomy (though the latter has not been examined in depth) (Sun et al., 2020; DeGennaro et al., 2013).

In the vinegar fly Drosophila melanogaster, development of the adult olfactory system in Orco mutants appears normal (Larsson et. al., 2004). However, over time some populations of olfactory neurons in the antennae degenerate. This degeneration is initially evident in the olfactory neuron axons innervating the antennal lobe, which start to show signs of blebbing and retraction four to six days post eclosion (DPE) (Chiang et al., 2009). By 14 DPE, some but not all antennal olfactory neurons lose their cell bodies (Hueston et al., 2016). There is evidence that the cause of antennal olfactory neuron degeneration in the Orco mutant is due to a lack of neuronal activity (Chiang et al., 2009). Here, we investigated if neurodegeneration in Orco mutants also occurs in the Drosophila maxillary palp, a simpler olfactory organ with ~120 olfactory neurons.

To examine the effects of the loss of Orco on maxillary palp olfactory neurons, we performed whole-mount antibody staining on maxillary palps using the pan-neuronal marker, elav (Robinow and White, 1991). This marker should label all ~120 olfactory neurons in the palps, as well as a small population (~20) of presumed mechanosensory neurons (Singh and Nayak, 1985). Neuronal cell death should result in a reduction in the number of elav+ cells. Potential alternative approaches could be to use DAPI staining or the TUNEL assay to visualize cell loss and DNA fragmentation associated with cell death, among others (Meehan et al., 2015). We compared anti-elav antibody staining in wildtype and Orco² mutant flies at three timepoints: 3, 7, and 15 days post eclosion (DPE) (Fig 1A). In the wildtype, we found consistent neuronal cell counts across timepoints. The Orco² mutant palps of 3-day-old flies were indistinguishable from wildtype (Fig 1A). However, by 15 DPE, extensive loss of cell bodies was detectable in the Orco² mutants (Fig 1A). This neurodegeneration was evident at 7 DPE (middle column), suggesting that cell body loss in the maxillary palp occurs more quickly and potentially more broadly than in the antennae. Overall, Orco² mutants lose 60 – 70% of their maxillary palp neurons. These results are quantified in Fig 1B. Both 7 and 15 DPE Orco² mutants had significantly fewer elav+ cells compared to all wildtype timepoints as well as 3 DPE Orco² mutants (p ≤ 0.0002) but were not statistically significantly different from each other (p = 0.4055) (one-way ANOVA with Tukey’s HSD; see figure legend for F statistic and exact p values for all pairwise comparisons). There was no difference between any of the wildtype conditions (p > 0.99), nor between the 3 DPE Orco² mutant and any of the wildtype timepoints (p > 0.79).

We further examined if sub-populations of olfactory neurons in the maxillary palp might be resistant to degeneration. There are six different populations of olfactory neurons in the maxillary palp as defined by the odor-responsive OrX they express. Using available genetic reagents, we examined neuron degeneration in two of these populations in the Orco mutant background. We used the GAL4/UAS system (Brand and Perrimon, 1993) to GFP label the pb1A (Or42a-GAL4/UAS-mCD8:GFP) or pb2B (Or46a-GAL4/UAS-mCD8:GFP) olfactory neurons (Goldman et al., 2005) in wildtype or Orco² mutant maxillary palps. We examined flies 12 – 14 DPE to allow sufficient time for degeneration. Surprisingly, we found that the pb1A (Or42a-expressing) olfactory neuron population was largely spared from degeneration (Fig 1C, quantified in Fig 1D). In contrast, the pb2B (Or46a-expressing) olfactory neurons were almost all missing by 14 DPE (Fig 1C, quantified in Fig 1D). Those pb2B cells that remained showed signs of neuronal process fragmentation and blebbing, consistent with previously reported antennal neuron degeneration (Chiang et al., 2009). Prior work has confirmed that both these populations express Orco (Grabe et al., 2016; Larsson et al., 2004; Task et al., 2020). Our results suggest that at least one sub-population of Orco mutant olfactory neurons are resistant to activity-dependent cell death and represents the first quantified example of a specific olfactory neuron population escaping degeneration. While pb1A cells are presumably non-functional due to a lack of Orco, future experiments will be required to confirm that this is indeed the case, especially in light of recent studies showing co-expression of odorant receptors with other, non-Orco-dependent chemoreceptors in the same cells (McLaughlin et al., 2021; Task et al., 2020; Younger and Herre et al., 2020). This could be achieved by genetically silencing pb1A activity, as has previously been done in the antenna (Chiang et al., 2009). Interestingly, Orco-dependent cell death in the antennae does not involve caspase-dependent pathways and cannot be rescued by expression of the pan-caspase inhibitor p35 (Chiang et al., 2009). It remains to be determined if Orco mutant neurons in the maxillary palp similarly engage caspase-independent molecular mechanisms.

The olfactory system of insects represents a convenient model for examining how a lack of induced activity might affect the health of a sensory neuron (MacDonald et al., 2006; Chiang et al., 2009; Kazama et al., 2011; Hueston et al., 2016). As shown here, the maxillary palp presents a potentially favorable system for studying neurodegeneration and activity-dependent neuronal maintenance. In contrast to the antenna, neuronal cell body loss in Orco mutant palps is rapid (within 6 or 7 days post eclosion vs. 14 days post eclosion in antennae). The palp is a simpler olfactory organ, and neuronal changes are easier to study and quantify by whole-mount staining in the palps. We present evidence that at least one genetically definable population of olfactory neurons appears resistant to degeneration. Future experiments could examine if any of the other four olfactory sub-populations in the maxillary palp also demonstrate such resistance. Given the extensive genetic tools available in the Drosophila model system, maxillary palp olfactory neurons might be amenable to genetic screens aimed at investigating the molecular mechanisms underlying activity-dependent sensory degeneration.