Sexually Dimorphic Neurophysiology

How is sexual dimorphism regulated by co-expressed isoforms of male-specific transcription factors? It is well known that the development and function of dimorphic insect courtship circuits require Fru^M proteins, which are male-specific transcription factors alternatively spliced into three isoforms, Fru^MA, Fru^MB and Fru^MC. Most fru^M-positive neurons express all these variants; however, within a single cell, the specific roles for all three isoforms have never been systematically determined. Therefore, the functional significance of isoform co-expression remains unclear. Do co-expressed isoforms each play unique roles to jointly regulate sexual dimorphism? If so, how is the cooperation achieved at the molecular level?

To bridge this critical knowledge gap in neurobiology, we focused on the courtship-promoting olfactory receptor neurons (ORNs), which express Fru^M and exhibit male-specific, age-dependent response sensitization to aphrodisiac olfactory cues. We show that Fru^MA, Fru^MB, and Fru^MC are all expressed and differentially regulated with age in the courtship-promoting Or47b and Ir84a ORNs, and that each has a functional role (Zhang et al., Cell Reports, 2020).

Mechanistically, Fru^MB is upstream of PPK25, a DEG/ENaC member necessary for amplification of the pheromone responses, while Fru^MC is upstream of PPK23, which is in turn required for normal PPK25 function. Thus, co-expression of multiple Fru^M isoforms underlies functional synergy, achieved through cooperation of their respective downstream targets. In summary, our systematic investigation offers key mechanistic insights into how co-expressed Fru^M isoforms synergistically mediate male-specific neurophysiology. Moreover, it highlights an underexplored function of sex-specific factors beyond instructing development and connectivity.

Signal Amplification Mechanism

Insect olfactory receptors are ligand-gated ion channels which directly covert odor stimuli to neuronal activation. However, it remains controversial whether and how ionotropic input signals are amplified in insect olfactory receptor neurons (ORNs). In collaboration with Dr. Jing Wang at UCSD, we developed a novel stimulus paradigm (Ng et al, JoVE, 2017) and discovered an age-dependent increase of olfactory responses in the pheromone-sensitive Or47b ORNs (Lin et al, Neuron, 2016).

Building upon these findings, we recently identified a member of the Degenerin/Epithelial sodium channel family (DEG/ENaC), named Pickpocket 25 (PPK25), which is required for and can confer elevated olfactory responses in both types of courtship-promoting ORNs in male flies. Pharmacological and genetic manipulations indicate that PPK25 functions as a calcium gated transduction channel in the sensory cilia (Ng et al, Neuron, 2019). These results demonstrate that signal amplification can occur downstream of ionotropic odorant receptors.

Our findings further reveal the surprising complexity of insect olfactory transduction: not all ORNs expressing receptors of the same class employ identical transduction mechanisms. Rather, a common transduction channel can function downstream of two distinct classes of receptors. Taken together, our study challenges the dogma that signal amplification is mediated exclusively through metabotropic sensory receptors, and thus advances the current understanding of sensory transduction.

Morphometric Analysis of ORNs

To define the respective electrotonic properties of grouped ORNs, we seek to determine the ultrastructure of ORNs in a sensillum using serial block-face electron microscopy (SBEM) and 3D reconstruction imaging technologies. To achieve this latter goal, we collaborated with Dr. Mark Ellisman to develop a novel method that permits high-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues (Tsang et al., eLife, 2018). We found that lateral inhibition is predominantly asymmetric between compartmentalized olfactory receptor neurons (ORNs) in Drosophila. In most sensillum types, the large-spike ORNs exerts greater ephaptic influence onto its small-spike neighbors. The same neuron is also less susceptible to ephaptic influence, indicating that as a common feature, lateral inhibition is asymmetric between most grouped ORNs (Zhang et al., Nature Communications, 2019). Our study indicates that not all ORNs are equal in their circuit function, and that genetically predetermined morphometric differences between compartmentalized ORNs underlie their functional disparity.

Interaction without Synapses

Although electric field (ephaptic) effects are broadly observed in the nervous systems of many animal species, their precise functional impact has been notoriously difficult to illustrate. This reflects the inability to discriminate ephaptic effects from transmission mediated by conventional chemical synapses or gap junctions. To overcome this limitation, we use recordings from pairs of olfactory sensory hairs (sensilla) impaled by the same tungsten electrode to show that ephaptic interaction alone is sufficient to mediate lateral inhibition between compartmentalized olfactory receptor neurons (ORNs) in Drosophila (Zhang et al., Nature Communications, 2019). This unconventional lateral inhibition has a profound impact on neuronal spike activity and can thus regulate information processing at the earliest stages of olfactory coding.

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