Constructing and Tuning Excitatory Cholinergic Synapses: The Multifaceted Functions of Nicotinic Acetylcholine Receptors in Drosophila Neural Development and Physiology

Abstract

Nicotinic acetylcholine receptors (nAchRs) are widely distributed within the nervous system across most animal species. Besides their well-established roles in mammalian neuromuscular junctions, studies using invertebrate models have also proven fruitful in revealing the function of nAchRs in the central nervous system. During the earlier years, both in vitro and animal studies had helped clarify the basic molecular features of the members of the Drosophila nAchR gene family and illustrated their utility as targets for insecticides. Later, increasingly sophisticated techniques have illuminated how nAchRs mediate excitatory neurotransmission in the Drosophila brain and play an integral part in neural development and synaptic plasticity, as well as cognitive processes such as learning and memory. This review is intended to provide an updated survey of Drosophila nAchR subunits, focusing on their molecular diversity and unique contributions to physiology and plasticity of the fly neural circuitry. We will also highlight promising new avenues for nAchR research that will likely contribute to better understanding of central cholinergic neurotransmission in both Drosophila and other organisms.

Keywords: Drosophila; cholinergic neurotransmission; dendrite development; neural development; nicotinic acetylcholine receptor; synaptic plasticity; synaptogenesis.

FIGURE 1

The Drosophila nAchR is an evolutionarily conserved ligand-gated ion channel with prototypical motifs and secondary structures. (A) Phylogenetic comparison between nAchR genes of D. melanogaster (green) and humans (black) (Taken from Rosenthal et al., 2021). (B) High amino acid sequence similarity between animal nAchRs permits the modeling of the Drosophila alpha6 (Dα6) subunit (red), using the known human alpha4 subunit (CHRNA4) X ray crystal structure (blue) as a template (Sequence comparisons were made with the Phyre2 online tool and visualized by the software PyMOL 2.5). The secondary structures and overall topology are generally conserved between the two. The ligand nicotine is labeled green. The TM3-TM4 loop for both CHRNA4 and Dα6 is discontinuous. (C) Dα6 is shown in isolation and is color coded by residue position (Blue: N-terminus; Red: C-terminus). Major conserved motifs are labeled. The ligand nicotine is in black. (D) Schematic illustrations of the two stoichiometrically classed nAchR subtypes: homopentamers contain identical subunits whereas heteromers are composed of mixed subunits. The ligand, Ach in blue, interacts with the subunits’ interface.

FIGURE 2

Schematic diagram illustrating the mutated residues in nAchR subunits that confer insecticide resistance. Natural and lab-derived insecticide resistance in Drosophila often develops from amino acid substitutions (red dots) in a single nAchR subunit. Because these resistance-endowing mutations are found at varied locations in the mature protein, it is believed that resistance occurs via multiple mechanisms. Some, such as the Ser221 depicted in the framed panel on the left, directly interfere with the action of competitive agonists, like the neonicotinoids, at the ligand-binding site (Image taken from Shimada et al., 2020). In contrast, others are more distal and likely indirectly impact nAchR functions by modulatory activities (^aHomem et al., 2020; ^bIhara et al., 2014; ^cPerry et al., 2008; ^dSomers et al., 2015; ^eHikida et al., 2018; ^fShimada et al., 2020; ^gZimmer et al., 2016).

FIGURE 3

Physiological responses towards nAchR-mediated cholinergic neurotransmission. Schematic diagram illustrating the three types of physiological events which occur through nAchR-mediated cholinergic signaling. nAchR activation at the postsynaptic density results in rapid depolarization of the postsynaptic cell, increasing the probability of an action potential and signal propagation to downstream circuit components (Lee and O’Dowd, 1999). Slower, secondary changes are initiated by secold messenger systems and through integration with incoming inhibitory synaptic transmission as well as neuromodulatory input (Su and O’Dowd, 2003; Campusano et al., 2007; Kuehn and Duch, 2013).

FIGURE 4

Transcriptional and post-transcriptional mechanisms regulating nAchRs’ expression, maturation, synaptic integration and activity. Drosophila central neurons control the production and activity of nAchRs through distinct steps by integrating regulatory influences exerted by presynaptic activity or the internal state of the animal. A simplified illustration of potential events and current molecular findings related to the transcriptional and posttranslational regulation of nAchRs is shown.

FIGURE 5

Endogenous Dα6 expression pattern is revealed by endogenous tagging approach. Top: A knock-in Trojan-Gal4 gene trap in the Dα6 locus driving mCD8::GFP (white) expression. Bottom: anti-HA antibody staining on a CRISPR/Cas9 engineered Dα6::HA allele (white). Both methods reveal similar staining profiles in the third instar brain lobe. Right panels: Magnified views of brain regions proximal to the ventral lateral neurons (LNvs)(green). Both samples show the positive labeling of the larval optic lobe pioneer neurons (IOLPs, arrowhead) (Taken from Rosenthal et al., 2021).