InteractiveFly: GeneBrief

nicotinic Acetylcholine Receptor α5, nicotinic Acetylcholine Receptor α6 and nicotinic Acetylcholine Receptor α7

Biological Overview | References

Gene names - nicotinic Acetylcholine Receptor α5, nicotinic Acetylcholine Receptor α6 and nicotinic Acetylcholine Receptor α7

Synonyms -

Cytological map positions - 34F2-34F4, 30D1-30E1 and 18C2-18C3

Function - transmembrane channels

Keywords - neurotransmitter-gated ion-channel, CNS, nicotinic AChR co-assembly, dendritic growth, synaptic potential, integration of visual signals in a Drosophila motion detection pathway, optic lobe, insecticide resistance, escape behavior, RNA-mediated A-to-I pre-mRNA editing

Symbol - nAChRα5, nAChRα6 and nAChRα7

FlyBase IDs: FBgn0028875, FBgn0032151 and FBgn0086778

Genetic map positions - chr2L:14061321-14089456, chr2L:9796948-9886214 and chrX:19223380-19237995

Classification - Cation transporter family protein, Neurotransmitter-gated ion-channel ligand binding domain, Neurotransmitter-gated ion-channel transmembrane region

Cellular location - surface transmembrane

NCBI link for nAChRα5: EntrezGene
NCBI link for nAChRα6: EntrezGene
NCBI link for nAChRα7: EntrezGene

nAChRα5 orthologs: Biolitmine
nAChRα6 orthologs: Biolitmine
nAChRα7 orthologs: Biolitmine
Recent literature
Zimmer, C. T., Garrood, W. T., Puinean, A. M., Eckel-Zimmer, M., Williamson, M. S., Davies, T. G. and Bass, C. (2016). A CRISPR/Cas9 mediated point mutation in the α 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster. Insect Biochem Mol Biol 73: 62-69. PubMed ID: 27117524
Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. This study used the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. This mutation does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in mutant flies. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target-site mutation and provides an example of how recent advances in genome editing can be harnessed to study insecticide resistance.
Hahm, E. T., Nagaraja, R. Y., Waro, G. and Tsunoda, S. (2018). Cholinergic homeostatic synaptic plasticity drives the progression of Abeta-induced changes in neural activity. Cell Rep 24(2): 342-354. PubMed ID: 29996096
Homeostatic synaptic plasticity (HSP) is the ability of neurons to exert compensatory changes in response to altered neural activity. How pathologically induced activity changes are intertwined with HSP mechanisms is unclear. This study shows that, in cholinergic neurons from Drosophila, beta-amyloid (Abeta) peptides Abeta40 and Abeta42 (see Drosophila β amyloid protein precursor-like) both induce an increase in spontaneous activity. In a transgenic line expressing Abeta42, it was observed that this early increase in spontaneous activity is followed by a dramatic reduction in spontaneous events, a progression that has been suggested to occur in cholinergic brain regions of mammalian models of Alzheimer's disease. Evidence is presented that the early enhancement in synaptic activity is mediated by the Drosophila alpha7 nicotinic acetylcholine receptor (nAChR) and that, later, Abeta42-induced inhibition of synaptic events is a consequence of Dalpha7-dependent HSP mechanisms induced by earlier hyperactivity. Thus, while HSP may initially be an adaptive response, it may also drive maladaptive changes and downstream pathologies.
Malloy, C. A., Somasundaram, E., Omar, A., Bhutto, U., Medley, M., Dzubuk, N. and Cooper, R. L. (2019). Pharmacological identification of cholinergic receptor subtypes: modulation of locomotion and neural circuit excitability in Drosophila larvae. Neuroscience. PubMed ID: 31102763
In Drosophila melanogaster acetylcholine (ACh) is the neurotransmitter used in peripheral sensory neurons and is a primary excitatory neurotransmitter and neuromodulator within the central nervous system (CNS). The receptors are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs). A behavioral and electrophysiological approach was used to assess cholinergic modulation of locomotion and sensory-CNS-motor circuit excitability. Intact and semi-intact 3rd instar larvae were exposed to ACh receptor agonists and antagonists to observe their roles in behavior and regulation of neural circuit excitability and to investigate AChR pharmacological properties in vivo. This was combined with targeted AChR RNAi-mediated knockdown to identify specific receptor subtypes facilitating ACh modulation of circuit efficacy. A contribution by both mAChRs and nAChRs was identified in regulation of locomotor behavior and reveal they play a role in modulation of the excitability of a sensory-CNS-motor circuit. A conspicuous role was identified for mAChR-A and mAChR-C in motor neurons in modulation of their input-output efficacy.
Homem, R. A., Buttery, B., Richardson, E. E., Tan, Y., Field, L. M., Williamson, M. S. and Davies, T. G. E. (2020). Evolutionary trade-offs of insecticide resistance - the fitness costs associated with target-site mutations in the nAChR of Drosophila melanogaster. Mol Ecol. PubMed ID: 32510730
The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field-evolved mutation being reported to date. This is an arginine to threonine substitution at position 81 of the nAChR_β1 subunit in neonicotinoid resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target-site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.
Ihara, M., Furutani, S., Shigetou, S., Shimada, S., Niki, K., Komori, Y., Kamiya, M., Koizumi, W., Magara, L., Hikida, M., Noguchi, A., Okuhara, D., Yoshinari, Y., Kondo, S., Tanimoto, H., Niwa, R., Sattelle, D. B. and Matsuda, K. (2020). Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions. Proc Natl Acad Sci U S A 117(28): 16283-16291. PubMed ID: 32611810
The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. This study shows that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.
Eadaim, A., Hahm, E. T., Justice, E. D. and Tsunoda, S. (2020). Cholinergic Synaptic Homeostasis Is Tuned by an NFAT-Mediated alpha7 nAChR-K(v)4/Shal Coupled Regulatory System. Cell Rep 32(10): 108119. PubMed ID: 32905767
Homeostatic synaptic plasticity (HSP) involves compensatory mechanisms employed by neurons and circuits to preserve signaling when confronted with global changes in activity that may occur during physiological and pathological conditions. Cholinergic neurons, which are especially affected in some pathologies, have recently been shown to exhibit HSP mediated by nicotinic acetylcholine receptors (nAChRs). In Drosophila central neurons, pharmacological blockade of activity induces a homeostatic response mediated by the Drosophila α7 (Dα7) nAChR, which is tuned by a subsequent increase in expression of the voltage-dependent K(v)4/Shal channel. This study shows that an in vivo reduction of cholinergic signaling induces HSP mediated by Dα7 nAChRs, and this upregulation of Dα7 itself is sufficient to trigger transcriptional activation, mediated by nuclear factor of activated T cells (NFAT), of the K(v)4/Shal gene, revealing a receptor-ion channel system coupled for homeostatic tuning in cholinergic neurons.
Santalla, M., Pagola, L., Gomez, I., Balcazar, D., Valverde, C. A. and Ferrero, P. (2021). Smoking flies: Testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster. Biol Open. PubMed ID: 33431431
Studies about the relationship between substances consumed by humans and their impact on health, in animal models have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolating certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. This work explores the impact of tobacco consumption on a model of "smoking flies". These experiments were designed to provide information about the effects of tobacco consumption on cardiac physiology. Intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation, was assessed. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and alpha 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data would help to understand the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila would help to know the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.
Hao, S., Gestrich, J. Y., Zhang, X., Xu, M., Wang, X., Liu, L. and Wei, H. (2021). Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster. Front Neurosci 15: 653858. PubMed ID: 34975366
Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. This study reports that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. It was also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in Ca(2+) imaging experiments, relatively low doses of OA caused increased Ca(2+) levels in PTTH neurons, whereas higher doses led to decreased Ca(2+) levels. It was also demonstrated that a low dose of OA was conveyed through OA *betal-type receptors. Additionally, electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, these findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.
Martelli, F., Hernandes, N. H., Zuo, Z., Wang, J., Wong, C. O., Karagas, N. E., Roessner, U., Rupasinghe, T., Robin, C., Venkatachalam, K., Perry, T., Batterham, P. and Bellen, H. J. (2022). Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies. Elife 11. PubMed ID: 35191376
Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, this study showed that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit α 6 (nAChRα6), reducing the cholinergic response. The nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.
Korona, D., Dirnberger, B., Giachello, C. N. G., Queiroz, R. M. L., Popovic, R., Müller, K. H., Minde, D. P., Deery, M. J., Johnson, G., Firth, L. C., Earley, F. G., Russell, S. and Lilley, K. S. (2022). Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins. Elife 11. PubMed ID: 35575460
Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs, a CRISPR/Cas9 strategy was used to generate null alleles for all ten nAChR subunit genes in a common genetic background. Interactions of nAChR subunits with peptide neurotoxins were studied by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, the effects were determined of α-Bungarotoxin (α -Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. Pull-down assays were employed to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, the localisation is reported of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
Zhang, Y. C., Pei, X. G., Yu, Z. T., Gao, Y., Wang, L. X., Zhang, N., Song, X. Y., Wu, S. F. and Gao, C. F. (2022). Effects of nicotinic acetylcholine receptor subunit deletion mutants on insecticide susceptibility and fitness in Drosophila melanogaster. Pest Manag Sci. PubMed ID: 35576366
Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in insects and also the target site for many insecticides. Unfortunately, the effectiveness of these insecticides is diminishing as a consequence of the evolution of insecticide resistance. Further exploration of insecticide targets is important to sustainable pest management. In order to validate the role of nAChR subunits in insecticide susceptibility and test whether the subunit's absence imposes the fitness cost on insects, the susceptibility of eight nAChR subunit deletion mutants of Drosophila melanogaster to nine insecticides was tested. These findings highlighted the specific resistance of the Dα6 deletion mutant to spinosyns. Although triflumezopyrim, dinotefuran and imidacloprid are competitive modulators of nAChRs, differences in susceptibility of the insect with different deletion mutants suggested that the target sites of these three insecticides do not overlap completely. Mutants showed decreased susceptibility to insecticides, accompanied by a reduction in fitness. The number of eggs produced by Dα1(attP), Dα2(attP), Dβ2(attP) and Dβ3(attP) females was significantly lesser than that of the vas-Cas9 strain as the control. In addition, adults of Dα2(attP) , Dα3(attP) and Dα7(attP) strains showed lower climbing performance. Meanwhile, males of Dα3(attP) , Dα5(attP) , Dβ2(attP) and Dβ3(attP) , and females of Dβ2(attP) showed significantly shorter longevity than those of the vas-Cas9 strain. This study provides new insights into the interactions of different insecticides with different nAChRs subunit in D. melanogaster as a research model, it could help better understand such interaction in agricultural pests whose genetic manipulations for toxicological research are often challenging.
Liu, C. H., Chen, M. Y., Cheng, J., Chuang, T. N., Liu, H. P. and Lin, W. Y. (2022). Imidacloprid Impairs Glutamatergic Synaptic Plasticity and Desensitizes Mechanosensitive, Nociceptive, and Photogenic Response of Drosophila melanogaster by Mediating Oxidative Stress, Which Could Be Rescued by Osthole. Int J Mol Sci 23(17). PubMed ID: 36077576
Imidacloprid (IMD) is a widely used neonicotinoid-targeting insect nicotine acetylcholine receptors (nAChRs). However, off-target effects raise environmental concerns, including the IMD's impairment of the memory of honeybees and rodents. Although the down-regulation of inotropic glutamate receptor (iGluR) was proposed as the cause, whether IMD directly manipulates the activation or inhibition of iGluR is unknown. Using electrophysiological recording on fruit fly neuromuscular junction (NMJ), this study found that IMD of 0.125 and 12.5 mg/L did not activate glutamate receptors nor inhibit the glutamate-triggered depolarization of the glutamatergic synapse. However, chronic IMD treatment attenuated short-term facilitation (STF) of NMJ by more than 20%. Moreover, by behavioral assays, it was found that IMD desensitized the fruit flies' response to mechanosensitive, nociceptive, and photogenic stimuli. Finally, the treatment of the antioxidant osthole rescued the chronic IMD-induced phenotypes. It was clarified that IMD is neither agonist nor antagonist of glutamate receptors, but chronic treatment with environmental-relevant concentrations impairs glutamatergic plasticity of the NMJ of fruit flies and interferes with the sensory response by mediating oxidative stress.
Giordani, G., Cattabriga, G., Becchimanzi, A., Di Lelio, I., De Leva, G., Gigliotti, S., Pennacchio, F., Gargiulo, G. and Cavaliere, V. (2022). Role of neuronal and non-neuronal acetylcholine signaling in Drosophila humoral immunity. Insect Biochem Mol Biol 153: 103899. PubMed ID: 36596348
Acetylcholine (ACh) is one the major neurotransmitters in insects, whose role in mediating synaptic interactions between neurons in the central nervous system is well characterized. It also plays largely unexplored regulatory functions in non-neuronal tissues. This study demonstrates that ACh signaling is involved in the modulation of the innate immune response of Drosophila melanogaster. Knockdown of ACh synthesis or ACh vesicular transport in neurons reduced the activation of drosomycin (drs), a gene encoding an antimicrobial peptide, in adult flies infected with a Gram-positive bacterium. drs transcription was similarly affected in Drosophila α7 nicotinic acetylcholine receptor, nAChRalpha7 (D&al;pha;7) mutants, as well as in flies expressing in the nervous system a dominant negative form (Dα7(DN)) of this specific receptor subunit. Interestingly, Dα7(DN) elicited a comparable response when it was expressed in non-neuronal tissues and even when it was specifically produced in the hemocytes. Consistently, full activation of the drs gene required Dα7 expression in these cells. Moreover, knockdown of ACh synthesis in non-neuronal cells affected drs expression. Overall, these findings uncover neural and non-neural cholinergic signals that modulate insect immune defenses and shed light on the role of hemocytes in the regulation of the humoral immune response.
Pribbenow, C., Chen, Y. C., Heim, M. M., Laber, D., Reubold, S., Reynolds, E., Balles, I., FernAndez, D. V. A. T., Suarez-Grimalt, R., Scheunemann, L., Rauch, C., Matkovic, T., Rosner, J., Lichtner, G., Jagannathan, S. R. and Owald, D. (2022). Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila. Elife 11. PubMed ID: 36250621
In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. This study provides evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). The nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons (MBONs) for appetitive memory induction, but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely function downstream of α5 and the postsynaptic scaffold protein Dlg. This study shows that ostsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative (familiarity) memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.
Wan, X., Shen, P., Shi, K., Li, J., Wu, F. and Zhou, C. (2023). A Neural Circuit Controlling Virgin Female Aggression Induced by Mating-related Cues in Drosophila. Neurosci Bull. PubMed ID: 36941515
Females increase aggression for mating opportunities and for acquiring reproductive resources. Although the close relationship between female aggression and mating status is widely appreciated, whether and how female aggression is regulated by mating-related cues remains poorly understood. This study reports an interesting observation that Drosophila virgin females initiate high-frequency attacks toward mated females. 11-cis-vaccenyl acetate (cVA), a male-derived pheromone transferred to females during mating, was shown to promote virgin female aggression. A cVA-responsive neural circuit was subsequently reveal consisting of four orders of neurons, including Or67d, DA1, aSP-g, and pC1 neurons, that mediate cVA-induced virgin female aggression. It was also determined that aSP-g neurons release acetylcholine (ACh) to excite pC1 neurons via the nicotinic ACh receptor nAChRα7. Together, beyond revealing cVA as a mating-related inducer of virgin female aggression, these results identify a neural circuit linking the chemosensory perception of mating-related cues to aggressive behavior in Drosophila females.
Scott, J. G., Norris, R. H., Mertz, R. W., Dressel, A. E. and Loeb, G. (2023). Selection and characterization of spinetoram resistance in field collected Drosophila melanogaster. Pestic Biochem Physiol 194: 105508. PubMed ID: 37532361
Insecticides are commonly employed in vineyards to control vinegar flies and limit sour rot disease. Widespread resistance to available insecticides is having a negative impact on managing Drosophila melanogaster populations, rendering control of sour rot more difficult. An insecticide registered for use in vineyards to which resistance is not yet widespread (at least in New York and Missouri) is spinetoram. Spinetoram targets the nicotinic acetylcholine receptor α6, and mutations in α6 have been associated with resistance in some insects. The goals of this study were to select for a spinetoram resistant strain of D. melanogaster (starting with field collected populations), characterize the resistance, and identify the mutation responsible. After five selections a strain (SpinR) with >190-fold resistance was obtained. Resistance could not be overcome by insecticide synergists, suggesting an altered target site was involved. The α6 allele from the spinetoram resistant strain was cloned and sequenced and Ωa mutation was cloned and sequenced causing a glycine to alanine change at amino acid 301 (equivalent position to the G275E mutation found in some spinosad/spinetoram resistant insects). This mutation was found at low levels in field populations, but increased with each selection until it became homozygous in SpinR. How the identification of the spinetoram resistance mutation can be used for resistance management is discussed.
Petsakou, A., Liu, Y., Liu, Y., Comjean, A., Hu, Y., Perrimon, N. (2023). Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut. Nature, 623(7985):122-131. PubMed ID: 37722602 ID:
A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer. This study used the Drosophila midgut to investigate this and discovered that during regeneration a subpopulation of cholinergic neurons triggers Ca(2+) currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. It was found that downregulation of the conserved cholinergic enzyme Acetylcholine esterase in the gut epithelium enables acetylcholine from specific Eiger (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca(2+), which spreads in the epithelium through Innexin2-Innexin7 gap junctions, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel disease. Altogether, the conserved cholinergic pathway facilitates epithelial Ca(2+) currents that heal the intestinal epithelium. These findings demonstrate nerve- and bioelectric-dependent intestinal regeneration and advance current understanding of how a tissue returns to homeostasis after injury.


Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. This study demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 +, Dα6 + Dα7) was demonstrated to have substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments. These data demonstrate the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs (Lansdell, 2012).

>Nicotinic acetylcholine receptors (nAChRs) are excitatory neurotransmitter receptors that are found in both vertebrate and invertebrate species. In insects, nAChRs are expressed throughout the nervous system and are the site of action for economically important insecticides such as spinosyns and neonicotinoids (Miller, 2007; Jones, 2007). Detailed information is available concerning the structure of nAChRs, as a consequence of studies conducted with receptors purified from the electric organ of the marine ray Torpedo and from X-ray crystallographic studies conducted with nAChR fragments and also with the closely related acetylcholine binding protein. Nicotinic receptors are assembled from five subunits arranged around a central cation-selective pore (Taly, 2009; Albuquerque, 2009). Conventional agonists, such as acetylcholine, activate the receptor by binding at an extracellular site located at the interface between two subunits, although recent evidence indicates that nAChRs can also be activated by ligands binding to an allosteric transmembrane site (Lansdell, 2012).

Ten nAChR subunits (Dα1-Dα7 and Dβ1-Dβ3) have been identified in Drosophila and a similar number of subunits have been identified in other insect species (Miller, 2007; Jones, 2007). Despite considerable efforts, there has been only limited success in expressing insect nAChRs in artificial expressions systems and, where functional expression has been achieved, ion channel currents have tended to be small or have been generated in response to relatively high agonist concentrations. Experimental approaches that have had some success in overcoming problems associated with expression of insect nAChRs include the expression of subunit chimeras containing domains from other neurotransmitter receptors, co-expression of insect nAChRs with vertebrate subunits or a combination of these approaches. Co-expression with vertebrate nAChR subunits is an approach that has been used in the characterization of nAChR subunits cloned from insect pest species such as the aphid Myzus persicae and the brown planthopper Nilaparvata lugens. However, for most insect species for which nAChRs have been cloned, there have been no reports of successful heterologous expression. This includes nAChRs cloned from the honeybee Apis mellifera, diamondback moth Plutella xylostella, house fly Musca domestica, locust Locusta migratoria, mosquito Anopheles gambiae, red flour beetle Tribolium castaneum, silkworm Bombyx mori and tobacco hornworm Manduca sexta (Lansdell, 2012 and references therein).

RIC-3 is a nAChR-associated molecular chaperone that was originally characterised in the nematode Caenorhabditis elegans (Halevi, 2002) but has also been identified in several other species, including mammals and insects (Millar, 2008). It is a transmembrane protein that is able to enhance maturation (folding and assembly) of several nAChR subtypes (Millar, 2008). For example, co-expression of RIC-3 with the vertebrate nAChR α7 subunit enhances levels of functional expression in Xenopus oocytes (Halevi, 2002) and is able to facilitate the functional expression of α7 nAChRs in mammalian cell lines that are otherwise non-permissive for expression of α7. In some cell types it has been found that the α7 subunit can be expressed (subunit protein can be detected) but, in the absence of RIC-3, is unable to fold into a conformation that can be detected by radioligand binding or form functional nAChRs. In addition, some success has been achieved in overcoming difficulties associated with expression of insect nAChRs by the co-expression with RIC-3 (Lansdell, 2008; Watson, 2010; Lansdell, 2012).

The Dα5, Dα6 and Dα7 subunits of Drosophila show close sequence similarity to one another (53-63% amino acid identity; Grauso, 2002) and also have close similarity to the vertebrate nAChR α7 subunit (42-46% amino acid identity; Jones, 2009). Of the three Drosophila subunits, Dα5 and Dα7 have the closest sequence similarity to one another and Dα6 has the highest sequence similarity to the vertebrate α7 (Sattelle, 2005). In the present study, the molecular cloning of the Dα5 subunit, the only Drosophila nAChR subunit for which a full-length cDNA clone was not previously available. Heterologous expression studies with Dα5, Dα6 and Dα7 are described in three host cell types: Drosophila S2 cells, human tsA201 cells and Xenopus oocytes. Functional expression of several subunit combinations has been achieved in Xenopus oocytes and has enabled the pharmacological properties of recombinant nAChRs to be examined. Evidence is provided that demonstrates the ability of subunits to form both homomeric and heteromeric nAChRs. Of particular note is evidence that Dα5 can generate functional homomeric channels and that Dα7 can form both homomeric and heteromeric channels. This is no previous studies demonstrating the ability of Dα5 and Dα7 subunits to generate such recombinant nAChRs, either with subunits cloned from Drosophila or with analogous nAChR subunits from other insect species (Lansdell, 2012).

The Dα5, Dα6 and Dα7 subunits differ from other Drosophila nAChR subunits in their close sequence similarity to the vertebrate α7 nAChR subunit (Grauso, 2002; Littleton, 2000), a subunit that is notable for its ability to form both homomeric and heteromeric nAChRs. In addition to being one of the best characterised homomeric nAChRs, the vertebrate α7 subunit can co-assemble into heteromeric nAChRs by co-assembly with the α8 subunit (in avian species). Although an α8 subunit is not present in mammals, recent evidence indicates that the mammalian α7 subunit can also form functional heteromeric nAChRs by co-assembly with β2 (Lansdell, 2012).

Relatively limited information is available about the physiological roles of the Dα5, Dα6 and Dα7 subunits in Drosophila, or about the role of analogous subunits in other insect species. There is, however, evidence from studies of native nAChRs in Drosophila that Dα5 forms part of a nAChR that is sensitive to α-bungarotoxin (Wu, 2005), Dα6 forms part of the spinosad-sensitive nAChR (Perry, 2007) and that Dα7 is required for the visually-mediated cholinergic escape response (Fayyazuddin, 2006; Lansdell, 2012 and references therein).

Difficulties have been encountered in the efficient functional expression of insect nAChRs. This study reports the cloning of a full-length cDNA of the Drosophila Dα5 subunit corresponding to a previously described isoform B (Grauso, 2002). Other isoforms of Dα5 described previously (isoforms A and C) (Grasso, 2002) are a consequence of alternative splicing and have fewer exons than isoform B. Isoform A lacks exon 7, which codes for part of the second transmembrane domain, whilst isoform C lacks exon 5, which codes for the region containing the extracellular Cys-loop. The cloning of the Dα5 subunit was first reported in 2002 (Grauso, 2002) but no expression studies were described at that time. More recently, it has been reported that Dα5 does not generate functional homomeric nAChRs when expressed in Xenopus oocytes, even when co-expressed with RIC-3 (Watson, 2010). Functional expression was, however, reported in the same study when Dα5 was co-expressed with Dα6 and RIC-3. The present study has detected functional responses when Dα5 is co-expressed with Dα6 but, in contrast to the previous study (Grauso, 2002), evidence was obtained for the functional expression of homomeric Dα5 nAChRs. Similarly, it was demonstrated that Dα7 can form both homomeric and heteromeric nAChRs. There have been no previous reports of the successful functional expression of Dα7, as either a homomeric or a heteromeric nAChR. Given the difficulties encountered in obtaining reproducible functional expression of insect recombinant nAChRs, it is not surprising that there may be some apparent differences in subunit combinations found to generate functional receptors in this and previous studies, particularly since the focus of the most detailed previous study was the identification of a spinosyn-sensitive nAChRs (Lansdell, 2012).

Studies conducted in cell lines provided evidence that the pairwise combination Dα5+Dα6 generates a high affinity radioligand binding site, a finding that agrees with previous studies demonstrating functional expression of Dα5+Dα6 nAChRs in oocytes (Watson, 2010). Interestingly, no evidence was found of specific binding when Dα7 was co-expressed with Dα5 and Dα6 in the same cell lines. This lack of specific binding would seem to suggest that, in the two cell lines examined, co-assembly of Dα7 with the Dα5 and Dα6 subunit interferes with the formation of correctly assembled complexes. A somewhat similar situation was observed in oocytes, where expression of Dα7 alone generates functional nAChRs but it fails to do so when co-expressed with Dα6. This may reflect a tendency for Dα6 and Dα7 to assemble into non-functional complexes. The one situation where this tendency is not dominant is when all three subunits (Dα5+Dα6+Dα7) are co-expressed with RIC-3 in oocytes, where they are able to form a functional ‘triplet’ nAChR with high apparent affinity for acetylcholine (Lansdell, 2012).

The present findings suggest that the environment provided by the host cell exerts a substantial effect on the assembly of these nAChR subtypes, a phenomena that has been reported previously for other nAChRs. Previous studies by another research group (Watson, 2010) support the conclusion that co-assembly of Dα5+Dα6 nAChRs is somewhat inefficient. Not only was functional expression of the Dα5+Dα6 subunit combination found to be inconsistent in the previous study, but it also appeared to be dependent on the ratio of cRNAs injected. Perhaps this inconsistent functional expression reflects a tendency for some subunit combinations to assemble into non-functional complexes and that this may be more prevalent in certain subunit stoichiometries. It is possible that, in the native cellular environment, factors determining efficiency of subunit assembly and maturation may differ, perhaps as a consequence of a different array of endogenous chaperone proteins. This conclusion is supported by previous studies that have indicated that influence of RIC-3 on maturation of nAChRs is influenced by the host cell (Lansdell, 2008) and may help to explain the differences that were observed in the ability of some subunit combinations to assemble into nAChRs in different expression systems (Lansdell, 2012).

The data obtained from expression studies in Drosophila and human cell lines is broadly similar. However, successful expression in human cells required incubation at a temperature lower than they would normally be maintained at (25°C, rather than 37°C) [note: Drosophila S2 cells are routinely maintained at 25°C]. Previous studies have demonstrated that the folding and assembly of the nAChRs from insects (Lansdell, 1997) and from some other non-insect species, such as the cold-water ray Torpedo, can be influenced by temperature. This temperature dependence appears to be a consequence of inefficient protein folding and/or subunit assembly at higher temperatures. Previously, due to difficulties in expression of Dα6 and Dα7 nAChR subunits, this study examined the ability of subunit chimeras to assemble into complexes capable of binding 125I]-α-bungarotoxin. From such studies, it was possible to conclude that the Dα6 and Dα7 subunits were capable of heterometic co-assembly. In the present study the data from subunit chimeras is less clear cut. Although higher levels of 125I]-α-bungarotoxin were seen consistently when the Dα5 chimera was co-expressed with either the Dα6 and Dα7 chimeras, it was not clear in all cases whether this was greater than an additive effect. Nevertheless these findings are consistent with the conclusion that Dα5 is able to co-assemble into heteromeric complexes. For all subunit combinations examined, responses to acetylcholine were completely blocked by α-bungarotoxin, a finding that is consistent with previous studies conducted with native nAChRs purified from Drosophila which demonstrated that Dα5 is part of an α-bungarotoxin binding nAChR (Wu, 2005; Lansdell, 2012 and references therein).

As mentioned above, a previous study has reported the functional expression of heteromeric Dα5+Dα6 nAChRs (co-expressed with RIC-3) in Xenopus oocytes and also the inability of either Dα5 or Dα6 to form functional homomeric nAChRs (Watson, 2010). Significantly, the authors of this earlier study describe substantial difficulties in achieving reliable functional expression. In the present study, despite demonstrating the functional expression of several combinations of the Dα5, Dα6 and Dα7 subunits, a much lower success rate was encountered than is typically achieved with other nAChRs. In both transfected cell lines and in Xenopus oocytes, this study occasionally failed to detect evidence of radioligand binding or functional expression, despite success with other nAChRs that were expressed as positive controls (for example the mammalian α7 nAChR). The difficulties encountered may be associated with a tendency for these subunits to co-assemble into non-functional complexes. It is possible that this may reflect a requirement for additional chaperone proteins. Indeed, a study conducted with a C. elegans nAChR has demonstrated a requirement for three different chaperone proteins for efficient functional heterologous expression (Lansdell, 2012).

In summary, whereas it has been reported previously that Dα5 and Dα6 can form a functional heteromeric nAChR (albeit inefficiently) when expressed in Xenopus oocytes, this is the first evidence that either Dα5 or Dα7 can form functional homomeric nAChRs. It is also the first demonstration that Dα7 can form a functional heteromeric nAChR. Of particular interest is the evidence that the three subunits examined in this study can co-assemble to form a functional triplet (Dα5+Dα6+Dα7) nAChR with a high apparent affinity for acetylcholine (Lansdell, 2012).

Memory-relevant mushroom body output synapses are cholinergic

Memories are stored in the fan-out fan-in neural architectures of the mammalian cerebellum and hippocampus and the insect mushroom bodies. However, whereas key plasticity occurs at glutamatergic synapses in mammals, the neurochemistry of the memory-storing mushroom body Kenyon cell output synapses is unknown. This study demonstrates a role for acetylcholine (ACh) in Drosophila. Kenyon cells express the ACh-processing proteins ChAT and VAChT, and reducing their expression impairs learned olfactory-driven behavior. Local ACh application, or direct Kenyon cell activation, evokes activity in mushroom body output neurons (MBONs). MBON activation depends on VAChT expression in Kenyon cells and is blocked by ACh receptor antagonism. Furthermore, reducing nicotinic ACh receptor subunit expression in MBONs compromises odor-evoked activation and redirects odor-driven behavior. Lastly, peptidergic corelease enhances ACh-evoked responses in MBONs, suggesting an interaction between the fast- and slow-acting transmitters. Therefore, olfactory memories in Drosophila are likely stored as plasticity of cholinergic synapses (Barnstedt, 2016).

Despite decades of work on learning and memory and other functions of the MB, the identity of the fast-acting neurotransmitter that is released from the KCs has remained elusive. Much of the insect brain was considered to be cholinergic, but the MB was thought to be unique. Histological studies concluded that the MB did not express ChAT but that subsets of KCs contained glutamate, aspartate, or taurine. However, conclusive evidence that these molecules are released as neurotransmitters has not materialized (Barnstedt, 2016).

This study presents multiple lines of evidence that ACh is a KC transmitter. (1) KCs express the ChAT and VAChT proteins that synthesize and package ACh into synaptic vesicles, and the expression of these genes is required for MB-dependent learned behavior. (2) Stimulation of KCs triggers responses in MBONs that are similar to those evoked by direct ACh application. (3) Reducing ACh processing in KCs impairs KC-evoked responses in MBONs. (4) ACh- and KC-evoked responses in MBONs are both sensitive to antagonism of nicotinic ACh receptors. (5) Odor-evoked responses in MBONs are attenuated by reducing the expression of several nicotinic ACh receptor subunits. Taken together, these data provide compelling support that ACh is a major neurotransmitter released from Drosophila KCs (Barnstedt, 2016).

The anatomy of ACh-responsive MBONs suggests that many αβ, α'β', and γ lobe KCs are likely to be cholinergic. Calcium imaging may miss subtle or inhibitory effects, so it remains possible that subclasses of KC might also release or corelease other small molecule transmitters. It is, for example, notable that the MB neurons express an atypical putative vesicular transporter. Furthermore, taurine histology specifically labels the αβ core neurons. Anatomy suggests that αβ core and αβ surface outputs are pooled by MBONs with dendrites in the α lobe tip and throughout the β lobe, but that the dendrites of MBONs in the α lobe stalk preferentially innervate αβ surface neurons. It will be important to understand how ACh signals from different KCs are integrated by MBONs. The αβ and γ, but not α'β', KCs can corelease ACh with the sNPF neuropeptide. The current data raise the possibility that coreleased sNPF may facilitate ACh-evoked responses. sNPF drives autocrine presynaptic facilitation of certain olfactory sensory neurons in the adult fly. Conversely, sNPF decreased the resting membrane potential of larval motor neurons that ectopically express sNPFR. MBONs with dendrites in certain lobes therefore receive different combinations of transmitters and may vary in responding to sNPF (Barnstedt, 2016).

Finding that ACh is the KC transmitter has important implications for learning-relevant plasticity at KC-MBON synapses. Current models suggest that valence-specific and anatomically restricted reinforcing dopaminergic neurons drive presynaptically expressed plasticity between KCs and particular MBONs. Reward learning skews KC-MBON outputs toward driving approach by depressing the odor drive to MBONs that direct avoidance, whereas aversive learning enhances drive to avoidance by reducing drive to approach MBONs and increasing drive to avoidance pathways. The results here indicate that learning is represented as dopaminergic tuning of excitatory cholinergic KC-MBON synapses (Barnstedt, 2016).

Learning requires dopamine receptor function in the KCs, which implies a presynaptic mechanism of plasticity at the KC-MBON junction. Presynaptic plasticity of odor-activated KCs provides a simple means to retain odor specificity of memory in the highly convergent anatomy of the MB-where 2,000 KCs converge onto single or very few MBONs per zone on the MB lobes. The anatomically analogous mammalian cerebellar circuits, to which the insect MBs have been compared, exhibit presynaptic glutamatergic plasticity that is cAMP dependent. Finding that the KC transmitter is ACh suggests that cAMP-dependent mechanisms can modulate synaptic connections, regardless of transmitter identity. The MB KCs appear to be strikingly similar to the large parallel ensemble of cholinergic amacrine cells in the vertical lobe of the cuttlefish. These Cephalopod amacrine cells also share the same fan-out input and fan-in efferent anatomy of the Drosophila KCs, and plasticity occurs at the cholinergic connection between amacrine cells and downstream large efferent neurons (Barnstedt, 2016).

Work in the locust suggested that spike-timing-dependent plasticity (STDP) marks the relevant conditioned odor-activated KC-MBON synapses so that they are susceptible to reinforcing modulation. STDP relies on coincidence of pre- and postsynaptic activity and influx of postsynaptic Ca2+ through NMDA-type glutamate receptors. Recent work in Drosophila pairing odor presentation with dopaminergic neuron activation reported odor-specific synaptic depression at a KC-MBON junction that did not require postsynaptic MBON depolarization. It will be important to determine whether this holds for all DAN-MBON compartments or whether some learning-induced plasticity involves synaptic Ca2+ influx through an ACh-triggered nAChR, rather than the more traditional glutamate-gated NMDA receptors (Barnstedt, 2016).

This study identified roles for the Dα1, Dα4, Dα5, and Dα6 nAChR subunits in M4/6 MBONs. Reducing the expression of these subunits lowered odor-evoked signals in MBONs and converted naive odor avoidance into approach behavior. Dα5 and Dα6 subunits can form functional heteromeric channels in vitro. Different MBONs may express unique combinations of AChRs and therefore have characteristic physiological responses to KC-released ACh, as well as perhaps different learning rules and magnitudes of plasticity. Pre- or postsynaptically localized muscarinic AChRs could provide additional memory-relevant modulation (Barnstedt, 2016).

Beyond important roles in memory formation, consolidation, and expression, the MB- and DAN-directed modulation of specific MBON pathways has also been implicated in controlling hunger, thirst, temperature, and sleep/wake state-dependent locomotor behaviors. It will therefore be important to understand how plasticity of cholinergic KC transmission serves these discrete functions (Barnstedt, 2016).

Parallel Visual Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons

Neurotransmitter receptors and ion channels shape the biophysical properties of neurons, from the sign of the response mediated by neurotransmitter receptors to the dynamics shaped by voltage-gated ion channels. Therefore, knowing the localizations and types of receptors and channels present in neurons is fundamental to understanding of neural computation. This study developed two approaches to visualize the subcellular localization of specific proteins in Drosophila: the flippase-dependent expression of GFP-tagged receptor subunits in single neurons and 'FlpTag', a versatile new tool for the conditional labelling of endogenous proteins. Using these methods, the subcellular distribution of the receptors GluClα, Rdl, and Dα7 and the ion channels Para and Ih in motion-sensing T4/T5 neurons of the Drosophila visual system was investigated. A strictly segregated subcellular distribution of these proteins a sequential spatial arrangement of glutamate, acetylcholine, and GABA receptors was discovered along the dendrite that matched the previously reported EM-reconstructed synapse distributions (Fendl, 2020).

How neural circuits implement certain computations in order to process sensory information is a central question in systems neuroscience. In the visual system of Drosophila, much progress has been made in this direction: numerous studies examined the response properties of different cell-types in the fly brain and electron microscopy studies revealed the neuronal wiring between them. However, one element crucial to understanding is still missing; these are the neurotransmitter receptors used by cells at the postsynaptic site. This knowledge is essential since neurotransmitters and corresponding receptors define the sign and the time-course of a connection, that is whether a synapse is inhibitory or excitatory and whether the signal transduction is fast or slow. The same neurotransmitter can act on different receptors with widely differing effects for the postsynaptic neuron. Glutamate for instance is mainly excitatory, however, in invertebrates it can also have inhibitory effects when it acts on a glutamate-gated chloride channel, known as GluClα. Recently, it has also been shown that acetylcholine, usually excitatory, might also be inhibitory in Drosophila, if it binds to the muscarinic mAChR-A receptor. Hence, knowledge inferring the type of transmitter receptor at a synapse is essential for understanding of the way neural circuits process information (Fendl, 2020).

Moreover, voltage-gated ion channels shape synaptic transmission and the integration of synaptic inputs by defining the membrane properties of every neural cell type. The voltage-gated calcium channel cacophony, for instance, mediates influx of calcium ions that drives synaptic vesicle fusion at presynaptic sites. Voltage-gated sodium channels like Paralytic (Para) are important for the cell's excitability and the generation of sodium-dependent action potentials. The voltage-gated channel Ih influences the integration and kinetics of excitatory postsynaptic potentials. However, only little is known about how these channels are distributed in neurons and how this shapes the neural response properties (Fendl, 2020).

One of the most extensively studied neural circuits in Drosophila is the motion vision pathway in the optic lobe and the underlying computation for direction-selectivity. The optic lobe comprises four neuropils: lamina, medulla, lobula, and lobula plate. As in the vertebrate retina, the fly optic lobe processes information in parallel ON and OFF pathways. Along the visual processing chain, T4/T5 neurons are the first neurons that respond to visual motion in a direction selective way. T4 dendrites reside in layer 10 of the medulla and compute the direction of moving bright edges (ON-pathway). T5 dendrites arborize in layer 1 of the lobula and compute the direction of moving dark edges (OFF-pathway). The four subtypes of T4/T5 neurons (a, b, c, d), project axon terminals to one of the four layers in the lobula plate, each responding only to movement in one of the four cardinal directions, their preferred direction (Fendl, 2020).

How do T4/T5 neurons become direction-selective? Both T4 and T5 dendrites span around eight columns collecting signals from several presynaptic input neurons, each of which samples information from visual space in a retinotopic manner. The functional response properties of the presynaptic partners of T4/T5 have been described in great detail along with their neurotransmitter phenotypes. T4 dendrites receive glutamatergic, GABAergic and cholinergic input, whereas T5 dendrites receive GABAergic and cholinergic input only. These input synapses are arranged in a specific spatial order along T4/T5 dendrites (Fendl, 2020).

Which receptors receive this repertoire of different neurotransmitters at the level of T4/T5 dendrites? Recently, several RNA-sequencing studies described the gene expression pattern of nearly all cell-types in the optic lobe of the fruit fly including T4/T5 neurons. T4/T5 neurons were found to express numerous receptor subunits of different transmitter classes and voltage-gated ion channels at various expression strengths. However, RNA-sequencing studies do not unambiguously answer the above question for two reasons: mRNA and protein levels are regulated in complex ways via post-transcriptional, translational, and protein degradation mechanisms making it difficult to assign protein levels to RNA levels. Secondly, standard RNA-sequencing techniques cannot provide spatial information about receptor localizations, hence, they are not sufficient to conclude which transmitter receptors receive which input signal. Both shortcomings could in principle be overcome by antibody staining since immunohistochemical techniques detect neurotransmitter receptors at the protein level and preserve spatial information. However, high-quality antibodies are not available for every protein of interest and may have variable affinity due to epitope recognition. Furthermore, labeling ion channels via antibodies and ascribing expression of a given channel to a cell-type in dense neuronal tissue remains challenging. The disadvantages of the above techniques highlight the need for new strategies for labeling neurotransmitter receptors in cell types of interest (Fendl, 2020).

This study employed existing and generated new genetic methods to label and visualize ion channels in Drosophila. For endogenous, cell-type-specific labeling of proteins, a generalizable method called FlpTag was developed that expresses a GFP-tag conditionally. Using these tools, the subcellular distribution was determined of the glutamate receptor subunit GluClα, the acetylcholine receptor subunit Dα7, and the GABA receptor subunit Rdl in motion-sensing T4/T5 neurons. These receptor subunits were differentially localized between dendrites and axon terminals. Along the dendrites of individual T4/T5 cells, the receptor subunits GluClα, Rdl, and Dα7 reveal a distinct distribution profile that can be assigned to specific input neurons forming synapses in this area. Furthermore, it was demonstrated the generalizability of the FlpTag approach by generating lines for the metabotropic GABA receptor subunit Gaba-b-r1 and the voltage-gated ion channels para and Ih. The strategies described in this study can be applied to other cells as well as other proteins to reveal the full inventory and spatial distribution of the various ion channels within individual neurons (Fendl, 2020).

Neurotransmitter receptors are essential neuronal elements that define the sign and temporal dynamics of synaptic connections. For understanding of complex neural circuits, it is indispensable to examine which transmitter receptor types are used by the participating neurons and to which compartment they localize. This study developed FlpTag, a generalizable method for endogenous, cell-type-specific labeling of proteins. Alongside several GFP-tagged UAS-lines, the newly developed FlpTag lines were developed to explore the distribution of receptor subunits GluClα, Rdl, Dα7, Gaba-b-r1 and voltage-gated ion channels Para and Ih in motion-sensing T4/T5 neurons of the visual system of Drosophila. These ion channels were found to be localized to either the dendrite, the axonal fiber or the axon terminal. Even at the level of individual dendrites, GluClα, Rdl and Dα7 were differentially distributed precisely matching the locations where T4 and T5 neurons sample signals from their glutamatergic, cholinergic, or GABAergic input neurons, respectively (Fendl, 2020).

Working with Drosophila as model organism bears some unrivaled advantages when it comes to genetic tools. The MiMIC and FlyFos libraries, for instance, are large-scale approaches of enormous value for the fly community as they provide GFP-tagged protein lines for thousands of Drosophila genes including several neurotransmitter receptors and voltage-gated ion channels. Recently, Kondo expanded these existing libraries with T2A-Gal4 insertions in 75 neurotransmitter receptor genes that can also be exchanged by the fluorescent protein tag Venus (Kondo, 2020). While all these approaches tag genes at their endogenous locus, none of them are conditional, for example they cannot be applied in a cell-type-specific manner. Hence, ascribing the expression of the pan-neuronally tagged proteins to cell-types of interest are challenging in dense neuronal tissue (Fendl, 2020).

To overcome these difficulties, two conditional strategies were used for the investigation of membrane protein localizations in the cell types of interest, T4 and T5 neurons. First, GFP-tagged UAS-lines were developed for GluClα and Rdl, and an existing UAS-Dα7::GFP line was tested. As stated above, aberrant localization of overexpressed proteins can occur, however, this is not always the case. Overexpression of UAS-GluClα::GFP shows a similar receptor localization pattern as both MiMIC and FlpTag endogenous lines, thus, validating the use of UAS-GluClα::GFP for studying receptor distribution. Additionally, previous studies reported that the UAS-Dα7::GFP line showed proper localization of the acetylcholine receptor to endogenous synapses when compared to antibody stainings or endogenous Bruchpilot (Brp) puncta. This study confirmed confirmed this finding and further showed that Dα7::GFP presumably localizes only to cholinergic synapses. Overexpressing Dα7::GFP in a medulla neuron that is devoid of endogenous Dα7 demonstrated that Dα7::GFP localized to apparent cholinergic synapses. Hence, the UAS-Dα7::GFP line can be used to study the distribution of cholinergic synapses, but not the exact composition of cholinergic receptor subunits. A recent study showed that quantitatively the levels of the postsynaptic density protein PSD95 change when overexpressed, but qualitatively the localization is not altered. Altogether, this suggests that tagged overexpression lines can be used for studying protein localizations, but they have to be controlled carefully and drawn conclusions might be different for every line (Fendl, 2020).

Ideally, a tool for protein tagging should be both endogenous and conditional. This can be achieved by introducing an FRT-flanked STOP cassette upstream of the gene of interest which was engineered with an epitope tag or fluorescent protein. Only upon cell-type specific expression of Flp, the tagged protein will be expressed in a cell-type specific manner. This genetic strategy was utilized by two independent studies to label the presynaptic protein Brp, the histamine channel Ort and the vesicular acetylcholine transporter VAChT. Recently, a new approach based on the split-GFP system was utilized for endogenous, conditional labeling of proteins in two independent studies. However, all these aforementioned approaches are not readily generalizable and easily applicable to any gene of interest (Fendl, 2020).

The FlpTag strategy presented in this study overcomes these caveats by allowing for endogenous, conditional tagging of proteins and by offering a generalizable toolbox for targeting many genes of interest. Similar to the conditional knock-out tools FlpStop and FlipFlop, FlpTag utilizes a FLEx switch to conditionally control expression of a reporter gene, in this case GFP. Likewise, FlpTag also easily integrates using the readily available intronic MiMIC insertions. This study attempted to generate FlpTag lines for six genes, GluClα, Rdl, Dα7, Gaba-b-r1, para and Ih. Four out of these six lines yielded conditional GFP-tagged protein lines (GluClα, Gaba-b-r1, para, Ih). The FlpTag cassette was injected in MI02620 for Rdl and MI12545 for Dα7, but no GFP expression was detected across the brain. The MiMiC insertion sites used for Rdl and Dα7 seem to be in a suboptimal location for tagging the protein (Fendl, 2020).

As of now, there are MiMIC insertions in coding introns for more than 2800 genes available, which covers approximately 24% of neuronal genes. Additionally, the attP insertion sites generated in the study by Kondo provide possible landing sites for the FlpTag cassette for 75 neurotransmitter receptor genes (Kondo, 2020). Transmembrane proteins such as neurotransmitter receptors form complex 3D structures making fluorescent tagging especially difficult. Neither the MiMIC insertion sites, nor the target sites of the Kondo study at the C-terminus of several transmitter receptor genes, ensure a working GFP-tagged protein line. For genes of interest lacking a suitable MiMIC insertion site a homology directed repair (HDR) cassette was generated that utilizes CRISPR/Cas9-mediated gene editing to integrate the FlpTag cassette in any desired gene locus. The plasmid consists of the FlpTag cassette flanked by multiple cloning sites for the insertion of homology arms (HA). Through HDR the FlpTag cassette can be knocked-in into any desired locus. Taken together, the FlpTag cassette is a generalizable tool that can be integrated in any available attP-site in genes of interest or inserted by CRISPR-HDR into genes lacking attP landing sites. This allows for the investigation of the endogenous spatial distributions of proteins, as well as the correct temporal dynamics of protein expression (Fendl, 2020).

Further, the FlyFos project demonstrated that most fly lines with an extra copy of GFP-tagged protein-coding genes worked normally and GFP-tagged proteins could be imaged in living fly embryos and pupae. In principle, live-imaging of the GFP-tagged lines that were created could be performed during different developmental stages of the fruit fly. In general, the tools generated in this study can be used as specific postsynaptic markers, visualizing glutamatergic, GABAergic, and cholinergic synapses with standard confocal light microscopy. This extends the existing toolbox of Drosophila postsynaptic markers for studying the localization and development of various types of synapses (Fendl, 2020).

T4/T5 neurons combine spatiotemporal input from their presynaptic partners, leading to selective responses to one of the four cardinal directions. Numerous studies investigated the mechanisms underlying direction-selective responses in T4/T5 neurons, yet the computation is still not fully understood. At an algorithmic level, a three-arm detector model is sufficient to describe how direction-selective responses in T4/T5 neurons arise. This model relies on the comparison of signals originating from three neighboring points in space via a delay-and-compare mechanism. The central arm provides fast excitation to the neuron. While one flanking arm amplifies the central signal for stimuli moving along the preferred direction, the other inhibits the central signal for stimuli moving along the null direction of the neuron. Exploring the neurotransmitter receptors and their distribution on T4/T5 dendrites allows defining the sign as well as the temporal dynamics of some of the input synapses to T4/T5 (Fendl, 2020).

According to the algorithmic model, an excitatory, amplifying input signal on the distal side of T4/T5 dendrites was expected. This study found that T4 cells receive an inhibitory, glutamatergic input from Mi9 via GluClα, which, at first sight, seems to contradict expectation. However, since Mi9 has an OFF-center receptive field, this glutamatergic synapse will invert the polarity from Mi9-OFF to T4-ON. Theoretically, in darkness, Mi9 inhibits T4 via glutamate and GluClα, and this inhibition is released upon an ON-edge moving into its receptive field. The concomitant closure of chloride channels and subsequent increased input resistance in T4 cells results in an amplification of a subsequent excitatory input signal from Mi1 and Tm3. As shown by a recent modeling study, this biophysical mechanism can indeed account for preferred direction enhancement in T4 cells (Borst, 2018). Some studies failed to detect preferred direction enhancement in T4/T5 neurons and they proposed that the enhanced signal in PD seen in GCaMP recordings could be a result from a non-linear calcium-to-voltage transformation. If this was really the case, the role of Mi9 and GluClα must be reconsidered and future functional experiments will shed light onto this topic (Fendl, 2020).

Nevertheless, Strother (2017) showed that the RNAi- knock-down of GluClα in T4/T5 neurons leads to enhanced turning responses on the ball set-up for faster speeds of repeating ON and OFF edges (Strother, 2017). Although this observation cannot answer the question about preferred direction enhancement in T4 cells, it indicates that both T4 and T5 receive inhibitory input and that removal of such create enhanced turning responses at the behavioral level. In line with these observations, the glutamate receptor GluClα was also found in T4/T5 axon terminals. A possible functional role of these inhibitory receptors in the axon terminals could be a cross-inhibition of T4/T5 cells with opposite preferred directions via lobula plate intrinsic neurons (LPis). Glutamatergic LPi neurons are known to receive a cholinergic, excitatory signal from T4/T5 neurons within one layer and to inhibit lobula plate tangential cells, the downstream postsynaptic partners of T4/T5 neurons, via GluClα in the adjacent oppositely tuned layer. This mechanism induces a motion opponent response in lobula plate tangential cells and increases their flow-field selectivity. In addition, LPi neurons could also inhibit T4/T5 neurons presynaptically at their axon terminals via GluClα in order to further sharpen the flow-field selectivity of lobula plate tangential cells. Taken together, exploring the subcellular distribution of GluClα in T4/T5 neurons highlights its differential functional roles in different parts of these cell types (Fendl, 2020).

Secondly, the Dα7 signal in the center of T4/T5 dendrites discovered in this study, corresponds to ionotropic, cholinergic input from Mi1 and Tm3 for T4, and Tm1, Tm2 and Tm4 for T5. These signals correspond to the central, fast, excitatory arm of the motion detector model. As T4 and T5 express a variety of different ACh receptor subunits, the exact subunit composition and underlying biophysics of every cholinergic synapse on T4/T5 dendrites still awaits further investigations (Fendl, 2020).

Third, inhibition via GABA plays an essential role in creating direction-selective responses in both T4 and T5 neurons by providing null direction suppression. Computer simulations showed that direction selectivity decreases in T4/T5 motion detector models without this inhibitory input on the null side of the dendrite. This study shows that T4 and T5 neurons possess the inhibitory GABA receptor subunit Rdl mainly on the proximal base on the null side of their dendrites, providing the synaptic basis for null direction suppression. The metabotropic GABA receptor subunit Gaba-b-r1 was not detected in T4/T5 neurons using the newly generated FlpTag Gaba-b-r1 line. Finally, all of the receptor subunits GluClα, Rdl and Dα7 investigated in this study are ionotropic, fast receptors, which presumably do not add a temporal delay at the synaptic level. In the detector model described above, the two outer arms provide a slow and sustained signal, and such properties are already intrinsic properties of these input neurons. However, it cannot be excluded that slow, metabotropic receptor subunits for acetylcholine or GABA (e.g. Gaba-br2) which are also present in T4/T5 and could induce additional delays at the synaptic level (Fendl, 2020).

Furthermore, the subcellular distribution was investigated of the voltage-gated ion channels Para and Ih in T4/T5 neurons. Para, a voltage-gated sodium channel, was found to be distributed along the axonal fibers of both T4 and T5 neurons. As Para is important for the generation of sodium-dependent action potentials, it will be interesting for future functional studies to investigate, if T4/T5 really fire action potentials and how this shapes their direction-selective response. Further, Ih, a voltage-gated ion channel permeable for several types of ions, was detected in T4/T5 dendrites using the FlpTag strategy. Ih channels are activated at negative potentials below -50 mV and as they are permeable to sodium and potassium ions, they can cause a depolarization of the cell after hyperpolarization. Loss-of-function studies will unravel the functional role of the Ih channel for direction-selective responses in T4/T5 neurons (Fendl, 2020).

Since the ability to combine synaptic inputs from different neurotransmitters at different spatial sites is common to all neurons, the approaches described in this study represent an important future perspective for other circuits. The tools can be used to study the ion channels GluClα, Rdl, Dα7, Gaba-b-r1, para and Ih in any given Drosophila cell-type and circuit. Furthermore, the FlpTag tool box can be used to target many genes of interest and thereby foster molecular questions across fields (Fendl, 2020).

The techniques described in this study can be transferred to other model organisms as well, to study the distribution of different transmitter receptors. For instance, in the mouse retina - similar to motion-sensing T4/T5 neurons in the fruit fly - so-called On-Off direction-selective ganglion cells receive asymmetric inhibitory GABAergic inputs from presynaptic starburst amacrine cells during null-direction motion. A previous study investigated the spatial distribution of GABA receptors of these direction-selective ganglion cells using super-resolution imaging and antibody staining. Additionally, starburst amacrine cells also release ACh onto ganglion cells which contributes to the direction-selective responses of ganglion cells. Thus, mapping the distribution of ACh receptors on direction-selective ganglion cells will be the next important step to further investigate cholinergic transmission in this network (Fendl, 2020).

Overall, this study has demonstrated the importance of exploring the distributions of neurotransmitter receptors and ion channels for systems neuroscience. The distinct distributions in T4/T5 neurons discovered in this study and the resulting functional consequences expand knowledge of the molecular basis of motion vision. Although powerful, recent RNAseq studies lacked information about spatial distributions of transmitter receptors which can change the whole logic of wiring patterns and underlying synaptic signs. Future studies can use this knowledge to target these receptors and directly probe their role in functional experiments or incorporate the gained insights into model simulations. However, this study is only highlighting some examples of important neural circuit components: expanding the approaches described in this study to other transmitter receptors and ion channels, as well as gap junction proteins will reveal the full inventory and the spatial distributions of these decisive determinants of neural function within an individual neuron (Fendl, 2020).

The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain

The synaptic cleft, a crucial space involved in neurotransmission, is filled with extracellular matrix that serves as a scaffold for synaptic differentiation. However, little is known about the proteins present in the matrix and their functions in synaptogenesis, especially in the CNS. This study reports that Hikaru genki (Hig), a secreted protein with an Ig motif and complement control protein domains, localizes specifically to the synaptic clefts of cholinergic synapses in the Drosophila CNS. The data indicate that this specific localization is achieved by capture of secreted Hig in synaptic clefts, even when it is ectopically expressed in glia. In the absence of Hig, the cytoskeletal scaffold protein DLG accumulates abnormally in cholinergic postsynapses, and the synaptic distribution of acetylcholine receptor (AchR) subunits Dalpha6 and Dalpha7 significantly decreased. hig mutant flies consistently exhibited resistance to the AchR agonist spinosad, which causes lethality by specifically activating the Dalpha6 subunit, suggesting that loss of Hig compromises the cholinergic synaptic activity mediated by Dalpha6. These results indicate that Hig is a specific component of the synaptic cleft matrix of cholinergic synapses and regulates their postsynaptic organization in the CNS (Nakayama, 2014).

The matrix proteins Hasp and Hig exhibit segregated distribution within synaptic clefts and play distinct roles in synaptogenesis

The synaptic cleft is the space through which neurotransmitters convey neural information between two synaptic terminals. This space is presumably filled with extracellular matrix molecules involved in synaptic function or differentiation. However, little is known about the identities of the matrix components, and it remains unclear how these molecules organize the matrix in synaptic clefts. This study identified Hig-anchoring scaffold protein (Hasp), a Drosophila secretory protein containing CCP and WAP domains. Molecular genetic analysis revealed that Hasp diffuses extracellularly and is predominantly captured at synaptic clefts of cholinergic synapses. Furthermore, Hasp regulates levels of DLG and the nAChR subunits Dα6 and Dα7 at postsynaptic terminals. Hasp is required for trapping of another matrix protein, Hig, which is also secreted and diffused in the brain, at synaptic clefts of cholinergic synapses; however, Hig is dispensable for localization of Hasp at synaptic clefts. In addition, in the brains of triple mutants for the nAChR subunits Dα5, Dα6, and Dα7, the level of Hig, but not Hasp, was markedly reduced in synaptic regions, indicating that these nAChR subunits are required to anchor Hig to synaptic clefts. High-resolution microscopy revealed that Hasp and Hig exhibit segregated distribution within individual synaptic clefts, reflecting their differing roles in synaptogenesis. These data provide insight into how Hasp and Hig construct the synaptic cleft matrix and regulate the differentiation of cholinergic synapses, and also illuminate a previously unidentified architecture within synaptic clefts (Nakayama, 2016).

The synapse comprises presynaptic and postsynaptic terminals that are separated by a very narrow space, the synaptic cleft. Neurotransmitters traverse this extracellular space to convey neural information between the two terminals, a process that is essential for various neural functions. The synaptic cleft, which also serves as an interface that regulates the differentiation of synapses, is not simply an empty space; instead, it is filled with matrix proteins forming a scaffold that organizes membrane molecules on the synaptic terminals. To date, the matrix components in synaptic clefts have not been thoroughly identified, especially in the CNS (Nakayama, 2016).

Because synaptic function largely relies on neurotransmitter receptors localized at the postsynaptic membranes, the local density and efficiency of neurotransmitter receptors are critical for proper control of synaptic function. Previous studies showed that several proteins secreted into the extracellular space regulate clustering of neurotransmitter receptors. Agrin, found in vertebrates, is a proteoglycan that clusters AChR at neuromuscular junctions (NMJs). Multiple studies have investigated how Agrin released by motor neurons transmits the signal to various cytoplasmic proteins and eventually to AChR. In Caenorhabditis elegans, LEV-9 and OIG-4, which are released by muscles, promote clustering of AChR at NMJs. The long isoform of C. elegans Punctin/MADD-4, secreted by cholinergic motor neurons, clusters AChRs, whereas its short isoform, released by GABAergic motor neurons, clusters GABAA receptors at the NMJs. In Drosophila NMJs, which are mostly glutamatergic, clustering of glutamate receptors depends on the secreted protein Mind-the-Gap. In mice, Cbln1, which links Neurexin to the glutamate receptor GluD2 at cerebellar synapses, induces GluD2 clustering in culture cells. Thus, several secretory proteins involved in clustering receptors have been studied in cholinergic, GABAergic, and glutamatergic NMJs, as well as in glutamatergic synapses in the CNS. However, the molecular mechanisms underlying the differentiation of other types of synapses remain to be revealed. In addition, it remains unclear how the secreted proteins distribute and organize a matrix within an individual synaptic cleft (Nakayama, 2016).

Previous work identified the hikaru genki (hig) gene in a genetic screen for Drosophila mutants that exhibited reduced locomotor behavior. Hig, a secretory protein with one Ig domain and a maximum of five complement control protein (CCP) domains, localizes to the synaptic clefts of mature and nascent synapses in the brain. Hig localizes predominantly at synaptic clefts of cholinergic synapses in the CNS and regulates the levels of nAChR subunits and DLG, a Drosophila PSD-95 family member, in the postsynaptic terminals. Hig does not simply diffuse over the entire space of the synaptic cleft but, instead, is juxtaposed with the area of nAChR on the postsynaptic membrane. During synaptogenesis, Hig secreted from cholinergic or noncholinergic neurons or even from glia cells is captured in synaptic clefts of cholinergic synapses, suggesting that a specific mechanism is responsible for anchoring Hig to synaptic clefts (Nakayama, 2016).

This study identified Hasp (Hig-anchoring scaffold protein), a CCP domain-containing synaptic matrix protein predominantly localized at synaptic clefts of cholinergic synapses in the Drosophila brain. Hasp has a domain organization resembling that of LEV-9 of Caenorhabditis elegans. The data show that Hasp is required for the synaptic localization of Hig and nAChR subunits; however, Hig and nAChR subunits are not reciprocally required for Hasp localization. High-resolution microscopy revealed that Hig and Hasp are nonuniformly distributed in individual synaptic clefts, suggesting the presence of functionally distinct matrix compartments (Nakayama, 2016).

This study has revealed that Hasp, an matrix component, occupies cholinergic synaptic clefts. Both Hig and Hasp proteins contain multiple CCP domains, and the loss of either protein causes similar behavioral and molecular phenotypes, suggesting that both proteins are involved in the same process of synaptic development or function. Consistent with this, Hasp and Hig localize close to each other at cholinergic synapses. However, high-resolution imaging revealed that these proteins occupy distinct areas within synaptic clefts. These results provide novel insight into the molecular architecture of the synaptic cleft matrix in the CNS and suggest that each of the areas containing Hig or Hasp plays a distinct role in synaptogenesis (Nakayama, 2016).

Genetic analysis revealed that the roles of Hasp and Hig proteins in synaptic differentiation are not identical: although both proteins similarly affect the levels of nAChR subunits and DLG, Hasp is required for Hig to localize at the synaptic cleft, whereas Hig is dispensable for the synaptic localization of Hasp. These functional relationships raise the possibility that Hasp directly regulates the levels of nAChR subunits, as well as those of DLG, and simultaneously mediates anchoring of Hig at synapses. Alternatively, Hasp may only be involved in capture of Hig and regulates the distribution of the synaptic proteins as a secondary consequence of its main function. The data indicate that the altered levels of AChR subunits Dα6, Dα7, and DLG in hasp and hig single mutants and hasp hig double mutants are quantitatively similar, strongly suggesting that the primary role of Hasp is localizing Hig to the synaptic clefts. The close interaction between Hig and nAChR subunits was corroborated by genetic data showing that Dα5, Dα6, and Dα7 are redundantly required for localization of Hig, but not Hasp at synaptic clefts, and also by coimmunoprecipitation of Hig with Dα6 and Dα7. Thus, Hig and the nAChR subunits mutually interact for their synaptic distribution, and the physiologically important role of Hasp is localizing Hig at synaptic clefts (Nakayama, 2016).

In C. elegans, LEV-9, a Hasp homolog, LEV-10, a transmembrane protein containing CUB domains, and Oig-4, a secretory protein containing an Ig domain, are required for LAChR clustering; the absence of any of these proteins, including LAChR, causes the loss of all the other proteins on NMJs. In Drosophila, however, Hasp is localized normally at the synaptic cleft in the CNS when Hig or a subset of nAChR subunits is missing. This difference between the mechanisms underlying synaptic localization of LEV-9 and Hasp could be explained simply by evolutionary diversification among species, or alternatively by differences in synaptic architecture between NMJ and CNS synapses (Nakayama, 2016).

It has not yet been determined how Hasp localizes Hig at synaptic clefts. Hasp may either trap extracellularly diffusing Hig or prevent degradation of Hig localized at synaptic clefts. Hasp contains a WAP domain, which has been implicated in protease inhibition, implying that Hasp stabilizes Hig by preventing its degradation. However, immunoblot analysis indicated that the amounts of full-length and short form Hig polypeptides were unchanged in extracts from hasp mutants, suggesting instead that Hasp recruits Hig at synaptic clefts. Hasp and Hig occupy their respective areas, which may be completely separate or partly overlap with each other. This regional distribution suggests that a single Hasp molecule may not be sufficient to trap Hig. Rather, a number of Hasp molecules may construct a Hasp compartment, which could serve as a scaffold for Hig or a Hig-based compartment maintained within synaptic clefts. A previous study showed that C. elegans LEV-9 must be processed into fragments to cluster AChR at NMJs. Consistent with this, Hasp and Hig are processed to produce truncated polypeptides. Therefore, the patterns of Hig and Hasp staining observed in this study may represent the distribution of a mixture of Hig and Hasp fragments containing their respective N-terminal amino acid-sequences (the antigens used to raise the antibodies) and may not reflect the entire fragment distribution. Further studies are required to reveal the details of Hig and Hasp cleavage, as well as the distribution of the processed fragments in synaptic clefts (Nakayama, 2016).

Hig could regulate the accumulation of nAChR on postsynaptic membranes via either of two mechanisms. Hig has an Ig domain and a maximum of five CCP domains in its C-terminal half and the residual N-terminal half contains an RGD sequence, a putative integrin binding site. This domain organization can be used to form a scaffold complex that may physically interact with nAChR subunits and thereby either maintain clustering of the receptors on postsynaptic membranes or prevent their degradation. Alternatively, Hig may transduce signals through transmembrane proteins into the cytoplasm of postsynaptic terminals and induce clustering of nAChRs that move laterally on the membrane, as reported for Agrin-mediated AChR clustering (Nakayama, 2016).

Mutant analysis revealed that loss of Hig or Hasp resulted in an increase in the level of DLG, as well as a reduction in the levels of Dα6 and Dα7, indicating that Hig also affects the accumulation of cytoplasmic proteins in postsynaptic terminals. It is notable that PSD-95 family members in vertebrates are present at cholinergic synapses, where they function as scaffolds for AChR, as they do for glutamate receptors at glutamatergic synapses. Moreover, synaptic PSD-95 accumulation is increased by reduced synaptic activity and decreased by elevated activity via regulation of phosphorylation or palmitoylation in glutamatergic synapses. The increase of DLG in hasp mutant brains may reflect similar homeostatic regulation in the Drosophila cholinergic synapses: the reduced synaptic activity caused by the decrease in Dα6 and Dα7 levels may activate a compensatory mechanism by which DLG accumulates to a greater extent on postsynaptic membranes (Nakayama, 2016).

On the basis of the current data, a model is proposed that illustrates how the synaptic cleft matrix is constructed during synaptogenesis. During the early stages of synaptogenesis, when synaptic structures are immature, Hasp is secreted extracellularly, diffused, and trapped by an unknown molecule, occupying a particular space in the synaptic clefts of cholinergic synapses. The molecule involved in trapping Hasp may be a secretory or membrane protein localized specifically to the cholinergic synapses. During this and later stages, the Hasp-containing scaffold increases its volume by incorporating new Hasp molecules, and nAChR subunits start to accumulate on postsynaptic membranes. Following Hasp localization, secreted Hig molecules are continuously captured in the differentiating matrix architecture containing the Hasp scaffold, as well as maintained by nAChR subunits, thereby increasing the volume of the Hig-containing scaffold. Reciprocally, the Hig scaffold stabilizes nAChR subunits on the postsynaptic membranes by a physical interaction in synaptic clefts or signaling into the cytoplasm of postsynaptic terminals. In mature cholinergic synapses, the two scaffolding complexes divide synaptic clefts into compartments, reflecting their distinct roles in synaptic differentiation. To further understand the entire process of matrix construction, it will be important to identify other matrix components in the Hasp and Hig scaffold complexes, and especially the Hasp-anchoring molecules (Nakayama, 2016).

The specific localization of both Hig and Hasp at cholinergic synapses suggests that the molecular composition of synaptic matrix may be related to the type of synapse and the distinct complement of neurotransmitters and receptors. In mice, >30 genes encoding predicted CCP proteins are expressed in the CNS. One of these proteins, SRPX2, regulates the formation of glutamatergic synapses in the brain. Further work should attempt to elucidate how these CCP proteins participate in synaptogenesis and how their combinatorial repertoire is involved in the diversification of synaptic properties. Because synaptic clefts are the space through which neurotransmitters disperse, the molecular composition of the matrix may also affect the behavior of neurotransmitters, thereby influencing synaptic plasticity and the efficiency of neurotransmission. Further studies focusing on the matrix architecture of synaptic clefts may reveal novel aspects of synaptic differentiation and function (Nakayama, 2016).

Nicotinic acetylcholine receptor modulator insecticides act on diverse receptor subtypes with distinct subunit compositions

Insect nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of many insecticides, including neonicotinoids, which are the most widely used insecticides in the world. However, the development of resistance in pests and the negative impacts on bee pollinators affect the application of insecticides and have created a demand for alternatives. Thus, it is very important to understand the mode of action of these insecticides, which is not fully understood at the molecular level. This study systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunit mutants to eleven insecticides acting on nAChRs. The results showed that there are several subtypes of nAChRs with distinct subunit compositions that are responsible for the toxicity of different insecticides. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. Moreover, spinosyns may act exclusively on the α6 homomeric pentamers but not any other nAChRs. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for the management of resistance and the development of novel insecticides targeting these important ion channels (Lu, 2022).

Chemical insecticides have been widely used to control pests in the agriculture, horticulture, and forestry industries as well as homes and cities. They have also played a vital role in preventing the spread of human and animal vector-borne diseases. However, insecticide resistance is a serious worldwide problem for invertebrate pest control, and more than 600 different insect and mite species have become resistant to at least one insecticide. In addition, at least one case of resistance to more than 335 insecticides/acaricides has been documented. Therefore, there is great demand for effective insecticide resistance management (IRM) and the development of new pest control compounds. To address both issues, the mode of action of insecticides need to be determines, that is the molecular-level processes underlying the effects of insecticides (Lu, 2022).

A complete understanding of the mode of action of an insecticide requires knowledge of how it affects a specific target site within an organism. Although most insecticides have multiple biological effects, toxicity is usually attributed to a single major effect. For some insecticides, however, the exact molecular targets remain elusive. To ascribe whether a candidate protein is indeed the target for an insecticidal effect in vivo, it is not sufficient to demonstrate an in vitro biochemical interaction between an insecticide and a protein. Genetic evidence demonstrating an effect due to mutation of the candidate target must be obtained before a given protein can be identified as an insecticide target (Lu, 2022).

Neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) are remarkably effective at controlling agricultural pests, ectoparasites and arthropod vectors. They are taken up by the roots or leaves and translocated to all parts of the plant due to high systemic activity, making them effectively toxic to a wide range of sap-feeding and foliar-feeding insects. Thus, neonicotinoids account for 24% of the global insecticide market, which is the largest market share of all chemical classes. They act selectively on insect nicotinic acetylcholine receptors (nAChRs) as agonists compared with the mammalian-selective nicotine. Spinosyns are a naturally derived, unique family of macrocyclic lactones that act on insect nAChR in an allosteric fashion. In addition, sulfoximine sulfoxaflor, butenolide flupyradifurone and mesoionic triflumezopyrim are three newly developed insecticides that are also nAChR competitive modulators. It is expected that the market of all of the above nAChRs targeting insecticides that show excellent insect-to-mammalian selectivity will continue to grow. However, the molecular targets of neonicotinoids and other nAChR modulators remain unclear, mainly because the structure and assembly of native nAChRs in insects have not been clarified (Lu, 2022).

Cation-selective nAChRs are members of the Cys-loop ligand-gated ion channel superfamily responsible for rapid excitatory neurotransmission. The functional nAChRs are homo- or heteromeric pentamers of structurally related subunits arranged around a central ion-conducting pore. Each subunit has an extracellular N-terminal domain that contains six distinct regions (loops A-F) involved in ligand binding, four C-terminal transmembrane segments (TM1-TM4) and an intracellular loop between TM3 and TM4. nAChRs are divided into α-subunits possessing two adjacent cystine residues in loop C, while those subunits without this motif are termed nonα subunits. In vertebrates, 17 nAChR subunits have been identified, and they can coassemble to generate a diverse family of nAChR subtypes with different pharmacological properties and physiological functions. Insects have fewer nAChR subunits (10-12 subunits) according to the available genome data. Although coimmunoprecipitation studies have indicated potential associations of several subunits, the exact subunit composition of native insect nAChRs remains unknown. Unlike the vertebrate counterparts, heterologous expression of genuine arthropod α and β subunits was not successful until two groups recently found that three ancillary proteins are essential for robust expression of arthropod nAChR heteromers. Thus, for a long time, researchers have used hybrid receptors with insect α subunits and mammalian/avian β subunits to study the interaction of insecticides and receptors. However, such alternatives may not faithfully reflect all features of native nAChRs (Lu, 2022).

This study systematically examined the effects of eleven nAChR-targeting insecticides against ten (seven α and three β) Drosophila melanogaster subunit mutants. It was found that there are multiple subtypes of receptors with distinct subunit compositions that are responsible for the toxicity of different insecticides. Artificial activation/inhibition of subunit-expressing neurons also mimicked insecticide poisoning symptoms in pests. Elucidating the molecular targets of these economically important agrochemicals and the assembly of native nAChRs will be very helpful for resistance management and ecotoxicological evaluations of beneficial insects, such as predators and pollinators (Lu, 2022).

The Insecticide Resistance Action Committee (IRAC) classifies neonicotinoids, sulfoximines, butenolides and mesoionics according to their chemical similarity relations into subgroups 4A, 4C, 4D and 4E, respectively. However, the current results showed that sulfoxaflor and flupyradifurone may mainly act on the same nAChR subtype, which consists of α3 and β1 subunits, although other subunits may also be involved considering genetic redundancy. More importantly, it was found that neonicotinoids act on distinct nAChR subtypes and that such selectivity is not dependent on the aromatic heterocyclic (A) or the electron-withdrawing nitro or cyano moiety (X-Y), which is considered the key toxophore. Interestingly, the ring systems and the R2 substituents in the open-chain structures are the determining factors. For example, the α1, α2, β1 and β2 mutants showed similar levels of resistance to imidacloprid and thiacloprid (both have a five-membered ring), indicating that they mainly act on the same α1/α2/β1/β2 pentamer. This finding is consistent with previous ex vivo recording results and two recent reconstituted studies, which showed that both drugs act as partial agonists on the α1/α2/β1/β2 nAChR. Acetamiprid is structurally similar to thiacloprid with the cyanoimine pharmacophore, although the acyclic configuration changes its molecular target in vivo. It may act on the α1/β1/β2 nAChR, and again, electrophysiological studies have already indicated that acetamiprid is nearly a full agonist. Moreover, its potency on the recombinant louse α1/α2/β1/β2 nAChR is approximately 860-fold lower than that of thiacloprid. Although thiamethoxam has a six-membered ring, it is a prodrug without intrinsic nAChR activity until metabolized to the active form clothianidine in plants and insects. Therefore, thiamethoxam, clothianidine, dinotefuran and nitenpyram can be considered the same type, which has N-methyl substitutions in the R2 position and mainly acts on the α1/α3/β1 nAChR. Neonicotinoids are traditionally divided into nitroimines (NNO2), nitromethylenes (CHNO2) or cyanoimines (NCN), although the current findings indicate that they should be classified according to their major nAChR subtype targets (Lu, 2022).

Despite the widespread use of neonicotinoids for almost three decades, the first and only field-evolved target-site resistance mutation (R81T in nAChRβ1) was reported in 2011, and it has only been found in two species to date. This unusual phenomenon is consistent with the findings that the seven neonicotinoids actually act on multiple receptor types in vivo and that only the β1 mutant caused high resistance to all neonicotinoids. New nicotine-mimicking insecticides, such as sulfoxaflor and flupyradifurone, mainly act on another nAChR subtype that is distinct from neonicotinoids, indicating their potential use in insecticide resistance management (Lu, 2022).

Electrophysiological studies with native tissues or recombinant receptors showed that low concentrations of neonicotinoids can block nAChR, while higher concentrations can activate the receptor. Therefore, it is still unclear whether insecticidal activity is the consequence of nAChR inhibition or activation in vivo. This study found that transient artificial activation rather than inhibition of nAChR-expressing neurons was sufficient to induce neonicotinoid-like poisoning symptoms in flies. Thus, the overall effect of neonicotinoids is neuronal depolarization by activation of nAChR, which is more physiologically relevant (Lu, 2022).

Triflumezopyrim is the first member of a new class of mesoionic insecticides that act via inhibition of the orthosteric binding site of the nAChR. It was found that the α1/α2/β1/β2 nAChR could be its major target, similar to imidacloprid and thiacloprid, and all these mutants showed high resistance to triflumezopyrim. This finding is consistent with radioligand binding results showing that triflumezopyrim potently displaced [3H]imidacloprid with a Ki value of 43 nM based on membrane preparations from the aphid. Thermogenetic inhibition of neurons expressing α1, α2 and β2 also mimicked lethargic intoxication symptoms. Thus, to maintain the durability and effectiveness of this new powerful tool for the control of hopper species in rice, it is critical to avoid repeated use of triflumezopyrim with imidacloprid and thiacloprid (Lu, 2022).

Spinosyns, including spinosad and spinetoram, have been shown to act on a population of nAChRs that are not targeted by neonicotinoids, and the binding site is also distinct from the orthosteric site. The α6 subunit has been proposed as the main target of spinosyns since the field-evolved resistance to spinosad is associated with loss-of-function mutations of α6 loci in many pest insects. However, the involvement of other subunits, such as α5 and α7, which are phylogenetically close to α6, has not been clarified. Previous reports showed that α5 and α7 can form functional homomeric and heteromeric channels in vitro while α6 can only form heteromeric channels with α5 or α5/α7 together. It was then asked whether there was genetic redundancy among these evolutionarily conserved subunits. The α5, α7 and α5/α7 double mutants were all sensitive to spinetoram , indicating that spinosyns may exclusively act on the α6 homomeric nAChR, which is consistent with a recent report using spinosad. Thermogenetic activation of α6-expressing neurons also induced spinosyn-like poisoning symptoms in flies (Lu, 2022).

Current knowledge about the subunit composition of insect nAChRs is very limited. Immunoprecipitation data with subunit-specific antibodies showed that Drosophila α3 and β1 coassemble within the same receptor complex. Further studies from the same group indicated that α1/α2/β2 and β1/β2 may coassemble into the same receptor complex. Similar studies using the brown planthopper suggested that there are two populations of nAChRs that contain Drosophila-equivalent subunit combinations α1/α2/β1 and α3/β1/β2. These previous findings are partially confirmed by the present results because α3/β1, α1/α3/β1, α1/β1/β2 and α1/α2/β1/β2 could be the major receptor subtypes for the tested insecticides, indicating that the β1 subunit could be an indispensable component for all heteromecic pentamers. In addition, it was noticed that for some insecticides, different subunit mutations contribute in an asymmetrical manner to resistance. Therefore, there could be functional redundancy between some α-type subunits, and the existence of other potential receptor subtypes, such as α1/β1 and α3/β1/β2 cannot be excluded. The diversity of insect nAChRs and their druggability make them an extremely important target for insecticide development (Lu, 2022).

Growing evidence indicates that sublethal doses of neonicotinoids, such as imidacloprid, thiamethoxam and clothianidin negatively affect wild and managed bees, which are important pollinators in ecosystems and agriculture. They reduce reproduction and colony development, perhaps by impairing the foraging, homing and nursing behaviors of bees. These severe sublethal effects have led to heavy restrictions on the use of the above three neonicotinoids in Europe to protect bee pollinators. Sulfoxaflor and flupyradifurone are potential alternatives for neonicotinoids; however, their risk to bees is controversial. Therefore, it is critical to understand the mode of action of these insecticides inside bees. The core groups of nAChR subunits are highly conserved among different insects spanning ~300 million years of evolution, which is likely due to their essential roles in the nervous system. Most Drosophila nAChR subunit genes (except α5 and β3) have one-to-one orthologs in other insects, including honeybees and bumblebees, and the sequence identities between orthologs are also high. Thus, the expression, assembly and function of these receptors could be conserved between flies and bees, suggesting that the results will enable further studies about the ecotoxicology and risk assessment of these nAChR modulators (Lu, 2022).

Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development

Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest (Ca2+-responsive transactivator, a syntaxin-related nuclear protein that interacts with CREB-binding protein and is expressed in the developing brain) or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. Genetic manipulation, imaging and quantitative dendritic architecture analysis were combined in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dalpha7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter it was demonstrated that AP-1 transcriptional activity is downstream of Dalpha7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. These results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies (Vonhoff, 2013).

By combining genetic and neuroanatomical tools with imaging in a single-cell model, the adult MN5 in Drosophila, this study demonstrates that: (1) AP-1 is transcriptionally active during all stages of postembryonic motoneuron dendritic growth, (2) AP-1 and excitatory cholinergic inputs are required for normal dendrite growth in MN5, (3) AP-1 transcriptional activity is enhanced via a CaMKII-dependent mechanism by increased neural activity during pupal development but not in the adult, and (4) both activity and AP-1 can promote or inhibit dendritic branching, depending on the developmental stage. AP-1 is required for normal MN5 dendrite growth downstream of activity and CaMKII (Vonhoff, 2013).

Although AP-1 has been thought to regulate dendrite development in an activity-dependent manner via global changes in gene expression, probably in a calcium-dependent manner as described for CREB or Crest, direct evidence for this hypothesis was sparse (Vonhoff, 2013).

This study demonstrated that excitatory cholinergic input to MN5 and AP-1 transcriptional activity were required for normal dendrite growth of MN5 during pupal life. MN5 total dendritic length and branch numbers were significantly reduced (~50%) by inhibition of AP-1 [by Jbz (a dominant-negative form of Jun) expression] and in Dα nAChR mutants. Conversely, overexpression of AP-1 or increased MN5 excitability as induced by potassium channel knockdown (by EKI) increased dendritic branching (Duch, 2008). Clearly, AP-1 acted downstream of activity as inhibition of AP-1 by Jbz completely attenuated EKI (electrical knock-in) mediated dendritic growth and branching (Vonhoff, 2013).

A new AP-1 reporter was employed to measure activity-induced AP-1 transcriptional activity by imaging, and to gain insight into the pathway that might connect MN5 activity to AP-1-dependent transcription. Although the detection threshold of this reporter might be too low to detect small changes in AP-1 activity, sensitivity was sufficient to reliably report increased AP-1 activity following overexpression of fos and jun, inhibition of AP-1 transcriptional activity by Jbz expression, and changes in AP-1 activity as induced by various manipulations of cellular signaling. Therefore, the reporter was deemed suitable for testing changes in AP-1 transcriptional activity in MN5 (Vonhoff, 2013).

Targeted expression of TrpA1 channels in MN5 allowed the induction of firing in vivo by temperature shifts during selected developmental periods. Activation of MN5 during pupal life for 36 hours (P9 to adult) or longer (P5 to adult) caused significant increases in AP-1-induced nuclear GFP fluorescence. By contrast, in adults neither similar nor longer durations of TrpA1 activation resulted in any detectable increase in AP-1 reporter-mediated nuclear GFP fluorescence in MN5. Similarly, live imaging in semi-intact adult preparations did not reveal any detectable AP-1 activity upon acute TrpA1 activation for various durations. This indicated that activity-dependent AP-1 activation was restricted to pupal life. However, whether AP-1 activation in the adult MN5 occurred upon patterned activity was not tested. Spaced stimuli that reflect endogenous activity patterns are required for insect motoneuron axonal and dendritic development and can regulate mammalian neuron dendritic morphology. However, during flight, MN5 fires tonically at frequencies between 5 and 20 Hz, a pattern that is well reflected by temperature-controlled TrpA1 channel activation. Therefore, adult flight behavior is unlikely to induce AP-1 activity, which is involved in dendrite and synapse development (Freeman, 2010). This is consistent with the assumption that dendritic structure is fairly stable in the adult (Vonhoff, 2013).

cAMP and Jun N-terminal kinase (Jnk) have been implicated as potential links between activity and AP-1 activation. Cell culture studies on Drosophila larval motoneurons and giant neurons demonstrate a role of calcium. This study showed that Dα7 nAChRs, which are highly permeable to calcium, were required for normal MN5 dendritic growth. Combining genetic manipulation of Dα7 nAChRs, AP-1 and CaMKII with imaging of AP-1 reporter activity revealed that CaMKII was required downstream of Dα7 nAChRs to cause AP-1-dependent transcription. These data show that activity-dependent calcium influx through nAChRs might activate AP-1 during pupal life via a CaMKII-dependent mechanism in vivo. Activity and AP-1 can promote or inhibit dendritic growth during pupal life, depending on timing (Vonhoff, 2013).

In larval motoneurons, AP-1 is required for dendritic overgrowth as induced by artificially increased activity (Hartwig, 2008). In MN5, AP-1 is required downstream of nAChRs and CaMKII for normal dendritic growth. By contrast, premature expression of AP-1 in MN5 inhibited dendritic growth. These data were consistent with the hypothesis that timing is the crucial factor. First, P103.3 and D42 both caused similar overgrowth but exhibited fairly different expression patterns. Second, C380-GAL4 and Dα7 nAChR-GAL4 both inhibited MN5 dendrite growth but expressed in largely different sets of neurons. Therefore, the common factor of C380 and Dα7 nAChR on the one hand and D42 and P103.3 on the other hand was timing. Third, shifting the timing of C380-GAL4-driven AP-1 expression to later stages prevented dendritic defects. Fourth, imposed activity prior to P5 by TrpA1 activation also inhibited dendritic branching. Dendritic defects as induced by imposed premature activity were rescued by inhibition of AP-1 via Jbz expression in MN5 (Vonhoff, 2013).

MN5 early dendritic growth starts at early pupal stage 5 (P5), and expression of Dα7 nAChRs begins 2.5 hours later, at mid stage P5. Similarly, Xenopus optic tectal and turtle cortical neurons receive glutamatergic and GABAergic inputs as soon as the first dendrites are formed. In vertebrates, early synaptic inputs and neurotransmitters play essential roles in dendrite development. The current data are consistent with the hypothesis that the endogenous expression of nAChRs caused increased activity throughout the developing motor networks, which, in turn, upregulated AP-1-dependent transcription and dendritic growth via a CaMKII-dependent mechanism. During zebrafish spinal cord development, activity is required for strengthening functional central pattern generator (CPG) connectivity. As dendrites are the seats of input synapses to motoneurons, an activity-dependent component in motoneuron dendritic growth that follows early synaptogenesis might function to refine dendrite shape during the integration into the developing CPG (Vonhoff, 2013).

Inactivity-induced increase in nAChRs upregulates Shal K(+) channels to stabilize synaptic potentials

Long-term synaptic changes, which are essential for learning and memory, are dependent on homeostatic mechanisms that stabilize neural activity. Homeostatic responses have also been implicated in pathological conditions, including nicotine addiction. Although multiple homeostatic pathways have been described, little is known about how compensatory responses are tuned to prevent them from overshooting their optimal range of activity. This study found that prolonged inhibition of nicotinic acetylcholine receptors (nAChRs), the major excitatory receptors in the Drosophila CNS, resulted in a homeostatic increase in the Drosophila α7 (Dα7)-nAChR. This response then induced an increase in the transient A-type K+ current carried by Shaker cognate L (Shal; also known as voltage-gated K+ channel 4, Kv4) channels. Although increasing Dα7-nAChRs boosted miniature excitatory postsynaptic currents, the ensuing increase in Shal channels served to stabilize postsynaptic potentials. These data identify a previously unknown mechanism for fine tuning the homeostatic response (Ping, 2012).

Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway

Detecting motion is a feature of all advanced visual systems, nowhere more so than in flying animals, like insects. In flies, an influential autocorrelation model for motion detection, the elementary motion detector circuit (EMD), compares visual signals from neighboring photoreceptors to derive information on motion direction and velocity. This information is fed by two types of interneuron, L1 and L2, in the first optic neuropile, or lamina, to downstream local motion detectors in columns of the second neuropile, the medulla. Despite receiving carefully matched photoreceptor inputs, L1 and L2 drive distinct, separable pathways responding preferentially to moving 'on' and 'off' edges, respectively. Serial electron microscopy (EM) identifies two types of transmedulla (Tm) target neurons, Tm1 and Tm2, that receive apparently matched synaptic inputs from L2. Tm2 neurons also receive inputs from two retinotopically posterior neighboring columns via L4, a third type of lamina neuron. Light microscopy reveals that the connections in these L2/L4/Tm2 circuits are highly determinate. Single-cell transcript profiling suggests that nicotinic acetylcholine receptors mediate transmission within the L2/L4/Tm2 circuits, whereas L1 is apparently glutamatergic. It is proposed that Tm2 integrates sign-conserving inputs from neighboring columns to mediate the detection of front-to-back motion generated during forward motion (Takemura, 2011).

Given that both L2 and L4 express Choline acetyltransferase (Cha) and are thus genotypically qualified to synthesize acetylcholine and provide cholinergic input to Tm2, the expression of acetylcholine receptors in Tm2 was profiled. This proved more complex than for L2 and L4. In addition to Dα7 and Dβ1 nAcR shared with L2 and L4, Tm2 also expressed Dα1/2 and Dβ2 nAcR. The exclusive expression of nicotinic rather than muscarinic receptors (nAcR not mAcR) in Tm2 suggests that both L2 and L4 provide fast excitatory inputs to Tm2. It was also found that Tm2 expressed Cha but not VGlut, indicating that, like L2 and L4, Tm2 is also genotypically cholinergic. In summary, these data predict that both synaptic connections in the L2/L4/Tm2 network are mediated by excitatory acetylcholine systems, and therefore sign-conserving (Takemura, 2011).

While either the L1 or L2 channel alone can mediate rudimentary motion detection, each also responds differentially in walking flies, and in head-yaw assays the L2 pathway is preferentially tuned to front-to-back motion. Although the connections between L4 and L2 along the anteroposterior direction might account for this front-to-back preference, these connections are reciprocal so that information also flows from posterior to anterior, while L2's activity fails to reveal asymmetrical responses. Between L2's two targets, only Tm2 receives two additional L4 inputs from neighboring posterior columns; Tm1 does not. These L2/L4/Tm2 connections are highly determinate, underscoring a critical role in connecting neighboring L2 channels along the AP direction, in what is arguably the most important motion direction for flies since it occurs during forward flight. Interestingly, other flies have a Tm neuron closely resembling Drosophila's Tm2 morphologically, for example Tm1 in the calliphorid Phaenicia. This is proposed to receive L2 inputs, suggesting that an L2/L4/Tm2 network might be conserved in higher Diptera (Takemura, 2011).

Tm2 could conceivably serve as half of the EMD's multiplier stage, comparing the temporally delayed input from collateral L4s with the cognate signal from L2. However, electrophysiological investigations on calliphorid 'Tm1' neurons, which resemble morphologically Drosophila's Tm2, have yet to provide strong evidence for this role. An alternative interpretation is that the L2/L4/Tm2 network serves instead as a prefilter in the preprocessing stage while Tm2's output feeds into the multiplier stage. The topology and sign-conserving nature of L4/Tm2 connections suggest the spatial summation of neighboring visual signals, which could increase light sensitivity at the expense of spatial acuity. It has been suggested that under low luminance conditions, neighboring visual signals are pooled prior to their interaction at the multiplier stage, while at higher luminance levels nearest-neighbor interactions dominate motion detection. Alternatively, the L4/Tm2 connections could convert visual signals sampled from the hexagonal ommatidial array into an orthogonal coordinate upon which motion signals can be derived. Differentiating between these possibilities must await future investigations that combine genetic and electrophysiological approaches (Takemura, 2011).

A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dα6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dα6, providing convincing evidence that Dα6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dα6 alone, co-expression of Dα6 with an additional nAChR subunit, Dα5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR (Watson, 2010).

Dα6 knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad

A null mutation of the nicotinic acetylcholine receptor (nAChR) subunit Dα, in Drosophila melanogaster, confers 1181-fold resistance to a new and increasingly important biopesticide, spinosad. This study's molecular characterisation of a spinosad resistance mechanism identifies Dα6 as a major spinosad target in D. melanogaster. Although D. melanogaster is not a major field pest, target site resistances found in this species are often conserved in pest species. This, combined with the high degree of evolutionary conservation of nAChR subunits, suggests that mutations in Dα orthologues may underpin the spinosad resistance identified in several economically important field pests (Perry, 2007).

Host-cell specific effects of the nicotinic acetylcholine receptor chaperone RIC-3 revealed by a comparison of human and Drosophila RIC-3 homologues

RIC-3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). This study reports the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC-3 (DmRIC-3). Heterologous expression studies of alternatively spliced DmRIC-3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled-coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline-rich N-terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC-3 to influence nAChR maturation was examined in co-expression studies with human alpha7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC-3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC-3. In contrast, when expressed in a human cell line, human RIC-3 enhanced nAChR maturation more efficiently than DmRIC-3. The cloning and characterisation of 11 alternatively spliced DmRIC-3 isoforms has helped to identify domains influencing RIC-3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC-3 (Landsell, 2008).

Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector, pest and beneficial species

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dalpha2 and a vertebrate beta2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin (Jones, 2007).

The nicotinic acetylcholine receptor Dα7 is required for an escape behavior in Drosophila

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dalpha7 nicotinic acetylcholine receptor (nAChR) of Drosophila shows that it is required for the giant fiber-mediated escape behavior. The Dα7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, it was found that gfA1, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele of Dα7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dalpha7 nAChR in giant fiber-mediated escape in Drosophila (Fayyazuddin, 2006).

Using anatomical, behavioral and physiological techniques to analyze mutant alleles of a nAChR, Dα7, this receptor has been shown to be essential for the giant fiber-mediated escape response inDrosophila. Flies with mutations inDα7 do not jump in response to a “lights off” stimulus. Using electrophysiological and anatomical evidence, it was shown that the visual and mechanosensory inputs on the dendrites of the giant fiber are cholinergic, and that loss of Dα7 in the giant fiber is responsible for the behavioral deficit in the visually mediated escape response. Furthermore, the cholinergic synapse between the the peripherally synapsing interneuron (PSI) and dorsal longitudinal muscle motor neurons (DLMmn) is defective inDα7 mutants. Finally, this study found thatgfA1, a previously molecularly uncharacterized mutant isolated in a behavioral screen for giant fiber defects, is a missense mutant ofDα7 that shows diminished EPSPs at the PSI-DLMmn synapse (Fayyazuddin, 2006).

ThegfA1 mutant phenotype is caused by the amino acid substitution K46E in loop 2 of the ligand binding domain of Dα7. Interestingly, although mutations in loop 2 of the ligand binding domain of nAChRs have been the focus of a number of structure-function studies in recent years, this is the first mutant in this domain that has been linked to a genetic phenotype. This region has been implicated in coupling ligand binding to gating in a number of cys-loop receptors that include nAChRs, GABA receptors, and glycine receptors. Charge reversal mutations in K46 of bovine α7 nAChRs, the homolog of Dα7K46, show diminished responses to acetylcholine and can act in a dominant-negative fashion when coexpressed with wild-type receptors, similar to what was observed for the gfA1 mutation. The data show that the charge of Dα7K46 is critical to the functioning of this receptor and hence synapses mediated by Dα7 containing nAChRs (Fayyazuddin, 2006).

The giant fiber circuit has been a model for central circuits inDrosophila and has been studied in some detail using elegant experiments that revealed significant details about the circuit in the intact fly. The giant fiber output in the thorax activates at least three pathways: the tergotrochanteral motor (TT<) neuron via electrical synapses, the DLM motor neuron via an interneuron (PSI) that is itself electrically coupled to the giant fiber, and the tibial levator muscle motor neuron through a novel pathway that is not well characterized. This study show that the PSI-DLMmn synapse, which was previously suggested to be cholinergic. It is remarkable that redundancy does not extend to the circuit underlying one of the most important behaviors for the day-to-day survival of the fly, the giant fiber-mediated escape circuit. This fact suggests that particular properties of Dα7 may be selected for during evolution to endow certain qualities to the circuit. Further characterization of the biophysical properties of Dα7 both in vivo and in vitro should shed some light on how specializations at the synaptic level are implemented in the choice of neurotransmitter receptor (Fayyazuddin, 2006).

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila (Wu, 2005).

Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing

Genome analysis of the fruit fly reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in alpha-type nicotinic acetylcholine receptor subunits. The subunits are designated Dalpha5, Dalpha6, and Dalpha7. Cloning of the Dalpha5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dalpha6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dalpha6 involves exons encoding nAChR functional domains. The Dalpha6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dalpha6, four of which are also edited in the Dalpha6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels (Grauso, 2002).

In the mutant Drosophila dADAR- that completely lacks ADAR activity, site-specific A-to-I editing of all known pre-mRNA targets in Drosophila is abolished. RT-PCR on dADAR mutant RNA for the Dalpha6 gene showed only adenosine in all the seven editing sites identified, thus demonstrating that Dalpha6 editing is dADAR dependent and is abolished in the ADAR mutant fly. In mammals, editing by ADAR has been shown to occur within the context of predicted RNA secondary structure formed through interactions between exon and intron sequences. RNA secondary structure leading to base pairing between the main group of editing sites in Dalpha6 exon 5 and its downstream or upstream intron were sought. In both cases, the edited region seems to form base pairing within exon 5 itself. A similar result was obtained for the Fsp site in the para channel (Grauso, 2002).

The presence of two additional putative sites of editing, found only in the adult Dalpha6 EST clone (sites 1 and 2), suggests that some sites could be edited in a stage-specific manner. The existence of such developmentally regulated editing has been also demonstrated at two of the four editing sites in the D. melanogaster para channel transcript, the Ssp and Sfc sites. It is speculated that Dalpha6 alternative splicing of multiple exons could also be developmentally regulated, as recently found for the exon 4 region of the Drosophila Dscam pre-mRNA (Grauso, 2002). Most of the Dalpha6 residues changed by both alternative splicing and pre-mRNA editing are localized to key functional domains like the ligand-binding loops and TM2. The dADAR mutant fly, where editing is missing in a number of ligand and voltage-gated ion channels, exhibits various age-dependent behavioral deficits accompanied by neurodegeneration. The dADAR mutant has also been independently discovered in a genetic screen for mutants sensitive to hypoxia conditions. Electrophysiological recordings on primary culture of dADAR mutant neurons show that the para channel conductivity is altered in the mutant fly especially in oxygen deprivation conditions. This result clearly indicates that misediting of channels could result in neuronal activity defects. Thus the not-edited/edited Dalpha6 subunit-containing receptors could play a critical role in nervous system function and integrity. Because editing at some Dalpha6-specific sites occurs also in the homologous positions of the alpha7-2 H. virescens gene (sites 3–6), there is a high degree of evolutionary conservation of the pre-mRNA editing between two distantly related insect groups (moths and flies). This implies modifications introduced by editing are of functional relevance (Grauso, 2002).


Search PubMed for articles about Drosophila nicotinic Acetylcholine Receptor

Albuquerque, E. X., Pereira, E. F., Alkondon, M. and Rogers, S. W. (2009). Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89: 73-120. PubMed ID: 19126755

Barnstedt, O., Owald, D., Felsenberg, J., Brain, R., Moszynski, J. P., Talbot, C. B., Perrat, P. N. and Waddell, S. (2016). Memory-relevant mushroom body output synapses are cholinergic. Neuron 89: 1237-1247. PubMed ID: 26948892

Borst, A. (2018). A biophysical mechanism for preferred direction enhancement in fly motion vision. PLoS Comput Biol 14(6): e1006240. PubMed ID: 29897917

Duch, C., Vonhoff, F. and Ryglewski, S. (2008). Dendrite elongation and dendritic branching are affected separately by different forms of intrinsic motoneuron excitability. J Neurophysiol 100: 2525-2536. PubMed ID: 18715893

Fayyazuddin, A., Zaheer, M. A., Hiesinger, P. R. and Bellen, H. J. (2006). The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila. PLoS Biol 4: e63. PubMed ID: 16494528

Fendl, S., Vieira, R. M. and Borst, A. (2020). Parallel Visual Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons. Elife 9. PubMed ID: 33079061

Freeman, A., Bowers, M., Mortimer, A. V., Timmerman, C., Roux, S., Ramaswami, M. and Sanyal, S. (2010). A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila. Brain Res 1326: 15-29. PubMed ID: 20193670

Grauso, M., Reenan, R. A., Culetto, E. and Sattelle, D. B. (2002). Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing. Genetics 160: 1519-1533. PubMed ID: 11973307

Halevi, S., McKay, J., Palfreyman, M., Yassin, L., Eshel, M., Jorgensen, E. and Treinin, M. (2002). The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors. EMBO J 21: 1012-1020. PubMed ID: 11867529

Jones, A. K., Brown, L. A. and Sattelle, D. B. (2007). Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector, pest and beneficial species. Invert Neurosci 7: 67-73. PubMed ID: 17216517

Jones, A. K. and Sattelle, D. B. (2009). Diversity of insect nicotinic acetylcholine receptor subunits. Adv Exp Med Biol. 683: 25–43. PubMed ID: 20737786

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Biological Overview

date revised: 18 February 2024

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