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 links for nAChRα5: Precomputed BLAST | EntrezGene

NCBI links for nAChRα6: Precomputed BLAST | EntrezGene

NCBI links for nAChRα7: Precomputed BLAST | EntrezGene
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.


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).

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).

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).


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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

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

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

Lansdell, S. J., Collins, T., Yabe, A., Gee, V. J., Gibb, A. J. and Millar, N. S. (2008). Host-cell specific effects of the nicotinic acetylcholine receptor chaperone RIC-3 revealed by a comparison of human and Drosophila RIC-3 homologues. J Neurochem 105: 1573-1581. PubMed ID: 18208544

Lansdell, S. J., Schmitt, B., Betz, H., Sattelle, D. B. and Millar, N. S. (1997). Temperature-sensitive expression of Drosophila neuronal nicotinic acetylcholine receptors. J Neurochem 68: 1812-1819. PubMed ID: 9109505

Lansdell, S. J., Collins, T., Goodchild, J. and Millar, N. S. (2012). The Drosophila nicotinic acetylcholine receptor subunits Dalpha5 and Dalpha7 form functional homomeric and heteromeric ion channels. BMC Neurosci 13: 73. PubMed ID: 22727315

Littleton, J. T. and Ganetzky, B. (2000). Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 26: 35-43. PubMed ID: 10798390

Millar, N. S. (2008). RIC-3: a nicotinic acetylcholine receptor chaperone. Br J Pharmacol 153 Suppl 1: S177-183. PubMed ID: 18246096

Nakayama, M., Matsushita, F. and Hama, C. (2014). The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain. J Neurosci 34: 13872-13877. PubMed ID: 25319684

Nakayama, M., Suzuki, E., Tsunoda, S. and Hama, C. (2016). The matrix proteins Hasp and Hig exhibit segregated distribution within synaptic clefts and play distinct roles in synaptogenesis. J Neurosci 36: 590-606. PubMed ID: 26758847

Perry, T., McKenzie, J. A. and Batterham, P. (2007). A Dα6 knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad. Insect Biochem Mol Biol 37: 184-188. PubMed ID: 17244547

Ping, Y. and Tsunoda, S. (2012). Inactivity-induced increase in nAChRs upregulates Shal K(+) channels to stabilize synaptic potentials. Nat Neurosci 15: 90-97. PubMed ID: 22081160

Sattelle, D. B., Jones, A. K., Sattelle, B. M., Matsuda, K., Reenan, R. and Biggin, P. C. (2005). Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster. Bioessays 27: 366-376. PubMed ID: 15770687

Takemura, S. Y., Karuppudurai, T., Ting, C. Y., Lu, Z., Lee, C. H. and Meinertzhagen, I. A. (2011). Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway. Curr Biol 21: 2077-2084. PubMed ID: 22137471

Taly, A., Corringer, P. J., Guedin, D., Lestage, P. and Changeux, J. P. (2009). Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat Rev Drug Discov 8: 733-750. PubMed ID: 19721446

Vonhoff, F., Kuehn, C., Blumenstock, S., Sanyal, S. and Duch, C. (2013). Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development. Development 140: 606-616. PubMed ID: 23293292

Watson, G. B., Chouinard, S. W., Cook, K. R., Geng, C., Gifford, J. M., Gustafson, G. D., Hasler, J. M., Larrinua, I. M., Letherer, T. J., Mitchell, J. C., Pak, W. L., Salgado, V. L., Sparks, T. C. and Stilwell, G. E. (2010). A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression. Insect Biochem Mol Biol 40: 376-384. PubMed ID: 19944756

Wu, P., Ma, D., Pierzchala, M., Wu, J., Yang, L. C., Mai, X., Chang, X. and Schmidt-Glenewinkel, T. (2005). The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor. J Biol Chem 280: 20987-20994. PubMed ID: 15781463

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date revised: 10 April 2017

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