InteractiveFly: GeneBrief

Choline acetyltransferase & Vesicular acetylcholine transporter: Biological Overview | References

While the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles (SVs), accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. This study extend the role of the VAChT, which is nested within the first intron of the choline acetyltransferase gene (ChaT), to include regulating the transmitter content of SVs. Manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, it was found that reduction of the polyQ region, by one glutamine residue (13Q to 12Q), results in a significant increase in both amplitude and frequency of spontaneous cholinergic miniature EPSCs (mEPSCs) recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine (13Q to 14Q) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked SV release. Finally, a complete deletion of the polyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure SVs are filled to physiologically-appropriate levels remain unknown. This study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission (Vernon, 2019).

Vesicular loading of synaptic vesicles (SVs) is dependent on initial acidification mediated by the vATPase pump. This pump generates both a pH gradient (ΔpH) and a voltage gradient (ΔΨ) across the SV membrane. The relative requirement for these two components for loading is dependent on neurotransmitter: anionic transmitters such as glutamate rely more heavily on ΔΨ. Zwitterionic transmitters require both gradients, whereas, cationic transmitters (e.g., ACh) rely predominantly on ΔpH. Transport of ACh into a SV involves the exchange of two protons in an antiporter system using the proton-electrochemical gradient. The current model suggests that one proton is used to transport ACh into the SV lumen while the second proton is needed to re-orientate the VAChT substrate binding site back toward the cytoplasm (2H+ for 1ACh+). In situ, acidified SVs exhibit a pH ~1.4 units less than un-acidified SVs. Theoretically, the cholinergic SV lumen has the capacity to concentrate ACh by 100-fold relative to cytoplasmic levels (which range between 1 and 4 mM). However, the maximal reported accumulation of ACh in SVs has been found to saturate at ~4 mM, suggesting a rather dramatic (and unknown) limiting factor impedes loading (Vernon, 2019).

A key limiting factor may be copy number of functional transporter per SV. Murine and Drosophila NMJ and mammalian cell culture models suggests vesicular loading is altered following either genetic and/or pharmacological manipulation of transporter activity. However, it is notable that upregulation of VAChT expression fails to show effects to quantal size at either snake NMJ or Drosophila motoneurons that receive cholinergic excitation (Cash, 2016). An inability of increased transporter to affect SV loading is consistent with a set-point model of filling. This model posits that SVs fill to a predetermined level, independent of filling rate, which changes following manipulation of transporter expression level. It has been previously reported that transgenic expression of VAChT, which carries a single glutamine truncation in a C-terminal poly-glutamine (polyQ) region (13Q to 12Q), results in increased quanta of spontaneously released SVs at identified interneuron to motoneuron synapses (Cash, 2016). This region, therefore, may contribute to the mechanism that regulates SV loading (Vernon, 2019).

This study use electrophysiological characterization of cholinergic release at Drosophila larval and embryonic interneuron->motoneuron synapses to investigate the physiologic implications to SV loading when the VAChT C-terminal polyQ region is manipulated. In agreement with previously published literature, this study found that expression of a single glutamine truncation VAChT^12Q increases both amplitude and frequency of spontaneously released cholinergic miniature EPSCs (mEPSCs; i.e., individual SV release) recorded from aCC and RP2 motoneurons. Evoked synaptic currents also show an increased duration consistent with an increased ACh load. Conversely, this study further shows that CRISPR induced single amino acid extension of the polyQ region (VAChT^14Q) results in the opposite effect: reduced mEPSC amplitude and frequency and, moreover, an inability to support evoked release. CRISPR mediated deletion of the polyQ region (VAChT^ΔQ) has no effect on mEPSC kinetics suggesting that elongation or truncation of the VAChT polyQ region is more detrimental to cholinergic functioning than its removal (Vernon, 2019).

It is notable that although mEPSC amplitude is increased following expression of VAChT^12Q the effect to evoked spontaneous rhythmic current (SRCs) is limited to increased duration. It is speculated that this may be indicative that the postsynaptic nAChR receptor field is already fully saturated under endogenous conditions and heightened cholinergic tone, through VAChT^12Q upregulation, is thus restricted to increasing SRC duration. Similarly, it is speculated on why increased SRC duration is accompanied by a decrease in SRC frequency. A possible explanation is a homeostatic-type negative feedback mechanism which acts to dampen the activity of presynaptic interneurons that form the central pattern generator controlling locomotor output. Future experiments will be required to clarify these issues (Vernon, 2019).

The results suggest that the length of the polyQ domain is both deterministic for SV filling and for the probability of SV release. Reducing glutamines by one residue is sufficient to increase SV load and release probability and vice versa. Moreover, addition of a glutamine (14Q) is sufficient to remove the ability of the CNS to generate a rhythmic fictive locomotor pattern, which is reliant on evoked release. It is rationalized that VAChT^14Q disrupts cholinergic loading, generating partially-filled SVs that, in turn, prevent evoked synaptic release. By contrast, increasing SV loading (12Q) results in evoked release events of longer duration. These observations are in agreement with recent work using a light activated vATPase pump (pHoenix) localized to SVs (Rost, 2015). Rost used this tool to show that glutamatergic vesicles are only 'nearly full' under normal conditions (i.e., can be further filled) and, moreover, show thatvesicle load is proportional to release probability (Rost, 2015). The current data are supportive of this observation: only increased SV loading supports evoked release. Moreover, the results are also indicative of a set point model, in which vesicles can only release once they surpass a threshold load. This hypothesis proposes two distinct models of SV loading. The set-point model proposes a mechanism restricting the amount of neurotransmitter per vesicle to a fixed maximum, whereas, the steady state model suggests the amount of neurotransmitter that enters a SV is offset by leakage, but that both are independent variables that can autonomously change to produce SVs with variable levels of filling. The set point model is consistent with observations at the snake NMJ and Drosophila central neurons. Whereas, the steady state model better describes loading at murine and Drosophila NMJ and in mammalian cell culture models (Vernon, 2019).

Analysis of related Drosophila species reveal polyQ regions of differing lengths (e.g., nine in D. willistoni, 11 in D. simulans, and 15 in D. pseudoobscura). It is tempting to speculate that evolution may have manipulated the length of the polyQ region to alter SV content in these related species. However, recordings from aCC/RP2 in these related species show mEPSC amplitude is remarkably conserved. Thus, the predicted effect of SV loading due to change in polyQ length, across these related species, may have been abrogated by compensatory mutations in other regions of the VAChT. A comparative analysis may thus be useful to identify such regions for future study (Vernon, 2019).

The VAChT polyQ region is specific to insects. A BLAST search comparison shows no other insect neuronal vesicular transporter possesses a C-terminal polyQ domain. Mammalian VAChT possesses a di-leucine motif in the same approximate location to the insect polyQ domain. The di-leucine motif is well established as a trafficking region. Removal of the mammalian VAChT C-terminal tail, or specific mutation of the di-leucine motif, results in mislocalization of the transporter to the neuronal membrane. Mutant Htt protein containing a polyQ expansion from 20Q to 120Q was found to preferentially bind to SVs in murine axon terminals and, further, to displace the binding of Huntington-associated protein (HAP1) usually co-localized to SVs. 120Q mutants were also shown to reduce glutamate release suggesting a direct interaction between extended polyQ domains and synaptic release. HAP1 has also been shown to bind synapsin 1 which is critical for SV pool mobilization and formation. It is therefore theorized that the polyQ region in VAChT may play a similar role in trafficking the transporter to the SV, plasma membrane and/or SV pool formation (Vernon, 2019).

It is notable that complete removal of the VAChT polyQ region does not influence mEPSCs, although it does alter SRC kinetics (increasing their duration). This dichotomy may mirror an increasingly accepted molecular distinction between spontaneous (mEPSCs) and synchronous (SRC/EPSC) release modalities. Other work has shown, for example, that mEPSC release is maintained in the absence of the vesicle associated SNARE protein synaptobrevin, while evoked release is halted. Munc-13 has also be shown to influence the spatial localization of evoked release while having no effect on mEPSCs at C. elegans NMJ. These observations are predictive of a model in which multiple fusion complexes are physiologically separate and dependent on the modality of release. Moreover, a role for VAChT in SV release is indicated by a reported interaction between synaptobrevin and VAChT. A glycine to arginine substitution (G342R) in VAChT is sufficient to reduce cholinergic mediated larval motility in C. elegans, an effect that is rescued by a complimentary substitution of an isoleucine to an aspartate in synaptobrevin (Vernon, 2019).

VAChT^ΔQ mutants show early larval mortality (L1) despite being able to produce SRCs. This is further confused by the similarity in SRC kinetics with chaB19>VAChT^12Q which produce viable L3 larvae and adults. Early VAChT^ΔQ mortality is attributed to the lack of wild-type transporter present in the VAChT^ΔQ genetic background and may be consistent with cholinergic deficiencies presented in wider physiologic function. In humans, ChAT immunoreactivity and nAChR/mAChR expression is observed in non-neuronal epithelial, endothelial, mesothelial and immune cells and are shown to modulate multiple cellular processes including but not exclusive to, cellular migration and apoptosis, proliferation, anti/proinflammatory responses and histamine release. In insects, non-neuronal ACh has been shown to be heavily influential in reproduction and larval development and so it remains possible that VAChT modulation may alter wider, and currently unknown, physiologic aspects of larval development (Vernon, 2019).

The effects reported in this study relating to expression of VAChT^12Q (truncation) versus VAChT^14Q (expansion) were achieved using different experimental conditions. VAChT^12Q was tested using Gal4-based overexpression in an otherwise wild-type VAChT background, while VAChT^14Q was tested using a CRISPR mutant. This was because an attempt to make VAChT^12Q via CRISPR was unsuccessful. Thus, the results reported in this study must be tempered. Indeed, the co-presence of wild-type VAChT in VAChT^12Q upregulation may, to some extent, reduce the observed phenotype. Moreover, protein level, nor protein localization, was measured and thus the possibility remains that the VAChT^14Q mutation may affect expression levels and/or vesicular localization, which makes it difficult to reach firm conclusions about results obtained. However, it is not believed this detracts from the interpretation of the data presented within this study (Vernon, 2019).

Since the first demonstration of fixed quanta that describes spontaneous release of SVs, a key question of 'how does a SV know when it is full' remains to be answered. The polyQ region of the Drosophila VAChT, that is reported in this study, seemingly orchestrates the filling of cholinergic SVs at central synapses. Future studies to identify the function of this region, including identification of binding partners, provide optimism for understanding how SVs monitor their fill state (Vernon, 2019).

Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development

The remarkable diversity of neurons in the nervous system is generated during development, when properties such as cell morphology, receptor profiles and neurotransmitter identities are specified. In order to gain a greater understanding of neurotransmitter specification this study profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons in vivo at three developmental time points. 86 differentially expressed transcription factors were identified that are uniquely enriched, or uniquely depleted, in a specific neurotransmitter type. Some transcription factors show a similar profile across development, others only show enrichment or depletion at specific developmental stages. Profiling of Acj6 (cholinergic enriched) and Ets65A (cholinergic depleted) binding sites in vivo reveals that they both directly bind the ChAT locus, in addition to a wide spectrum of other key neuronal differentiation genes. It was also shown that cholinergic enriched transcription factors are expressed in mostly non-overlapping populations in the adult brain, implying the absence of combinatorial regulation of neurotransmitter fate in this context. Furthermore, the data underlines that, similar to Caenorhabditis elegans, there are no simple transcription factor codes for neurotransmitter type specification (Estacio-Gómez, 2020).

Neurotransmitter identity is a key property of a neuron that needs to be tightly regulated in order to generate a properly functioning nervous system. This study has investigated the dynamics and extent of transcription factor specificity in fast-acting neurotransmitter neuronal types in Drosophila. The transcription state of cholinergic, GABAergic and glutamatergic neurons was profiled in the developing and adult brain of Drosophila. Enriched Pol II occupancy was observed at the relevant neurotransmitter synthesis genes and other genes associated with the activity of the specific types. The monoamine neurotransmitter related genes Vmat, DAT and Tdc2 are enriched in glutamatergic neurons, which is not unprecedented, as monoamine populations can also be glutamatergic (Aguilar, 2017; Trudeau, 2018). Cholinergic, GABAergic, serotonergic and dopaminergic receptors are enriched in embryonic GABAergic neurons relative to the other two fast-acting neurotransmitter types, which correlates with GABAergic interneurons acting as integrative components of neural circuits. The enrichment of MAP kinase pathway regulators in cholinergic neurons is intriguing, suggesting that this signalling pathway may have a specific role in these neurons. This is supported by a recent study showing that MAP kinase signalling acts downstream of Gq-Rho signalling in C. elegans cholinergic neurons to control neuron activity and locomotion (Estacio-Gómez, 2020).

Importantly, this study has uncovered and highlighted transcription factors and non-coding RNAs differentially expressed between these types. Some of these are expected based on previous studies in Drosophila, including acj6 (cholinergic) (Lee, 2002) and Dbx (GABAergic) (Lacin, 2019). Also, studies in other model organisms fit with the current findings, for example, cholinergic enriched knot, whose orthologue, UNC-3 (C. elegans), is a terminal selector for cholinergic motor neuron differentiation. In addition, RFX, the vertebrate orthologue of Rfx, which was identified as glutamatergic enriched, can increase the expression of the neuronal glutamate transporter type 3. However, this study has identified many differentially expressed transcription factors that have not had their role studied with respect to neurotransmitter specification, or cases where there is supportive, but not direct, evidence for a role in neurotransmitter specification. For instance, vertebrate neuronal precursors expressing Nkx2.1 (HGTX orthologue) predominantly generate GABAergic interneurons, and a polyalanine expansion in ARX (hbn orthologue) causes remodelling and increased activity of glutamatergic neurons in vertebrates. Acj6 is expressed in a subset of cholinergic neurons and Dbx in a subset of GABAergic neurons. None of the enriched transcription factors identified in this study are expressed in all of the neurons of a particular neurotransmitter type. This highlights that, similar to C. elegans, there are no simple transcription factor codes for neurotransmitter type specification in Drosophila (Estacio-Gómez, 2020).

Uniquely enriched factors are candidates for promoting a neurotransmitter fate, and a number of them were tested for their ability to reprogram neurons on a global scale in embryos. No obvious changes were observed, however, this is not particularly surprising considering the importance of cellular context for the reprogramming of neuronal properties. Successful reprograming may require intervention at a specific time point (e.g., at the progenitor stage), the co-expression of appropriate co-factors, and/or to exclusively target a neuronal subpopulation within each neurotransmitter type. Future work could investigate these factors in specific and relevant lineages, to shed light on important contextual information (Estacio-Gómez, 2020).

The majority of transcription factors identified as directly regulating neurotransmitter fate act in a positive manner, whereas only a handful of studies describe the role of repressors. Incoherent feedforward loops exist in C. elegans, where terminal selectors activate repressors, which feedback onto effector genes. In vertebrates, both Neurogenin 2 and Tlx3 are required for the specification of certain glutamatergic populations but also act to repress GABAergic fate. Whether this is direct repression of Glutamic acid decarboxylase (Gad) genes (required for the synthesis of GABA), or indirectly, through another transcription factor, is unclear. This study has identified several transcription factors that are expressed in two neurotransmitter types, but absent from the other. These include apterous (ap), Ets65A (long transcripts) and orthopedia (otp), which this study hypothesises to be candidate repressors, given their absence from cells with a specific neurotransmitter identity. Profiling of Ets65A-PA binding in vivo, reveals that it directly binds ChAT, and therefore has the potential to directly regulate cholinergic fate. Similar to the candidate activators, ectopic expression of these candidates did not show any obvious repression of the respective neurotransmitter genes, however, again, this might be because they can only act as a repressor in specific contexts (e.g. when a co-repressor is present), or that they regulate genes associated with specific types but do not directly regulate neurotransmitter identity (Estacio-Gómez, 2020).

The development of single cell RNA-seq (scRNA-seq) technology has led to the profiling of several Drosophila tissues, including the whole adult brain, the central adult brain and the adult optic lobes. This study has mined the whole adult brain data (Davie, 2018) to compare and investigate the cholinergic enriched factors that this study has identified in adult brains. The enrichment of these transcription factors (compared to GABAergic and glutamatergic neurons) is also observed in the scRNAseq data. Furthermore, it was discovered that the cholinergic cells that these factors are expressed in are almost non-overlapping. This is an intriguing finding, as it suggests that these factors, if they are indeed acting to promote/maintain cholinergic fate, they are not acting together in this context. This scenario maybe different during development, where specification is occurring, and it will be interesting to test this when high coverage scRNAseq data is available for the third instar larval brain. This study observed more differentially expressed transcription factors in the L3 larval stage (58) compared to the embryo (40) or adults (33). This may reflect the existence of both the functioning larval nervous system (built during embryogenesis) and the developing adult nervous system at this stage. While both the embryo and larval data are similar on a global scale, Pol II occupancy and chromatin accessibility in the adult brain is less correlated. It is currently unclear whether this is due to adult VNCs being absent from the profiling experiments, or differences between immature and fully mature neurons, such as overall lower transcriptional activity in adults. Previous work has shown that global chromatin accessibility distribution in adult neurons is distinct from larval neurons, which may account for some of these differences (Estacio-Gómez, 2020).

Apart from the neurotransmitter synthesis genes, the chromatin accessibility of the different neuronal types, at a given stage, is surprisingly similar, as demonstrated in embryos. The enriched accessibility is not just restricted to the gene bodies of the neurotransmitter genes, and peaks are present upstream (Gad1) and downstream (VGlut), which are likely enhancers. Accessibility at the ChAT gene is clearly higher in cholinergic neurons at the embryonic and adult stages, however, in third instar larvae, the difference is less pronounced. This could reflect increased plasticity at this stage, possibly linked to the dramatic remodelling of larval neurons during metamorphosis, or that this accessibility across the types is due to non-specific expression of the VAChT gene that overlaps with ChAT at its 5' end. While a subset of transcription factors display obvious contrasts in Pol II occupancy, the same transcription factors have no observable, or minor, differences in accessibility. This could be due to transcription factors being expressed at relatively lower levels and/or that they are only expressed in a subset of the cells, therefore the difference is less prominent (Estacio-Gómez, 2020).

Evidence is emerging for the roles of miRNAs in generating neuronal diversity, including the differentiation of taste receptor neurons in worms and dopaminergic neurons in vertebrates. This study found the enriched expression of mir-184 in GABAergic cells, which is intriguing, as mir-184 has been shown to downregulate GABRA3 (GABA-A receptor) mRNA (possibly indirectly) in vertebrate cell lines, and may be a mechanism to help prevent GABAergic neurons self-inhibiting. Furthermore, mir-87 has enriched RNA polymerase II occupancy in cholinergic neurons, and when mutated causes larval locomotion defects in Drosophila (Estacio-Gómez, 2020).

Acj6 is expressed in adult cholinergic neurons, whilst Ets65A-PA is expressed in non-cholinergic adult neurons. However, despite this, they bind a large number of common target genes. This includes 20% (101/493) of all genes annotated for a role in 'neuron projection development' (GO:0031175). This is quite striking, especially as this is in the adult, where there is virtually no neurogenesis or axonogenesis. However, this may reflect dendritic re-modelling processes, or a requirement of neurons to continuously express transcription factors, even after development, to maintain their fate. The acj6 orthologues, unc-86 and Brn3a are both required to maintain the fate of specific cholinergic populations, and transcriptional networks that specific Tv1/Tv4 neurons in Drosophila are also required to maintain them in the adult. Therefore, the binding of Acj6 and Ets65A-PA to developmental genes and ChAT in adult neurons could be required for the continued activation (and repression) of genes governing neuronal identity. MAP kinase signalling genes are enriched in cholinergic neurons and Ets65A-PA specifically binds MAP kinase signalling genes, making it tempting to speculate that Ets65A-PA acts to repress cholinergic specific genes such as ChAT and MAP kinase genes. These Acj6 and Ets65A-PA data also emphasize the diverse set of neuronal differentiation genes a single transcription factor could regulate (Estacio-Gómez, 2020).

The precise synthesis and utilisation of neurotransmitters ensures proper information flow and circuit function in the nervous system. The mechanisms of specification are lineage specific, predominantly through the action of transcription factors. This study has provided further insights into the complement of different transcription factors that regulate neurotransmitter identity throughout development. Furthermore, this study identified the genomic binding of a known activator, and a candidate repressor, of cholinergic fate in the adult, emphasizing the broad spectrum of neural identity genes that they could be regulating outside of neurotransmitter use. Given the strong evidence for conserved mechanisms controlling neurotransmitter specification, these data will be a useful resource for not just researchers using Drosophila but other model systems too. Continued work to elucidate the mechanisms, co-factors and temporal windows in which these factors are acting will be fundamental in gaining a comprehensive understanding of neurotransmitter specification (Estacio-Gómez, 2020).

Cholinergic activity is essential for maintaining the anterograde transport of Choline Acetyltransferase in Drosophila

Cholinergic activity is essential for cognitive functions and neuronal homeostasis. Choline Acetyltransferase (ChAT), a soluble protein that synthesizes acetylcholine at the presynaptic compartment, is transported in bulk in the axons by the heterotrimeric Kinesin-2 motor. Axonal transport of soluble proteins is described as a constitutive process assisted by occasional, non-specific interactions with moving vesicles and motor proteins. This study reports that an increase in the influx of Kinesin-2 motor and association between ChAT and the motor during a specific developmental period enhances the axonal entry, as well as the anterograde flow of the protein, in the sensory neurons of intact Drosophila nervous system. Loss of cholinergic activity due to Hemicholinium and Bungarotoxin treatments, respectively, disrupts the interaction between ChAT and Kinesin-2 in the axon, and the episodic enhancement of axonal influx of the protein. Altogether, these observations highlight a phenomenon of synaptic activity-dependent, feedback regulation of a soluble protein transport in vivo, which could potentially define the quantum of its pre-synaptic influx (Dey, 2018).

Axonal transport of ChAT has been extensively studied in the various organisms and neuron types. Estimates of accumulated ChAT activity at the ligature of rat sciatic nerve suggested that the enzyme flows anterogradely at an average rate of ~1.2 mm/day. Using the high-resolution FRAP this study estimated a much faster max flow rate (1.8 μm/s or ~155 mm/day) in the axons of intact lch5 neurons of Drosophila larvae, as compared to an earlier estimate (0.97 µ/s or ~83 mm/day) obtained from the short interneurons of the ventral ganglion. A similar disparity in rates has been reported for the other slow axonal cargoes such as neurofilaments, CaMKII, Synapsin, and Actin. This apparent discrepancy in the rate estimates is a consequence of spatiotemporal characteristics of the transport which are reflected in the assaying paradigms and acquisition parameters. Besides the 76-79 h AEL interval in the third instar stage, another episode of ChAT influx was observed in lch5 axons during 52-56 h AEL in the second instar stage. Assuming that ChAT transport episode is restricted to an hourly interval during each molt, the effective flow rate during a 24 h molting period would be 3.5 mm/day which correlates well to transport characteristics of ChAT as a slow rate component. These results are obtained from fully ensheathed functional neurons connected to the native circuitry at different developmental stages. Thus, it also provided near endogenous characteristics of the transport. With the improved observation capability, it was found that the temporal parameters of the ChAT transport are consistent in both the small interneurons of ventral ganglion, as well as in the mature lch5 neurons, suggesting that the episodic nature of the ChAT transport is an intrinsic property (Dey, 2018).

ChAT was reported to bind directly to the C-terminal tail domain of the Kinesin-2α subunit in vitro. Kinesin-2 is essential for two distinct aspects of the transport process - entry into the axons and for conferring the anterograde bias observed at 78 h AEL. The FRAP and FRET assay further suggested that the episodic movement of the bulk of ChAT is initiated through a transient association with the Kinesin-2 motor throughout the neuron. Although the motor was present in the axon all throughout, the association was limited to an hourly interval or less during late larval development. Studies showed that temporal switching of association from Kinesin-3 (Unc-104) to Dynein shifts the transport of Rab3 vesicles from anterograde to retrograde in the DD neurons of C. elegans. This change in the modality of Rab3 vesicle transport in the DD neurons was associated with synapse restructuring induced by cyclin, CYY-1 and the cyclin-dependent kinase, CDK548. The ChAT transport episodes appear to closely follow the molting cycle, which is induced through the surge of Juvenile Hormone (20-Hydroxyecdysterone) in Drosophila larvae. Considering the timescale of the transport modulation, certain post-translational modifications such as phosphorylation could enhance the affinity between ChAT and Kinesin-2. For example, Calmodulin Kinase II (CaMKII)-mediated phosphorylation of Kinesin-2 tail is suggested to increased the transport of N-Cadherin towards the synapses. A preliminary motif scan also revealed putative Casein Kinase II-mediated phosphorylation sites in ChAT and the tail domains of Kinesin-2. Therefore, certain developmental signaling cues could trigger the episodic interaction between ChAT and Kinesin-2 through phosphorylation or other post-translational modifications (Dey, 2018).

Kinesin-2 plays an essential role in the anterograde movement of ChAT in Drosophila and mouse axons. Apart from ChAT, both Rab4, and Acetylcholinesterase (AChE) are transported by Kinesin-2 in axons, and functions of these three proteins are implicated in synapse homeostasis. Above data indicates that direct interaction between the motor and ChAT for brief duration induced the episodic flow towards synapse, and synaptic activity is essential for this interaction. Blocking the acetylcholine synthesis through the HC3 treatment, and activation of the post synaptic neurons through the inhibition of actylcholine receptors by BTX, respectively, was found to reduce the axonal entry and disrupt the episodic nature of ChAT transport within a short time, indicating that synaptic activity regulates axonal entry through a fast retrograde communication. Neuronal depolarization and synaptic activity have also been suggested to regulate the axonal transport either directly or indirectly, for example, the activity-dependent synaptic capture of Dense Core Vesicles, the depolarization-triggered redistribution of κ-opioid receptor mRNA, and effect on mitochondrial trafficking due to activity-induced changes in the intracellular calcium and ATP3,4,55. In the context of axonal injury, the earliest communication between the distal end of the axon and the soma is established by ionic fluxes. Such an event is conjectured to modulate the signaling molecules and transcriptional profile of the neuron, activate redistribution of mRNA and nuclear proteins, and alter the structural characteristics of the AIS via retrograde signaling complex. Thus, continued stimulation of lch5 neurons during larval stages together with certain post-translational modifications induced by the developmental cues could define the episodic postsynaptic feedback and interactions between ChAT and Kinesin-2. Subsequently, the enhanced transport could enhance the synaptic contacts in the ventral ganglion (Dey, 2018).

Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS

The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. This study generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. No cases were found of neurons using more than one neurotransmitter, but it was found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, this work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. This detailed transmitter usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior (Lacin, 2019).

The ventral nerve cord (VNC) of Drosophila melanogaster is home to circuits coding for vital behaviors, such as walking, jumping, and flight. It is composed of about 16,000 neurons, all of which arise from a set of segmentally repeated 30 paired and one unpaired neural stem cells (Neuroblasts [NBs]). NBs generate unique progeny via undergoing two rounds of proliferation: a brief embryonic and an extended postembryonic phase. Embryonic neurogenesis generates the neurons of the larval CNS and many of these cells are then remodeled to function in the adult CNS. 90-95% of the adult neurons, however, are adult-specific and arise during the post-embryonic phase of neurogenesis (Lacin, 2019).

The VNC contains only NBs that show a Type I pattern of proliferation. Each NB divides repeatedly via asymmetric cell division to renew itself and to generate a secondary precursor cell, called a ganglion mother cell (GMC). Each GMC, in turn, divides terminally to form two neurons, each of which acquires a unique identity due to the presence or absence of active Notch signaling. Within a NB progeny, the Notch-ON neurons are called the 'A' hemilineage; their Notch-OFF siblings are called the 'B' hemilineage (Lacin, 2019).

By progressing through the temporal transcriptional cascade, Hunchback -> Kruppel -> Pdm, NBs generate diverse 'A' and 'B' neurons during the early embryonic phase. Subsequently, NBs express Castor and Grainyhead in late embryonic stages and many NBs maintain this Castor/Grainyhead expression into the postembryonic stages. Correlated with this shared gene expression, neurons of a hemilineage ('A' or' B') that are born during the late embryonic and early postembyonic stages often adopt similar fates. Recent studies characterized the morphologies of postembryonic hemilineages in their immature states in the larva and mature states in the adult. These studies revealed that in the larva, the immature neurons of each hemilineage cluster together and extend their initial processes as a bundle to the same region and that after metamorphosis, in the adult, they continue to be clustered and share common anatomical and functional features (Lacin, 2019).

In addition to similar morphology, neurons within a postembryonic hemilineage share patterns of transcription factor expression. In the larval VNC, each hemilineage cluster can be identified with a specific combination of transcription factor expression (Lacin, 2014). Interestingly, vertebrate homologs of many of these hemilineage-specific transcription factors are expressed in the spinal cord and are required for fate determination. For example, in flies, the combinatorial expression of Lim3, Islet, and Nkx6 is observed uniquely in hemilineage 15B, which is composed of leg motor neurons (Lacin, 2014). In mice, homologs of these three factors are essential for the identity of spinal motor neurons. Indeed, interneurons in the vertebrate spinal cord are also organized into discrete cardinal classes that share developmental origins, transcription factor and neurotransmitter expression, and functional roles. The fly VNC appears to be organized in an analogous manner, with the developmental origins of neuronal clusters providing the basis for their transcription factor expression and functional properties. The relationship between specific stem cells and neurotransmitter expression in their progeny, though, has yet to be resolved (Lacin, 2019).

Studies on grasshoppers and Manduca sexta showed that clusters of GABAergic interneurons were based on their lineage of origin. Likewise, cholinergic and glutamatergic neurons are also typically found as clusters in the VNC and the brain consistent with a shared lineage. In the fly, as a prelude to studies that seek to dissect the developmental basis of behavior, neurotransmitter usage was comprehensively mapped across the VNC to determine how neurotransmitter selection relates to lineage identity. A similar comprehensive neurotransmitter map was generated for the C. elegans nervous system and proved to be beneficial in identifying regulatory mechanisms that control neurotransmitter identity and circuit assembly. By using molecular and genetic tools, this study mapped neurotransmitter usage in all hemilineages in the adult fly VNC. The results revealed that, as found in the neurons of the vertebrate cardinal classes, all neurons within a fly hemilineage use the same neurotransmitter. In agreement with earlier findings (Harris, 2015), this study further shows that hemilineages are not just developmental units but also functional units that drive animal behavior (Lacin, 2019).

The adult VNC is made up primarily from 34 hemilineages -- clusters of lineally related, segmentally repeated, postembryonic-born neurons. Previous work systematically characterized the development and neuronal morphologies of these hemilineages, and showed that neurons within a hemilineage adopt similar fates, evident from their immature axonal projection and transcription factor expression. This study has systematically mapped the neurotransmitter choice of most VNC neurons by studying all of the postembryonic hemilineages. Surprisingly, it was found that neurotransmitter code in the VNC is simple in that all neurons within a hemilineage use the same neurotransmitter, thus transmitter identity is determined at the stem cell level. These results further support earlier findings that hemilineages represent functional units, both in terms of anatomy and now neurotransmitter chemistry (Lacin, 2019).

The statement that all neurons within a hemilineage use the same neurotransmitter excludes the neurons that are born during early embryonic neurogenesis. As mentioned earlier, neurons born during this time are highly diverse and might use a different neurotransmitter than the rest of the neurons in the hemilineage. For example, both NB4-2 and NB5-2 generate glutamatergic motor neurons from their early embryonic divisions, but their postembryonic progenies (both A and B hemilineages) are purely GABAergic. Similarly, glutamatergic U/CQ motor neurons, which are born in early embryonic stages share the same hemilineage with the cholinergic 3A neurons. Thus, neuronal fates within a hemilineage can be dramatically different when embryonic and postembryonic neurons are compared. It is believed that the reason why postembryonic hemilineages in the VNC are homogenous in terms of neuronal fate is due to the expansion of particular, later-born neuronal classes as neuronal lineages became larger during evolution of more derived insects to accommodate more complex behaviors such as flight. Since all insect species have similar sets of NBs, new behaviors (e.g., flight) appear to have evolved via changes in the number of neurons generated by each NB, but not changes in the number of NBs. Moreover, the Notch mediated asymmetric division enabled the insect to have two distinct clonal populations of neurons (hemilineages) from a single NB. Interestingly, only 34 of 50 potential hemilineages are used in the adult fly VNC while 16 of them are eliminated by apoptosis. Thus, flies have the potential to acquire novel behaviors by simply resurrecting hemilineages that are fated to die (Lacin, 2019).

At least within the thorax, the hemilineages express the same transmitter regardless of their segment of residence. This conservation was expected for the hemilineages that contribute to the leg neuropils, since neuron numbers and the projections of these cells appear similar across the different thoracic segments. On the other hand, the hemilineages innervating the dorsal, flight-related neuropils have segment specific organization and show dramatically different axonal projections depending on their segmental location. For example, 7B neurons in each segment have unique projection and appear to execute distinct behaviors. Despite these differences, 7B neurons use acetylcholine in every segment. These results show that the neurotransmitter fate is tightly linked to the lineage origin and that the segmental diversification of the 7B neurons with the evolution of the derived flight system of flies may have had to occur with this transmitter constraint (Lacin, 2019).

It is expected that most hemilineages in the fly brain are also homogenous in terms of neurotransmitter expression as large neuronal clusters were observed expressing the same neurotransmitter. However, some complex brain hemilineages that have diverse neuronal populations might have different neurotransmitter expression as it was shown for the lAL lineage (Lacin, 2019).

This study has also extended earlier transcription factor expression studies in immature neurons of larval stages into the mature neurons of the adult. The expression of many transcription factors is maintained into adult stages and can be used to mark specific hemilineages in the adult. However, some transcription factors are expressed transiently during development. For example, Dbx marks many immature 3B neurons in the larva, but its expression disappears in these neurons after pupa formation. From the expression analysis of the limited number of transcription factors examined in this study, no factor was found that specifically marked all neurons of a specific neurotransmitter type. However, a few transcription factors, whose expression tightly correlated with the neurotransmitter fate, were found. For example, Dbx expression is restricted to GABAergic neurons, even though Dbx does not appear to promote the GABA fate by itself as GABAergic fate is unaltered in response to Dbx loss or misexpression. Similarly, it was found that Unc-4 expression is restricted to cholinergic lineages among the postembryonic lineages in the VNC; however, in the brain Unc-4 is expressed in glutamatergic lineages in addition to cholinergic lineages, suggesting that different parts of the CNS might use the same transcription factor for different fates via utilizing different cofactors. Supporting this, it was found that none of the GABAergic lineages in the VNC are marked with Lim3, which was shown to be expressed in most GABAergic neurons of the fly optic lobe and required for their GABAergic identity. The reverse scenario where neurons acquire the same neurotransmitter identity via different transcriptional regulatory networks is also commonly observed. For example in the C. elegans nervous system, distinct combinations of 13 transcription factors are responsible for VGlut expression in 25 different glutamatergic neuron classes (Serrano-Saiz, 2013). Thus, transcription factors act together combinatorially rather than individually to specify neurotransmitter fate (Lacin, 2019).

This study did not find any correlation between the Notch state of the neurons and neurotransmitter identity, an observation made in the optic lobe. Any neurotransmitter type can be observed in both 'A' and 'B' hemilineages. It is also noted that NB4-2 (progenitor of 13A/B) and NB5-2 (progenitor of 6A/B) are the only two NBs in which both the 'A' and 'B' hemilineages use the same neurotransmitter, GABA (Lacin, 2019).

Interestingly, the neurotransmitter pattern of Drosophila hemilineages appears to be conserved in other insect species. For example, based on location and morphology, it was deduced that sibling 'Kl' and 'Km' GABAergic clusters of the moth, Manduca Sexta, are homologous to the 13A and 13B hemilineages, respectively, and the 'M' cluster is likely homologous to the 6A, 6B, and 5B neurons, which form a large cluster GABAergic in the posterior thoracic ganglia of the fly. Similar GABAergic clusters were also observed in the nerve cords of grasshopper and silverfish. Interestingly, like the Drosophila VNC, the grasshopper nerve cord contains two clusters of En+GABA+ neurons, named 'A' and 'B' groups, which are likely homologous to 0A and 6B neurons, respectively (Lacin, 2019).

Unexpectedly, this study detected ChAT transcripts in many GABAergic and glutamatergic neurons, most of which are members of lineages 5B and 11B (GABAergic) and 14A and 15B (glutamatergic). The low levels of ChAT transcripts and the lack of ChAT immunostainings in these cells suggested that ChAT transcripts are actively degraded and not translated. It is possible that these neurons produce acetylcholine but only in certain conditions for example during development or under stress. Indeed, neurotransmitter switching has been observed in many neurons of vertebrates, however, most of these switches involve aminergic neurotransmitters (Spitzer, 2015; Lacin, 2019 and references therein).

Another possibility for the presence of ChAT transcripts in noncholinergic neurons is that it is a remnant of a neurotransmitter switch that might have happened during evolution. 15B neurons are a good candidate for such a possibility. 15B motor neurons are glutamatergic like all other fly motor neurons, while all vertebrate and some invertebrate (e.g, C. elegans and Aplysia) motor neurons are cholinergic, raising the possibility that motor neurons of the common ancestor used acetylcholine. Thus, the ChAT expression in 15B motor neurons might be a vestige from the cholinergic ancestry of motor neurons (Lacin, 2019).

Overexpression of the vesicular acetylcholine transporter disrupts cognitive performance and causes age-dependent locomotion decline in Drosophila

Acetylcholinergic (ACh) neurotransmission is essential for key organismal functions such as locomotion and cognition. However, the mechanism through which ACh is regulated in the central nervous system is not fully understood. The vesicular acetylcholine transporter (VAChT) mediates the packaging and transport of ACh for exocytotic release and is a critical component of the ACh release machinery. Yet its precise role in the maintenance of cholinergic tone remains a subject of active investigation. This study use the overexpression of VAChT as a tool to investigate the role of changes in ACh exocytosis on the regulation of synaptic activity and its downstream consequences. Yhe effect was measured of an increase in VAChT expression on locomotion and cognitive performance as well as on organismal survival across the lifespan. The surprising finding is reported that increased VAChT expression results in a significantly shorter lifespan in comparison to control flies. Moreover, constructs overexpressing VAChT demonstrate an age-dependent decrease in locomotion performance. Importantly, this study reports clear deficits in learning and memory which wetr measured through a courtship conditioning assay. Together, these data provide evidence for the adverse effects of overexpression of the vesicular acetylcholine transporter in the maintenance of normal behavioral abilities in Drosophila and demonstrates for the first time a role for ACh in the regulation of organismal survival (Showell, 2020).

The genetic architecture of ovariole number in Drosophila melanogaster: Genes with major, quantitative, and pleiotropic effects

Ovariole number has a direct role in the number of eggs produced by an insect, suggesting that it is a key morphological fitness trait. Many studies have documented the variability of ovariole number and its relationship to other fitness and life-history traits in natural populations of Drosophila. However, the genes contributing to this variability are largely unknown. A genome-wide association study of ovariole number was conducted in a natural population of flies. Using mutations and RNAi-mediated knockdown, the effects of twenty-four candidate genes on ovariole number was confirmed, including a novel gene, anneboleyn (formerly CG32000), that impacts both ovariole morphology and numbers of offspring produced. Pleiotropic genes were identified that regulated ovariole number traits and sleep and activity behavior. While few polymorphisms overlapped between sleep parameters and ovariole number, thirty-nine candidate genes were nevertheless in common. The effects of seven genes on both ovariole number and sleep were verified: bin3, blot, CG42389, kirre, slim, VAChT, and zfh1. Linkage disequilibrium among the polymorphisms in these common genes was low, suggesting that these polymorphisms may evolve independently (Lobell, 2017).

Transgenic line for the identification of cholinergic release sites in Drosophila melanogaster

The identification of neurotransmitter type used by a neuron is important for the functional dissection of neuronal circuits. In the model organism Drosophila melanogaster, several methods for discerning the neurotransmitter systems are available. This study expanded the toolbox for the identification of cholinergic neurons by generating a new line FRT-STOP-FRT-VAChT::HA that is a conditional tagged knock-in of the VAChT gene in its endogenous locus. Importantly, in comparison to already available tools for the detection of cholinergic neurons, the FRT-STOP-FRT-VAChT::HA allele also allows for identification of the subcellular localization of the cholinergic presynaptic release sites in a cell-specific manner. The newly generated FRT-STOP-FRT-VAChT::HA line was used to characterize the Mi1 and Tm3 neurons in the fly visual system and found that VAChT is present in the axons of the both cell types, suggesting that Mi1 and Tm3 neurons provide cholinergic input to the elementary motion detectors, the T4 neurons (Pankova, 2017).

Immunolocalization of the vesicular acetylcholine transporter in larval and adult Drosophila neurons

Vesicular acetylcholine transporter (VAChT) function is essential for organismal survival, mediating the packaging of acetylcholine (ACh) for exocytotic release. However, its expression pattern in the Drosophila brain has not been fully elucidated. To investigate the localization of VAChT, an antibody against the C terminal region of the protein was developed; this antibody recognizes a 65KDa protein corresponding to VAChT on an immunoblot in both Drosophila head homogenates and in Schneider 2 cells. Further, the expression is reported of VAChT in the antennal lobe and ventral nerve cord of Drosophila larva, and the expression was confirmed of the protein in mushroom bodies and optic lobes of adult Drosophila. Importantly, it was shown that VAChT co-localizes with a synaptic vesicle marker in vivo, confirming previous reports of the localization of VAChT to synaptic terminals. Together, these findings help establish the vesicular localization of VAChT in cholinergic neurons in Drosophila and presents an important molecular tool with which to dissect the function of the transporter in vivo (Boppana, 2017).

Studies in rodents have shown the expression of VAChT in the brain. However, until now, few reports have analyzed the expression of VAChT across multiple regions in the brain of Drosophila. This study shows that VAChT is expressed in the medulla in discrete punctae in adult flies. Moreover, the results also demonstrate for the first time that VAChT is expressed in the CNS and VNC of Drosophila larva. Importantly, this study confirms previously published reports that VAChT is expressed in optic lobe lamina and in the mushroom body in adult Drosophila. Together, these findings delineate the expression of VAChT in the adult and shows for the first time its expression during Drosophila larval development (Boppana, 2017).

Studies in mice have explored the function of VAChT in animal behavior and physiology. Other studies using the rodent model have exploited VAChT as a marker for cholinergic neurons without studying its role in neuronal function per se. By contrast, only a handful of studies in Drosophila have directly assessed VAChT expression in cholinergic neurons. Indeed, the antibody used in this study is consistent with those prior studies of neuronal VAChT expression; labeling cholinergic neurite projections in the lamina and forming punctae at both the basal and the top of the lamina. The identity of these neurons have been established anatomically to be L4 monopolar and Cha-Tan neurons. Moreover, this study confirmed that VAChT is expressed both in the KCs and in the PN axons that innervate them. In addition, this study reports the intriguing finding that VAChT is also expressed in the lateral horn which together with MBs form higher olfaction centers in the fly brain. The expression of VAChT in this area provides spatial evidence for functional studies describes by others of a key role for ACh release in synaptic physiology and behavior in the olfactory circuit. Importantly, the confirmation of VAChT expression in MBs contributes to helping to resolve a long standing debate about whether or not KCs themselves are cholinergic (Boppana, 2017).

In larvae, the antennal lobe is among the most striking structures labelled by the anti-VAChT antibody. This is the first report of the labeling of VAChT in the antennal lobe during development. Moreover, the presence of VAChT along nerve tracks of the midline in the abdominal segment has also not been described previously. The punctae-like expression of VAChT is reminiscent of the expression of synaptotagmin along the ventral nerve cord in Drosophila embryo and larvae. From these findings, it is suggested that this antibody could serve as a marker for cholinergic neurons in the optic lobe, antennal lobe and projection neuron nerve terminals (Boppana, 2017).

Co-staining of anti-VAChT and anti-CSP2 confirmed the synaptic terminal localization of VAChT. Localized both along the axons and in distinct synaptic terminal sites, both antibodies strongly mark laminal neuron projections; an unsurprising finding, giving the role of ACh in the function of the photoreceptor cells of the visual system. VAChT appears to preferentially stain neurite projections and synaptic sites while ChAT-GFP labels cell bodies more strongly than it does axonal processes. This data is in agreement with reports in rodents that VAChT more reliably labels synaptic terminals than does ChAT. It is noted that VAChT does not co-localize with ChAT-GFP in several instances, and it is speculated that this is due to the limitation of ChAT-GFP as a marker for ACh neurons. Similarly, it is noted that there appeared to be more localization of VAChT to cholinergic cell bodies in the larvae than in the adult. This phenomenon may be due to the time it takes for synaptic vesicles to mature and migrate to the terminal (Boppana, 2017).

This study describes the expression of VAChT in the brain of larval and adult Drosophila. The finding that VAChT is expressed in MBs provides support for the idea that ACh is the neurotransmitter released by MBs, contributing to the resolution of a long standing controversy. Moreover, strong expression of VAChT in the larval antennal lobe suggests that the key role for cholinergic neurotransmission that has been described in the adult is likely to also occur in during larval development. These efforts have been aided by the generation of a VAChT antibody which is suggested as a marker for cholinergic neurons in the delineation of the neuronal function of VAChT (Boppana, 2017).

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 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).

Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing

Single identified neurons were removed from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently (Liu, 2016).

This study tested if the analysis of the transcriptome of single neurons removed from the ventral nerve cord of living brains can be used to identify new molecules directing neuronal network formation. Single MP2 neurons were harvested at a time when axonal guidance draws to a close and the first evoked action potentials can be recorded. Focus was placed on MP2 neurons because of their common origin and their two very distinct fates, an anterior projecting cholinergic interneuron versus a posterior projecting interneuron destined for apoptosis. The chosen time point of cell removal, stage 17, a time when neurons are nearly fully differentiated and when neuronal networks become functional, should increase the possibility of identifying transcripts involved in network function and to exclude transcripts controlling cell determination. Indeed, the transcripts that were discovered are involved in cell contact, cellular maintenance and epigenetic control of chromatin states. Due to their different fates a vast array of transcriptional differences between the two MP2 siblings were expected, yet only 7 out of 2631 transcripts, which are differentially expressed (threshold 1.4x). This number of differentially expressed transcripts is dependent on the high stringency filters applied and will also vary in the course of development. Yet, the lineage of MP2 neurons also sets them apart from all other interneurons in the lateral CNS because their precursor only divides once, generating neurons directly and not via a transient ganglion mother cell (gmc). Preliminary results comparing the transcriptome of sibling interneurons from a neuroblast (Nb) following a conventional division pattern (Nb5-2) reveal a much higher diversity. In contrast to MP2, which divides only once, Nb5-2 can undergo up to 20 divisions, generating 20 gmc’s and 40 neurons. During these divisions a cascade of temporal factors controls changes in neuronal identity. Applying the same stringent criteria as in the MP2 analysis, two interneurons removed from the same Nb5-2 lineage differ in 569 transcripts out of 1226 (46,2%). Comparing interneurons from different Nbs (Nb6-1 vs Nb7-1) increases molecular diversity even further, 633 out of 1232 transcripts are differentially expressed (51.4%). These preliminary results are in line with previous publications reporting a high molecular diversity of interneurons. The low number of differential expressed transcripts between MP2 neurons seems to be the exception (Liu, 2016).

One transcript expressed higher in dMP2 than vMP2 encodes the SRSF protein B52, which is the closest Drosophila orthologue to human SRSF6. SRSF proteins were first characterised as pre-mRNA alternative and constitutive splicing factors. In addition to their role in splicing, several SRSF proteins have been shown to regulate transcription elongation, RNA export, decay, translation, and for B52, transcriptional regulation. Interestingly, SRSF4, one of the human orthologues of B52 expressed in the CNS, has been shown to be involved in alternative splicing of Tau and the origin of Frontotemporal Dementia. This study studied the function of B52 in neural development in Drosophila in more detail. B52 is maternally deposited and expressed throughout the CNS. Since the transcriptome of MP2 siblings were examined at the end of embryogenesis, it was not expected that B52 is a crucial factor in cell fate determination. Indeed neural depletion of B52 function by pan-neural expression of a RNA aptamer or in subsets of neuroblasts and their progeny does not create gross morphological defects. Yet, it was discovered that functional depletion of B52 affects axonal growth and the pre-mRNA splicing and biosynthesis of ChAT (Liu, 2016).

One of the B52 splicing targets identified by genomic systematic evolution of ligands by exponential enrichment (SELEX)50 and co-immunoprecipitation is the BTB/POZ transcription factor longitudinal lacking, (lola), which controls axon extension via the transcription of spire, an actin nucleation protein. Interference with B52 function in dMP2 increases axonal growth. Whereas increased expression of B52 does not alter axonal growth suggesting a saturation of the B52 target in the wild type condition. Thus it is speculated that B52 may control axonal growth via splicing of lola for which at least 25 different isoforms have been reported (Liu, 2016).

In contrast to lola pre-mRNA, ChAT pre-mRNA has not yet been identified as a B52 splicing target. Therefore it cannot be excluded that the observed splicing regulation of ChAT pre-mRNA is indirectly mediated via a yet unknown B52 target. In the embryo, depletion of B52 function by the expression of UAS-BBS results in accumulation of unspliced ChAT pre-mRNA and consequently a reduction in ChAT protein synthesis and reduced acetylcholine synthesis. Cholinergic interneurons are the only excitatory input for embryonic motor neurons. During the transition from uncoordinated muscle twitches to coordinated peristaltic muscle contractions, the shape of motorneuronal dendrites is fine tuned by presynaptic contact as well as acetylcholine release. In ChAT mutants, larvae do not hatch and motorneuronal dendrites overgrow. A reduction of ChAT synthesis and acetylcholine synthesis as seen upon embryonic B52 functional depletion may therefore cause the observed increase in uncoordinated movements and the hatching delay (Liu, 2016).

At the end of their life B52 homozygous mutant larvae are smaller than their heterozygous siblings and appear paralysed. Both phenotypes might be explained by deficient Ecdysone signalling. A reduction in larval size is not unexpected because the Ecdysone receptor (EcR)q and many ecdysone inducible transcripts have been identified as B52 splicing targets. Larvae without EcR function do not moult, remain small and gradually become immobile and insensitive to touch (Liu, 2016).

Pre-mRNA splicing depends on the stoichiometry of snRNPs and splice factors. In addition, phosphorylation of SRSF proteins defines their binding specificity, function and localisation. The accumulation of unspliced ChAT pre-mRNA caused by the depletion of B52 function in embryos and by targeted expression of B52 in 2nd instar larvae, two opposing manipulations, support that stoichiometry plays an important role for efficient splicing. Yet, changes in stoichiometry appear insufficient to explain the efficient splicing of Cha introns 4-7 in B52ΔS2249 mutant larvae, which show a severe reduction of B52 mRNA. The different splicing phenotypes between mutant and functionally depleted larvae may be due to partial redundancy between B52 and the second SRSF protein in Drosophila, Asf/SRSF2. Target specificity of B52 and Asf proteins shows some overlap51. Therefore the increase in artificial B52 binding sites with the expression of the BBS aptamer may not only titrate out B52 but also Asf leading to a functional depletion of both SRSF proteins. In contrast, in B52 homozygous mutants, the loss of B52 protein can still be compensated by the presence of Asf protein (Liu, 2016).

This work shows that functional depletion of B52 reduces ChAT pre-mRNA splicing and ChAT protein synthesis although the exact regulation of ChAT pre-mRNA by B52 awaits further investigation. The lower expression of B52 in the cholinergic vMP2 than in the non-cholinergic dMP2 may be a compromise allowing vMP2 to still splice ChAT pre-mRNA efficiently and continue axonal growth (Liu, 2016).

Bipolar disorder (BD) is a devastating mental disease, in which the patient fluctuates between depressive and maniac mood swings. Elevated acetylcholine levels are the trigger for the depressive phases. A recent study tried to identify transcriptional changes in the dorsolateral prefrontal cortex in postmortem brains of patients who suffered from bipolar disorder. Only five differentially expressed genes and 12 differentially expressed transcripts were discovered. Interestingly, one of the transcripts encodes for the SRSF5 protein, an orthologue of B52, which was significantly downregulated in BD patients. This study shows that depletion of B52 function decreases ChAT protein, and subsequently acetylcholine levels. Therefore, not only are ChAT and SRSF proteins widely conserved throughout the animal kingdom but also the mechanism controlling splicing of ChAT pre-mRNA by SRSF proteins might be conserved (Liu, 2016).

Central cholinergic synaptic vesicle loading obeys the set-point model in Drosophila

Experimental evidence shows that neurotransmitter release, from presynaptic terminals, can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through change in the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine into SVs at cholinergic synapses. This study investigated how the VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster by using an insecticidal compound, 5Cl-CASPP, to block VAChT and by transgenic overexpression of VAChT in cholinergic interneurons. Decreasing VAChT activity produces a decrease in spontaneous SV release with no change to quantal size and no decrease in the number of vesicles at the active zone. This suggests that many vesicles are lacking in neurotransmitter. Overexpression of VAChT leads to increased frequency of SV release, but again with no change in quantal size or vesicle number. This indicates that loading of central cholinergic SVs obeys the "set-point" model, rather than the "steady-state" model that better describes loading at the vertebrate neuromuscular junction. However, this study shows that expression of a VAChT polymorphism lacking one glutamine residue in a COOH-terminal polyQ domain leads to increased spontaneous SV release and increased quantal size. This effect spotlights the poly-glutamine domain as potentially being important for sensing the level of neurotransmitter in cholinergic SVs (Cash, 2016).

Using a Drosophila central synapse, this study has investigated in vivo how VAChT regulates cholinergic transmission. Decreased VAChT activity was shown to leads to decreased spontaneous quantal release frequency. Increased VAChT activity, by contrast, leads to increased frequency of spontaneous release with no change to amplitude or number of SVs at the active zone, suggestive of an increased probability of SV release (Cash, 2016).

Decreased functional VAChT causes a reduction in spontaneous quantal release frequency but not quantal size. This is in agreement with studies at Drosophila and snake NMJs, where decreased vesicular transporter results in decreased frequency but not amplitude of miniature excitatory junctional potentials. However, many studies in mice and rats also link decreased VAChT with decreased transmitter load. A possible explanation for this apparent difference is that Drosophila cholinergic SVs usually contain only one VAChT, and so each SV is either loaded with ACh or empty if the VAChT is blocked by 5Cl-CASPP. This stduy reports no change in vesicle number or size at the active zone, which suggests that there may be empty vesicles that undergo recycling, as has been previously reported. This is supported by a decrease in total quanta released after bafilomycin treatment (Cash, 2016).

When VAChT activity is increased, a clear increase is seen in frequency with no change in amplitude of spontaneous quantal release. An increase in mini frequency with increased VAChT is in good agreement with a study in Xenopus spinal neurons. Moreover, VGLUT overexpression at the Drosophila NMJ results in a modest increase in quantal release (Daniels, 2004). However, these studies, and another in rat, consistently report an increase in transmitter load of the SVs, which supports the steady-state model of SV filling. Based on the current observations, SV loading of ACh at central synapses in Drosophila is consistent with a set-point model, which caps the amount of uptake regardless of transporter activity (Williams, 1997). Williams suggests the set point model is achieved through a feedback mechanism. The observation that, following removal of a single glutamine residue from a 13-glutamine polyQ domain, mini amplitude is significantly increased indicates that this region may be involved in that feedback mechanism. An alternative is that VAChT-Q, which is a naturally occurring polymorphism, obeys the steady-state model. Either way, it would seem that the current data do not support one clear mechanism, raising the possibility that SV filling may differ between species and perhaps even within a species where such polymorphisms are expressed (Cash, 2016).

This study further showed that increased spontaneous release frequency is not likely caused by an increase in the number of vesicles at the active zone or active zone density. This corroborates previous work that shows VAChT plays a second role: facilitating SV mobilization or fusion. In Caenorhabditis elegans, an interaction between VAChT and SV release machinery has been reported (Sandoval, 2006). A glycine-to-arginine amino acid change at position 347 disrupts an interaction with synaptobrevin, a vesicle-associated membrane protein that is pivotal for exocytosis. The glycine at position 347 is well conserved and is present in Drosophila VAChT. However, a glycine-to-arginine substitution at this position in the Drosophila VAChT seemingly results in a nonfunctional protein that cannot rescue the VAChT null (Cash, 2016).

This study presents supporting data for a dual role for VAChT in SV filling and release. Moreover, it provides evidence to suggest that the polyQ domain of Drosophila VAChT has a role in determining SV transmitter load. Mammals, including humans, rats, and mice, have a di-leucine motif at residues 485-486 within the cytoplasmic COOH-terminal. This di-leucine motif has been reported to be important for localizing VAChT to the SV membrane and also to play a role in endocytosis after neurotransmitter release through an interaction with the AP-2 complex. Drosophila VAChT does not have a di-leucine motif, but, unlike the mammalian VAChT sequences mentioned, has a 13-residue polyQ domain at the COOH-terminal. Extended polyQ domains are associated with diseases such as Huntington's and spinocerebellar ataxia. Little is known about the normal function of polyQ domains, but functions may include protein-protein interactions, transcriptional regulation, and RNA binding and signaling. This poses the possibility that the polyQ domain may be responsible for VAChT localization and endocytosis in Drosophila. It has been suggested that the number of glutamines may be of importance. It is possible, therefore, that VAChT-Q may be transported more efficiently to the SV membrane. In D. melanogaster the VAChT-Q tested in this study is a naturally occurring polymorphism identified during cloning of the Drosophila VAChT by Sluder (2012) and subsequently confirmed by sequencing PCR products from cDNA libraries (Cash, 2016).

A BLAST search for similar amino acid sequences and predicted sequences of VAChT found that many Drosophila species contain polyQ domains in the same region as D. melanogaster, but that the length varied from 15 in D. pseudoobscura to 5 in D. grimshawi. Other insects that also have a polyQ domain in the same region include the housefly Musca domestica, which has a nine-residue polyQ domain, and three Anopheles mosquitoes (A. sinensis, A. gambiae, and A. darlingi with 7, 10, and 7 glutamines, respectively). Ant, moth, bee, and butterfly species found during the search did not contain a polyQ of more than two glutamines in the same region. The presence of the polyQ domain in three malaria-transmitting mosquito species but not other insects identifies this region as a possible target for insecticides to control these disease-carrying insects (Cash, 2016).

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. This study shows that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution (Sadananda, 2012).

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. This study describes a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. The vesicular acetylcholine transporter is confidently assigned as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway, and it is anticipated that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family (Sluder, 2012).

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (ChaT). To isolate Vacht mutants, an F(2) genetic screen was performed and mutations were identified that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht¹ and Vacht²). The other is a new temperature-sensitive allele of Cha (Cha(ts3)). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. All deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht¹ is the embryonic stage, whereas that for Vacht² is the larval stage. Viable first-instar larvae homozygous for Vacht² showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus (Kitamoto, 2000).

Deficits in the vesicular acetylcholine transporter alter lifespan and behavior in adult Drosophila melanogaster

The neurotransmitter acetylcholine (ACh) is involved in critical organismal functions that include locomotion and cognition. Importantly, alterations in the cholinergic system are a key underlying factor in cognitive defects associated with aging. One essential component of cholinergic synaptic transmission is the vesicular ACh transporter (VAChT), which regulates the packaging of ACh into synaptic vesicles for extracellular release. Mutations that cause a reduction in either protein level or activity lead to diminished locomotion ability whereas complete loss of function of VAChT is lethal. While much is known about the function of VAChT, the direct role of altered ACh release and its association with either an impairment or an enhancement of cognitive function are still not fully understood. It was hypothesized that point mutations in Vacht cause age-related deficits in cholinergic-mediated behaviors such as locomotion, and learning and memory. Using Drosophila melanogaster as a model system, several mutations within Vacht were studied and their effect on survivability and locomotive behavior were observed. A weak hypomorphic Vacht allele was found that shows a differential effect on ACh-linked behaviors. It was also demonstrated that partially rescued Vacht point mutations cause an allele-dependent deficit in lifespan and defects in locomotion ability. Moreover, using a thorough data analytics strategy to identify exploratory behavioral patterns, new paradigms were introduced for measuring locomotion-related activities that could not be revealed or detected by a simple measure of the average speed alone. Together, these data indicate a role for VAChT in the maintenance of longevity and locomotion abilities in Drosophila and additional measurements of locomotion are provided that can be useful in determining subtle changes in Vacht function on locomotion-related behaviors (White, 2020).

Structure and organization of the Drosophila cholinergic locus

The Drosophila cholinergic locus is composed of two distinct genetic functions: choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter (VAChT), the synaptic vesicle membrane protein that pumps transmitter into vesicles. Both genes share a common first exon and the remainder of the VAChT gene contains a single coding exon residing entirely within the first intron of ChAT. RNase protection analysis indicates that all Drosophila VAChT specific transcripts contain the shared first exon and suggests common transcriptional control for ChAT and VAChT. Similar types of genomic organization have been evolutionarily conserved for cholinergic loci in nematodes and vertebrates, and may operate to ensure coordinate expression of these functionally related genes in the same cells. The relative levels of Drosophila ChAT and VAChT mRNA differ, however, in different tissues or in Cha mutants, indicating that independent regulation of ChAT and VAChT transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5'- and 3'-untranslated regions (UTR). The 5'-UTR contains a GTG ChAT translational initiation codon along with three other potential ATG initiation codons. These features of the VAChT 5'-UTR region suggest that a ribosome scanning model may not be used for VAChT translation initiation (Kitamoto, 1998).

Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori

Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, the cholinergic gene expression in a specific subset of neurons is unknown in insects except for Drosophila melanogaster. This study analysed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. Multiple enhancer regions were found that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. Dibutyryl-cAMP, an analogue of cAMP, influences the expression of ChAT in B. mori. Tissue-specific expression analysis of transcriptional factors identified potential candidates that control the cholinergic gene locus expression. This investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect (Banzai, 2021).

Modular organization of Cis-regulatory control information of neurotransmitter pathway genes in Caenorhabditis elegans

This study explored the cis-regulatory logic that dictates gene expression in specific cell types in the nervous system. We focus on a set of eight genes involved in the synthesis, transport, and breakdown of three neurotransmitter systems: acetylcholine (unc-17 /VAChT, cha-1 /ChAT, cho-1/ChT, and ace-2 /AChE), glutamate (eat-4 /VGluT), and gamma-aminobutyric acid (unc-25 /GAD, unc-46 /LAMP, and unc-47 /VGAT). These genes are specifically expressed in defined subsets of cells in the nervous system. Through transgenic reporter gene assays, the cellular specificity of expression of all of these genes was found to be controlled in a modular manner through distinct cis-regulatory elements, corroborating the previously inferred piecemeal nature of specification of neurotransmitter identity. This modularity provides the mechanistic basis for the phenomenon of 'phenotypic convergence,' in which distinct regulatory pathways can generate similar phenotypic outcomes (i.e., the acquisition of a specific neurotransmitter identity) in different neuron classes. Cases of enhancer pleiotropy were identified, in which the same cis-regulatory element is utilized to control gene expression in distinct neuron types. A cis-regulatory allele of the vesicular acetylcholine transporter, unc-17 /VAChT, was engineered to assess the functional contribution of a 'shadowed' enhancer. A selective loss was observed of unc-17 /VAChT expression in one cholinergic pharyngeal pacemaker motor neuron class and a behavioral phenotype that matches microsurgical removal of this neuron. This analysis illustrates the value of understanding cis-regulatory information to manipulate gene expression and control animal behavior (Serrano-Saiz, 2020).

Past work has defined the modular nature of neurotransmitter identity specification in C. elegans from two different perspectives. First, distinct terminal selector-type transcription factors were found to be required to specify the same neurotransmitter identity in different neuron types. In several cases, it was explicitly shown that this specificity is achieved through the binding of the respective transcription factor to defined cis-regulatory elements within neurotransmitter pathway genes. Second, reporter constructs that capture distinct regions of the loci that encode for neurotransmitter pathway genes were shown to be selectively expressed in distinct subsets of ACh, glutamate, GABA, or monoaminergic neurons, respectively. This study has expanded the analysis of cis-regulatory control regions of neurotransmitter pathway genes, thereby corroborating the largely modular control of neurotransmitter identity. However, not every neuron type is controlled by an independent, dedicated module, since the same cis-regulatory element can be employed by the same transcription factor in different cell types, likely by acting in the context of a distinct combinatorial code of transcription factors. Hence, cell type-specific gene expression profiles are controlled by combinations of different types of regulatory constellations (Serrano-Saiz, 2020).

This and past analysis clearly shows that distinct, modular regulatory inputs control the expression of neurotransmitter pathway systems in distinct neuron types (see Illustrations of the organization of cis-regulatory elements for neuron type-specific gene expression). Apart from using transgenic reporter gene assays, this study provides a striking example of this modularity by engineering a cis-regulatory allele of the unc-17/VAChT locus in which a small, phylogenetically conserved motif was mutated. This manipulation not only leads to a selective loss of unc-17/VAChT expression in a single neuron class, but also causes the phenotype that one would expect from dysfunctionality of this neuron (Serrano-Saiz, 2020).

Nevertheless, past work, as well as the work presented in this study, also demonstrates that regulatory elements that drive expression in distinct neuron types can be difficult to disentangle, i.e., even small cis-regulatory elements still drive expression in multiple neuron classes. Does this observation contrast with the modular control model and support a singular control model? Theoretically, a more finely grained mutational analysis may still separate distinct elements for distinct cell types. However, there are already clear cases where it is known that the same transcription factor acts in distinct cells to specify, for example, their cholinergic identity, seemingly in support of the singular model. However, in many if not all cases, this singular transcription factor may act in combination with different sets of cell type-specific partner proteins. For example, the COE-type transcription factor unc-3 controls unc-17/VAChT and cho-1/ChT expression in VNC motor neurons and their presynaptic command interneurons, which are all cholinergic and specified by unc-3. Mutation of the unc-3-binding site leads to loss of expression in all these neuron classes. Similarly, a regulatory element from the unc-17/VAChT locus shows expression in the IL2, URA, and URB neuron classes, all of which require unc-86 for their proper neurotransmitter identity acquisition. Likewise, a regulatory element from the eat-4 locus is expressed in the AIM and PHC neuron classes, both of which require unc-86 for their glutamatergic identity specification. Cis-regulatory elements that control expression of GABA pathway genes in the RIS and AVL neuron classes (both controlled by the nhr-67 and lim-6 transcription factors) can also not be easily separated, while cis-regulatory elements for GABAergic VNC motor neurons (which are dependent on the unc-30 transcription factor) can be separated from RIS and AVL. These examples demonstrate that the modularity of cis-regulatory control does not need to manifest itself by the existence of distinct, completely separable cis-regulatory elements that drive expression in distinct neuron types. Rather, the same cis-regulatory element and its cognate transcription factor may be deployed in different cell types because transcription factors operate in distinct, cell type-specific combinations (Serrano-Saiz, 2020).

Enhancers that are employed to drive expression in different cell types have been termed 'pleiotropic enhancers'. Such pleiotropy has been mostly inferred from genome-wide binding profiles of histone modification marks that indicate (but do not prove) enhancer activity at a relatively coarse level. Specifically, marks of enhancer activity can be found in the same location in distinct tissue types. The underlying mechanistic basis for enhancer pleiotropy is often not clear. The cis-regulatory analysis conducted in this and previous papers not only supports the concept of enhancer pleiotropy in an orthogonal and more granular manner, but also strongly hints at its mechanistic basis. Different neuron types utilize the same cis-regulatory element because this regulatory element contains binding sites for the same trans-acting factor (Serrano-Saiz, 2020).

While the unc-3 case provides the clearest evidence for the same cis-regulatory motif to be employed in different neuron types, this analysis also reveals that a transcription factor required for acquisition of the same neurotransmitter identity in distinct neuron types does not necessarily always act through the same cis-regulatory element. For example, UNC-86 operates in more than a dozen different neuron types to specify neurotransmitter identity. It appears that UNC-86 functions through separable cis-regulatory elements in the unc-17 locus to enable cholinergic gene expression in different cell types. These distinctive types of engagement of UNC-86 with its target promoters are a likely reflection of distinct molecular partnerships that UNC-86 may engage in (Serrano-Saiz, 2020).

In several neurotransmitter pathway genes, the existence is noted of separable elements that drive expression in the same neuron types. For example, RME motor neuron expression is controlled by separable cis-regulatory elements in the unc-25/GAD locus. This is akin to the 'shadow enhancers' that have been observed to drive the expression of developmental control genes in Drosophila. One immediate question that arises from such a constellation is whether this is an indication of true enhancer redundancies. Complete redundancy would predict that deletion of one of these elements would result in no expression defect because of the existence of the other element. Alternatively, the sufficiency of isolated elements to drive expression could be a result of the multicopy nature of concatamerized C. elegans transgenes. In the natural genomic context, several separable cis-regulatory elements may need to cooperate to turn on expression in a specific cell type. This notion was tested explicitly through mutating a small conserved motif that is located in one of two separate cis-regulatory elements of the unc-17 locus, which each drive expression in the same set of pharyngeal neurons. Completely penetrant defects in unc-17 expression were found in a single neuron class, thereby indicating that, in their endogenous context, a cis-regulatory element may be required even if another element produces, in the context of a multicopy array, the same pattern of expression. This finding is not without precedent. Two separate cis-regulatory elements were defined in the cog-1 homeobox gene locus that are alone sufficient to drive expression in the ASE neuron. Nevertheless, a cis-regulatory allele of cog-1 was isolated from a mutant screen in which only one of those two elements is mutated. These examples illustrate an important limitation for the interpretation of reporter gene analysis: the expression of isolated elements may demonstrate that an element is sufficient to produce an expression pattern, but they do not prove that such an element is required in its normal genomic context (Serrano-Saiz, 2020).

Pioneering work by Eric Davidson and colleagues in the sea urchin embryo revealed that 'nuts and bolts' differentiation genes, i.e., genes that do not encode regulatory factors, but rather 'function' proteins that endow a cell with specific anatomical or functional properties, are largely controlled by positive regulatory inputs, i.e., by transcriptional activators. This cis-regulatory analysis of neurotransmitter pathway genes provides further support for this notion. If the expression patterns of neurotransmitter pathway genes were frequently shaped by repressive mechanisms, successive deletion of regulatory elements should have revealed ectopic expression of reporter genes. While such derepression was observed in some rare cases, repression does not appear to be a major mechanism in shaping the expression of neurotransmitter pathway genes. The cis-regulatory analysis of other neuronal differentiation genes, undertaken by this laboratory over the years , also provides only limited evidence in favor of repression being an important mechanism to shape neuron type-specific gene expression profiles. This is not to say that transcriptional repression plays no role in shaping the terminal differentiation program. Several studies have revealed that transcriptional repression is used in a number of distinct cellular contexts (sensory neurons and motor neurons) to establish subtle, but important molecular differences between members of the same neuron class [e.g., differences between left vs. right ASE neurons or differences between different VNC motor neuron classes (Serrano-Saiz, 2020).

Lastly, the dissection of cis-regulatory elements of genes that define specific phenotypic traits of distinct neuron types (i.e., neurotransmitter identities) highlights the underlying mechanistic basis of phenotypic convergence. In evolutionary theory, phenotypic convergence refers to independent evolution of similar traits in different organisms. This terminology has recently been applied to the acquisition of the phenotypic trait of neurotransmitter identity. In both C. elegans and Drosophila, distinct transcription factors control the same phenotypic trait in distinct neuron types (Serrano-Saiz, 2013; Konstantinides, 2018; Kratsios and Hobert 2018). Cis-regulatory analyses like the ones described here demonstrate that it is the cis-regulatory control regions of neurotransmitter pathway genes that integrate distinct regulatory inputs to turn on a common neurotransmitter trait in distinct neuron types.

Search PubMed for articles about Drosophila ChAT or VAChT

Aguilar, J. I., Dunn, M., Mingote, S., Karam, C. S., Farino, Z. J., Sonders, M. S., Choi, S. J., Grygoruk, A., Zhang, Y., Cela, C., Choi, B. J., Flores, J., Freyberg, R. J., McCabe, B. D., Mosharov, E. V., Krantz, D. E., Javitch, J. A., Sulzer, D., Sames, D., Rayport, S. and Freyberg, Z. (2017). Neuronal depolarization drives increased dopamine synaptic vesicle loading via VGLUT. Neuron 95(5): 1074-1088 e1077. PubMed ID: 28823729

Banzai, K. and Izumi, S. (2021). Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori. Insect Mol Biol. PubMed ID: 34549831

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

Boppana, S., Kendall, N., Akinrinsola, O., White, D., Patel, K. and Lawal, H. (2017). Immunolocalization of the vesicular acetylcholine transporter in larval and adult Drosophila neurons. Neurosci Lett 643:76-83. PubMed ID: 28188850

Cash, F., Vernon, S. W., Phelan, P., Goodchild, J. and Baines, R. A. (2016). Central cholinergic synaptic vesicle loading obeys the set-point model in Drosophila. J Neurophysiol 115(2): 843-850. PubMed ID: 26655826

Daniels, R. W., Collins, C. A., Gelfand, M. V., Dant, J., Brooks, E. S., Krantz, D. E. and DiAntonio, A. (2004). Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content. J Neurosci 24(46): 10466-10474. PubMed ID: 15548661

Davie, K., Janssens, J., Koldere, D., De Waegeneer, M., Pech, U., Kreft, L., Aibar, S., Makhzami, S., Christiaens, V., Bravo Gonzalez-Blas, C., Poovathingal, S., Hulselmans, G., Spanier, K. I., Moerman, T., Vanspauwen, B., Geurs, S., Voet, T., Lammertyn, J., Thienpont, B., Liu, S., Konstantinides, N., Fiers, M., Verstreken, P. and Aerts, S. (2018). A single-cell transcriptome atlas of the aging Drosophila brain. Cell 174(4): 982-998 e920. PubMed ID: 29909982

Dey, S. and Ray, K. (2018). Cholinergic activity is essential for maintaining the anterograde transport of Choline Acetyltransferase in Drosophila. Sci Rep 8(1): 8028. PubMed ID: 29795337

Estacio-Gómez, A., Hassan, A., Walmsley, E., Le, L. W. and Southall, T. D. (2020). Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development. Biol Open 9(5). PubMed ID: 32493733

Harris, R. M., Pfeiffer, B. D., Rubin, G. M. and Truman, J. W. (2015). Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system. Elife 4. PubMed ID: 26193122

Kerk, S. Y., Kratsios, P., Hart, M., Mourao, R. and Hobert, O. (2017). Diversification of C. elegans motor neuron identity via selective effector gene repression. Neuron 93(1): 80-98. PubMed ID: 28056346

Kitamoto, T., Wang, W. and Salvaterra, P. M. (1998). Structure and organization of the Drosophila cholinergic locus. J Biol Chem 273(5): 2706-2713. PubMed ID: 9446576

Kitamoto, T., Xie, X., Wu, C. F. and Salvaterra, P. M. (2000). Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster. J Neurobiol 42(2): 161-171. PubMed ID: 10640324

Kratsios, P. and Hobert, O. (2018). Nervous system development: flies and worms converging on neuron identity control. Curr Biol 28(19): R1154-R1157. PubMed ID: 30300603

Konstantinides, N., Kapuralin, K., Fadil, C., Barboza, L., Satija, R. and Desplan, C. (2018). Phenotypic convergence: distinct transcription factors regulate common terminal features. Cell 174(3): 622-635 e613. PubMed ID: 29909983

Lacin, H., Zhu, Y., Wilson, B. A. and Skeath, J. B. (2014). Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system. Development 141(5): 1011-1021. PubMed ID: 24550109

Lacin, H., Chen, H. M., Long, X., Singer, R. H., Lee, T. and Truman, J. W. (2019). Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS. Elife 8. PubMed ID: 30912745

Lee, M. H. and Salvaterra, P. M. (2002). Abnormal chemosensory jump 6 is a positive transcriptional regulator of the cholinergic gene locus in Drosophila olfactory neurons. J Neurosci 22(13): 5291-5299. PubMed ID: 12097480

Liu, B. and Bossing, T. (2016). Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing. Sci Rep 6: 34952. PubMed ID: 27725692

Lobell, A. S., Kaspari, R. R., Serrano Negron, Y. L. and Harbison, S. T. (2017). The genetic architecture of ovariole number in Drosophila melanogaster: Genes with major, quantitative, and pleiotropic effects. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28550012

Pankova, K. and Borst, A. (2017). Transgenic line for the identification of cholinergic release sites in Drosophila melanogaster. J Exp Biol 220(Pt 8):1405-1410. PubMed ID: 28167805

Rost, B. R., Schneider, F., Grauel, M. K., Wozny, C., Bentz, C., Blessing, A., Rosenmund, T., Jentsch, T. J., Schmitz, D., Hegemann, P. and Rosenmund, C. (2015). Optogenetic acidification of synaptic vesicles and lysosomes. Nat Neurosci 18(12): 1845-1852. PubMed ID: 26551543

Sadananda, A., Hamid, R., Doodhi, H., Ghosal, D., Girotra, M., Jana, S. C. and Ray, K. (2012). Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse. Traffic 13(7): 979-991. PubMed ID: 22486887

Sandoval, G. M., Duerr, J. S., Hodgkin, J., Rand, J. B. and Ruvkun, G. (2006). A genetic interaction between the vesicular acetylcholine transporter VAChT/UNC-17 and synaptobrevin/SNB-1 in C. elegans. Nat Neurosci 9(5): 599-601. PubMed ID: 16604067

Showell, S. S., Martinez, Y., Gondolfo, S., Boppana, S. and Lawal, H. O. (2020). Overexpression of the vesicular acetylcholine transporter disrupts cognitive performance and causes age-dependent locomotion decline in Drosophila. Mol Cell Neurosci 105: 103483. PubMed ID: 32217162

Serrano-Saiz, E., Poole, R. J., Felton, T., Zhang, F., De La Cruz, E. D. and Hobert, O. (2013). Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins. Cell 155(3): 659-673. PubMed ID: 24243022

Serrano-Saiz, E., Gulez, B., Pereira, L., Gendrel, M., Kerk, S. Y., Vidal, B., Feng, W., Wang, C., Kratsios, P., Rand, J. B. and Hobert, O. (2020). Modular organization of Cis-regulatory control information of neurotransmitter pathway genes in Caenorhabditis elegans. Genetics 215(3): 665-681. PubMed ID: 32444379

Sluder, A., Shah, S., Cassayre, J., Clover, R., Maienfisch, P., Molleyres, L. P., Hirst, E. A., Flemming, A. J., Shi, M., Cutler, P., Stanger, C., Roberts, R. S., Hughes, D. J., Flury, T., Robinson, M. P., Hillesheim, E., Pitterna, T., Cederbaum, F., Worthington, P. A., Crossthwaite, A. J., Windass, J. D., Currie, R. A. and Earley, F. G. (2012). Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action. PLoS One 7(5): e34712. PubMed ID: 22563457

Spitzer, N. C. (2015). Neurotransmitter switching? No surprise. Neuron 86(5): 1131-1144. PubMed ID: 26050033

Trudeau, L. E. and El Mestikawy, S. (2018). Glutamate cotransmission in cholinergic, GABAergic and monoamine systems: contrasts and commonalities. Front Neural Circuits 12: 113. PubMed ID: 30618649

Vernon, S. W., Goodchild, J. and Baines, R. A. (2019). A poly-glutamine region in the Drosophila VAChT dictates fill-level of cholinergic synaptic vesicles. eNeuro 6(1). PubMed ID: 30847389

White, D., de Sousa Abreu, R. P., Blake, A., Murphy, J., Showell, S., Kitamoto, T. and Lawal, H. O. (2020). Deficits in the vesicular acetylcholine transporter alter lifespan and behavior in adult Drosophila melanogaster. Neurochem Int 137: 104744. PubMed ID: 32315665

Williams, J. (1997). How does a vesicle know it is full? Neuron 18(5): 683-686. PubMed ID: 9182793

date revised: 27 August 2020

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