Ca2+ imaging of synaptic compartments using subcellularly targeted GCaMP8f in Drosophila.

GCaMP8f is a sensitive genetically encoded Ca2+ indicator that enables imaging of neuronal activity. Here, we present a protocol to perform Ca2+ imaging of the Drosophila neuromuscular junction using GCaMP8f targeted to pre- or postsynaptic compartments. We describe ratiometric Ca2+ imaging using GCaMP8f fused to mScarlet and synaptotagmin that reveals Ca2+ dynamics at presynaptic terminals. We then detail "quantal" imaging of miniature transmission events using GCaMP8f targeted to postsynaptic compartments by fusion to a PDZ-binding motif. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Han et al.,2 Perry et al.,3 and Han et al.4.

Tissue-specific knockout in Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions in animal development. However, this approach has been successfully applied in only a small number of Drosophila tissues. The Drosophila motor nervous system is an excellent model system for studying the biology of neuromuscular junction (NMJ). To expand tissue-specific CRISPR to the Drosophila motor system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of this toolkit by knocking out known genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. Using these tools, we discovered an essential role for SNARE pathways in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating the retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to lease extracellular vesicles at the NMJ.

Excess glutamate release triggers subunit-specific homeostatic receptor scaling.

Ionotropic glutamate receptors (GluRs) are targets for modulation in Hebbian and homeostatic synaptic plasticity and are remodeled by development, experience, and disease. We have probed the impact of synaptic glutamate levels on the two postsynaptic GluR subtypes at the Drosophila neuromuscular junction, GluRA and GluRB. We first demonstrate that GluRA and GluRB compete to establish postsynaptic receptive fields, and that proper GluR abundance and composition can be orchestrated in the absence of any synaptic glutamate release. However, excess glutamate adaptively tunes postsynaptic GluR abundance, echoing GluR scaling observed in mammalian systems. Furthermore, when GluRA vs. GluRB competition is eliminated, GluRB becomes insensitive to glutamate modulation. In contrast, GluRA is now homeostatically regulated by excess glutamate to maintain stable miniature activity, where Ca2+ permeability through GluRA receptors is required. Thus, excess glutamate, GluR competition, and Ca2+ signaling collaborate to selectively target GluR subtypes for homeostatic regulation at postsynaptic compartments.

Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." Here, we used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated ∼2-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.SIGNIFICANCE STATEMENT "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how input-specific synaptic diversity is achieved, which could have implications for neurologic disorders that involve changes in synaptic function.

PIKFYVE inhibition mitigates disease in models of diverse forms of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.

A glutamate receptor C-tail recruits CaMKII to suppress retrograde homeostatic signaling.

Presynaptic homeostatic plasticity (PHP) adaptively enhances neurotransmitter release following diminished postsynaptic glutamate receptor (GluR) functionality to maintain synaptic strength. While much is known about PHP expression mechanisms, postsynaptic induction remains enigmatic. For over 20 years, diminished postsynaptic Ca2+ influx was hypothesized to reduce CaMKII activity and enable retrograde PHP signaling at the Drosophila neuromuscular junction. Here, we have interrogated inductive signaling and find that active CaMKII colocalizes with and requires the GluRIIA receptor subunit. Next, we generated Ca2+-impermeable GluRs to reveal that both CaMKII activity and PHP induction are Ca2+-insensitive. Rather, a GluRIIA C-tail domain is necessary and sufficient to recruit active CaMKII. Finally, chimeric receptors demonstrate that the GluRIIA tail constitutively occludes retrograde homeostatic signaling by stabilizing active CaMKII. Thus, the physical loss of the GluRIIA tail is sensed, rather than reduced Ca2+, to enable retrograde PHP signaling, highlighting a unique, Ca2+-independent control mechanism for CaMKII in gating homeostatic plasticity.

Rapid homeostatic modulation of transsynaptic nanocolumn rings.

Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, we found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. We reveal distinct phases of structural transsynaptic homeostatic plasticity, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy.

Botulinum neurotoxin accurately separates tonic vs. phasic transmission and reveals heterosynaptic plasticity rules in Drosophila.

In developing and mature nervous systems, diverse neuronal subtypes innervate common targets to establish, maintain, and modify neural circuit function. A major challenge towards understanding the structural and functional architecture of neural circuits is to separate these inputs and determine their intrinsic and heterosynaptic relationships. The Drosophila larval neuromuscular junction is a powerful model system to study these questions, where two glutamatergic motor neurons, the strong phasic-like Is and weak tonic-like Ib, co-innervate individual muscle targets to coordinate locomotor behavior. However, complete neurotransmission from each input has never been electrophysiologically separated. We have employed a botulinum neurotoxin, BoNT-C, that eliminates both spontaneous and evoked neurotransmission without perturbing synaptic growth or structure, enabling the first approach that accurately isolates input-specific neurotransmission. Selective expression of BoNT-C in Is or Ib motor neurons disambiguates the functional properties of each input. Importantly, the blended values of Is+Ib neurotransmission can be fully recapitulated by isolated physiology from each input. Finally, selective silencing by BoNT-C does not induce heterosynaptic structural or functional plasticity at the convergent input. Thus, BoNT-C establishes the first approach to accurately separate neurotransmission between tonic vs. phasic neurons and defines heterosynaptic plasticity rules in a powerful model glutamatergic circuit.

One domain to rule them all: "In synapse" reconstitution of core active zone functions.

Active zones are colossal and complex molecular machines that transform electrical signals into rapid neurotransmitter secretion. In this issue of Neuron, Tan et al. (2022) elegantly distill central functions of this synaptic apparatus by tethering a small domain of the scaffold RIM near Ca2+ channels.

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter.

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Autocrine inhibition by a glutamate-gated chloride channel mediates presynaptic homeostatic depression.

Homeostatic modulation of presynaptic neurotransmitter release is a fundamental form of plasticity that stabilizes neural activity, where presynaptic homeostatic depression (PHD) can adaptively diminish synaptic strength. PHD has been proposed to operate through an autocrine mechanism to homeostatically depress release probability in response to excess glutamate release at the Drosophila neuromuscular junction. This model implies the existence of a presynaptic glutamate autoreceptor. We systematically screened all neuronal glutamate receptors in the fly genome and identified the glutamate-gated chloride channel (GluClα) to be required for the expression of PHD. Pharmacological, genetic, and Ca2+ imaging experiments demonstrate that GluClα acts locally at axonal terminals to drive PHD. Unexpectedly, GluClα localizes and traffics with synaptic vesicles to drive presynaptic inhibition through an activity-dependent anionic conductance. Thus, GluClα operates as both a sensor and effector of PHD to adaptively depress neurotransmitter release through an elegant autocrine inhibitory signaling mechanism at presynaptic terminals.

Engineering skeletal muscle tissues with advanced maturity improves synapse formation with human induced pluripotent stem cell-derived motor neurons.

To develop effective cures for neuromuscular diseases, human-relevant in vitro models of neuromuscular tissues are critically needed to probe disease mechanisms on a cellular and molecular level. However, previous attempts to co-culture motor neurons and skeletal muscle have resulted in relatively immature neuromuscular junctions (NMJs). In this study, NMJs formed by human induced pluripotent stem cell (hiPSC)-derived motor neurons were improved by optimizing the maturity of the co-cultured muscle tissue. First, muscle tissues engineered from the C2C12 mouse myoblast cell line, cryopreserved primary human myoblasts, and freshly isolated primary chick myoblasts on micromolded gelatin hydrogels were compared. After three weeks, only chick muscle tissues remained stably adhered to hydrogels and exhibited progressive increases in myogenic index and stress generation, approaching values generated by native muscle tissue. After three weeks of co-culture with hiPSC-derived motor neurons, engineered chick muscle tissues formed NMJs with increasing co-localization of pre- and postsynaptic markers as well as increased frequency and magnitude of synaptic activity, surpassing structural and functional maturity of previous in vitro models. Engineered chick muscle tissues also demonstrated increased expression of genes related to sarcomere maturation and innervation over time, revealing new insights into the molecular pathways that likely contribute to enhanced NMJ formation. These approaches for engineering advanced neuromuscular tissues with relatively mature NMJs and interrogating their structure and function have many applications in neuromuscular disease modeling and drug development.

Tissue-Specific Ribosome Profiling in Drosophila.

Robust mechanisms exist that serve to dynamically regulate the translation of mRNA into proteins across heterogeneous tissues. These processes ensure timely generation of proteins in quantities that scale with the demands of specific cell types. Importantly, this translational regulation occurs with spatiotemporal precision and is capable of recalibration as conditions change. Aberrant regulation of translation contributes to and exacerbates a wide range of diseases. Although dynamic control of translation is an essential and fundamental process shared by organisms, specific tissues and cell types can be differentially impacted by circumstances that challenge and impair basal translation, highlighting the heterogeneous nature of translational regulation. To understand how translation is differentially regulated during changing environments and across specific cells and tissues, methods capable of profiling translation in specific tissues and cells are crucial. Here, we describe a method for profiling genome-wide translation in specific tissues or cell types in Drosophila melanogaster, in which we combine ribosome affinity purification with ribosome profiling to enable a simplified protocol for robust analysis of translation in specific tissues.

Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly.

As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. Here, we identified a "molecular choreography" coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, we here provide direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with postsynaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes.

Synaptic homeostats: latent plasticity revealed at the Drosophila neuromuscular junction.

Homeostatic signaling systems are fundamental forms of biological regulation that maintain stable functionality in a changing environment. In the nervous system, synapses are crucial substrates for homeostatic modulation, serving to establish, maintain, and modify the balance of excitation and inhibition. Synapses must be sufficiently flexible to enable the plasticity required for learning and memory but also endowed with the stability to last a lifetime. In response to the processes of development, growth, remodeling, aging, and disease that challenge synapses, latent forms of adaptive plasticity become activated to maintain synaptic stability. In recent years, new insights into the homeostatic control of synaptic function have been achieved using the powerful Drosophila neuromuscular junction (NMJ). This review will focus on work over the past 10 years that has illuminated the cellular and molecular mechanisms of five homeostats that operate at the fly NMJ. These homeostats adapt to loss of postsynaptic neurotransmitter receptor functionality, glutamate imbalance, axonal injury, as well as aberrant synaptic growth and target innervation. These diverse homeostats work independently yet can be simultaneously expressed to balance neurotransmission. Growing evidence from this model glutamatergic synapse suggests these ancient homeostatic signaling systems emerged early in evolution and are fundamental forms of plasticity that also function to stabilize mammalian cholinergic NMJs and glutamatergic central synapses.

The auxiliary glutamate receptor subunit dSol-1 promotes presynaptic neurotransmitter release and homeostatic potentiation.

Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). Here, we identify an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. We find that dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, our data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, we propose that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse.

Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments.

Neurons must establish and stabilize connections made with diverse targets, each with distinct demands and functional characteristics. At Drosophila neuromuscular junctions (NMJs), synaptic strength remains stable in a manipulation that simultaneously induces hypo-innervation on one target and hyper-innervation on the other. However, the expression mechanisms that achieve this exquisite target-specific homeostatic control remain enigmatic. Here, we identify the distinct target-specific homeostatic expression mechanisms. On the hypo-innervated target, an increase in postsynaptic glutamate receptor (GluR) abundance is sufficient to compensate for reduced innervation, without any apparent presynaptic adaptations. In contrast, a target-specific reduction in presynaptic neurotransmitter release probability is reflected by a decrease in active zone components restricted to terminals of hyper-innervated targets. Finally, loss of postsynaptic GluRs on one target induces a compartmentalized, homeostatic enhancement of presynaptic neurotransmitter release called presynaptic homeostatic potentiation (PHP) that can be precisely balanced with the adaptations required for both hypo- and hyper-innervation to maintain stable synaptic strength. Thus, distinct anterograde and retrograde signaling systems operate at pre- and post-synaptic compartments to enable target-specific, homeostatic control of neurotransmission.

Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration.

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions in which Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). Although wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which time NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.

Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling.

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we design a forward genetic screen to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, we find that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, we show that Peflin, a Ca2+-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment. Our study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses.