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.

Presynaptic Mitochondrial Volume and Packing Density Scale with Presynaptic Power Demand.

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. Neurons have presumably optimized the volume of their bioenergetic machinery to ensure these power demands are met, but the relationship between presynaptic power demands and the volume available to the bioenergetic machinery has never been quantified. Here, we estimated the power demands of six motor nerve terminals in female Drosophila larvae through direct measurements of neurotransmitter release and Ca2+ entry, and via theoretical estimates of Na+ entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (∼66 nmol·min-1·µl-1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol·min-1·µl-1 at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min-1·µl-1 of cytosol at rest, rising to 4.36 during activity. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.SIGNIFICANCE STATEMENT The remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here, we exploit the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the first in vivo estimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.

Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of both Drosophila and mice to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. KEY POINTS: Neurons commonly fire in bursts imposing highly volatile demands on the bioenergetic machinery that generates ATP. Using a computational approach, we built profiles of presynaptic power demand at the level of single action potentials, as well as the transition from rest to sustained activity. Phosphagen systems are known to buffer ATP levels in muscles and we demonstrate that phosphagen kinases, which support such phosphagen systems, also localize to mitochondria in motor nerve terminals of fruit flies and mice. By knocking down phosphagen kinases in fruit fly motor nerve terminals, and using fluorescent reporters of the ATP:ADP ratio, lactate, pH and Ca2+ , we demonstrate a role for phosphagen kinases in stabilizing presynaptic ATP levels. These data indicate that the maintenance of phosphagen systems in motor neurons, and not just muscle, could be a beneficial initiative in sustaining musculoskeletal health and performance.

Computational modeling predicts ephemeral acidic microdomains in the glutamatergic synaptic cleft.

At chemical synapses, synaptic vesicles release their acidic contents into the cleft, leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction as glutamate, ATP, and protons (H+) were released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase activity and predicts substantial cleft acidification but only for fractions of a millisecond after neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 ms because the plasma membrane calcium-ATPase removes H+ from the cleft in exchange for calcium ions from adjacent pre- and postsynaptic compartments, thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis have little influence on the magnitude of acidification. Phosphate but not bicarbonate buffering is effective at suppressing the magnitude and time course of the acid spike, whereas both buffering systems are effective at suppressing cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH transients accompanying neurotransmission.

Neuronal Glutamatergic Synaptic Clefts Alkalinize Rather Than Acidify during Neurotransmission.

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, we observed alkaline spikes of over 1 log unit during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only ∼0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+ movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H+ antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+ channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.SIGNIFICANCE STATEMENT Neurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.

Endogenous Tagging Reveals Differential Regulation of Ca2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression.

Neurons communicate through Ca2+-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca2+ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca2+ channels during homeostatic plasticity. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca2+ influx during PHD is achieved through functional adaptions to pre-existing Ca2+ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.SIGNIFICANCE STATEMENT Presynaptic Ca2+ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca2+ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca2+ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca2+ channels at individual active zones within 10 min. We propose that differential regulation of Ca2+ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication.

Na+ /H+ exchange via the Drosophila vesicular glutamate transporter mediates activity-induced acid efflux from presynaptic terminals.

KEY POINTS: Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid-extruding protein when deposited in the plasmamembrane during exocytosis. The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms. A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals. ABSTRACT: Neuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pHcyto ) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid-extruding protein when deposited on the plasmamembrane.

The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.

Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice.

Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1(-/-) mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTgSod1(-/-) mice compared with control animals, whereas muscles of age-matched Sod1(-/-) mice displayed 30-40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1(-/-) mice were absent in SynTgSod1(-/-) mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1(-/-) mice but not in SynTgSod1(-/-) mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1(-/-) mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.

Ultrastructural analysis reveals mitochondrial placement independent of synapse placement in fine caliber C. elegans neurons.

Neurons rely on mitochondria for an efficient supply of ATP and other metabolites. However, while neurons are highly elongated, mitochondria are discrete and limited in number. Due to the slow rates of diffusion over long distances it follows that neurons would benefit from an ability to control the distribution of mitochondria to sites of high metabolic activity, such as synapses. It is assumed that neurons' possess this capacity, but ultrastructural data over substantial portions of a neuron's extent that would allow for tests of such hypotheses are scarce. Here, we mined the Caenorhabditis elegans electron micrographs of John White and Sydney Brenner and found systematic differences in average mitochondrial length (ranging from 1.3 to 2.4 µm), volume density (3.7% to 6.5%) and diameter (0.18 to 0.24 µm) between neurons of different neurotransmitter type and function, but found limited differences in mitochondrial morphometrics between axons and dendrites of the same neurons. Analyses of distance intervals found mitochondria to be distributed randomly with respect to presynaptic specializations, and an indication that mitochondria were displaced from postsynaptic specializations. Presynaptic specializations were primarily localized to varicosities, but mitochondria were no more likely to be found in synaptic varicosities than non-synaptic varicosities. Consistently, mitochondrial volume density was no greater in varicosities with synapses. Therefore, beyond the capacity to disperse mitochondria throughout their length, at least in C. elegans, fine caliber neurons manifest limited sub-cellular control of mitochondrial size and distribution.

Mitochondrial free Ca²âº levels and their effects on energy metabolism in Drosophila motor nerve terminals.

Mitochondrial Ca²âº uptake exerts dual effects on mitochondria. Ca²âº accumulation in the mitochondrial matrix dissipates membrane potential (ΔΨm), but Ca²âº binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²âº ([Ca²âº]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²âº]m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²âº indicators with a 20-fold difference in Ca²âº affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²âº indicator (D4cpv) calibrated against known Ca²âº levels. Matrix pH (pHm) and ΔΨm were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²âº]m was 0.22 ± 0.04 µM, but it rose to ~26 µM (24.3 ± 3.4 µM with rhod-FF/rhod-5N and 27.0 ± 2.6 µM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²âº]m coincided with a rapid elevation in pHm and was followed by an after-stimulus ΔΨm hyperpolarization. However, pHm decreased and no ΔΨm hyperpolarization was observed in response to lower levels of [Ca²âº]m, up to 13.1 µM. These data indicate that surprisingly high levels of [Ca²âº]m are required to stimulate presynaptic mitochondrial energy metabolism.

Cytosolic calcium coordinates mitochondrial energy metabolism with presynaptic activity.

Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pHm), inner membrane potential (Δψm), and NAD(P)H levels ([NAD(P)H]m) increase within seconds of nerve stimulation. The elevations of pHm, Δψm, and [NAD(P)H]m indicate an increased capacity for ATP production. Elevations in pHm were blocked by manipulations that blocked mitochondrial Ca2+ uptake, including replacement of extracellular Ca2+ with Sr2+ and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca2+ that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca2+ levels ([Ca2+]c) between MNs when each fires at its endogenous rate. The average [Ca2+]c level (329±11 nM) was slightly above the average Ca2+ affinity of the mitochondria (281±13 nM). In summary, we show that when MNs fire at endogenous rates, [Ca2+]c is driven into a range where mitochondria rapidly acquire Ca2+. As we also show that Ca2+ stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca2+]c levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs.

Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo.

All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pH(cyto)) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pH(cyto) shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pH(cyto). Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤ 4 s) trains of action potentials but did buffer slow (~60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca(2+) increase upon stimulation, and partial inhibition of the plasma membrane Ca(2+)-ATPase, a Ca(2+)/H(+) exchanger, attenuated pH(cyto) shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (~0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pH(cyto) shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pH(cyto) shifts cannot be dismissed as artifacts of ex vivo preparations.

Differential control of presynaptic CaMKII activation and translocation to active zones.

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca(2+) mobilization from the endoplasmic reticulum (ER) and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca(2+) release. Here these issues are addressed in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. We report that neuronal excitation acts with ER Ca(2+) stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca(2+) provided by different presynaptic ER Ca(2+) release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca(2+) in the terminal, while translocation to AZs requires Ca(2+) microdomains generated by IP3Rs.

Presynaptic mitochondria in functionally different motor neurons exhibit similar affinities for Ca2+ but exert little influence as Ca2+ buffers at nerve firing rates in situ.

Mitochondria accumulate within nerve terminals and support synaptic function, most notably through ATP production. They can also sequester Ca(2+) during nerve stimulation, but it is unknown whether this limits presynaptic Ca(2+) levels at physiological nerve firing rates. Similarly, it is unclear whether mitochondrial Ca(2+) sequestration differs between functionally different nerve terminals. We addressed these questions using a combination of synthetic and genetically encoded Ca(2+) indicators to examine cytosolic and mitochondrial Ca(2+) levels in presynaptic terminals of tonic (MN13-Ib) and phasic (MNSNb/d-Is) motor neurons in Drosophila, which, as we determined, fire during fictive locomotion at approximately 42 Hz and approximately 8 Hz, respectively. Mitochondrial Ca(2+) sequestration starts in both terminals at approximately 250 nM, exhibits a similar Ca(2+)-uptake affinity (approximately 410 nM), and does not require Ca(2+) release from the endoplasmic reticulum. Nonetheless, mitochondrial Ca(2+) uptake in type Is terminals is more responsive to low-frequency nerve stimulation and this is due to higher cytosolic Ca(2+) levels. Since type Ib terminals have a higher mitochondrial density than Is terminals, it seemed possible that greater mitochondrial Ca(2+) sequestration may be responsible for the lower cytosolic Ca(2+) levels in Ib terminals. However, genetic and pharmacological manipulations of mitochondrial Ca(2+) uptake did not significantly alter nerve-stimulated elevations in cytosolic Ca(2+) levels in either terminal type within physiologically relevant rates of stimulation. Our findings indicate that presynaptic mitochondria have a similar affinity for Ca(2+) in functionally different nerve terminals, but do not limit cytosolic Ca(2+) levels within the range of motor neuron firing rates in situ.

Calcium-dependent phosphorylation regulates neuronal stability and plasticity in a highly precise pacemaker nucleus.

Specific types of neurons show stable, predictable excitability properties, while other neurons show transient adaptive plasticity of their excitability. However, little attention has been paid to how the cellular pathways underlying adaptive plasticity interact with those that maintain neuronal stability. We addressed this question in the pacemaker neurons from a weakly electric fish because these neurons show a highly stable spontaneous firing rate as well as an N-methyl-D-aspartate (NMDA) receptor-dependent form of plasticity. We found that basal firing rates were regulated by a serial interaction of conventional and atypical PKC isoforms and that this interaction establishes individual differences within the species. We observed that NMDA receptor-dependent plasticity is achieved by further activation of these kinases. Importantly, the PKC pathway is maintained in an unsaturated baseline state to allow further Ca(2+)-dependent activation during plasticity. On the other hand, the Ca(2+)/calmodulin-dependent phosphatase calcineurin does not regulate baseline firing but is recruited to control the duration of the NMDA receptor-dependent plasticity and return the pacemaker firing rate back to baseline. This work illustrates how neuronal plasticity can be realized by biasing ongoing mechanisms of stability (e.g., PKC) and terminated by recruiting alternative mechanisms (e.g., calcineurin) that constrain excitability. We propose this as a general model for regulating activity-dependent change in neuronal excitability.

Synaptic homeostasis on the fast track.

Synaptic homeostasis is a phenomenon that prevents the nervous system from descending into chaos. In this issue of Neuron, Frank et al. overturn the notion that synaptic homeostasis at Drosophila NMJs is a slow developmental process. They report that postsynaptic changes are offset within minutes by a homeostatic increase in neurotransmitter release that requires the presynaptic Ca(2+) channel Cacophony.

Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Neto-α Controls Synapse Organization and Homeostasis at the Drosophila Neuromuscular Junction.

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.