Synaptic Release Sites Research Articles

Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

Lead exposure induced transgenerational developmental neurotoxicity by altering genome methylation in Drosophila melanogaster

Heavy metal toxicity is a significant global health concern, with particular attention given to lead (Pb) exposure due to its adverse effects on cognitive development, especially in children exposed to low concentrations. While Pb neurotoxicity has been extensively studied, the analysis and molecular mechanisms underlying the transgenerational effects of Pb exposure-induced neurotoxicity remain poorly understood. In this study, we utilized Drosophila, a powerful developmental animal model, to investigate this phenomenon. Our findings demonstrated that Pb exposure during the developmental stage had a profound effect on the neurodevelopment of F0 fruit flies. Specifically, we observed a loss of correlation between the terminal motor area and muscle fiber area, along with an increased frequency of the β-lobe midline crossing phenotype in mushroom bodies. Western blot analysis indicated altered expression levels of synaptic vesicle proteins, with a decrease in Synapsin (SYN) and an increase in Bruchpilot (BRP) expression, suggesting changes in synaptic vesicle release sites. These findings were corroborated by electrophysiological data, showing an increase in the amplitude of evoked excitatory junctional potential (EJP) and an increase in the frequency of spontaneous excitatory junctional potential (mEJP) following Pb exposure. Importantly, our results further confirmed that the developmental neurotoxicity resulting from grandparental Pb exposure exhibited a transgenerational effect. The F3 offspring displayed neurodevelopmental defects, synaptic function abnormalities, and repetitive behavior despite lacking direct Pb exposure. Our MeDIP-seq analysis further revealed significant alterations in DNA methylation levels in several neurodevelopmental associated genes (eagle, happyhour, neuroglian, bazooka, and spinophilin) in the F3 offspring exposed to Pb. These findings suggest that DNA methylation modifications may underlie the inheritance of acquired phenotypic traits resulting from environmental Pb exposure.

An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation

Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca2+/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.

Large-scale interrogation of retinal cell functions by 1-photon light-sheet microscopy

Visual processing in the retina depends on the collective activity of large ensembles of neurons organized in different layers. Current techniques for measuring activity of layer-specific neural ensembles rely on expensive pulsed infrared lasers to drive 2-photon activation of calcium-dependent fluorescent reporters. We present a 1-photon light-sheet imaging system that can measure the activity in hundreds of neurons in the exvivo retina over a large field of view while presenting visual stimuli. This allows for a reliable functional classification of different retinal cell types. We also demonstrate that the system has sufficient resolution to image calcium entry at individual synaptic release sites across the axon terminals of dozens of simultaneously imaged bipolar cells. The simple design, large field of view, and fast image acquisition make this a powerful system for high-throughput and high-resolution measurements of retinal processing at a fraction of the cost of alternative approaches.

Interaction of Munc13-1 and RIM that contributes to the formation/maintenance of synaptic vesicle release sites

Interaction of Munc13-1 and RIM that contributes to the formation/maintenance of synaptic vesicle release sites

Interactions of active zone proteins with Munc13-1 that contribute to the formation of synaptic vesicle release sites

Interactions of active zone proteins with Munc13-1 that contribute to the formation of synaptic vesicle release sites

Endocannabinoid Signaling Contributes to Experience-Induced Increase of Synaptic Release Sites From Parvalbumin Interneurons in Mouse Visual Cortex.

During postnatal development of the visual cortex between eye-opening to puberty, visual experience promotes a gradual increase in the strength of inhibitory synaptic connections from parvalbumin-positive interneurons (PV-INs) onto layer 2/3 pyramidal cells. However, the detailed connectivity properties and molecular mechanisms underlying these developmental changes are not well understood. Using dual-patch clamp in brain slices from G42 mice, we revealed that both connection probability and the number of synaptic release sites contributed to the enhancement of synaptic strength. The increase of release site number was hindered by dark rearing from eye-opening and rescued by 3-days re-exposure to the normal visual environment. The effect of light re-exposure on restoring synaptic release sites in dark reared mice was mimicked by the agonist of cannabinoid-1 (CB1) receptors and blocked by an antagonist of these receptors, suggesting a role for endocannabinoid signaling in light-induced maturation of inhibitory connectivity from PV-INs to pyramidal cells during postnatal development.

Modeling Hippocampal CA1 Gabaergic Synapses of Audiogenic Rats.

Wistar Audiogenic Rats (WARs) are genetically susceptible to sound-induced seizures that start in the brainstem and, in response to repetitive stimulation, spread to limbic areas, such as hippocampus. Analysis of the distribution of interevent intervals of GABAergic inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells showed a monoexponential trend in Wistar rats, suggestive of a homogeneous population of synapses, but a biexponential trend in WARs. Based on this, we hypothesize that there are two populations of GABAergic synaptic release sites in CA1 pyramidal neurons from WARs. To address this hypothesis, we used a well-established neuronal computational model of a CA1 pyramidal neuron previously developed to replicate physiological properties of these cells. Our simulations replicated the biexponential trend only when we decreased the release frequency of synaptic currents by a factor of six in at least 40% of distal synapses. Our results suggest that almost half of the GABAergic synapses of WARs have a drastically reduced spontaneous release frequency. The computational model was able to reproduce the temporal dynamics of GABAergic inhibition that could underlie susceptibility to the spread of seizures.

Regional Variation in Striatal Dopamine Spillover and Release Plasticity.

Recent optical observations of dopamine at axon terminals and kinetic modeling of evoked dopamine responses measured by fast scan cyclic voltammetry (FSCV) support local restriction of dopamine diffusion at synaptic release sites. Yet, how this diffusion barrier affects synaptic and volume transmission is unknown. Here, a deficiency in a previous kinetic model's fitting of stimulus trains is remedied by replacing an earlier assumption that dopamine transporters (DATs) are present only on the outer side of the diffusion barrier with the assumption that they are present on both sides. This is consistent with the known distribution of DATs, which does not show obvious DAT-free zones proximal to dopamine release sites. A simultaneous multifitting strategy is then shown to enable unique model fits to sets of evoked dopamine FSCV responses acquired in vivo or in brain slices. This data analysis technique permits, for the first time, the calculation of the fraction of dopamine which spills over from what appears to be the perisynaptic space, as well as other parameters such as dopamine release, release plasticity, and uptake. This analysis shows that dopamine's diffusion away from its release sites is remarkably hindered (τ = 5 s), but dopamine responses are rapid because of DAT activity. Furthermore, the new analysis reveals that uptake inhibitors can inhibit dopamine release during a stimulus train, apparently by depleting the releasable pool. It is suggested that ongoing uptake is critical for maintaining ongoing synaptic dopamine release and that the previously reported and also herein claimed increase of the initial dopamine release of some uptake inhibitors might be an important mechanism in addiction. Finally, brain mapping data reveal that the diffusion barrier is conserved, but there are variations in perisynaptic uptake, volume transmission, and release plasticity within the rat striatum. Therefore, an analysis paradigm is developed to quantify previously unmeasured features of brain dopaminergic transmission and to reveal regional functional differences among dopamine synapses.

In Vitro Configuration of Munc13-1 Bridging of Phospholipid Bilayers at Resting Conditions

Neurotransmitter release is an intricate process involving multiple proteins that govern synaptic vesicle exocytosis. While SNAREs drive membrane fusion, chaperone proteins regulate their function, ensuring fusion happens in a precise and controllable manner. Munc13-1 is an elongated, multi-domain protein participating in docking through plasma membrane interactions via the N-terminal C1, C2B domains and vesicle tethering via the C-terminal C2C domain and SNARE priming by opening Syntaxin-1A, thus promoting SNARE-pin formation. In addition, it was recently proposed that Munc13-1 is a core component of molecular architecture at the synaptic vesicle release site due to its ability to form a supramolecular assembly (clusters). Stability of these clusters could imply ordered spatial arrangement of Munc13-1 molecules but to date, no structural evidence has been reported. Here we report the spatial arrangement of the truncated version of Munc13-1 consisting of C1, C2B, MUN (Δ1408-1442, EF) and C2C domains (Munc13L) on the surface of negatively charged phospholipid membranes. Using cryo-tomography, we found that Munc13L forms hexagonal 2D crystal in Ca2+ free conditions, bridging two membrane bilayers. Subtomograms extracted from the observed crystal were aligned and averaged to yield a 3D map at 14 Å resolution. Fitting of existing crystal structures of C1, C2B and MUN domains revealed that Munc13L exists in two conformational states in the crystal. One form is extended, connecting two bilayers to form a triskelion-like structure by the interaction of C-terminal ends of MUN domains, while the other form is supporting the extended form using the interaction of its C2C domain with MUN region of the supported molecule. These interactions are novel and require further investigation to determine the physiological role and its transformation when activated by calcium.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Cell type-specific super-resolution imaging reveals an increase in calcium-permeable AMPA receptors at spinal peptidergic terminals as an anatomical correlate of inflammatory pain.

Spinal hyperexcitability is a key event in the development of persistent pain, and arises partly from alterations in the number and localization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors. However, determining precisely where these changes occur is challenging due to the requirement for multiplex labelling and nanoscale resolution. The recent development of super-resolution light microscopy provides new tools to address these challenges. Here, we apply combined confocal/direct STochastic Optical Reconstruction Microscopy (dSTORM) to reveal changes in calcium-permeable subunits of AMPA-type glutamate receptors (GluA1) at identified spinal cord dorsal horn (SCDH) peptidergic axon terminals in a model of inflammatory pain. L4/5 lumbar spinal cord was collected from adult male C57BL/6J mice 24 hours after unilateral hind paw injection of saline or 1% carrageenan (n = 6/group). Tissue was immunolabelled for markers of peptidergic axon terminals (substance P; SP), presynaptic active zones (Bassoon), and GluA1. Direct stochastic optical reconstruction microscopy revealed a 59% increase in total GluA1 immunolabelling in the SCDH in the carrageenan group, which was not detected by confocal microscopy. Cell type-specific analyses identified a 10-fold increase in GluA1 localized to SP structures, and identified GluA1 nanodomains that scaled with behavioural hypersensitivity, and were associated with synaptic release sites. These findings demonstrate that dSTORM has the sensitivity and power to detect nanoscale anatomical changes in the SCDH, and provides new evidence for synaptic insertion of GluA1-AMPA-Rs at spinal peptidergic nociceptive terminals in a model of inflammatory pain.

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology.

In the central nervous system, a pair of neurons often formmultiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Prominent Postsynaptic and Dendritic Exocytosis of Endogenous BDNF Vesicles in BDNF-GFP Knock-in Mice.

Brain-derived neurotrophic factor (BDNF) is a secreted messenger molecule that is crucial for neuronal function and induction of synaptic plasticity. Although altered availability of BDNF underlies many neurological deficits and neurodegenerative disorders, secretion dynamics of endogenous BDNF are unexplored. We generated a BDNF-GFP knock-in (KiBE) mouse, in which GFP-labeled BDNF is expressed under the control of the unaltered endogenous mouse BDNF gene regulatory elements. This KiBE mouse model enables for the first time live cell imaging analysis of endogenous BDNF dynamics. We show that BDNF-GFP release and biological activity in vivo are unaffected by the GFP tag, since homozygous KiBE mice, which lack wild-type BDNF, are healthy and have a normal life expectancy. STED superresolution microscopy shows that 70% of BDNF-GFP vesicles in KiBE mouse neurites are localized in dendrites, being typically 200nm away from synaptic release sites. Live cell imaging in hippocampal slices also reveals prominent targeting of endogenous BDNF-GFP vesicles to dendrites. Fusion pore opening and cargo release of dendritic BDNF vesicles start within 30s after a strong depolarizing stimulus and continue for > 100s thereafter, revealing an astonishingly delayed and prolonged release of endogenous BDNF.

Nanoscale molecular landscape of the synaptic vesicle release site in the hippocampus

Nanoscale molecular landscape of the synaptic vesicle release site in the hippocampus

Coupling the Structural and Functional Assembly of Synaptic Release Sites.

Information processing in our brains depends on the exact timing of calcium (Ca2+)-activated exocytosis of synaptic vesicles (SVs) from unique release sites embedded within the presynaptic active zones (AZs). While AZ scaffolding proteins obviously provide an efficient environment for release site function, the molecular design creating such release sites had remained unknown for a long time. Recent advances in visualizing the ultrastructure and topology of presynaptic protein architectures have started to elucidate how scaffold proteins establish “nanodomains” that connect voltage-gated Ca2+ channels (VGCCs) physically and functionally with release-ready SVs. Scaffold proteins here seem to operate as “molecular rulers or spacers,” regulating SV-VGCC physical distances within tens of nanometers and, thus, influence the probability and plasticity of SV release. A number of recent studies at Drosophila and mammalian synapses show that the stable positioning of discrete clusters of obligate release factor (M)Unc13 defines the position of SV release sites, and the differential expression of (M)Unc13 isoforms at synapses can regulate SV-VGCC coupling. We here review the organization of matured AZ scaffolds concerning their intrinsic organization and role for release site formation. Moreover, we also discuss insights into the developmental sequence of AZ assembly, which often entails a tightening between VGCCs and SV release sites. The findings discussed here are retrieved from vertebrate and invertebrate preparations and include a spectrum of methods ranging from cell biology, super-resolution light and electron microscopy to biophysical and electrophysiological analysis. Our understanding of how the structural and functional organization of presynaptic AZs are coupled has matured, as these processes are crucial for the understanding of synapse maturation and plasticity, and, thus, accurate information transfer and storage at chemical synapses.

Activation of RHO-1 in cholinergic motor neurons competes with dopamine signalling to control locomotion.

The small GTPase RhoA plays a crucial role in the regulation of neuronal signalling to generate behaviour. In the developing nervous system RhoA is known to regulate the actin cytoskeleton, however the effectors of RhoA-signalling in adult neurons remain largely unidentified. We have previously shown that activation of the RhoA ortholog (RHO-1) in C. elegans cholinergic motor neurons triggers hyperactivity of these neurons and loopy locomotion with exaggerated body bends. This is achieved in part through increased diacylglycerol (DAG) levels and the recruitment of the synaptic vesicle protein UNC-13 to synaptic release sites, however other pathways remain to be identified. Dopamine, which is negatively regulated by the dopamine re-uptake transporter (DAT), has a central role in modulating locomotion in both humans and C. elegans. In this study we identify a new pathway in which RHO-1 regulates locomotory behaviour by repressing dopamine signalling, via DAT-1, linking these two pathways together. We observed an upregulation of dat-1 expression when RHO-1 is activated and show that loss of DAT-1 inhibits the loopy locomotion phenotype caused by RHO-1 activation. Reducing dopamine signalling in dat-1 mutants through mutations in genes involved in dopamine synthesis or in the dopamine receptor DOP-1 restores the ability of RHO-1 to trigger loopy locomotion in dat-1 mutants. Taken together, we show that negative regulation of dopamine signalling via DAT-1 is necessary for the neuronal RHO-1 pathway to regulate locomotion.

Homeostatic Feedback Modulates the Development of Two-State Patterned Activity in a Model Serotonin Motor Circuit in Caenorhabditis elegans.

Neuron activity accompanies synapse formation and maintenance, but how early circuit activity contributes to behavior development is not well understood. Here, we use the Caenorhabditis elegans egg-laying motor circuit as a model to understand how coordinated cell and circuit activity develops and drives a robust two-state behavior in adults. Using calcium imaging in behaving animals, we find the serotonergic hermaphrodite-specific neurons (HSNs) and vulval muscles show rhythmic calcium transients in L4 larvae before eggs are produced. HSN activity in L4 is tonic and lacks the alternating burst-firing/quiescent pattern seen in egg-laying adults. Vulval muscle activity in L4 is initially uncoordinated but becomes synchronous as the anterior and posterior muscle arms meet at HSN synaptic release sites. However, coordinated muscle activity does not require presynaptic HSN input. Using reversible silencing experiments, we show that neuronal and vulval muscle activity in L4 is not required for the onset of adult behavior. Instead, the accumulation of eggs in the adult uterus renders the muscles sensitive to HSN input. Sterilization or acute electrical silencing of the vulval muscles inhibits presynaptic HSN activity and reversal of muscle silencing triggers a homeostatic increase in HSN activity and egg release that maintains ∼12-15 eggs in the uterus. Feedback of egg accumulation depends upon the vulval muscle postsynaptic terminus, suggesting that a retrograde signal sustains HSN synaptic activity and egg release. Our results show that egg-laying behavior in C. elegans is driven by a homeostat that scales serotonin motor neuron activity in response to postsynaptic muscle feedback.SIGNIFICANCE STATEMENT The functional importance of early, spontaneous neuron activity in synapse and circuit development is not well understood. Here, we show in the nematode Caenorhabditis elegans that the serotonergic hermaphrodite-specific neurons (HSNs) and postsynaptic vulval muscles show activity during circuit development, well before the onset of adult behavior. Surprisingly, early activity is not required for circuit development or the onset of adult behavior and the circuit remains unable to drive egg laying until fertilized embryos are deposited into the uterus. Egg accumulation potentiates vulval muscle excitability, but ultimately acts to promote burst firing in the presynaptic HSNs which results in egg laying. Our results suggest that mechanosensory feedback acts at three distinct steps to initiate, sustain, and terminate C. elegans egg-laying circuit activity and behavior.

Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse.

Synapses have high energy demands which increase during intense activity. We show that presynaptic terminals can utilise extracellular glucose or lactate to generate energy to maintain synaptic transmission. Reducing energy substrates induces a metabolic stress: presynaptic ATP depletion impaired synaptic transmission through a reduction in the number of functional synaptic vesicle release sites and a slowing of vesicle pool replenishment, without a consistent change in release probability. Metabolic function is compromised in many pathological conditions (e.g. stroke, traumatic brain injury and neurodegeneration). Knowledge of how synaptic transmission is constrained by metabolic stress, especially during intense brain activity, will provide insights to improve cognition following pathological insults. The synapse has high energy demands, which increase during intense activity. Presynaptic ATP production depends on substrate availability and usage will increase during activity, which in turn could influence transmitter release and information transmission. We investigated transmitter release at the mouse calyx of Held synapse using glucose or lactate (10, 1 or 0mm) as the extracellular substrates while inducing metabolic stress. High-frequency stimulation (HFS) and recovery paradigms evoked trains of EPSCs monitored under voltage-clamp. Whilst postsynaptic intracellular ATP was stabilised by diffusion from the patch pipette, depletion of glucose increased EPSC depression during HFS and impaired subsequent recovery. Computational modelling of these data demonstrated a reduction in the number of functional release sites and slowed vesicle pool replenishment during metabolic stress, with little change in release probability. Directly depleting presynaptic terminal ATP impaired transmitter release in an analogous manner to glucose depletion. In the absence of glucose, presynaptic terminal metabolism could utilise lactate from the aCSF and this was blocked by inhibition of monocarboxylate transporters (MCTs). MCT inhibitors significantly suppressed transmission in low glucose, implying that lactate is a presynaptic substrate. Additionally, block of glycogenolysis accelerated synaptic transmission failure in the absence of extracellular glucose, consistent with supplemental supply of lactate by local astrocytes. We conclude that both glucose and lactate support presynaptic metabolism and that limited availability, exacerbated by high-intensity firing, constrains presynaptic ATP, impeding transmission through a reduction in functional presynaptic release sites as vesicle recycling slows when ATP levels are low.