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Proton concentration can change within the cleft during synaptic activity due to vesicular release and Ca²⁺extrusion from cellular compartments. These changes within the synaptic cleft can impact neural activity by proton-dependent modulation of ion channel function. The pH transient differs in magnitude and direction between synapses, requiring different synapse types to be measured to generate a complete understanding of this mechanism and its impacts on physiology. With a focus on the mouse neuromuscular junction (NMJ), the recently published “Postsynaptic Calcium Extrusion at the Mouse Neuromuscular Junction Alkalinizes the Synaptic Cleft” measured synaptic cleft pH at a cholinergic synapse and found a biphasic pH transient. The study demonstrated that the changes in proton concentration found were due to postsynaptic signaling when measuring pH at the muscle membrane, despite the expectation of a presynaptic contribution. This result suggests a diffusional barrier within the NMJ isolates pH transients to presynaptic versus postsynaptic compartments. Generating a Donnan equilibrium that impacts protons, evidence suggests the basal lamina may be a key regulator of pH at the NMJ. Exploring synaptic pH, proton regulating factors, and downstream pH transient effects at presynaptic versus postsynaptic membranes may lead to new insight for a variety of diseases. 

ABSTRACT Neurons are almost exclusively cultured in media containing glucose at much higher concentrations than found in the brain. To test whether these “standard” hyperglycemic culture conditions affect neuronal respiration relative to near‐euglycemic conditions, we compared neuronal cultures grown with minimal glial contamination from the hippocampus and cortex of neonatal C57BL/6NCrl mice in standard commercially available media (25 mM Glucose) and in identical media with 5 mM glucose. Neuronal growth in both glucose concentrations proceeded until at least 14 days in vitro, with similar morphology and synaptogenesis. Neurons grown in high glucose were highly dependent on glycolysis as their primary source of ATP, measured using ATP luminescence and cellular respirometry assays. In contrast, neurons grown in 5 mM glucose showed a more balanced dependence on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), greater reserve mitochondrial respiration capacity, and increased mitochondrial population relative to standard media. Our results show that neurons cultured in artificially high glucose‐containing media preferentially use glycolysis, opposite to what is known for neurons in vivo as the primary pathway for ATP maintenance. Changes in gene and protein expression levels corroborate these changes in function and additionally suggest that high glucose culture media increases neuronal inflammation. We suggest using neuronal culture systems in 5 mM glucose to better represent physiologically relevant neuronal respiration.image 

Neurotransmission is shaped by extracellular pH. Alkalization enhances pH-sensitive transmitter release and receptor activation, whereas acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. Previous work has shown that the synaptic cleft can either acidify because of synaptic vesicular release and/or alkalize because of Ca²⁺extrusion by the plasma membrane ATPase (PMCA). The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain ambiguous. We set out to elucidate the extracellular pH transients that occur at this cholinergic synapse under near-physiological conditions and identify their sources. We monitored pH-dependent changes in the synaptic cleft of the mouse levator auris longus using viral expression of the pseudoratiometric probe pHusion-Ex in the muscle. Using mice from both sexes, a significant and prolonged alkalization occurred when stimulating the connected nerve for 5 s at 50 Hz, which was dependent on postsynaptic intracellular Ca²⁺release. Sustained stimulation for a longer duration (20 s at 50 Hz) caused additional prolonged net acidification at the cleft. To investigate the mechanism underlying cleft alkalization, we used muscle-expressed GCaMP3 to monitor the contribution of postsynaptic Ca²⁺. Activity-induced liberation of intracellular Ca²⁺in muscle positively correlated with alkalization of the synaptic cleft, whereas inhibiting PMCA significantly decreased the extent of cleft alkalization. Thus, cholinergic synapses of the mouse NMJ typically alkalize because of cytosolic Ca²⁺liberated in muscle during activity, unless under highly strenuous conditions where acidification predominates. SIGNIFICANCE STATEMENTChanges in synaptic cleft pH alter neurotransmission, acting on receptors and channels on both sides of the synapse. Synaptic acidification has been associated with a myriad of diseases in the central and peripheral nervous system. Here, we report that in near-physiological recording conditions the cholinergic neuromuscular junction shows use-dependent bidirectional changes in synaptic cleft pH—immediate alkalinization and a long-lasting acidification under prolonged stimulation. These results provide further insight into physiologically relevant changes at cholinergic synapses that have not been defined previously. Understanding and identifying synaptic pH transients during and after neuronal activity provides insight into short-term synaptic plasticity synapses and may identify therapeutic targets for diseases. 

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca²⁺) uptake supports NVU function by buffering Ca²⁺and stimulating energy production. However, excessive mitochondrial Ca²⁺uptake causes toxic mitochondrial Ca²⁺overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca²⁺uptake and efflux in the brain are mediated by the mitochondrial Ca²⁺uniporter complex (MCU_cx) and sodium/Ca²⁺/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCU_cxinhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca²⁺overloading. These findings suggest that combining MCU_cxinhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCU_cxinhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCU_cx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues. 

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 femaleDrosophilalarvae, we observed alkaline spikes of over 1 log unit during fictive locomotionin 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 STATEMENTNeurotransmission 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. 

A considerable amount of energy is expended following presynaptic activity to regenerate electrical polarization and maintain efficient release and recycling of neurotransmitter. Mitochondria are the major suppliers of neuronal energy, generating ATP via oxidative phosphorylation. However, the specific utilization of energy from cytosolic glycolysis rather than mitochondrial respiration at the presynaptic terminal during synaptic activity remains unclear and controversial. We use a synapse specialized for high-frequency transmission in mice, the calyx of Held, to test the sources of energy used to maintain energy during short activity bursts (<1 s) and sustained neurotransmission (30–150 s). We dissect the role of presynaptic glycolysis versus mitochondrial respiration by acutely and selectively blocking these ATP-generating pathways in a synaptic preparation where mitochondria and synaptic vesicles are prolific, under near-physiological conditions. Surprisingly, if either glycolysis or mitochondrial ATP production is intact, transmission during repetitive short bursts of activity is not affected. In slices from young animals before the onset of hearing, where the synapse is not yet fully specialized, both glycolytic and mitochondrial ATP production are required to support sustained, high-frequency neurotransmission. In mature synapses, sustained transmission relies exclusively on mitochondrial ATP production supported by bath lactate, but not glycolysis. At both ages, we observe that action potential propagation begins to fail before defects in synaptic vesicle recycling. Our data describe a specific metabolic profile to support high-frequency information transmission at the mature calyx of Held, shifting during postnatal synaptic maturation from glycolysis to rely on monocarboxylates as a fuel source. NEW & NOTEWORTHY We dissect the role of presynaptic glycolysis versus mitochondrial respiration in supporting high-frequency neurotransmission, by acutely blocking these ATP-generating pathways at a synapse tuned for high-frequency transmission. We find that massive energy expenditure is required to generate failure when only one pathway is inhibited. Action potential propagation is lost before impaired synaptic vesicle recycling. Synaptic transmission is exclusively dependent on oxidative phosphorylation in mature synapses, indicating presynaptic glycolysis may be dispensable for ATP maintenance. 

AbstractMotor 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 inDrosophila larvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of bothDrosophila 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) inDrosophilalarval 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.image Key pointsNeurons 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.