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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. 

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.