Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jun 24;40(26):4972-4980.
doi: 10.1523/JNEUROSCI.2945-19.2020. Epub 2020 May 19.

Spike Activity Regulates Vesicle Filling at a Glutamatergic Synapse

Dainan Li  1 , Yun Zhu  1 , Hai Huang  2   3
Affiliations

Spike Activity Regulates Vesicle Filling at a Glutamatergic Synapse

Dainan Li et al. J Neurosci. .

Abstract

Synaptic vesicles need to be recycled and refilled rapidly to maintain high-frequency synaptic transmission. However, little is known about the control of neurotransmitter transport into synaptic vesicles, which determines the contents of synaptic vesicles and the strength of synaptic transmission. Here, we report that Na+ substantially accumulated in the calyx of Held terminals of juvenile mice of either sex during high-frequency spiking. The activity-induced elevation of cytosolic Na+ activated vesicular Na+/H+ exchanger, facilitated glutamate loading into synaptic vesicles, and increased quantal size of asynchronous released vesicles but did not affect the vesicle pool size or release probability. Consequently, presynaptic Na+ increased the EPSCs and was required to maintain the reliable high-frequency signal transmission from the presynaptic calyces to the postsynaptic medial nucleus of the trapezoid body (MNTB) neurons. Blocking Na+/H+ exchange activity decreased the postsynaptic current and caused failures in postsynaptic firing. Therefore, during high-frequency synaptic transmission, when large amounts of glutamate are released, Na+ accumulated in the terminals, activated vesicular Na+/H+ exchanger, and regulated glutamate loading as a function of the level of vesicle release.SIGNIFICANCE STATEMENT Auditory information is encoded by action potentials (APs) phase-locked to sound frequency at high rates. A large number of synaptic vesicles are released during high-frequency synaptic transmission; accordingly, synaptic vesicles need to be recycled and refilled rapidly. We have recently found that a Na+/H+ exchanger expressed on synaptic vesicles promotes vesicle filling with glutamate. Here, we showed that when a large number of synaptic vesicles are released during high-frequency synaptic transmission, Na+ accumulates in axon terminals and facilitates glutamate uptake into synaptic vesicles. Na+ thus accelerates vesicle replenishment and sustains reliable synaptic transmission.

Keywords: axon terminal; calyx of Held; glutamate; spiking; synaptic vesicle; vesicular glutamate transport.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Presynaptic spikes control Na+ concentration. A, Maximum intensity montage of a P11 calyx of Held recorded using two-photon microscopy at 32°C. The cell was filled with SBFI and Alexa Fluor 594, while the Alexa Fluor 594 signal is displayed. B, Spikes were evoked at 100 Hz for 10 s by afferent fiber stimulation. C, Corresponding Na+ increases were detected at both the preterminal axon heminode (red) and calyceal terminal (blue). D, Summary plot of cytosolic Na+ increase at the axon and terminal in P8–P12 calyces at 32°C. E, F, Similar to A, C, while a P14 calyx was recorded at 37°C. G, Summary plot of cytosolic Na+ increase at the axon and terminal in P13–P16 calyces at 35–37°C; *p < 0.05, **p < 0.01. Error bars, ±SEM.
Figure 2.
Figure 2.
Presynaptic Na+ regulates the EPSC amplitude. A, Pairs of presynaptic APs induced by afferent fiber stimulation when 0 mm (left), 10 mm (middle), or 40 mm (right) Na+ was present in the presynaptic pipette solution. Traces recorded within 2 min of break-in (black) and after 15 min (red) of recording are superimposed. B, Postsynaptic currents in response to the presynaptic APs. C, left, Time course of the normalized amplitudes of the first EPSC during paired-pulse recordings. Each point represents the average of 1-min recordings. Right, Relative amplitudes of the first EPSC after 15-min dialysis of the calyces with different Na+ concentrations. Amplitudes were normalized to the amplitudes measured within 2 min of break-in. D, Presynaptic Na+ does not affect the paired-pulse ratio. Statistical significance was assessed using a two-tailed, paired Student's t test; **p < 0.01, ***p < 0.001. Error bars, ±SEM.
Figure 3.
Figure 3.
Presynaptic Na+ regulates EPSC amplitude in response to presynaptic depolarization. A, B, Presynaptic calcium currents (A) and corresponding postsynaptic responses (B) induced by pairs of 1-ms presynaptic depolarizations from –80 to +10 mV when 0 mm (left), 10 mm (middle), or 40 mm (right) Na+ was present in the presynaptic pipette solutions. Recordings made within 2 min of break-in (black) and after 15 min (red) are superimposed. C, left, Time course of the changes in EPSC amplitude. Each point represents the average of 1-min recordings. Right, Relative amplitudes of the first EPSC at 15–20 min of recordings with different presynaptic Na+ concentrations. Amplitudes were normalized to the amplitudes measured within 2 min of break-in. D, Presynaptic Na+ does not affect the paired-pulse ratio in response to presynaptic depolarization. Statistical significance was assessed using a two-tailed, paired Student's t test; **p < 0.01, ***p < 0.001. Error bars, ±SEM.
Figure 4.
Figure 4.
Presynaptic Na+ does not affect the readily releasable pool size or release probability. A, B, Sampled presynaptic Ca2+ currents (ICa; A) and Cm responses (B) induced by a 1-ms (black) or 30-ms (red) depolarizations from –80 to +10 mV with presynaptic pipette solutions containing 0 mm (left), 10 mm (middle), or 40 mm (right) Na+. The corresponding membrane conductance (Gm) and series conductance (Gs) are shown to confirm the recording stability. C, Group data show that the presynaptic [Na+] does not affect the Cm responses or release probability. Error bars, ±SEM.
Figure 5.
Figure 5.
Presynaptic Na+ regulates aEPSC amplitude. A, Example trace of prolonged period of asynchronous release (aEPSCs) followed by the initial fast EPSC evoked by a single presynaptic stimulation. The insert shows an expanded trace of the asynchronous release (blue). B, Example traces of asynchronous events within 2 min (black) and >10 min (red) after presynaptic break-in to 0, 10, or 40 mm Na+ pipette solution. The bottom traces show the recordings before (black) and >10 min after (red) application of 8-Br-cAMP; 10 μm H-89 was present in the postsynaptic pipette solution to inhibit 8-Br-cAMP-induced kinase activation. C, Bar graphs of the change in aEPSC amplitude when presynaptic pipette solution, as well as before and after application of 8-Br-cAMP. Statistical significance was assessed using a two-tailed paired Student's t test; **p < 0.01, ***p < 0.001. Error bars, ±SEM.
Figure 6.
Figure 6.
Presynaptic Na+ level contributes to reliable synaptic transmission. A, Example traces of 50 presynaptic APs immediately (black) and 10 min (red) after break-in with 0, 10, and 40 mm Na+ in the presynaptic pipette solution. APs were evoked by 200-Hz afferent fiber stimulation. B, Postsynaptic spiking in MNTB principal neurons in response to the presynaptic firing in A. C, Raster plots of spikes evoked by 200-Hz, 250-ms stimulus trains repeated with 60-s intervals. Presynaptic spikes are shown at the top in red. D, Summary plots of average postsynaptic firing probability in response to 50 presynaptic APs at 200 Hz. E, Overall change in the postsynaptic firing probability between the first 2 min and after 10 min, with 0, 10, or 40 mm presynaptic Na+. Statistical significance was assessed using a two-tailed paired Student's t test; *p < 0.05, **p < 0.01. Error bars, ±SEM.
Figure 7.
Figure 7.
NHE activity is required for reliable synaptic signaling. A, Example traces of EPSC recordings before and 10 min after incubation of 100 μm EIPA on 20-Hz presynaptic stimulation. B, EIPA decreased EPSC amplitude. C, Example traces of postsynaptic AP recordings before and 10 min after applying EIPA (100 μm). D, EIPA reduced the reliability of postsynaptic firing. Statistical significance was assessed using a two-tailed paired Student's t test; **p < 0.01. Error bars, ±SEM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amara SG, Fontana AC (2002) Excitatory amino acid transporters: keeping up with glutamate. Neurochem Int 41:313–318. 10.1016/s0197-0186(02)00018-9 - DOI - PubMed
    1. Balmer TS, Trussell LO (2016) Quantum disentanglement: electrical analysis of the complex roles of ions in filling vesicles with glutamate. Neuron 90:667–669. 10.1016/j.neuron.2016.05.011 - DOI - PubMed
    1. Barnes-Davies M, Forsythe ID (1995) Pre- and postsynaptic glutamate receptors at a giant excitatory synapse in rat auditory brainstem slices. J Physiol 488:387–406. 10.1113/jphysiol.1995.sp020974 - DOI - PMC - PubMed
    1. Bender KJ, Ford CP, Trussell LO (2010) Dopaminergic modulation of axon initial segment calcium channels regulates action potential initiation. Neuron 68:500–511. 10.1016/j.neuron.2010.09.026 - DOI - PMC - PubMed
    1. Berret E, Kim SE, Lee SY, Kushmerick C, Kim JH (2016) Functional and structural properties of ion channels at the nerve terminal depends on compact myelin. J Physiol 594:5593–5609. 10.1113/JP272205 - DOI - PMC - PubMed

Publication types

LinkOut - more resources