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Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents

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Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents The Journal of Neuroscience, November 9, 2016 • 36(45):11559–11572 • 11559 Development/Plasticity/Repair Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents Stephen G. Clarke,1,2 XMatthew S. Scarnati,1 and XKenneth G. Paradiso1 1Department of Cell Biology and Neuroscience and 2Graduate Program in Biomedical Engineering, Rutgers University, Piscataway, New Jersey 08854 At chemical synapses, presynaptic action potentials (APs) activate voltage-gated calcium channels, allowing calcium to enter and trigger neurotransmitter release. The duration, peak amplitude, and shape of the AP falling phase alter calcium entry, which can affect neu- rotransmitter release significantly. In many neurons, APs do not immediately return to the resting potential, but instead exhibit a period of depolarization or hyperpolarization referred to as an afterpotential. We hypothesized that presynaptic afterpotentials should alter neurotransmitter release by affecting the electrical driving force for calcium entry and calcium channel gating. In support of this, presynaptic calcium entry is affected by afterpotentials after standard instant voltage jumps. Here, we used the mouse calyx of Held synapse, which allows simultaneous presynaptic and postsynaptic patch-clamp recording, to show that the postsynaptic response is affected significantly by presynaptic afterpotentials after voltage jumps. We therefore tested the effects of presynaptic afterpoten- tials using simultaneous presynaptic and postsynaptic recordings and AP waveforms or real APs. Surprisingly, presynaptic afterpoten- tialsafterAPstimulididnotaltercalciumchannelresponsesorneurotransmitterreleaseappreciably.WeshowthattheAPrepolarization time course causes afterpotential-induced changes in calcium driving force and changes in calcium channel gating to effectively cancel each other out. This mechanism, in which electrical driving force is balanced by channel gating, prevents changes in calcium influx from occurring at the end of the AP and therefore acts to stabilize synaptic transmission. In addition, this mechanism can act to stabilize neurotransmitter release when the presynaptic resting potential changes. Key words: action potential; afterpotential; calcium current; neurotransmission; presynaptic; synapse Significance Statement The shape of presynaptic action potentials (APs), particularly the falling phase, affects calcium entry and small changes in calcium influx can produce large changes in postsynaptic responses. We hypothesized that afterpotentials, which often follow APs, affect calcium entry and neurotransmitter release. We tested this in calyx of Held nerve terminals, which allow simultaneous recording of presynaptic calcium currents and postsynaptic responses. Surprisingly, presynaptic afterpotentials did not alter calcium cur- rent or neurotransmitter release. We show that the AP falling phase causes afterpotential-induced changes in electrical driving force and calcium channel gating to cancel each other out. This mechanism regulates calcium entry at the end of APs and therefore stabilizes synaptic transmission. This also stabilizes responses when the presynaptic resting potential changes. Introduction activation, channel gating, and the electrical driving force for Changes in the shape, duration, and amplitude of the action po- calcium entry (Pattillo et al., 1999; Geiger and Jonas, 2000; tential (AP) affect calcium entry by affecting calcium channel Bischofberger et al., 2002; Debanne, 2004; Wu et al., 2004; Bean, 2007; Kole et al., 2007; Hoppa et al., 2014). In addition, relatively small changes in presynaptic calcium influx can produce large Received Jan. 7, 2016; revised Aug. 29, 2016; accepted Sept. 14, 2016. changes in the postsynaptic response (Bollmann et al., 2000; Sch- Author contributions: K.G.P. designed research; S.G.C., M.S.S., and K.G.P. performed research; S.G.C., M.S.S., and neggenburger and Neher, 2000; Ferna´ndez-Chaco´n R et al., 2001; K.G.P. analyzed data; K.G.P. wrote the paper. Eggermann et al., 2012). Therefore, the shape, duration, and am- This work was supported by the National Institutes of Health (Grant R00 NS051401-42 to K.G.P. We thank Dr. L. Milescu and Dr. D. Robinson for critical reading of this manuscript and helpful comments and Dr. M. Plummer for helpful discussions on this work. The authors declare no competing financial interests. Correspondence should be addressed to Kenneth G. Paradiso, Department of Cell Biology and Neuroscience, DOI:10.1523/JNEUROSCI.0066-16.2016 Rutgers University, 604 Allison Rd., Nelson D423, Piscataway, NJ 08854. E-mail: [email protected]. Copyright © 2016 the authors 0270-6474/16/3611559-14$15.00/0 11560 • J. Neurosci., November 9, 2016 • 36(45):11559–11572 Clarke et al. • A Mechanism That Stabilizes Presynaptic Calcium Currents plitude of the presynaptic AP are critical components in deter- Materials and Methods mining the strength of synaptic transmission. Brain slice preparation. Brain slices from postnatal day 8 (P8) to P12 CD1 In many neurons, the repolarization phase of the AP does not albino mice (Charles River Laboratories) of either sex were used in every return directly to the resting potential. Instead, afterpotentials experiment described. We also used P8–P12 C57BL/6 mice (Charles (Gasser and Erlanger, 1930) often occur at the end of the AP River Laboratories) of either sex in some of the dual presynaptic and repolarization such that the membrane potential remains tran- postsynaptic recording experiments. There were no differences in the siently at a level slightly above or below the resting potential experimental results for the two strains of mice. The animals were housed in a facility approved by the Association for Assessment and Accredita- (Bean, 2007; Debanne et al., 2011). Afterpotentials can facilitate tion of Laboratory Animal Care International and protocols used for (Paradiso and Wu, 2009; Lewis and Raman, 2014) or suppress AP handling and care were reviewed by the Rutgers University Animal Care firing (Kim et al., 2007; Paradiso and Wu, 2009). A brief afterhy- and Facilities Committee. Slice thickness varied from 150 to 170 ␮M. perpolarization (AHP) can reduce the time required for voltage- Throughout the process of dissection and slicing, the brain was main- gated ion channels to recover from inactivation (Rudy and tained in a low-calcium artificial CSF (aCSF) solution at 1–2°C consisting McBain, 2001; Kopp-Scheinpflug et al., 2011), which can allow of the following (in mM): 125 NaCl, 25 NaHCO3, 25 glucose, 3 myo- higher firing frequencies. In addition, afterpotentials should af- inositol, 2.5 KCl, 2 Na-pyruvate, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 MgCl , and 0.1 CaCl , pH 7.3, when oxygenated with carbogen gas (95% fect calcium channel currents because the majority of calcium 2 2 oxygen, 5% carbon dioxide). Once produced, slices were transferred to a entry occurs during the repolarization phase of the AP (Borst and chamber maintained at 37°C for 30–45 min in normal calcium aCSF Sakmann, 1998, 1999; Pattillo et al., 1999; Bischofberger et al., solution with the same composition listed above except for 1 mM MgCl2 2002; Yang and Wang, 2006). This was tested in a study of so- and2mM CaCl2. This same solution was used as our recording solution matic calcium currents, in which it was shown that AP broaden- (see below). Experiments were performed at room temperature for 4–6 ing at any time during the repolarization phase resulted in an h after the incubation period. increased amount of calcium influx (Pattillo et al., 1999). Inter- Electrophysiology. To optimize the quality of voltage-clamp and estingly,a5mVhyperpolarization was also tested and found to current-clamp recordings, presynaptic terminals were patched at, or in produce little to no change in the voltage-gated calcium current, close proximity to, the axonal heminode. Patch-clamp recordings were conducted using an EPC10 USB double patch-clamp amplifier using which the investigators attributed to a possible balance between PatchMaster software (HEKA; Harvard Bioscience). A parasagittal slice the changes of calcium influx and calcium channel gating. This orientation was used in all presynaptic voltage-clamp experiments to suggests that afterdepolarizations (ADPs) could affect the pre- limit axonal length to minimize space-clamp errors in our recordings. synaptic calcium entry and that AHPs may have a small effect or This ensured a more uniform control over the terminal with minimal possibly no effect on calcium entry. Given the demonstrated ef- delay. To help determine the characteristics of the patched cell after fects on calcium currents, presynaptic afterpotentials could affect recordings, such as axon length and patch location relative to the axon neurotransmitter release. heminode, fluorescent dye (0.10 mg/ml Lucifer yellow) was typically To determine how afterpotentials affect the presynaptic added to the presynaptic pipette solutions. For the dual recordings that calcium channel currents, we used the calyx of Held nerve used current-clamp configuration in the presynaptic terminal, coronal slice orientation was used. Patch pipettes were produced from thick- terminal, which is located in the auditory brainstem (Barnes- walled borosilicate glass, 2.0 mm outer diameter, 1.16 mm inner diame- Davies and Forsythe, 1995; Borst et al., 1995). The size of this ter (Sutter Instruments). Pipettes with a resistance of 2.5–5 M⍀ were
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