Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission. SIGNIFICANCE STATEMENT The presence and functional role of MTs in the presynaptic terminal are controversial. Here, we demonstrate that MTs are present near SVs in calyceal presynaptic terminals and that MT depolymerization specifically prolongs the slow-recovery component of EPSCs from short-term depression. In contrast, F-actin depolymerization specifically prolongs fast-recovery component. Depolymerization of MT or F-actin has no direct effect on SV exocytosis/endocytosis or basal transmission, but significantly impairs the fidelity of high-frequency transmission, suggesting that presynaptic cytoskeletal filaments play essential roles in SV replenishment for the maintenance of high-frequency neurotransmission. synaptic vesicles Introduction The cytoskeleton, comprising filamentous actin (F-actin), microtubules (MTs), and intermediate filaments, supports the cell architecture and functions. During neuronal development, MTs and F-actin play crucial roles in cell motility, axonal growth, and organelle/protein transport to growth cones ( Arnette et al., 2016 ). However, their functional roles in developed synapses remain unestablished. Among cytoskeletal elements, F-actin is localized at the distal end of presynaptic axons ( Hirokawa et al., 1989 ; Saitoh et al., 2001 ). Functionally, it is thought to regulate neurotransmitter release ( Morales et al., 2000 ), mediate endocytosis of synaptic vesicles (SVs; Watanabe et al., 2013 ; Delvendahl et al., 2016 ; Wu et al., 2016 ), and promote the recovery of synaptic responses from activity-dependent short-term depression (STD; Cole et al., 2000 ; Sakaba and Neher, 2003 ) via fast SV replenishment ( Lipstein et al., 2013 ) and clearance of used SVs from release sites ( Hosoi et al., 2009 ; Lee et al., 2012 ). Compared with F-actin, much less is known about the presynaptic functional roles of MTs, except that they are involved in the transport of mitochondria and presynaptic elements such as those of active zones (AZs) or SVs ( Hirokawa et al., 2010 ; Melkov and Abdu, 2018 ). Previous electron microscopy (EM) studies at the frog neuromuscular junction (NMJ) reported that MTs anchoring SVs are directing toward AZs ( Gray 1978 , 1983 ; Hirokawa et al., 1989 ). Likewise, at the Drosophila NMJ, the MT-associated protein Futsch ( Hummel et al., 2000 ) links MT to AZs, thereby supporting neurotransmitter release ( Lepicard et al., 2014 ). However, at lamprey or chick embryonic synapses, MTs do not colocalize with SVs ( Smith et al., 1970 ; Bird, 1989 ). At the calyx of Held in adult cats, MTs are observed in presynaptic terminal swellings, but not in the SV pool ( Perkins et al., 2010 ). In a recent imaging study in cultured calyceal presynaptic terminals, MTs are shown to be present within terminal swellings and depolymerization of MTs by nocodazole treatment impaired long-distance SV movements between swellings, whereas depolymerization of F-actin had no effect ( Guillaud et al., 2017 ). However, in cultured hippocampal synapses, interbouton SV trafficking is reportedly blocked by an F-actin depolymerizing drug ( Darcy et al., 2006 ). To address whether MTs play any functional role in presynaptic terminals, we first examined their localization in the calyx of Held of rodent brainstem using confocal and stimulated emission depletion (STED) microscopy. We then depolymerized MTs in slices, using vinblastine, and examined whether it affects synaptic functions. For comparisons, we also depolymerized F-actin using latrunculin A, and re-examined its effects on synaptic functional properties ( Sakaba and Neher, 2003 ), newly at physiological temperature (PT; 37°C) and at calyces of Held in posthearing rats. Our results revealed differential contributions of F-actin and MTs, specifically to fast and slow recovery of EPSCs from STD without cross talk, suggesting that these cytoskeletal elements independently contribute to recycling of SVs to maintain high-frequency neurotransmission at this fast synapse. Materials and Methods All experiments were performed in accordance with guidelines of the Physiological Society of Japan and animal experiment regulations at Okinawa Institute of Science and Technology Graduate University. Slice preparation and solutions for electrophysiological recordings. Brainstems were isolated from Wistar rats of either sex, at the age of postnatal day 13–16, after decapitation under isoflurane anesthesia. Transverse brainstem slices (175–250 μm in thickness), containing the medial nucleus of the trapezoid body (MNTB), were cut using a vibroslicer (VT1200S, Leica) in ice-cold solution containing the following (m m ): 200 sucrose, 2.5 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 6 MgCl 2 , 10 glucose, 3 myo -inositol, 2 sodium pyruvate, and 0.5 sodium ascorbate, at pH 7.4, when bubbled with 95% O 2 and 5% CO 2 , and 310–320 mOsm. Before recordings, slices were incubated for 1 h at 37°C in artificial CSF (aCSF) containing the following (m m ): 125 NaCl, 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, 3 myo -inositol, 2 sodium pyruvate, 0.5 sodium ascorbate, 1.25 NaH 2 PO 4 , and 26 NaHCO 3 , at pH 7.4, when bubbled with 95% O 2 and 5% CO 2 , and 310–315 mOsm. For recording EPSCs, the aCSF contained 10 μ m bicuculline methiodide (Sigma-Aldrich) and 0.5 μ m strychnine hydrochloride (TCI) to block GABA A and glycine receptors, respectively. The pipette solution for recording postsynaptic currents contained the following (m m ): 110 CsF, 30 CsCl, 10 HEPES, 5 EGTA, 1 MgCl 2 , and 5 QX314-Cl, at pH 7.3–7.4, adjusted with CsOH, and 300–320 mOsm, unless otherwise noted. For presynaptic membrane capacitance measurements, the aCSF contained 10 m m tetraethylammonium chloride (TCI), 0.5 m m 4-aminopyridine (Nacalai Tesque), 1 μ m tetrodotoxin (Nacalai Tesque), 10 μ m bicuculline methiodide, and 0.5 μ m strychnine hydrochloride. The pipette solution for presynaptic membrane capacitance ( C m ) measurements contained the following (m m ): 105 Cs gluconate, 30 CsCl, 10 HEPES, 0.5 EGTA, 12 disodium phosphocreatinine, 3 Mg-ATP, 0.3 Na 2 -GTP, and 1 MgCl 2 , at pH 7.3–7.4, adjusted with CsOH, and 315–320 mOsm. For simultaneous presynaptic and postsynaptic action potential (AP) recording, the presynaptic pipette solution contained the following (m m ): 110 potassium gluconate, 30 KCl, 5 EGTA, 12 disodium phosphocreatine, 10 l -glutamate, 1 MgCl 2 , 3 Mg-ATP, and 0.3 Na 2 -GTP, at pH 7.3–7.4, adjusted with KOH, and 315–320 mOsm. The postsynaptic pipette solution contained the following (m m ): 120 potassium gluconate, 30 KCl, 5 EGTA, 12 disodium phosphocreatine, 1 MgCl 2 , 3 Mg-ATP, 0.3 Na 2 -GTP, and 1 l -arginine, at pH 7.3–7.4 adjusted with KOH, 315–320 mOsm). To depolymerize MTs, slices were incubated for 20–60 min at PT (35–37°C) with vinblastine sulfate (50 μ m ; Wako) dissolved in aCSF. To depolymerize F-actin, slices were incubated with latrunculin A (20 μ m ; Wako) at PT for 60 min. Fluorescence imaging. For fixed tissue imaging, the following primary antibodies were used: Anti-VGluT1 guinea pig antiserum (1:2000; AB5905, Millipore; RRID: AB_2301751 ); anti-synaptophysin rabbit polyclonal antiserum (1:250; catalog #101002, Synaptic Systems; RRID: AB_887905 ); anti-α-tubulin mouse monoclonal clone DM1A (1:250; catalog #T9026, Sigma-Aldrich; RRID: AB_477593 ); and anti-β3-tubulin rabbit antiserum (1:1000; product #2200, Sigma-Aldrich; RRID: AB_262133 ). Secondary antibodies were goat IgG conjugated with Invitrogen Alexa Fluor 488, 568, or 647 (Thermo Fisher Scientific). For fixed tissue imaging, acute brainstem slices (250 μm) were cut (see above) and fixed with 4% paraformaldehyde in PBS for 30 min at 37°C and overnight at 4°C. On the following day, slices were rinsed three times in PBS, permeabilized in PBS containing 0.5% Triton X-100 (Tx-100; Nacalai Tesque) for 30 min and blocked in PBS containing 3% bovine serum albumin (BSA; Sigma-Aldrich) and 0.05% Tx-100 for 45 min. Slices were incubated overnight at 4°C with primary antibody diluted in PBS 0.05% Tx-100, 0.3% BSA. On the next day, slices were rinsed three times with PBS containing 0.05% Tx100 for 10 min and incubated with corresponding secondary antibody diluted in PBS 0.05% Tx-100, 0.3% BSA for 1 h at room temperature (RT). Slices were further rinsed three times in PBS 0.05% Tx-100 for 10 min and finally were washed in PBS for another 10 min. Before mounting, the nucleus was stained with Life Technologies NucBlue (Thermo Fisher Scientific) in PBS for 20 min according to manufacturer instruction. For confocal imaging, slices were then directly mounted on glass slides (Matsunami) using a mounting medium (Ibidi) and sealed using nail polish. For STED microscopy, slices were sequentially incubated in PBS containing 10%, 20%, and 50% of 2,2′-thiodiethanol (TDE) for 1 h each, followed by washing three times in 97% TDE solution for 10 min each, and mounted on glass slides using TDE mounting reagent (Abberior). For live imaging of silicon-rhodamine (SiR)-tubulin-stained slices, acute brainstem slices (250 μm) were incubated with SiR-tubulin (1 μ m ; Cytoskeleton) at 37°C according to manufacturer instructions. The slices were then mounted onto a 35 mm Ibidi dish and immobilized using a platinum grid holder, then incubated in aCSF containing 50 μ m vinblastine at 37°C. HeLa cells, cultured in DMEM 10% FBS and grown in a 35 mm Ibidi dish for 3 d, were incubated with 1 μ m SiR-tubulin or SiR-actin at 37°C and 5% CO 2 . Culture medium was replaced with Tyrode's solution before vinblastine or latrunculin A treatment and observation. Confocal images were acquired on a laser scanning microscope (LSM 780, Carl Zeiss) equipped with a Plan-apochromat 63×, oil-immersion objective with a numerical aperture (NA) of 1.4 and excitation laser lines (wavelengths: 405, 488, 561, and 633 nm). For quantifying fluorescence intensity levels, the region of interest was delimited to calyceal terminals, and background fluorescence was subtracted using ImageJ software. Super-resolution imaging was performed on STED 3× TCS SP8 microscope (Leica) equipped with an HC PL APO CS2 100×, 1.4 NA oil-immersion objective (Leica) and tunable white laser excitation line, and with depletion laser lines (wavelengths: 592, 660, and 775 nm). STED images were deconvoluted using Huygens software (Leica). Three-dimensional reconstruction of STED 3× confocal stacks was performed in Imaris 9.2 with filament tracer and measurement pro plugins (Bitplane/Oxford Instruments) to estimate the distance between identified vesicles and microtubules. Electrophysiological recordings and data analysis. The calyx of Held presynaptic terminals and postsynaptic MNTB principal cells were visually identified with a 40× water-immersion objective attached to an upright microscope (BX51WI, Olympus). All experiments were performed at PT. EPSCs were evoked in MNTB principle neurons by afferent fiber stimulation using a bipolar tungsten electrode, positioned on the axon bundles halfway between the midline and MNTB region. For whole-cell recording of EPSCs, MNTB principal neurons were voltage clamped at a holding potential of −70 mV. Postsynaptic pipettes were pulled for a resistance of 2–3 MΩ and had a series resistance of 4–10 MΩ, which was compensated by 40–70% for a final value of 3 MΩ. STD of EPSCs was induced by a train of 30 stimuli at 100 Hz, and recovery from STD was monitored from EPSCs evoked by stimulations at different intervals (0.02–30 s). The recovery time course was fit by a double exponential function, from which fast time constants (τ fast ) and slow time constants (τ slow ) were measured. Presynaptic membrane capacitance measurements at calyces of Held were made as previously described ( Sun et al., 2002 ; Yamashita et al., 2005 ) except that recordings were made at PT in the present study instead of RT. Calyceal terminals were voltage clamped at a holding potential of −80 mV, and single-pulse step to +10 mV (20 ms in duration) was applied for inducing presynaptic Ca 2+ currents. The rate of endocytosis was evaluated from the 50% decay time of membrane capacitance change. Data analysis. Electrophysiological data were acquired at a sampling rate of 50 kHz using an EPC-10 patch-clamp amplifier controlled by PatchMaster software (HEKA), after on-line filtering at 5 kHz and subsequently analyzed off-line using IGOR Pro 6.22 (WaveMetrics), Excel 2010 (Microsoft), Origin Pro 8.6 (Origins Laboratory), SPSS (IBM), and Prism 6 (GraphPad Software). Data fitting was performed using the least-squares method (single or double exponential). Imaging data were analyzed using Las AF Lite (Leica), ZEN (Zeiss), and ImageJ. All values are given as the mean ± SEM, and 95% confidence intervals on the difference of the means were considered statistically significant by two-tailed unpaired t test or one-way ANOVA with a post hoc Scheffé's test ( p < 0.05). Results Colocalization of microtubules with SVs at the calyx of Held presynaptic terminals Before addressing the functional roles of MTs in presynaptic terminals, we examined the localization of MTs in the calyx of Held presynaptic terminals in the brainstem of juvenile rats (postnatal day 13–16) using confocal and STED microscopy. After tissue fixation and permeabilization to washout-free tubulin, immunofluorescence staining of calyceal terminals, using specific antibodies against α- or β3-tubulin, revealed tubulin-polymer bundles, running along axons and extending into calyceal terminals surrounding postsynaptic MNTB neurons ( Fig. 1 A–C ). Using STED, tubulin-polymer bundles were clearly observed in proximity with SVs labeled with vesicular glutamate transporter 1 (VGluT1, Fig. 1 D , E ) or synaptophysin ( Fig. 1 F ). At high magnifications, immunofluorescent tubulin signals were seen to partially overlap with those of SVs labeled with VGluT1 ( Fig. 1 G , H ). Three-dimensional reconstruction of STED confocal stacks and analyses of SV distance from MTs indicated that 59% of SVs are localized on or alongside the MT lattice within 100 nm in the whole terminal ( Fig. 2 A , B ). In presynaptic swellings, 36% of SVs were localized within 100 nm of MTs with an average ± SEM distance of 44 ± 2.5 nm ( n = 106 SVs), whereas 64% of SVs were distributed >100 nm away from MTs with an average distance of 408 ± 16 nm ( n = 106; Fig. 2 B ). These results indicate that MTs are extended and spread into calyceal swellings and suggest that MTs are partially colocalized with a subset of glutamatergic SVs in the nerve terminal.
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