Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem

Miae Jang; Elizabeth Gould; Jie Xu; Eun Jung Kim; Jun Hee Kim

doi:10.7554/eLife.42156

Jang, Miae; Gould, Elizabeth; Xu, Jie; Kim, Eun Jung; Kim, Jun Hee

2019-04-18 00:00:00

RESEARCH ARTICLE Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem 1 1 1,2 1 1 Miae Jang , Elizabeth Gould , Jie Xu , Eun Jung Kim , Jun Hee Kim * The Department of Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, United States; Children’s Medical Center, The Second Xiangya Hospital, Central South University, Changsha, China Abstract Neuron–glia communication contributes to the fine-tuning of synaptic functions. Oligodendrocytes near synapses detect and respond to neuronal activity, but their role in synapse development and plasticity remains largely unexplored. We show that oligodendrocytes modulate neurotransmitter release at presynaptic terminals through secretion of brain-derived neurotrophic factor (BDNF). Oligodendrocyte-derived BDNF functions via presynaptic tropomyosin receptor kinase B (TrkB) to ensure fast, reliable neurotransmitter release and auditory transmission in the +/– developing brain. In auditory brainstem slices from Bdnf mice, reduction in endogenous BDNF significantly decreased vesicular glutamate release by reducing the readily releasable pool of 2+ glutamate vesicles, without altering presynaptic Ca channel activation or release probability. Using conditional knockout mice, cell-specific ablation of BDNF in oligodendrocytes largely recapitulated this effect, which was recovered by BDNF or TrkB agonist application. This study highlights a novel function for oligodendrocytes in synaptic transmission and their potential role in the activity-dependent refinement of presynaptic properties. DOI: https://doi.org/10.7554/eLife.42156.001 *For correspondence: [email protected] Introduction Competing interests: The The formation of complex neuronal networks requires the experience-dependent establishment and authors declare that no remodeling of synapses. The precise control of synaptic function depends not only on neurons, but competing interests exist. also on glial cells. Immature oligodendrocytes, located near synapses, make functional synapses with Funding: See page 23 neurons, express neurotransmitter receptors (Bergles et al., 2000; Lin and Bergles, 2004; Berret et al., 2017), and secrete neurotrophic factors such as BDNF (Bagayogo and Dreyfus, Received: 21 September 2018 2009). Thus, oligodendrocytes are in a prime position to participate in bi-directional communication. Accepted: 17 April 2019 Published: 18 April 2019 In this study, we address whether oligodendrocytes can modulate synaptic function through activity- dependent BDNF signaling. Reviewing editor: Dwight E BDNF regulates neuronal survival and growth in the developing central nervous system (CNS; Bergles, Johns Hopkins Alderson et al., 1990; Hohn et al., 1990; Rodriguez-Te´bar et al., 1990) and is extensively involved University School of Medicine, United States in synaptic transmission and plasticity in various brain regions (Kang and Schuman, 1995; Levine et al., 1995; Carmignoto et al., 1997). For example, activity-dependent BDNF secretion is Copyright Jang et al. This involved in long-term synaptic plasticity in the hippocampus (Harward et al., 2016; Vignoli et al., article is distributed under the 2016; Ga¨rtner and Staiger, 2002). It is known that BDNF signaling regulates vesicular glutamate terms of the Creative Commons Attribution License, which release at presynaptic terminals (Pozzo-Miller et al., 1999; Tyler and Pozzo-Miller, 2001), but, permits unrestricted use and because of the low expression of both intracellular and extracellular BDNF in most brain areas, little redistribution provided that the is known regarding the source and the exact location of action of BDNF at synapses and, specifically, original author and source are its presynaptic effects. credited. Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 1 of 26 Research article Neuroscience Glial cells modulate synaptic properties and activities through the secretion of BDNF. Astrocytes recycle BDNF and are involved in the stabilization of long-term synaptic plasticity (Vignoli et al., 2016). Microglial BDNF is also an important regulator of synaptic plasticity and function during early brain development (Parkhurst et al., 2013). In the CNS, oligodendrocytes express and secrete BDNF, and BDNF secretion is regulated by activation of glutamate receptors (Bagayogo and Drey- fus, 2009). We recently showed that immature oligodendrocytes have the capacity to sense neuro- nal activity and receive glutamatergic inputs in the auditory brainstem (Berret et al., 2017), suggesting that neuron-oligodendrocyte communication occurs through chemical signaling. BDNF may thus be a bi-directional signaling factor between oligodendrocytes and neurons. In this study, we investigated the effects of oligodendroglial BDNF on synaptic functions and the mechanisms whereby oligodendroglial BDNF regulates neurotransmitter release at the presynaptic +/– terminal using mice with reduced BDNF levels (Bdnf ) and with an oligodendrocyte-specific condi- tional knockout (cKO) of Bdnf. We studied the synaptic functions of oligodendroglial BDNF at the synapse between the calyx of Held terminal and the medial nucleus of the trapezoid body (MNTB) neuron in the auditory brainstem, which is an oligodendrocyte- and synapse-rich brain region. Using immunofluorescence microscopy, electrophysiology, electron microscopy, and in vivo auditory brain- stem response (ABR) tests, we found that oligodendroglial BDNF is critical for determining the read- ily releasable pool (RRP) of glutamate vesicles and actively participates in glutamate release at the calyx terminals. The results suggest that oligodendrocytes are involved in synaptic transmission and plasticity specifically through BDNF signaling in the developing auditory brainstem region. Results BDNF and glutamatergic synapses in the auditory brainstem Immunostaining using the neuronal marker MAP2 and the oligodendroglial marker, Olig1, in brain- stem sections from wild-type (WT) mice showed that BDNF is highly expressed in MNTB principal neurons (Figure 1A). It is of note that oligodendrocytes located close to the calyx–MNTB neuron synapse also expressed BDNF. BDNF expression was notably decreased in all cell types in the +/ MNTB in Bdnf mice at P21 (Figure 1A). To examine the effect of endogenous BDNF on fast gluta- matergic transmission in the auditory brainstem, we recorded miniature excitatory post-synaptic cur- +/ rents (mEPSCs) from MNTB principal neurons in brainstem slices from P16–20 WT and Bdnf mice (Figure 1B). There was no significant difference in the amplitude or kinetics, including rise and decay +/– times, of mEPSCs in WT and Bdnf mice (amplitude: 39.9 ± 2.51 pA, n = 13 vs 33.4 ± 2.92 pA, n = 12, respectively, p=0.10; rise time: 0.3 ± 0.01 ms, n = 13 vs 0.3 ± 0.02 ms, n = 12, respectively, p=0.37; decay time: 0.6 ± 0.03 ms, n = 13 vs 0.7 ± 0.07 ms, n = 12, respectively, p=0.58, unpaired t-test; Figure 1C–E). In addition, the frequency of mEPSCs was not statistically different +/ (2.7 ± 0.69 Hz, n = 8 vs 2.4 ± 0.41 Hz, n = 11 in WT and Bdnf mice, respectively; p=0.76, unpaired t-test; Figure 1F). However, the amplitude of evoked EPSCs (eEPSCs) triggered by afferent fiber +/ +/ stimulation was significantly smaller in Bdnf mice (3.1 ± 0.31 nA, n = 17 in Bdnf mice vs 5.9 ± 0.35 nA, n = 9 in WT; p<0.0001, unpaired t-test; Figure 1G,H). To examine the changes in postsynaptic receptor kinetics or in asynchronous release, we analyzed the decay of eEPSCs. The line corresponding to eEPSC decay was well fit as a single exponential with a time constant tau (t) +/ =1.0 ± 0.07 ms (n = 9) in WT and 0.9 ± 0.05 ms (n = 17) in Bdnf mice, which were not significantly different (p=0.31, unpaired t-test; Figure 1G,I). These results suggest that a reduction in endoge- nous BDNF results in impaired glutamatergic transmission, which is caused by alterations in presyn- aptic properties rather than postsynaptic components. To test the effect of reduced BDNF on presynaptic properties, we examined the paired pulse +/ ratio (PPR), which was similar in both groups (0.8 ± 0.02, n = 9 in WT and 0.8 ± 0.03, n = 17 in Bdnf mice; p=0.93, unpaired t-test; Figure 2A). This indicates that a reduction in endogenous BDNF 2+ does not alter the Ca -dependent release probability at presynaptic terminals. Next, we examined the short-term depression and the RRP size of available glutamate vesicles at presynaptic terminals +/ in WT and Bdnf mice. During a train of stimuli at 100 Hz (50 pulses), the amplitude of eEPSCs dis- played strong depression, falling to ~20% of the initial amplitude near the end of the train in both +/ WT and Bdnf mice (Figure 2B). There was no notable difference in short-term depression +/ between WT and Bdnf mice (n = 7 vs 12). To predict the RRP size, the release probability (P ), Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 2 of 26 20 pA 2 nA Research article Neuroscience WT Bdnf+/- MAP2/Olig1 BDNF MAP2/Olig1 BDNF WT Bdnf+/- 0.5 s C D E 1.5 F 60 0.6 8 0.4 1.0 0.5 20 0.2 0.0 0.0 0 0 WT Bdnf+/- WT Bdnf+/- WT Bdnf+/- WT Bdnf+/- G H I 10.0 *** 1.5 Bdnf+/- Scaled Bdnf+/- WT 7.5 1.0 5.0 0.5 2.5 Fitted Fitted 5 ms 0.0 0.0 WT Bdnf+/- WT Bdnf+/- Figure 1. Reduction in endogenous BDNF impairs synaptic transmission at the calyx of Held synapse. (A) Representative immunolabeled images for +/– endogenous BDNF expression (green) in the MNTB principal neurons (MAP2, blue) and oligodendrocytes (Olig1, red) in WT and Bdnf mice at postnatal day (P)21. Images shown are representative of results from n = 5 mice per group. Scale bars, 20 mm. (B) Representative traces of mEPSCs from +/– MNTB neurons in WT (black) and Bdnf mice (gray) at P16–20. (C–F) Quantification of the amplitude (C), rise time (D), decay time (E), and frequency +/– +/– (F) of mEPSCs from WT and Bdnf mice. (G) A single EPSC evoked by afferent fiber stimulation in WT (black) and Bdnf (gray) mice. The decay time +/– constant (t, red) was obtained by single exponential fitting after normalizing the amplitude of EPSCs from Bdnf mice. (H, I) Summary of the +/– amplitude (H) and decay time constant (I) of eEPSCs from WT and Bdnf mice. Data are shown as the mean ± s.e.m. ***p<0.001 (unpaired t-test). DOI: https://doi.org/10.7554/eLife.42156.002 and the synaptic vesicle replenishment rate, we used two variants of the cumulative analysis of EPSC trains (Figure 2C,D). In the Elmqvist and Quastel (EQ) method (Elmqvist and Quastel, 1965), the RRP size was estimated by fitting a line to the linear portion of these data (corresponding to the sec- ond through the fourth EPSC) and extrapolating to the x axis, we measured the total equivalent Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 3 of 26 Amplitude (pA) Rise time (ms) Decay time (ms) eEPSC (nA) τ (ms) Frequency (Hz) 1 nA 1 nA 2 nA Research article Neuroscience Bdnf+/- WT 1.0 0.8 0.6 0.4 0.2 20 ms 0.0 WT Bdnf+/- Bdnf+/- WT WT Bdnf+/- 100 ms 0 100 200 300 400 500 Time (ms) C D ** E F G 120 0.6 0.8 8 WT WT Bdnf+/- Bdnf+/- 0.6 80 0.4 60 0.4 40 0.2 40 0.2 20 20 0 0.0 0.0 0 10 20 30 40 50 WT Bdnf+/- Bdnf+/- WT Bdnf+/- WT 0 20 40 60 80 100 Stimulus number Cumulative eEPSC (nA) After tetanus WT After tetanus 100 Hz, 3 s W T (Tetanus) Bdnf+/- 2.0 1.5 1.0 Bdnf+/- 2 0.5 0 50 100 WT Bdnf+/- Time (sec) 5 ms Figure 2. Reduction in endogenous BDNF alters presynaptic properties at the calyx terminals. (A) Representative traces of EPSCs evoked by paired- +/– pulse stimulation from WT (black) and Bdnf (gray) mice (at P16, left). Summary of the PPR (right). (B) Trains of eEPSCs at 100 Hz stimulation in WT +/– +/– (black) and Bdnf (gray) mice (left). Normalized amplitude of eEPSCs relative to the first eEPSC amplitude in WT and Bdnf mice (right). (C) Plot of +/– eEPSC amplitudes against the amplitude of the cumulative eEPSC in WT and Bdnf mice. Plots were linearly fitted from the second through the fourth +/– cumulative eEPSCs (red line for WT and blue line for Bdnf ), which were estimated by back-extrapolated linear fits to the x axis to estimate the RRP. +/– (D, E) Summary of the cumulative eEPSC size and the release probability (P ) using the EQ method in WT and Bdnf mice. (F) Plot of the cumulative Figure 2 continued on next page Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 4 of 26 eEPSC (nA) Cumulative EPSCs (nA) Normalized eEPSC Pr Cumulative EPSCs (nA) Amplitude (nA) Paired pulse ratio Normalized eEPSC (%) (PPR) Pr Research article Neuroscience Figure 2 continued +/– eEPSC against stimulus number in WT and Bdnf mice. A line fit to the steady-state points is back-extrapolated to the y-axis to estimate the RRP. (G) +/– Summary of the release probability (P ) using the SMN method in WT and Bdnf mice. (H) Left, EPSCs evoked at 30 s before and after tetanic +/– stimulation (100 Hz, 3 s) from WT (top) and Bdnf (bottom) mice. Right, plot of normalized eEPSC amplitude after the tetanus relative to the eEPSC +/– amplitude before the tetanus. (I) Summary of the amplitude of eEPSCs before and after the tetanus from WT and Bdnf mice. Data are shown as the mean ± s.e.m. *p<0.05; **p<0.01 (unpaired t-test; paired t-test). DOI: https://doi.org/10.7554/eLife.42156.003 EPSC at the beginning of the train, which indirectly indicates the available pool of vesicles for release (Taschenberger et al., 2002; Kushmerick et al., 2006). We plotted the eEPSC amplitudes during a train versus their cumulative amplitudes at the end of a train (Figure 2C), which were 91.5 ± 9.46 nA +/ (n = 6) in WT and 46.3 ± 6.89 nA (n = 13) in Bdnf mice (p=0.0016, unpaired t-test; Figure 2D). +/ The forward extrapolation linear fits revealed that calyces in Bdnf mice had a much smaller RRP +/ of glutamate vesicles as compared with WT mice (17.6 ± 2.73 nA in Bdnf mice, n = 11 vs 33.4 ± 3.31 nA in WT, n = 5; p=0.004, unpaired t-test; Figure 2C). The RRP divided by the mEPSC amplitude (Figure 1C) approximately estimates the number of vesicles, which was reduced in in +/ +/ Bdnf mice (~837 vesicles in WT and ~527 vesicles in Bdnf mice). There was no significant differ- +/ ence in Pr, determined as the slop of the linear fit (0.25 ± 0.035 in Bdnf mice, n = 10 vs 0.26 ± 0.037 in WT, n = 6; p=0.8582, unpaired t-test; Figure 2E). In the Schneggenburger-Meyer-Neher (SMN) method (Schneggenburger et al., 1999), EPSC amplitudes from trains are plotted cumulatively against the stimulus number (Figure 2F). A line fit to the steady-state points (the last 10 of 50 points) is back-extrapolated to the y-axis, and the y-inter- cept divided by the mEPSC amplitude estimates the RRP size. This analysis also revealed that calyces +/ in Bdnf mice had a much smaller RRP of glutamate vesicles as compared with WT mice +/ (9.8 ± 1.29 nA in Bdnf mice, n = 11 vs 19.4 ± 1.71 nA in WT, n = 5; p=0.0008, unpaired t-test; Figure 2F). Conversely, the release probability (P ), which is calculated by dividing the amplitude of +/– the first eEPSC by the RRP size, was not different in WT and Bdnf mice (0.35 ± 0.02, n = 5 vs 0.38 ± 0.05, n = 11, respectively, p=0.73, unpaired t-test; Figure 2G). Another interesting finding +/ was the reduced replenishment rate of vesicles in Bdnf mice, which was estimated by the slope of +/ the linear fit (0.61 ± 0.10 in Bdnf mice, n = 11 vs 1.39 ± 0.19 in WT, n = 5; p=0.0018, unpaired t-test; Figure 2F), indicating that BDNF signaling plays a role in the replenishment of vesicles at the calyx terminal. The values of RRP obtained by the SMN method were smaller than those obtained by the EQ method, because this RRP was measured as the pool decrement during stimulation, whereas the RRP estimated using the EQ Method indicates the size of a pre-existing pool of vesicles (Neher, 2015). Taken together, these results show that a reduction in endogenous BDNF decreased +/ the pool of glutamate vesicles available for release at the beginning of a train in Bdnf mice, sug- gesting that BDNF is important for determining the RRP at presynaptic terminals. An increase in the RRP of vesicles can contribute to short-term plasticity such as post-tetanic potentiation (PTP; Habets and Borst, 2005; Regehr, 2012). Thus, we tested whether a decrease in the RRP caused by reduction of BDNF alters PTP at the calyx synapse (Figure 2H,I). Tetanic stimula- tion (100 Hz, 3 s) increased the amplitude of eEPSCs from 5.6 ± 1.01 pA to 8.0 ± 0.96 pA in WT (n = 5; p=0.02, paired t-test), indicating a PTP induction, whereas there was no significant increase +/ in the eEPSC amplitude after tetanus in Bdnf mice (2.8 ± 0.37 pA to 2.7 ± 0.39 pA, n = 9; p=0.05, paired t-test). Taken together, BDNF controls synaptic plasticity as well as neurotransmitter release by regulating the RRP at the calyx terminal. BDNF signaling and exocytosis of vesicular glutamate We next evaluated the regulatory effect of endogenous BDNF on the exocytosis of vesicular neuro- 2+ influx at presynaptic terminals by measuring the membrane capacitance jump transmitter and Ca 2+ +/ (DC ) and voltage-activated Ca channel current (I ) in P9–13 calyces from WT and Bdnf mice m Ca 2+ (Figure 3A). Depolarization induced Ca currents and, consequently, DC in a pulse duration– +/ dependent manner (2 to 20 ms). DC in Bdnf mice was much smaller than that in WT (DC after a m m +/ 20 ms depolarization: 93.4 ± 8.82 fF, n = 11 in Bdnf vs 148 ± 11.32 fF, n = 11 in WT mice; p=0.0013, unpaired t-test; Figure 3A,B). However, the membrane capacitances of calyces from WT +/ and Bdnf mice were similar (20 ± 1.5 pF, n = 10 vs 21.5 ± 1.35 pF, n = 9, respectively, p=0.43, Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 5 of 26 100 fF 200 fF 0.4 nA Research article Neuroscience A WT Bdnf+/- 2 ms 3 ms 20 ms 5 ms 10 ms 40 ms 100 pA 10 ms WT WT WT B C D Bdnf+/- Bdnf+/- Bdnf+/- ** ** 0 5 10 15 20 10 20 30 0 5 10 15 20 25 Cm (pF) Duration (ms) Duration (ms) Bdnf+/- WT -80 -60 -40 -20 20 40 60 mV -0.2 -0.4 WT Bdnf+/- 100 ms -0.8 I (nA) Ca G H I WT * WT Bdnf+/- Bdnf+/- 6 Y6 0.5 s 0 0.5 1 1.5 2 WT Bdnf+/- Time (sec) +/– Figure 3. Exocytosis of vesicular glutamate is decreased at the calyx terminals in Bdnf mice. (A) Representative traces of membrane capacitance (C ; 2+ +/– top) and Ca current (I ; middle) induced by 2-, 3-, 5-, 10-, and 20 ms depolarization (bottom) from P9–13 calyx terminals in WT (black) and Bdnf Ca (gray) mice. Capacitance within 50 ms after depolarization is not shown to avoid artifacts. (B) Summary of capacitance changes (DC ), which are plotted +/– against the depolarization duration in WT and Bdnf mice. (C) Scatter plot: DC (elicited by 20 ms depolarization)is plotted against the corresponding +/– resting C for each calyx terminal. The squares indicate the mean value. The black and gray lines are linearly fit from each dot for WT and Bdnf mice, Figure 3 continued on next page Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 6 of 26 Cm (fF) Cm (fF) Cm (%) QI (pC/pF) Ca Cm (fF) Research article Neuroscience Figure 3 continued 2+ respectively. (D) Summary of Ca current charge (QI ), which is plotted against the depolarization duration for each genotype. (E) Representative Ca +/– traces of I induced by a 200 ms step-like depolarization (from –80 to 60 mV, D10 mV) in WT (black; n = 5) and Bdnf (gray; n = 5) mice. (F) The I–V Ca 2+ relationship for voltage-activated Ca channels at the calyx terminal for each genotype is also shown. (G) Examples of C (top) induced by the train of +/– 20 depolarizing pulses (10 ms, 10 Hz; bottom) from –80 to 0 mV in WT (black; n = 11) and Bdnf (gray; n = 9) mice. (H) Summary of the normalized accumulated capacitance jump (SDC ) relative to the stimulation time for each genotype. Data were normalized relative to the capacitance jump induced by the first 10 ms depolarization. (I) Summary of the SDC after the train of 20 depolarizing pulses (at 2 s) in each genotype. Data are shown as the mean ± s.e.m. *p<0.05; **p<0.01 (unpaired t-test). DOI: https://doi.org/10.7554/eLife.42156.004 The following figure supplement is available for figure 3: Figure supplement 1. Reduction of endogenenous BDNF does not affect endocytosis in the calyx of Held. DOI: https://doi.org/10.7554/eLife.42156.005 unpaired t-test; Figure 3C), indicating that the sizes of the calyx terminals are similar, and thus the difference in DC was not due to the terminal size. In addition, plotting DC (for the 20 ms depolari- m m zation) as a function of the C showed the distribution of C as estimated by linear regression analy- m m 2 2 +/– sis (R = 0.1 in WT, n = 9; R = 0.06 in Bdnf mice, n = 9; Figure 3C). Despite their similar resting +/ values for C , DC was much smaller in calyces from Bdnf mice as compared with comparably m m sized calyces from WT. However, a positive correlation between the resting C and DC suggest m m that larger calyces release more vesicles in both genotypes (Figure 3C). Next, we examined the 2+ effects of BDNF reduction on presynaptic Ca channel and I related to changes in exocytosis of Ca glutamate vesicles. There was no significant difference in the presynaptic I charge (QI ) in Ca Ca +/ response to depolarizing pulses. The smaller DC in calyces from Bdnf mice was not attributed to 2+ alterations in presynaptic Ca currents. In WT calyces, a 20 ms depolarization induced a I of Ca +/ 6.5 ± 0.53 pC/pF (n = 10), whereas in Bdnf calyces, I was 5.9 ± 0.96 pC/pF (n = 9; p=0.50, Ca unpaired t-test; Figure 3D). In addition, the current–voltage relationship (I–V) curve for these volt- 2+ +/ age-activated Ca channels at the calyx terminal in WT and Bdnf mice (at P10-12) exhibited a similar pattern with the peak current of 625 ± 48.3 pA vs 614 ± 16.5 pA at 10 mV in WT and +/ Bdnf mice, respectively (n = 15 vs n = 15; Figure 3E,F). Furthermore, we examined presynaptic action potential evoked by afferent fiber stimulation, and there was no significant difference in 2+ amplitude and half-with of presynaptic action potential, which is associated with Ca channel activa- +/ tion and release probability (amplitude, 122.5 ± 7.8 mV in WT, n = 3 vs 124.6 ± 4.4 mV in Bdnf , +/ n = 4, p=0.8082 and half-width, 318 ± 41.2 ms in WT, n = 3 vs 283 ± 45.5 ms in Bdnf , n = 4, p=0.6123, unpaired t-test, data not shown). These data suggest that a reduction in endogenous BDNF decreases exocytosis of vesicular glutamate, but this reduction is not associated with changes 2+ 2+ in Ca influx via Ca channels at the presynaptic terminals. To confirm whether BDNF regulates the RRP and exocytosis of glutamate vesicles at presynaptic terminals, we assessed the vesicle pool size by gradually depleting the RRP using 20 depolarizing pulses (10 ms, 10 Hz). The accumulated capacitance jump (SDC ), which is a measure of the sum of +/ available glutamate vesicles released by stimulation, was significantly smaller in Bdnf mice (260 ± 54.1 fF, n = 9) relative to WT (508 ± 86.2 fF, n = 11; p=0.04, t-test; Figure 3G–I). This difference, however, was not associated with the endocytosis rate, which was similar in both groups +/ (19.5 ± 2.75 s, n = 10 in WT vs 17.3 ± 1.91 s, n = 9 in Bdnf mice; p=0.51, unpaired t-test; Fig- ure 3—figure supplement 1). Therefore, the decreased SDC resulted mainly from the reduction in +/– the RRP size in Bdnf mice. Taken together, these results suggest that the endogenous BDNF level is directly involved in glutamatergic transmission based on its ability to determine the RRP size at the presynaptic terminal. BDNF–TrkB signaling and RRP size We examined whether BDNF function in determining the RRP is mediated by endogenous BDNF at the terminal or by BDNF derived from neighboring cells, which activate TrkB signaling at presynaptic terminals. TrkB was expressed at the calyx terminal and axon, which was immunolabelled with VGluT1 and detected by Alexa 568 dye filling during presynaptic whole-cell recording (Figure 4A). We directly activated TrkB using its agonist, 7,8-dihydroxyflavone (7,8-DHF), which binds to the TrkB extracellular domain and activates TrkB-mediated downstream signaling (Jang et al., 2010; Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 7 of 26 1 nA 2 nA 1 nA 1 nA Research article Neuroscience TrkB VGluT1 TrkB Alexa/TrkB B C +7,8-DHF 7,8-DHF WT WT Bdnf+/- 20 ms +7,8-DHF 1.0 0.8 Bdnf+/- 0.6 0.4 0.2 0.0 Bdnf+/- 20 ms WT D E +7,8-DHF 7,8-DHF 0 min 7 min 5 min 10 min Bdnf+/- -2.0 5 ms -2.5 2 -3.0 30 min 0 min 10 min -3.5 0 200 400 600 Bdnf+/- Time (sec) F G Bdnf+/- 7,8-DHF Linear fit Bdnf+/- +7,8-DHF 0 20 40 60 Cumulative eEPSC (nA) 100 ms Figure 4. The activation of TrkB rescues decreased glutamate release at the calyx terminal. (A) Expression of TrkB (green) and VGluT1 (red) at calyx terminals in the MNTB from WT mice (P20). The calyx terminal and axon (arrows, P12 WT mice), filled with Alexa 568 during whole-cell recording, expressed TrkB (green). Scale bars, 10 mm. (B) Representative traces of eEPSCs in response to paired-pulse stimulation in the absence or presence of +/– 7,8-DHF for WT (n = 5) and Bdnf (n = 17) mice. (C) Summary of the effect of 7,8-DHF on the eEPSC amplitude (top) and the PPR (bottom). (D) Top, Figure 4 continued on next page Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 8 of 26 eEPSC (nA) Paired pulse ratio eEPSC (nA) eEPSC (nA) eEPSC (nA) Research article Neuroscience Figure 4 continued example of eEPSCs at 0, 5, 7, and 10 min after the acute application of 7,8-DHF. Bottom, the amplitude of eEPSCs is plotted against time after 7,8-DHF application. (E) Summary of the acute effect of 7,8-DHF on eEPSCs at different time points. (F) Representative traces of the eEPSC train at 100 Hz from +/– Bdnf mice in the absence (gray; n = 11) or presence of 7,8-DHF (blue; n = 7). (G) Plot of eEPSC amplitude against the amplitude of the cumulative +/– eEPSC from Bdnf mice in the absence (gray) or presence of 7,8-DHF (blue). The black and red lines represent the linear fit from the second through fourth cumulative eEPSCs. Data are shown as the mean ± s.e.m. *p<0.05 (unpaired t-test; paired t-test). DOI: https://doi.org/10.7554/eLife.42156.006 Marongiu et al., 2013), to determine whether this activation rescues the impaired RRP size and glu- +/– tamate release at the calyx synapses in Bdnf mice. In WT mice, the pre-application of 20 mM 7,8- DHF to brainstem slices (30 min) had no effect on the amplitude (6.6 ± 0.53 nA for control vs 6.3 ± 0.70 nA for 7,8-DHF, n = 5, p=0.70, unpaired t-test) or PPR (0.75 ± 0.03 for control vs +/ 0.74 ± 0.01 for 7,8-DHF, n = 5, p=0.86, unpaired t-test) of eEPSCs (Figure 4B,C). In Bdnf mice, TrkB activation using 7,8-DHF significantly increased the amplitude of eEPSCs (from 2.8 ± 0.54 nA, n = 8 to 4.5 ± 0.40 nA, n = 17, p=0.02, unpaired t-test) without changing the PPR (0.7 ± 0.05 for con- trol, n = 8 vs 0.8 ± 0.03 for 7,8-DHF, n = 17, p=0.58, unpaired t-test; Figure 4B,C). Acute application +/ of 7,8-DHF also increased the amplitude of eEPSCs during the 10 min after its application in Bdnf mice in a time-dependent manner (2.8 ± 0.97 nA for control, n = 5 vs 3.9 ± 1.14 nA for 7,8-DHF at 10 min vs, n = 5, p=0.023, paired t-test; Figure 4D,E). In addition, the pre-application of 7,8-DHF significantly increased the cumulative eEPSC size (from 45.9 ± 7.9 nA, n = 11 to 75.2 ± 9.7 nA, n = 6; +/– p=0.03, unpaired t-test) and partially restored the RRP in Bdnf mice (from 17.6 ± 2.73 nA, n = 11 to 27.8 ± 3.64 nA, n = 7; p=0.03; unpaired t-test, Figure 4F,G). These findings suggest that the down-regulation of BDNF–TrkB signaling at the presynaptic terminal impairs the RRP size and gluta- matergic transmission in the MNTB. Neighboring oligodendrocytes and their BDNF signal Our results suggested that local BDNF signaling from neighboring cells around presynaptic terminals is critical for determining the RRP at presynaptic terminals during postnatal development. We inves- tigated whether glial cells, specifically oligodendrocytes, are the source of this BDNF signaling. A number of oligodendrocytes are apposed to the calyx synapse in the MNTB during postnatal devel- opment (Figures 1A and 5A; Berret et al., 2017). To study the specific role of oligodendrocytes in presynaptic functions as BDNF providers, we generated Bdnf cKO mice, in which BDNF was specifi- cally deleted in CNPase-expressing oligodendrocytes using the Cre/loxP system (Figure 5B). To con- cre cre firm the specificity of the Cnp line, Cnp mice were crossed to a GCaMP6f-GFP mouse (or tdTomato reporter) as a reporter line. GFP+ cells were positive for Olig2 and were present next to the calyx synapse, but did not express MAP2, NeuN, and GFAP expression in the MNTB (Figure 5— cre figure supplement 1). This confirms that Cnp is specific to oligodendrocytes and is not expressed in neurons or astrocytes in the MNTB of the auditory brainstem. In addition, CNP+ cells were posi- tive for CC1, but negative for PDGFRa, indicating that most CNP+ cells in the MNTB are pre-myeli- nating oligodendrocytes beyond the precursor stage (Figure 5—figure supplement 1). Bdnf cKO cre fl/fl cre mice (Cnp :Bdnf ; Figure 5B) were generated by crossing Cnp mice with mice containing a fl/fl floxed allele of BDNF (Bdnf ). To further confirm the oligodendrocyte-specific depletion of BDNF, oligodendrocytes were isolated via the fluorescent activated cell sorting (FACS) using an O1 anti- body, which is specific to oligodendrocytes, or Cnp- driven GCaMP6f-GFP (GFP). Utilizing quantita- tive PCR, we confirmed that the sorted O1+ or GFP+ fraction expressed a substantial level of Bdnf cre fl/+ in control mice (Cnp :Bdnf ), whereas the O1+ or GFP+ fraction from Bdnf cKO mice showed sig- nificantly reduced level of Bdnf (Figure 5—figure supplement 2). Using presynaptic terminal record- ings, we compared Bdnf cKO mice with control mice to examine how oligodendroglial BDNF affects presynaptic properties (Figure 5C). The deletion of BDNF in oligodendrocytes significantly decreased exocytosis of glutamate vesicles at the calyx terminal in brainstem slices from Bdnf cKO mice. In P9–12 Bdnf cKO mice, DC in response to a 2-, 3-, 5-, 10-, 20-, or 40 ms depolarization was much smaller than in control mice (for 20 ms, 123 ± 20 fF for Bdnf cKO, n = 19 vs 266 ± 35.4 fF for the control, n = 20; p=0.0016, unpaired t-test, Figure 5D,E). Longer depolarization induced a larger DCm and 40 ms- pulse exhib- ited saturation of DC in both control and cKO calyces. DC resulting from 2 ms depolarization was m m Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 9 of 26 100 fF Research article Neuroscience B C Alexa568 Alexa568/ MAP2 bp DIC Alexa (Calyx) 600 Bdnf floxed 400 Bdnf WT fl/+ fl/fl CNP Cre 500 CNP WT Cre-/- Cre+/- D E Control cKO cKO 2 ms 3 ms 5 ms 10 ms 20 ms ** ** 5 ms * 0 10 20 30 40 10 ms time (ms) F G H I Contr ** Control -90 -60 -30 30 60 Contr cKO cKO (mV) 600 -0.2 10 4 Y6 -0.4 Contr 200 cKO 5 2 cKO 0.5 s 0 0 0 5 10 15 20 Control cKO Control cKO -0.7 (nA) Duration(ms) Figure 5. Removal of endogenous BDNF from oligodendrocytes affects exocytosis of vesicular glutamate at the presynaptic terminal. (A) Confocal images of oligodendrocytes filled with Alexa 568 using whole-cell recording and MNTB principal neurons, which were immunolabeled with MAP2, from cre fl/fl a WT mouse (P10). (B) Conditional deletion of BDNF in oligodendrocytes (Cnp : Bdnf ). Genotyping PCR using genomic DNA from control and Bdnf cre fl/+ cre fl/fl cKO mice, which are Cnp : Bdnf and Cnp : Bdnf , respectively. (C) DIC and fluorescence images of the patched calyx terminal filled with Alexa568. Oligodendrocyte (red arrow) was located in close to the calyx synapse in the MNTB. Yellow asterisk indicates MNTB principal neuron. (D) 2+ Representative traces for membrane capacitance (C ; top) and Ca current (I ; bottom) induced by 2-, 3-, 5-, 10-, and 20 ms depolarization (bottom) m Ca from P10–12 calyx terminals in control (black) and Bdnf cKO (red) mice. Scale: 200 fF (top) and 500 pA (bottom), respectively (E) Depolarization duration plotted against DC for control (black; 2 ms, n = 23; 3 ms, n = 16; 5 ms, n = 23; 10 ms, n = 27; 20 ms, n = 24; 40 ms, n = 3) and Bdnf cKO mice (red; 2 ms, n = 25; 3 ms, n = 10; 5 ms, n = 24; 10 ms, n = 25; 20 ms, n = 23; 40 ms, n = 4). (F) Summary of the resting C in WT and Bdnf cKO mice. (G) The 2+ plot of depolarization duration versus Ca current charge (QI ) was generated from data as in (D) for both genotypes. (H) Left: Representative traces Ca of I induced by a 100 ms step-like depolarization (from –80 to 60 mV, D10 mV) in control (black; n = 4) and Bdnf cKO mice (red; n = 5). Right: The I–V Ca 2+ relationship for voltage-activated Ca channels at the calyx terminal for each genotype is also shown. (I) Summary of the SDC after the train of 20 depolarizing pulses (at 2 s) for each genotype. Data are shown as the mean ± s.e.m. *p<0.05; **p<0.01 (unpaired t-test). DOI: https://doi.org/10.7554/eLife.42156.007 The following figure supplements are available for figure 5: cre Figure supplement 1. The specificity of the Cnp line. DOI: https://doi.org/10.7554/eLife.42156.008 cre fl/fl Figure supplement 2. Specific reduction of BDNF in OLs in Cnp : Bdnf mice. DOI: https://doi.org/10.7554/eLife.42156.009 difficult to resolve in ~50% of Bdnf cKO calyces. In both genotypes, the C (15 ± 1.9 pF, n = 14 for 2+ control vs 15 ± 3.5 pF, n = 17 for Bdnf cKO; Figure 5F) and the Ca influx during depolarizing pulses were similar (the QI for 20 ms was 5.1 ± 0.45 pC/pF, n = 13 for the control vs 5.5 ± 0.39 Ca pC/pF, n = 11 for the Bdnf cKO; p=0.45, unpaired t-test; Figure 5G), indicating that the loss of oli- 2+ godendroglial BDNF impairs vesicular exocytosis without a change in Ca channel activation, similar Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 10 of 26 Control cKO Cm (pF) QI (pC/pF) Ca Cm (fF) Cm (fF) Research article Neuroscience +/– to what was observed in Bdnf mice. In addition, the current–voltage relationship (I–V) curve for 2+ these voltage-activated Ca channels at the calyx terminal in control and Bdnf cKO mice (at P10-12) exhibited a similar pattern with the peak current of 633 ± 35.4 pA vs 540 ± 107.1 pA at 10 mV in control and Bdnf cKO mice, respectively (n = 4 vs 5; Figure 5H). Furthermore, we assessed the SDC during 20 pulses of a 10 ms depolarization at 10 Hz, a protocol that gradually depletes the RRP and thus reflects the RRP size. The SDC evoked by 20 depolarizing pulses was reduced in the Bdnf cKO mice (107 ± 19.14 fF, n = 10) as compared with that in the control (525 ± 134 fF, n = 8; p=0.006, unpaired t-test; Figure 5I). There was no difference in the endocytosis rate (19.7 ± 8.86 s, n = 7 for the control vs 20 ± 5.28 s, n = 9 for Bdnf cKO; p=0.97, unpaired t-test; data not shown). These findings suggest that oligodendrocytes are critically involved in determining the presynaptic RRP and vesicular glutamate release through BDNF signaling during postnatal development. Role of oligodendroglial BDNF in glutamatergic transmission We examined the role of oligodendroglial BDNF in glutmatergic transmission in the immature (P10- P12, before hearing onset, Figure 6A) and mature calyx synapses (P16-P20, after hearing onset, Figure 6B) during postnatal development. The amplitude of eEPSCs was significantly smaller in both immature and mature Bdnf cKO mice (2.4 ± 0.53 nA, n = 6 in Bdnf cKO vs 4.9 ± 0.85 nA, n = 5 in control at P10-12; p=0.032, unpaired t-test; Figure 6A,C, and 1.6 ± 0.37 nA, n = 11 in Bdnf cKO vs 6.1 ± 0.51 nA, n = 9 in control at P16-20; p<0.0001, unpaired t-test; Figure 6B,E). In both immature and mature synapses, there was no difference in PPR (Figure 6D,F). Next, we examined the RRP size of available glutamate vesicles and its release probability at presynaptic terminals in control and Bdnf cKO mice at different ages (P10-12 vs P16-20). Using the EQ method, calyces in Bdnf cKO mice had a much smaller RRP of glutamate vesicles as compared with control mice (9.8 ± 0.58 nA in Bdnf cKO mice, n = 6 vs 20.1 ± 2.57 nA in control at P10-12, n = 3; p=0.0238, Mann-Whitney test; Figure 6G and 9.6 ± 3.95 nA in Bdnf cKO mice, n = 6 vs 34.6 ± 3.39 nA in control at P16-20, n = 9; p=0.0004, unpaired t-test; Figure 6I). Conversely, the P was not different in both immature and mature control and Bdnf cKO mice (0.33 ± 0.026 in Bdnf cKO mice, n = 6 vs 0.32 ± 0.012 in control at P10-12, n = 3; p=0.7619, Mann-Whitney test; Figure 6H and 0.41 ± 0.038 in Bdnf cKO mice, n = 4 vs 0.38 ± 0.026 in control at P16-20, n = 9; p=0.4459, unpaired t-test; Figure 6J). In addition, the SMN method analysis showed a reduction in the RRP and the replenishment rate of RRP in Bdnf cKO mice, without significant difference in P (Figure 6—figure supplement 1). A deletion of BDNF from oligodendrocytes around the calyx synapses significantly impaired the RRP and glutamate release at immature and mature calyx synapses in Bdnf cKO mice, suggesting that oligodendroglial BDNF is important for regulating glutamatergic transmission in the auditory brainstem before and after hearing onset. Oligodendrocytes and calyx terminal vesicle regulation To visualize changes in the presynaptic RRP and to quantify the number of glutamate vesicles at the active zone at the calyx terminal, we performed ultrastructural analysis of the calyx–MNTB neuron synapse (at P10-12 and P20) using electron microscopy (EM). Within individual active zones of the calyx terminals, an average of 2–3 docked vesicles was observed in control mice, whereas there were fewer docked vesicles or an absence of docked vesicles at the active zones in the Bdnf cKO mice (Figure 7A). In immature calyx synapses at P10-12, the number of docked vesicles located within 10 nm from the presynaptic active zone membrane was 2.1 ± 0.24 vesicles (62 active zones of three individual cells), whereas in the Bdnf cKO mice the number of docked vesicles was significantly reduced to1.5 ± 0.18 vesicles (67 active zones of five individual cells, p=0.0285, unpaired t-test, Figure 7B, Figure 7—figure supplement 1). To test whether oligodendroglial BDNF influences the development of calyces, we assessed the size of calyces using 3D reconstruction of confocal images of the calyx terminals from control and Bdnf cKO mice (at P10-12) after presynaptic recordings. The volume of the calyx terminal was not significantly different in Bdnf cKO mice (1378 ± 143.7 mm , n = 7 for control and 1199 ± 146 mm , n = 6 for Bdnf cKO; p=0.3308, Mann-Whitney test; Figure 7— figure supplement 2). This result was consistent with the membrane capacitance (C ) measurement; there was no difference (15 ± 1.9 pF, n = 14 for control vs 15 ± 3.5 pF, n = 17 for Bdnf cKO, Figure 5F). In mature calyx synapses at P20, 2.7 ± 0.14 vesicles were located within 10 nm and 20.2 ± 0.73 vesicles were within 200 nm of the active zone of the calyx terminal in the control Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 11 of 26 Research article Neuroscience A B Immature calyx synapse (P10-12) Mature calyx synapse (P16-20) Control Control 1 nA 1 nA 20 ms 20 ms Bdnf cKO Bdnf cKO C D *** E F 8 1.0 10 1.0 8 0.8 0.8 0.6 6 0.6 0.4 4 0.4 0.2 0.2 0 0.0 0 0.0 Control cKO Control cKO Control cKO Control cKO Immature calyx synapse (P10-12) Mature calyx synapse (P16-20) G H I J *** 0.5 30 0.5 6 50 0.4 0.4 5 5 20 40 0.3 0.3 3 0.2 3 0.2 10 20 2 2 0.1 0.1 0.0 0 0 0 0.0 0 10 20 30 40 50 60 0 20 40 60 80 100 Control cKO Control cKO Control cKO Control cKO Cumulative eEPSC (nA) Cumulative eEPSC (nA) Figure 6. Oligodendroglial BDNF critically regulates glutamatergic transmission in the MNTB. (A, B) Representative traces of EPSCs evoked by paired- pulse stimulation from immature calyx synapse (at P10-12, A) and mature calyx synapse (at P16-20, B) in control (black) and Bdnf cKO (red) mice. (C–F) Summary of the amplitude of EPSCs and the PPR from immature calyx synapses (C, D) and mature calyx synapses (E, F). (G–H) Using the EQ method, plot of eEPSC amplitudes against the amplitude of the cumulative eEPSC in immature calyx synapses from control (black) and Bdnf cKO (red) mice. Right: Summary of the RRP size, which was estimated by back-extrapolated linear fits to the x axis. (G) Summary of the release probability (P , H). (I–J) Summary of the RRP size (I) and the P (J) in mature calyx synapses from control (black) and Bdnf cKO (red) mice. Data are shown as the mean ± s.e.m. *p<0.05; ***p<0.001 (unpaired t-test; paired t-test). Figure 6 continued on next page Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 12 of 26 eEPSC (nA) Amplitude (nA) (nA) PPR Pr eEPSC (nA) Amplitude (nA) (nA) PPR Pr Research article Neuroscience Figure 6 continued DOI: https://doi.org/10.7554/eLife.42156.010 The following figure supplement is available for figure 6: Figure supplement 1. RRP and replenishment rate of calyces in cKO. DOI: https://doi.org/10.7554/eLife.42156.011 (counted in 166 active zones from four cells; Figure 7A,C). In Bdnf cKO mice, 1.0 ± 0.12 and 19.1 ± 0.75 vesicles were located within 10 nm and 200 nm of the active zone, respectively, (196 active zones from five individual cells; <10 nm, p<0.0001; <200 nm, p=0.26, unpaired t-test; Figure 7C). Thus, the number of docked vesicles was significantly decreased in both immature and mature calyces in the Bdnf cKO mice. These anatomical changes in presynaptic terminals strongly indicate that oligodendroglial BDNF signaling is important for determining the RRP and specifically for mobilizing glutamate vesicles at the presynaptic terminal during postnatal development. We next tested whether activation of presynaptic TrkB using an agonist can recover the reduced number of docked vesicles at the active zone in Bdnf cKO mice. Auditory brainstem slices from con- trol and Bdnf cKO mice (at P20) were prepared for EM imaging after 30 min pre-treatment with 7,8- DHF (20 mM) as described in Figure 4. Application of 7,8-DHF recovered the reduced number of docked vesicles to 2.2 ± 0.22 within 10 nm of the active zone (63 active zones from three individual cells). There was no change in the number of docked vesicles within 200 nm of the active zone (Figure 7A,C). This result indicates that activation of BDNF-TrkB signaling rescues the docking defect or impaired mobilization of vesicles at the active zone, resulting in recovery of the reduced RRP in Bdnf cKO mice. Oligodendroglial BDNF and presynaptic BDNF–TrkB signaling We next tested whether extracellular application of BDNF or 7,8-DHF can recover the impaired glu- tamate vesicle release at presynaptic terminals in Bdnf cKO mice. The pre-application of BDNF (100 ng/ml) to brainstem slices for 30 min increased DC in response to depolarizing pulses at the calyx terminal from Bdnf cKO mice. After 20 ms depolarizing pulses, DC was much larger at calyces after BDNF application (200 ± 12.72 fF, n = 5) relative to untreated terminals from Bdnf cKO mice (93.8 ± 23.51 fF, n = 13; p=0.04, unpaired t-test; Figure 8A,B). There were no corresponding changes in QI in treated and untreated terminals (for 20 ms pulses, 5.1 ± 0.47 pC/pF, n = 13 vs Ca 5.8 ± 0.65 pC/pF, n = 5, respectively; p=0.42, unpaired t-test; Figure 8B). Interestingly, the applica- tion of BDNF had no effect on DC and QI in control calyces with a normal RRP (Figure 8—figure m Ca supplement 1). In addition, the direct activation of TrkB also rescued the impaired RRP and gluta- mate release at the calyx terminal in Bdnf cKO mice. After 20 ms depolarization pulses, DC was much larger at calyces in the presence of 7,8-DHF as compared with those from Bdnf cKO without the 7,8-DHF application (177.6 ± 12.72 fF, n = 5 vs 93.8 ± 23.51 fF, n = 13, respectively; p=0.04, unpaired t-test; Figure 8A,B). There was no change in the QI in the presence of 7,8-DHF (for 20 Ca ms pulses, 5.1 ± 0.47 pC/pF, n = 13; p=0.42, unpaired t-test; Figure 8B). Furthermore, the SDC induced by 20 pulses of 10 ms depolarization at 10 Hz was significantly increased by ~100% in the presence of 7,8-DHF in the Bdnf cKO (107 ± 19.14 fF, n = 10 in the absence of 7,8-DHF vs 328 ± 60 fF, n = 7 in the presence of 7,8-DHF; p=0.001, unpaired t-test; Figure 8C–E). The extracellular BDNF application also partially restored the SDC to 399 ± 120 fF in the Bdnf cKO (n = 6; p=0.008, unpaired t-test; Figure 8E). Thus, the activation of BDNF–TrkB signaling by the application of BDNF or 7,8-DHF partially recovered the impaired RRP and exocytosis at the presynaptic terminal in the Bdnf cKO. These findings suggest that oligodendrocyte-derived BDNF activates presynaptic TrkB signaling, which modulates the RRP and enhances glutamatergic transmission in the MNTB during postnatal development. Oligodendroglial BDNF and auditory functions To assess how loss of oligodendroglial BDNF and subsequent synaptic dysfunction influence audi- tory functions along the central auditory system, we measured auditory brainstem responses (ABRs), which represent the summed synchronized activity of neurons in the auditory pathway (Kim et al., 2013), in control and Bdnf cKO mice (P20–25). In both, the ABR waveform consisted of five distinct Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 13 of 26 Research article Neuroscience Mature calyx synapse Control cKO cKO + 7,8 DHF * * B C Immature calyx synapse Mature calyx synapse Docked vesicles Docked vesicles Clustered vesicles *** *** 8 50 1 2 0 0 Control cKO Control cKO cKO Control cKO cKO + 7,8 DHF + 7,8 DHF Figure 7. Loss of oligodendroglial BDNF reduces the number of docked vesicles at active zones of the calyx terminals. (A) EM images of the calyx terminal in the MNTB in control (left) and cKO (middle), and 7,8-DHF treatment on cKO mice (right) at P20. Higher magnification of a presynaptic terminal showed the active zones and synaptic vesicles. The active zones are the dense and dark sites in contact with the MNTB cell membrane (white arrows). Yellow asterisks indicate the docked vesicles within 10 nm of the active zone. The clustered vesicles were located within 200 nm of the active zones. Scale bars, 100 nm. (B) Summary of the number of docked vesicles in immature calyx terminals from control (black) and cKO (red) mice at P10. (C) Summary of the number of docked vesicles (left) and clustered vesicles (right) at active zones for mature calyx terminals from control (black), cKO (red), and 7,8-DHF treatment on cKO mice (blue) at P20. Data are shown as the mean ± s.e.m. *p<0.05; ***p<0.001 (unpaired t-test). DOI: https://doi.org/10.7554/eLife.42156.012 The following figure supplements are available for figure 7: Figure supplement 1. EM image of the immature calyx terminals in the MNTB in control and cKO mice at P10. DOI: https://doi.org/10.7554/eLife.42156.013 Figure supplement 2. 3D reconstructions of the calyx terminal show reduced terminal volume in Bdnf cKO mice. DOI: https://doi.org/10.7554/eLife.42156.014 peaks (herein referred to as waves I–V) during the 6 ms following a click stimulus and each wave cor- responds to electrical responses from the auditory nerve (wave I) and the ascending auditory path- way (e.g. cochlea nucleus, the superior olivary complex, lateral lemniscus, and inferior colliculus; wave II-V). There was no difference in the threshold of ABRs in response to click stimulation in con- trol and Bdnf cKO mice (42.8 ± 2.39 dB vs 42.8 ± 3.04 dB, n = 19 vs 14, respectively; Figure 9A,B). Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 14 of 26 Vesicle number Vesicle number Vesicle number 100 fF 100 fF Research article Neuroscience A B cKO +BDNF +7,8-DHF +7,8-DHF cKO +BDNF 0 0 50 ms 0 5 10 15 20 0 5 10 15 20 Duration(ms) Duration(ms) +7,8-DHF C E +BDNF cKO cKO ** +7,8-DHF +7,8-DHF ** Y6 Y6 0.5 s Control cKO 0 0.5 1 1.5 2 Time (sec) Figure 8. Application of extracellular BDNF or 7,8-DHF partially rescues the reduced exocytosis at calyx terminals in Bdnf cKO mice. (A) Representative traces of C (top) and I (middle) induced by 20 ms depolarization from –80 to 0 mV (bottom) at calyx terminals in Bdnf cKO mice (P9–13, red) in the m Ca presence of BDNF (100 ng/ml; green) or 7,8-DHF (20 mM; blue). (B) The duration of depolarizing pulses was plotted versus DC (left) and QI (right) for m Ca terminals from Bdnf cKO slices in the absence (red) and the presence of BDNF (green) or 7,8-DHF (blue). (C) Representative traces of C (top) induced by the train of 20 depolarizing pulses (10 ms, 10 Hz; bottom) from –80 to 0 mV in terminals from Bdnf cKO mice in the absence (red) or presence of 7,8- DHF (blue). (D) Summary of the normalized SDC relative to the stimulation time in the absence (red) or presence of 7,8-DHF (blue). (E) Summary of SDC of calyx terminals after the train of 20 depolarizing pulses (at 2 s) in the control slices (black) and in Bdnf cKO slices in the absence (red) and in the presence of BDNF (green) or 7,8-DHF (blue). Data are shown as the mean ± s.e.m. *p<0.05; **p<0.01 (unpaired t-test). DOI: https://doi.org/10.7554/eLife.42156.015 The following figure supplement is available for figure 8: Figure supplement 1. BDNF application does not affect presynaptic I and exocytosis at the calyx of terminal in control. Ca DOI: https://doi.org/10.7554/eLife.42156.016 In addition, the latency of wave I, and the time difference between wave I and wave IV, indicating central conduction, did not show significant difference in Bdnf cKO mice. We did not observe a sig- nificant difference in the amplitude of wave I, whereas the amplitudes of ABR waves II–IV were signif- icantly reduced in Bdnf cKO mice (Figure 9A,B). In particular, the amplitude of wave III, which reflects the summed neuronal activities of the superior olivary complex, was significantly reduced in the range of click intensities from 55 dB to 85 dB in Bdnf cKO mice (at 75 dB, 2.6 ± 0.19 mV, n = 21 for control and 1.7 ± 0.16 mV, n = 16 for the Bdnf cKO; p=0.002, unpaired t-test; Figure 9B). There was no significant difference in the latency of wave I, indicating peripheral conduction, and in central conduction, which was estimated by the time difference between wave IV and wave II (Figure 9B). These ABRs indicate that neuronal activity and synaptic synchrony in central auditory nuclei are impaired in Bdnf cKO mice. Taken together, the ABRs suggest that endogenous oligodendroglial BDNF regulates the synchrony of synaptic activities and critically influences auditory transmission during postnatal development. Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 15 of 26 Cm (%) Cm (fF) Cm (fF) QI (pC/pF) Ca 1 V Research article Neuroscience A B Wave I Wave II C Latency I 2.0 1.2 I II III IV V 1.0 1.5 Control 0.8 1.0 0.6 0.4 0.5 0.2 0.0 0.0 Control cKO Control cKO Control cKO Wave III Wave IV Latency IV-II * 2.5 cKO 4.5 2.0 ** 4.0 3.5 1.5 2.0 3.0 2.5 1.0 2.0 1.5 1.5 0.5 1.0 0.5 1 ms 1.0 0.0 0.0 Control cKO Control cKO Control cKO Figure 9. The absence of oligodendroglial BDNF impairs the auditory function of Bdnf cKO mice. (A) Examples of the ABRs in a control (black) and a Bdnf cKO mouse (red, both at P25), were recorded in response to a click stimulus of sound (75 dB). Roman numerals indicate peak waves I to V. (B) Summary of the amplitude of waves I to IV in response to click stimulus (75 dB), and the latency of wave I and the latency between wave II and IV in control (black) and Bdnf cKO mice (red). Data are shown as the mean ± s.e.m. *p<0.05; **p<0.01 (unpaired t-test). DOI: https://doi.org/10.7554/eLife.42156.017 Discussion Several forms of cell–cell communication influence synapse formation and pruning. In particular, glial cells are actively involved in synaptic pruning or refinement either during development or in response to brain injury (Chung and Barres, 2012; Karimi-Abdolrezaee and Billakanti, 2012; Schafer et al., 2012). Notably, glial-secreted factors play a critical role in synaptic maturation (Parkhurst et al., 2013; Christopherson et al., 2005; Kucukdereli et al., 2011). BDNF, a neurotro- phic factor, is secreted from glial cells and involved in activity-dependent synaptic plasticity (Zhang and Poo, 2002; Lu, 2003). Oligodendrocytes are considered an important source of BDNF during early postnatal development (Byravan et al., 1994; Dai et al., 2003). Here, we found that oli- godendrocyte-derived BDNF is critical for determining the RRP size and exocytosis of glutamate vesicles at the presynaptic terminal in the developing brainstem. BDNF–TrkB signaling at the presynaptic terminal +/ In this study, we found that a reduction in endogenous BDNF in Bdnf and Bdnf cKO mice 2+ channel activation. The impaired glutamatergic transmission without altering presynaptic Ca results are comparable to a previous study in the inner ear showing the deletion of endogenous 2+ BDNF significantly reduced exocytosis of glutamate vesicles but did not affect Ca currents in cochlear hair cells of mice (Zuccotti et al., 2012). Furthermore, we found that the application of BDNF or a TrkB agonist (7,8-DHF, 20 mM) led to partial recovery of the reduction in the RRP and in +/ exocytosis of vesicular glutamate in Bdnf and Bdnf cKO, but there was no significant effect in WT or control mice (Figures 7 and 8, Figure 8—figure supplement 1). However, a previous study in the MNTB of the rat brainstem showed that exogenous BDNF application reduces glutamate release by 2+ slowing down presynaptic Ca channel activation and inhibiting exocytosis and endocytosis (Baydyuk et al., 2015). These conflicting findings may result from the differences between species, ages, or BDNF application method. In particular, the timing of the BDNF signal induction may differ- entially modulate synapse function as either acute or chronic applications of BDNF can differentially modulate synaptic plasticity (Sherwood and Lo, 1999; Schildt et al., 2013; Guo et al., 2018). Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 16 of 26 Amplitude ( V) Amplitude ( V) Amplitude ( V) Amplitude ( V) Time (ms) Time (ms) Research article Neuroscience Exogenous administration of BDNF to brain slices has limitations. Depending on administration time, exogenous application could result in the non-specific binding effect of BDNF to presynaptic 2+ 2+ Ca channels, resulting in inhibition of Ca channel activation rather than through BDNF-TrkB sig- 2+ naling. Further studies are required to determine the effect of exogenous BDNF on Ca channel subtypes expressed in the presynaptic terminal and what aspects of BDNF signaling generate differ- ential responses. The mechanisms underlying the presynaptic effects of BDNF-TrkB signaling remain elusive. Acti- vation of TrkB leads to the induction of a combination of downstream signaling pathways, including the mitogen-activated protein kinase (MAPK), the PLC pathway, and the phosphatidylinositol 3- kinase (PI3K) pathway, that could modulate synaptic vesicles at the presynaptic terminal (Yoshii and Constantine-Paton, 2010; Reichardt, 2006). The acute and local effects of oligodendrocyte-derived 2+ BDNF on the RRP could be mediated by the increases of intracellular Ca levels, which may depend on the activation of the PLC pathway (Matsumoto et al., 2001; Reichardt, 2006). Recent studies 2+ demonstrated that BDNF-induced rise in intracellular Ca concentration at the presynaptic terminal 2+ was mediated by Ca influx through TRPC3 channels, resulting in a transient increase in spontane- 2+ ous glutamate release (Cheng et al., 2017), and/or release of Ca from intracellular stores (Amaral and Pozzo-Miller, 2012). Oligodendrocyte-derived BDNF in the MNTB of the auditory brainstem during early postnatal development It is important to identify the source of BDNF release at the synapse to understand how BDNF func- tions and acquires target specificity. BDNF increases by ~10-fold in the mouse CNS in the first 3 postnatal weeks (Kolbeck et al., 1999; Tao et al., 1998). Although the major source of BDNF in the adult brain appears to be neurons (Hofer et al., 1990; Rauskolb et al., 2010), BDNF is frequently detected in oligodendrocytes, astrocytes, and microglia in the developing brain (Dougherty et al., 2000). BDNF expression is observed in auditory brainstem nuclei in the mouse from P6, and its expression follows the protracted period of development in the auditory pathway, with expression beginning in the ventral cochlear nucleus and continuing to the MNTB and then to the medial supe- rior olive and the lateral superior olive (Wiechers et al., 1999; Hafidi, 1999). Glial cells may partici- pate in modulating synaptic structure and function during the development of the auditory circuitry by providing a permissive environment through the secretion of BDNF. Oligodendrocytes populate the MNTB prior to astrocytes, indicating oligodendrocytes have a primary role in the maturation of synapses during MNTB development. During the early postnatal weeks, oligodendrocytes are pres- ent throughout the auditory brainstem including the MNTB nuclei (as they were at birth), whereas GFAP-positive astrocytes appear in the MNTB during the second postnatal week (Dinh et al., 2014). Our immunostaining results, which are consistent with this previous study, showed that there is a greater oligodendrocyte population in the MNTB as compared with GFAP-positive astrocytes by P8 and that most oligodendrocytes are located in proximity to the calyx synapse (data not shown). This study demonstrated the presence of BDNF in oligodendrocytes in the MNTB of mouse brain- stem during early postnatal development. Oligodendrocytes expressed BDNF in the MNTB of the cre auditory brainstem (Figure 1A), and isolated O1+ or Cnp -driven GCaMP6f-GFP cells expressed a substantial amount of Bdnf mRNA (Figure 5—figure supplement 2). Previous study demonstrated that oligodendrocytes release BDNF in response to glutamate application (Bagayogo and Dreyfus, 2009). Oligodendrocyte processes contact the calyx terminal, which releases glutamate, before forming a myelin sheath during early development (Figure 5A). This suggests that glutamate-medi- ated signaling between oligodendrocytes and the calyx synapses induces BDNF release from oligo- dendrocytes to increase synaptic strength. Due to the biochemical nature of BDNF, it is thought to act locally at the synapse with limited diffusion within the micrometer range (Horch and Katz, 2002; Sasi et al., 2017). Oligodendrocytes likely exert a direct impact through the localized secretion of BDNF to the calyx synapses. The results demonstrate oligodendrocytes actively participate in bidi- rectional neuron–glia communication at the calyx synapse through BDNF-dependent signaling dur- ing early postnatal development. cre This study utilizes Cnp to generate a Bdnf deletion specifically in oligodendrocytes. A recent cre study reported that Cnp -driven YFP reporter signal was detected in 5.5% of NeuN+ neurons, sug- cre gesting a potential limitation on the specificity of recombination of Cnp mouse line cre (Tognatta et al., 2017). To address the specificity of Cnp in the MNTB, we have analyzed reporter Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 17 of 26 Research article Neuroscience expression and verified a specific reduction of Bdnf in isolated oligodendrocytes. Using two reporter lines, Rosa-GCaMP6f-GFP and Rosa-tdTomato, we identified that <5% of neurons in the MNTB cre expressed Cnp -driven reporter in early postnatal ages (P10- P20). In addition, using FACS, iso- lated GCaMP6f-GFP+ cell population contains high levels of Olig2 mRNA with very low levels of cre Kcc2 mRNA, a neuronal marker (Figure 5—figure supplement 2). The majority (>95%) of Cnp expressing cells in the MNTB are oligodendrocytes as shown through immunohistochemistry. We demonstrated that GCaMP6f-GFP+ cells have detectable Bdnf mRNA, which was significantly reduced in Bdnf cKO mice. There was no significant difference in the level of Bdnf mRNA in the GFP or an O1 fraction, which considered as a non-oligodendroglial population, although there was a trend toward lower Bdnf mRNA in the O1 or GFP fraction. There is the possibility that the cre small percentage of neurons, affected by Cnp , is sufficient to reduce global levels of Bdnf and impact on the synaptic phenotype in the cKO. In cultured neurons, the effect of BDNF within a syn- apse has been observed to occur within a distance of 4.5 mm (Horch and Katz, 2002). Thus, BDNF reduction in a small portion of neurons (<5%) is unlikely to have widespread effects or global impact on the synaptic phenotype observed in the cKO. We interpret that functional alterations of the calyx synapse were caused by the loss of BDNF in oligodendrocytes, which constitute the majority of CNP-expressing cells. Bidirectional signaling between oligodendrocytes and nerve terminals In cultured oligodendrocytes, the activation of glutamate receptors and the phospholipase C path- way enhances the release of dense-core vesicles containing BDNF (Bagayogo and Dreyfus, 2009), suggesting that release of BDNF from oligodendrocytes depends on neuronal activity and is medi- ated by neuron–oligodendrocyte interactions. Our recent study demonstrated that a sub-population of oligodendrocytes interacts with neurons via synapses and displays action potentials in response to intensive neuronal activities in the auditory brainstem (Berret et al., 2017). It is intriguing to specu- late that bidirectional signaling occurs between oligodendrocytes and nerve terminals at synapses, in which glutamatergic inputs from neurons trigger oligodendrocytes to release BDNF, and then oli- godendrocyte-derived BDNF binds to presynaptic TrkB, and finally modulates the glutamate vesicle pool at the nerve terminal. These findings indicate that oligodendrocytes may modulate synaptic plasticity in an activity-dependent manner. An increase in the RRP of vesicles could also contribute to short-term plasticity such as PTP (Habets and Borst, 2005; Regehr, 2012). We show that the +/– reduction of global BDNF significantly impairs the induction of PTP at the calyx synapse in Bdnf mice (Figure 2). Oligodendrocytes can regulate synaptic strength and plasticity at the calyx synapse by modulating the RRP size through BDNF signaling. Oligodendrocytes that are closely apposed to synapses thus monitor and sense synaptic activity and modulate synaptic plasticity at the presynaptic terminals. In the case of the calyx synapse, this occurs through BDNF–TrkB signaling, which may rep- resent an efficient way for oligodendrocytes to find active nerve terminals and to assist in maintain- ing synaptic activities. During this critical window of development, when activity-dependent synaptic refinement can occur along the auditory nervous system, oligodendrocytes actively participate in synaptic transmission and plasticity through BDNF signaling in the developing brain. Materials and methods Key resources table Reagent type (species) or Source or Additional resource Designation reference Identifiers information tm1Jae Genetic reagent B6.129S4-Bdnf /J The Jackson stock no.:002266 PMID:8139657 (M. musculus) Laboratory tm3Jae Genetic reagent Bdnf /J The Jackson stock no.:004339 Rios et al., 2001 (M. musculus) Laboratory Cre Genetic reagent Cnp mice Dr. Klaus Nave MGI no: 3051635 Lappe-Siefke (M. musculus) (Max Planck et al., 2003 Institute) Continued on next page Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 18 of 26 Research article Neuroscience Continued Reagent type (species) or Source or Additional resource Designation reference Identifiers information Genetic reagent GCaMP6f The Jackson stock no.:024105 Dr. Paukert, (M. musculus) Laboratory UTHSCSA Genetic reagent tdTomato The Jackson stock no.:007909 Dr. Paukert, (M. musculus) Laboratory UTHSCSA Antibody Mouse monoclonal Millipore MAB5540 1:500 anti-Olig1 Antibody Mouse monoclonal Millipore MAB3418 1:200 anti-MAP2 Antibody Mouse monoclonal Millipore MAB377 1:200 anti-NeuN Antibody Rabbit polyclonal DAKO Z033429 1:500 anti-GFAP Antibody Mouse monoclonal Millipore OP80 1:200 anti-CC1 Antibody Rabbit monoclonal Abcam 109186 1:100 anti-Olig2 Antibody Rat monoclonal Abcam AB90967 1:300 anti-PDGFRa Antibody Rabbit polyclonal Bioss BS4989R 1:100 anti-BDNF Antibody Mouse monoclonal Santa Cruz sc-136990 1:50 anti-TrkB Antibody Guinea pig Millipore AB5905 1:1000 polyclonal anti-VGluT1 Chemical 7,8-Dihydroxyflavone Sigma D5446 20 mM compound, drug (7,8-DHF) Chemical BDNF Millipore GF301 100 ng/ml compound, drug Chemical TEA-Cl Sigma T2265 10 mM compound, drug Chemical 4-AP Sigma 275875 0.1 mM compound, drug Chemical TTX TOCRIS 1078 1 mM compound, drug Chemical QX314 bromide TOCRIS 1014 4 mM compound, drug Chemical Bicuculline TOCRIS 130 10 mM compound, drug Chemical Strychnine Sigma S8753 2 mM compound, drug Animals All animal procedures were performed in accordance with the guidelines approved by the University of Texas Health Science Center, San Antonio (UTHSCSA) Institutional Animal Care and Use Commit- +/ +/ tee protocols. BDNF heterozygous (Bdnf ) mice were generated by crossing Bdnf mice tm1Jae (B6.129S4-Bdnf /J; The Jackson Laboratory) with WT mice (C57B[L]6/J). The offspring were gen- otyped with a standard PCR assay. Primer sequences were as follows: forward, 5’-ATGCGTACC TGACTTTCTCCTTCT-3’; reverse, 5’-ACTGGGTGCTCAGGTACTGGTTGT-3’, which amplify a 280 bp +/– and 350 bp fragment for Bdnf mice and a 280 bp fragment for WT. cre fl/fl fl/fl To create the cKO mice (Cnp : Bdnf ), mice carrying the floxed allele of Bdnf (Bdnf ; The cre Jackson Laboratory; Rios et al., 2001) were crossed to Cnp heterozygous mice (Lappe- Siefke et al., 2003). The constitutive KO allele is obtained after Cre-mediated recombination by Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 19 of 26 Research article Neuroscience cre fl/fl crossing Cnp mice with Bdnf mice to obtain the deletion of Bdnf only in CNPase-expressing cells (Lappe-Siefke et al., 2003; Rios et al., 2001). Genotypes of all mice were determined by PCR fl/fl analysis of tail genomic DNA using the appropriate primers: for Bdnf , forward, 5’-TGTGATTGTG TTTCTGGTGAC-3’ and reverse, 5’-GCCTTCATGCAACCGAAGTATG-3’, which amplifies a 487 bp cre (floxed Bdnf allele) and a 437 bp (Bdnf WT allele) fragment; for Cnp , forward, 5’-GCCACACA TTCCTGCCCAAGCTC-3’ and reverse 1, 5’-GTCGCCACGCTGTCTTGGGCTCC-3’, and reverse 2, 5’- cre CTCCCACCGTCAGTACGTGAGAT-3’, which amplifies a 400 bp (Cnp WT allele) and 550 bp (Cnp cre fl/+ allele) fragment. Control mice (Cnp : Bdnf ) were identified by PCR amplification of a 400 bp, cre fl/fl 437 bp, and 487 bp fragment, whereas Bdnf cKO mice (Cnp : Bdnf ) were identified by PCR amplification of a 400 bp, 550 bp, and 487 bp fragment. Recombination efficiency in oligodendro- cre cytes in the Cnp mice was determined by transgenic crosses to the GCaMP6f reporter mouse (provided by Dr. Paukert, UTHSCSA or purchased from Jackson Laboratory). All mice were housed in the institutional animal facilities on a 12 hr light/dark cycle. Mice of both sexes aged P8–25 were used for all experiments. Slice preparation Transverse brainstem slices containing the MNTB were prepared from P9–18 mouse pups. After rapid decapitation of the mice, the brains were quickly removed from the skull and immediately immersed in ice-cold low-calcium artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 3 MgCl , 0.1 CaCl , 25 glucose, 25 NaHCO , 1.25 NaH PO , 0.4 ascorbic acid, three myo- 2 2 3 2 4 inositol, and 2 Na-pyruvate, pH 7.3–7.4 when bubbled with carbogen (95% O , 5% CO ; osmolarity 2 2 of 310–320 mOsm). Then, 200-mm-thick sections were collected using a Vibratome (VT1200S, Leica, Germany). Slices were incubated in a chamber that contained normal aCSF bubbled with carbogen at 35˚C for 30 min and then were kept at room temperature. The normal aCSF was the same as the low-calcium aCSF, except that 3 mM MgCl and 0.1 mM CaCl were replaced with 1 mM MgCl and 2 2 2 2 mM CaCl . Electrophysiology Whole-cell patch-clamp recording was carried out on postsynaptic principal neurons and presynaptic calyx of Held terminals in the MNTB using an EPC-10 amplifier controlled by PATCHMASTER soft- ware (HEKA Elektronik, Lambrecht/Pfalz, Germany). Slices were visualized using an infrared differen- tial interference contrast microscope (AxoExaminer, Zeiss, Oberkochen Germany) with a 63 water immersion objective and a CMOS camera (Hamamatsu Photonics, Hamamatsu, Japan). During experiments, slices were perfused with normal aCSF solution at 2 ml/min at room temperature. Presynaptic recording 2+ To measure presynaptic Ca currents (I ) and changes in membrane capacitance (DC ), the boro- Ca m silicate glass pipettes were filled with a solution containing the following (in mM): 130 Cs-methane- sulfonate, 10 CsCl, five sodium phosphocreatine, 10 HEPES, 0.05 BAPTA, 10 TEA-Cl, 4 Mg-ATP, and 0.3 GTP, pH adjusted to 7.3 with CsOH. When filled with the intracellular solution, the pipettes had an open pipette resistance of 4–6 MW. Series resistance was <20 MW before compensation and <10 2+ MW with compensation. Presynaptic Ca currents were analyzed after leak subtraction using a ‘tra- ditional’ p/4 stimulus train in the EPC10-Patchmaster. For identification and morphological analyses, intracellular solutions were supplemented with 50 mM Alexa 568 (Life Technologies, USA). Extracellu- + + lar aCSF solution contained 10 mM TEA-Cl, 0.1 mM 4-AP, and 1 mM TTX to block K and Na chan- nels, respectively. Postsynaptic recording For recordings of eEPSCs, the pipettes were filled with a solution containing the following (in mM): 130 Cs-methanesulfonate, 10 CsCl, five sodium phosphocreatine, 10 HEPES, 5 EGTA, 10 TEA-Cl, 4 Mg-ATP, and 0.3 GTP, pH adjusted to 7.3 with CsOH. To this solution, we added 4 mM QX-314 bro- mide to block the voltage-activated Na current. Extracellular aCSF solution contained 10 mM bicu- culline and 2 mM strychnine to block GABA and glycine receptors, respectively. The holding potential was –70 mV in the voltage-clamp mode. Patch electrodes had resistances of 4–5 MW. Series resistance was <20 MW, with 80% compensation. Afferent fibers of the calyx of Held synapses Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 20 of 26 Research article Neuroscience were stimulated with a bipolar electrode (Frederic Haer, Bowdoinham, ME) placed near the midline of the MNTB. An Iso-Flex stimulator driven by a Master 10 pulse at 1.2-fold threshold (<15 V con- stant voltage) was used. Data were analyzed off-line and displayed with Igor Pro (Wavemetrics, Lake Oswego, OR). Differences were considered statistically significant when p-values were <0.05 by a Student’s t-test (GraphPad Prism, US). Data are shown as the mean ± s.e.m. Immunostaining Slices used for patch-clamp analysis or fresh brainstem slices (~200 mm thick) were fixed with 4% (w/ v) paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Free-floating slices were blocked in 4% goat serum and 0.3% (w/v) Triton X-100 in PBS for 1 hr and then were incubated with primary antibody overnight at 4˚C. The following primary antibodies were used: mouse anti-Olig1 (1:500; Millipore, MAB5540), mouse anti-MAP2 (1:200; Millipore, MAB3418), mouse anti-NeuN (1:200; Milli- pore, MAB377), rabbit anti-GFAP (1:500; DAKO, Z033429), mouse anti-CC1 (1:200, Millipore, OP80), mouse anti-NeuN (1:600, Millipore, MAB377), anti-Olig2 (1:100, Abcam, 109186), rat anti- PDGFRa (1:300, abcam, AB90967), rabbit anti-BDNF (1:100; Bioss, BS4989R), mouse anti-TrkB (1:50; Santa Cruz, sc-136990), and guinea pig anti-VGluT1 (1:1000; Millipore, AB5905). Tissues were then incubated with different Alexa-conjugated secondary antibodies (1:500; Invitrogen) for 2 hr at room temperature. After three rinses with PBS, slices were coverslipped using mounting medium with 4 ,6- diamidino-2-phenylindole (DAPI; Vectashield; Vector Laboratories) to counterstain cell nuclei. Stained slices were viewed on a confocal laser-scanning microscope (Zeiss LSM-510) at 488, 568, and 633 nm using 40 or 60 oil immersion objective. Transmission EM Animals were anesthetized and intracardially perfused with normal saline. Brains were removed and 400-mm-thick samples of brainstem MNTB area were dissected out followed by primary fixation in 1% glutaraldehyde/4% paraformaldehyde. Further processing was performed by the UTHSCSA Elec- tron Microscopy Lab. Briefly, each brainstem was post-fixed with 1% Zetterqvist’s buffered osmium tetroxide, dehydrated, and embedded in PolyBed resin at 80˚C in an oven. Tissue containing the MNTB, which is innervated by calyces of Held, was cut into 90 nm ultrathin sections and placed on copper grids. The sections were then stained with uranyl acetate and Reynold’s lead citrate. The samples were imaged on a JEOL 1400 electron microscope using Advanced Microscopy Techniques software. The calyx of Held terminals contacting cell bodies of MNTB principal neurons were recog- nizable as a cluster of cells located medially in the superior olivary complex (Taschenberger et al., 2002). A total of 166–196 synapses were analyzed from five animals for each group (control and Bdnf cKO). The number of docked vesicles per the active zone was measured for each synapse at a final magnification of 80,000. The active zone was defined as the dark presynaptic density contact- ing the postsynaptic density. Docked and clustered vesicles were defined as those within 10 nm and 200 nm of the presynaptic active zone, respectively (Satzler et al., 2002; Taschenberger et al., 2002). ABR recordings ABR recordings were performed as described (Kim et al., 2013). Briefly, mice were anesthetized with 4% isoflurane and maintained with 2% isoflurane during recording (1 l/min O flow rate). ABR recordings were carried out in a sound attenuation chamber (Med Associates, Albans, VT). Body temperature of mice was maintained at 37˚C using a heating pad. Subdermal needle electrodes for recording were placed on the top of the head (active), ipsilateral mastoid (reference), and contralat- eral mastoid (ground). The electrical potential differences between the vertex and the mastoid elec- trodes were amplified and filtered (100–5000 Hz), and a recording window of 10 ms starting at the onset of click sound stimulation was distally sampled at 40-ms intervals. Acoustic stimuli were gener- ated by the Auditory Evoked Potentials Workstation (Tucker-Davis Technologies [TDT], Alachua, FL). Closed-field click stimuli were delivered to the left ear using a series of square waves (0.1 ms dura- tion) through TDT Multi-Field Magnetic Speakers placed 10 cm away from the left ear canal. A repe- tition rate of sound stimuli of 16/s was transmitted through a 10 cm length of plastic tubing (Tygon; 3.2 mm outer diameter). Sound intensities ranged from 90 to 20 dB with 5 dB decrements and responses to 512 sweeps were averaged. The lowest sound intensity that produced a reproducible Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 21 of 26 Research article Neuroscience waveform was interpreted as the threshold. Free-field pure tone stimuli were taken at frequencies of 8, 12, 16, 24, and 32 kHz at 70 to 20 dB in decrements of 5 dB. Fluorescent activated cell sorting (FACS) Cre fl/fl Cre fl/+ Bdnf cKO mice (Cnp : Bdnf , n = 3) and control mice (Cnp : Bdnf , n = 2) were used for FACS Cre +/- Cre experiments. Cnp : Rosa-GCAMP6f-GFP (n = 3) and Cnp heterozygous controls (n = 3) were used. A cell suspension was generated from the brainstem using enzymatic (papain, 48 U/mL, P1325, Sigma) and mechanical titration. Dissected brainstem was incubated in dissociation media (145 mM NaCl, 5 mM KCl, 20 mM Hepes, 1 mM Na-pyruvate, 2 mM EDTA, pH 7.2) with papain and DNase (10 mM, 10104159001, Sigma) for 20 min. The tissue was spun down at 300 x g for 5 min and resuspended in 1 ml of dissociation media with DNase (10 mM). Mechanical titration was performed using a 1000 ul pipette tip followed by a 200 ul pipette tip. After dissociation, the suspension was fil- tered and spun down at 300x g for 7 min. The cells were resuspended in 400 ml of dissociation media. 50 ml were set aside for a no primary antibody control. 1 ml anti-O1 antibody (MAB1327, R and D systems) was added to the remaining 350 ml cell suspension (dilution 1:350) and cells were incubated on ice for 25 min. Cells were washed with 1 ml of dissociation media and spun at 300x g for 7 min. Cells were resuspendend in 400 ml dissociation solution with secondary antibody (Alexa 488 anti-mouse IgM, 1:500) and incubated for 25 min. Cells were washed with 1 ml of dissociation media, spun at 300x g for 7 min, and resuspended in 400 ml of dissociation solution. Sorting was per- formed on a BD FACSARIA III (BD Biosciences) in the Flow Cytometry Facility at UT Health San Anto- nio with funding from University and the NIH (NCI P30 CA054174). BD FACS Diva software 8.0.1 was used to visualize forward scatter and side scatter to determine cell population and perform dou- blet discrimination. 488-labeled O1+ cells were selected, yielding a population of 7,000–25,000 cells. Cre +/- In Cnp : Rosa-GCAMP6f-GFP mice, GFP+ cells produced a population of 50,000–75,000 cells. Negative cells were also collected with a total of 200,000–1,000,000 cells. Cells were kept on ice prior to RNA isolation. Quantitative polymerase chain reaction (qPCR) RNA isolation was performed using an RNAqueous kit with no modifications to procedure (AM1931, Thermofisher). This kit includes DNase treatment. RNA was quantified using a nanodrop (ND-1000, Thermofisher). Purity was assessed by A260/A280 ratios with values ranging from 1.83 to 1.98. 100 ng of RNA was utilized for each reverse transcription (RT) reaction. RT was performed using Super- script III First-strand synthesis with 1 ml Oligo (dT) primers, 1 ml 10 mM dNTP mix (180180–051, Invi- trogen). cDNA was stored at 20C for up to 48 hr. qPCR was performed using the PowerUp SYBR green master mix (A25742, Thermofisher). Primers were used at 0.25 mM. Reactions were manually loaded into optical plates with covers (plates:4309849, covers:4360954, Applied Biosystems). The plate was spun for 30 s. qPCR was performed using 7900HT Fast Real-Time PCR system (Applied Biosystems), data was analyzed using SDS v2.4 (Applied Biosystems) and the determined CT was used for analysis. No outliers were removed. No template controls (NTC) as well as no RT controls did not produce determinable CT values. Gapdh was chosen as a reference. Technical replicates were done in triplicate. Delta CT (mean – mean ) was used to determine Delta Delta CT Gene GAPDH (Delta CT -Delta CT ) or (Delta CT -Delta CT ), relative gene expression was calculated 2^(- Pos Neg cko CTL deltadeltaCT) and normalized to 100%. Mouse primers used follows: Olig2 Forward: 5’-CAAATCTAATTCACATTCGGAAGGTTG Olig2 Reverse: 5’-GACGATGGGCGACTAGACACC Kcc2 Forward: 5’-GGGCAGAGAGTACGATGGC Kcc2 Reverse: 5’-TGGGGTAGGTTGGTGTAGTTG BDNF Forward: 5’-TCGTTCCTTTCGAGTTAGCC BDNF Reverse: 5’-TTGGTAAACGGCACAAAAC GAPDH Forward: 5’-AGTATGACTCCACTCACGGCAA GAPDH Reverse: 5’-TCTCGCTCCTGGAAGATGGT Statistics All statistical analyses were performed in GraphPad Prism. For electrophysiology, the n equals the number of individual whole-cell recordings. All electrophysiological experiments were performed in Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 22 of 26 Research article Neuroscience at least seven independent slices from at least seven individual animals. The in vivo ABR test was performed in at least 20 individual controls and at least 15 individual Bdnf cKO mice. Data were ana- lyzed off-line and displayed with Igor Pro (Wavemetrics, Lake Oswego, OR). a values were set to 0.05, and all comparisons were two-tailed. To compare two groups, unpaired t-test or Mann-Whit- ney U test was carried out. Differences were considered statistically significant when p-values were <0.05 by a Student’s t-test or Mann-Whitney U test (GraphPad Prism). Data are shown as the mean ± standard error of the mean (s.e.m.) Data availability The authors declare that all data generated or analyzed in this study are available within the article. Acknowledgements cre We would like to thank Drs. Klaus Nave and Manzoor Bhat for providing the Cnp mouse line. This work was supported by a grant from the National Institute on Deafness and Other Communication Disorders (NIDCD; R01 DC03157) to J H Kim. Additional information Funding Funder Grant reference number Author National Institute on Deafness R01 DC03157 Jun Hee Kim and Other Communication Disorders The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Miae Jang, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft; Elizabeth Gould, Data curation, Validation, Visualization; Jie Xu, Data curation, Formal analysis, Validation, Investigation; Eun Jung Kim, Data curation, Formal analysis, Validation, Investigation, Visualization; Jun Hee Kim, Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing Author ORCIDs Jun Hee Kim http://orcid.org/0000-0003-0207-8410 Ethics Animal experimentation: All animal procedures were performed in accordance with the guidelines approved by the University of Texas Health Science Center, San Antonio (UTHSCSA) Institutional Animal Care and Use Committee protocols (#140045x). Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.42156.020 Author response https://doi.org/10.7554/eLife.42156.021 Additional files Supplementary files Transparent reporting form DOI: https://doi.org/10.7554/eLife.42156.018 Jang et al. eLife 2019;8:e42156. DOI: https://doi.org/10.7554/eLife.42156 23 of 26 Research article Neuroscience Data availability All data generated or analysed during this study are included in the manuscript and supporting files. References Alderson RF, Alterman AL, Barde YA, Lindsay RM. 1990. Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 5:297–306. DOI: https://doi.org/10. 1016/0896-6273(90)90166-D, PMID: 2169269 Ca2+ Ca2+ Amaral MD, Pozzo-Miller L. 2012. Intracellular stores and influx are both required for BDNF to rapidly increase quantal vesicular transmitter release. Neural Plasticity 2012:203536. 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Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem

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Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem

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Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem

Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem

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